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https://f1000research.com/articles/4-445/v1
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31 Jul 15
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{
"type": "Research Article",
"title": "Sex-difference affects disease progression in the MRMT-1 model of cancer-induced bone pain",
"authors": [
"Sarah Falk",
"Tamara Al-Dihaissy",
"Laura Mezzanotte",
"Anne-Marie Heegaard",
"Tamara Al-Dihaissy",
"Laura Mezzanotte",
"Anne-Marie Heegaard"
],
"abstract": "An overwhelming amount of evidence demonstrates sex-induced variation in pain processing, and has thus increased the focus on sex as an essential parameter for optimization of in vivo models in pain research. Mammary cancer cells are often used to model metastatic bone pain in vivo, and are commonly used in both males and females. Here we demonstrate that compared to males, females have an increased capacity for recovery following inoculation of MRMT-1 mammary cells, thus potentially causing a sex-dependent bias of the progression of the pain state.",
"keywords": [
"Cancer-induced bone pain",
"sex differences",
"in vivo model",
"x-ray",
"pain behavior",
"bioluminescence"
],
"content": "Introduction\n\nA crucial step in translational research is development of animal models that can accurately mimic the human condition in relation to both symptoms and underlying mechanisms1,2. To understand the molecular mechanism of complex human disease, and thereby create disease-specific treatment, the models need to be reproducible and with a high predictive value, requiring a detailed knowledge of the expected variation in the models used. Cancer-induced bone pain is a highly complex pain state involving cancer cells, bone cells, immune cells as well as neuronal and non-neuronal processing in the periphery and at spinal and supraspinal levels3. Numerous studies have reported on variation in the more than 45 animal models that have been used to model the pain state4. The most common variations are related to cell lines, species or injection site used4, and in addition we have previously demonstrated that sex does also affect the model5, thereby emphasizing the need to consider sex bias.\n\nThe majority of preclinical pain research is still performed using male animals, despite an overwhelming amount of both human and animal data demonstrating significant sex variations6–8. Conversely, within the field of bone cancer pain, preclinical researchers have had more focus on the issue, likely due to a more intuitive choice of sex, based on the origin of the cancer cells used. A meta-analysis of studies on cancer-induced bone pain demonstrated that 49% of the studies used males, 22% used females and 29% did not report the sex of the animals used4. These numbers are indeed more balanced than the numbers previously reported, revealing that 79% of all animal studies published in PAIN from 1996 to 2005 were using male animals, whereas only 8% used females8. However, in this study we demonstrate that not only is sex an integral factor to be considered when choosing an animal model, intrinsic sex-dependent variation has to be carefully analyzed in order to avoid bias. The data presented in this study suggest, that in the MRMT-1-Luc2 carcinoma cell model of metastatic bone cancer pain, a sex-dependent bias related to recovery is skewing the behavioral responses observed in females to a greater extent compared to males, potentially masking effects in studies where female animals are used to model the pain state.\n\n\nMethods\n\nExperiments were performed on 10 male and 10 female Sprague-Dawley rats (Taconic M&B, Denmark) group-housed in cages under a 12-h alternating light/dark cycle with ad libitum access to food and water. In addition, unpublished data from four previous independent in-house experiments were analyzed; these included a total of 16 females and 42 males, and were performed as described in this method section (see Figure 4C). The only difference with respect to methods is that three experiments were performed prior to transfection of the MRMT-1 cells line, and this study and one of the prior experiments were conducted after transfection of the MRMT-1 cells line. For all animals bodyweight and general health was monitored throughout the studies. All experiments were approved by the Danish Animal Experiments Inspectorate under the Danish Ministry of Food, Agriculture and Fisheries (2014-15-0201-00031), and were carried out in accordance to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain9.\n\nGeneration of MRMT1 cells expressing reporter proteins was achieved by genomic integration of the Luc2-copGFP reporter gene construct using lentiviral transduction. The lentiviral vector contains the EF1α promoter sequence driving the equimolar expression of a codon-optimized firefly luciferase (Luc2) gene and a copepod green fluorescent protein (copGFP) thanks to the presence of a T2A sequence. Vector production and cell transduction were performed under appropriate biosafety level conditions (ML-II) in accordance with the National Biosafety Guidelines and Regulations for Research on Genetically Modified Organisms. Procedures and protocols were reviewed and approved by the LUMC Biosafety Committee (GMO permit 08-129). Vector production and transduction procedures have been described in detail elsewhere10. In brief, cells were seeded in 24-well plate at a cell density of 7.5×104 cells/well and maintained in a humidified incubator at 37°C and 5% CO2. After attachment was accomplished, the cells were transduced using a MOI (multiplicity of infection) of 1. Subsequently cells were FACS sorted for high expression of copGFP and cell culture was expanded for experiments.\n\nSyngeneic MRMT1-Luc2 rat mammary gland carcinoma cells (Leiden University medical Center, The Nederlands) were cultured in RPMI 1640 medium without phenol red (Invitrogen, Paisley, UK/Nærum, Denmark) supplemented with 10% heat-inactivated foetal bovine serum, 1% L-glutamine and 2% penicillin/streptomycin (Invitrogen, Paisley, UK/Nærum, Denmark). On the day of surgery, MRMT1-Luc2 carcinoma cells were released by brief exposure to 0.1% w/v trypsin-EDTA and centrifuged in medium for 3 min at 1200rpm. The pellet was washed twice with Hanks’ balanced salt solution (HBSS) without calcium, magnesium or phenol red (Invitrogen, Paisley, UK/Nærum, Denmark) and centrifuged for 3 min at 1200rpm. Cells were re-suspended in HBSS and kept on ice until use.\n\nThe inoculation of carcinoma cells was performed modified from 11 as previously described12. Hence, following induction of isoflurane anaesthesia (induction 4%, maintenance 1.5–2%) the animal was placed with the abdominal side up. A small incision was made in a shaved and disinfected area on the anterior-medial surface of the tibia and the tibia carefully exposed. A hole was made in the tibia with a 0,7mm dental drill through which a thin polyethylene tube was fed 1cm into the proximal intramedullary cavity. Animals were injected with 5×103 MRMT1-Luc2 carcinoma cells in 10µl HBSS. Following removal of the tube, the hole was plugged with bone restorative material (IRM, Dentsply, Surrey, UK/Vallensbæk, Denmark). The wound was irrigated with saline and closed with two metal clips. The animals were placed under a heat source until fully awake. Postoperative analgesia was administrated by injection of Rimadyl (s.c. 5mg/kg, Pfizer, Denmark) and application of 2% Lidocaine gel (AstraZeneca, Denmark) to the wound.\n\nBehavioral responses were assessed prior to surgery and on day 7, 10, 14, 17, 21 and 23. All animals were introduced to all behavioral tests 2–3 times prior to the start of the experiment. Animals reaching humane endpoint before day 23 (n=7) (predefined as limb use score 0) were euthanized by decapitation following brief exposure to isoflurane, and the behavioral scores from the last day carried forward for data analysis.\n\nVon Frey test. Mechanical hypersensitivity, detected by von Frey filaments (North Coast Medicinal, Inc., Camino Arroyo, Gilroy, CA, USA), was used as an indicator of early pain behavior. The threshold was determined by the up and down method, as described previously13. Briefly, rats were placed in acrylic enclosures on a wire mesh floor and allowed to acclimatize for minimum 30 min. Starting at 6 g, filaments ranging from 0.4–26 g were applied to the plantar surface of the hind paw with a minimum of 3 min intervals between two stimulations. A stimulus was recorded as positive if paw withdrawal was observed within 3 s of stimulation with a given filament; if no response was observed it was recorded as negative. Following a positive response, the next stimulation was performed with a filament with a decreased bending strength, whereas a negative response was followed with stimulation with a filament with increased bending strength. According to the original protocol, optimal threshold calculation by this method requires 6 responses in the immediate vicinity of the 50% threshold, therefore recording of the 6 data points did not begin until the response threshold was first crossed (positive response changes to negative response or inverse). The 2 responses detecting the threshold were designated as the first 2 responses of the series of 6. In cases where continuous negative responses were observed to the maximum of the stimulus set, a value of 26 g was set as the cut-off and used as withdrawal threshold for data analysis. In all other cases the resulting pattern of positive and negative responses was calculated into a 50% response withdrawal threshold using the formula: 50% g threshold = (10Xf+kδ)/(10,000), where Xf = value (log unit) of the final von Frey hair used; k = tabular value for the pattern of positive/negative responses; and δ = mean difference (in log units) between filaments.\n\nWeight-bearing test. Rats were placed in the incapacitance tester (MJS Technology Ltd., Buntingford, Herfordshire, UK) and allowed to acclimatize until calm. Measurements were performed over a period of 4 s and in triplicates. An average weight-bearing ratio was subsequently calculated as the amount of weight placed on the cancer-bearing leg divided by the total amount of weight placed on both legs and used for data analysis.\n\nLimb use test. Rats were allowed to move freely in a transparent plastic cage without bedding (500 mm × 300 mm × 500 mm). Following 10 min of acclimation, each animal was observed for 3 min and assigned a limb use score from 3 to 0 as follows: 3: Normal use of leg, 2: mild or insignificant limping, 1: significant limping, 0: significant limping and part lack of use.\n\nAnimals were anesthetized in an induction chamber with 2.5% isoflurane (Isobar Vet; 100%, Nomeco, Copenhagen, DK). D-Luciferin (PerkinElmer, Skovlunde, Denmark) dissolved in PBS was administered by intraperitoneal injection (40mg/kg). 10 min after injection, animals were placed on their back in a nose cone in a Lumina XR instrument (Caliper Life Sciences, Teralfene, Belgium) and anesthesia was maintained with a 2.5% isoflurane/oxygen mix. Image capture was performed with binning: M(4), F/stop: 1 and exposure time from 1 s to 60 s according to the power signal. The signal was adjusted according to the exposure time prior to data analysis. For each animal, an average of three images was used for analysis. Between each capture, the animal was repositioned to minimize bias caused by placement of the animals in the machine. Bioluminescence images were analyzed using IVIS Imaging Software (Living Image©, version 4.0.0.9801; Caliper Life Sciences, Teralfene, Belgium). For each image, the threshold was adjusted to 35% of the signal, and the readout was measured in total flux, photos/s.\n\nX-Ray images were captured subsequent to the bioluminescence images. The severity of bone degradation was analyzed using ImageJ (ImageJ 1.47v). Each X-Ray image was calibrated to a standard aluminum wedge. The mean grayscale value of a standard region of interest within the trabecular bone of the proximal tibia was measured and the average of two corresponding background regions in the soft tissue proximate to tibia was subtracted. The grayscale value was translated into millimeter aluminum equivalents (mmAl) according to the standard wedge and used as estimate of the relative bone density of the distal femur. Data analysis was blinded for sex of the animals.\n\nAnimals with no bioluminescence signal or lack of osteolysis, hence no active cancer growth, were excluded from the active state group. All animals included in this study were assigned to an active or restored state based on presence of bioluminescence signal and osteolysis (active) or lack of bioluminescence signal and osteolysis (restored). Four previous in-house experiments were reanalyzed with regard to active and restored state based on presence bioluminescence signal and osteolysis or lack of bioluminescence signal and osteolysis, or based on osteolysis or lack of osteolysis alone. One study was similarly using bioluminescence as exclusion criteria, whereas the data from the three previous studies was analyzed with respect to lack of osteolysis. Lack of osteolysis was based on the last measure day, usually day 21–23, whereas one experiment was terminated at day 14, however on this day 10 out of 11 animals showed clear osteolysis.\n\nNo adverse events were observed during the experiment.\n\n\nStatistical analysis\n\nAll analyses were blinded for the researchers. Analyses were performed using GraphPad Prism 6, (GraphPad Software, CA, USA), and for all data a 95% confidence interval was used as a measure of statistical significance. All data are expressed as mean ± standard error of mean (S.E.M.). In vitro analysis of bioluminescence signal was analyzed with linear regression. Behavioral, bone degradation and tumor progression data were analyzed using two-way repeated measure ANOVA followed by Bonferroni post-hoc test to compare baseline values to each additional time point, and in addition to compare females to males. Analysis of odds ratio of cancer clearance was tested using Chi-square test. Level of significance for all tests was set at */#p < 0.05, **/##p < 0.01, ***/###p < 0.001, ****/####p < 0.0001.\n\n\nResults\n\nThe bioluminescence signal was evaluated to validate successful transfection of Luc2 into the MRMT-1 carcinoma cells. In vitro, the signal increased linearly with the number of cells, p <0.0001 (Figure 1A). In vivo, i.p. administration of D-Luciferin induced a robust plateau of the bioluminescence signal 5 min after injection and lasting approximately 20min (Figure 1B). Luc2 was hence successfully transfected into the carcinoma cells, linearly reflecting the number of living cells in vitro, and produced a robust signal in vivo.\n\n(A) In vitro quantification of bioluminescence signal. Linear regression demonstrated that the signal increased linearly with the number of cells (dotted line), p<0.0001. (B) In vivo quantification. The bioluminescence signal increased during the initial 5min following i.p. injection. After 5min the signal reached a stable plateau lasting for approximately 20min. Results from two experiments.\n\nPain behavior was quantified by detection of mechanical hypersensitivity, as well as limb use and weight-bearing deficits on days 7, 10, 14, 17, 21 and 23. Compared to baseline level, a significant decline in the 50% withdrawal threshold, reflecting mechanical hypersensitivity, was observed in both females and males from day 10 to 21 (Figure 2A, Dataset 1: rawdata_vonfrey). Limb use scoring was significantly reduced only on day 23 for the females, but on day 17–23 for the males (Figure 2B, Dataset 2: rawdata_limbuse). Decline in weight-bearing ratio was significantly reduced on day 17 and 23 for the females and on day 17–23 for the males (Figure 2C, Dataset 3: rawdata_weight-bearing). No significant difference was observed between the sexes on any of the test days, however females tended to present with a less pronounced limb use and weight-bearing deficit in the later phase of disease progression, days 17–23 (Figure 2B and C).\n\n(A) A decrease in 50% withdrawal threshold was observed in both females and males as the disease progressed. (B and C) Males demonstrated a significant decrease in limb use and weight-bearing ratio on day 17–23. In contrast, females showed significant decrease limb use only on day 23, and decreased weight-bearing on day 17 and 23. (D) A significant decrease in relative bone density was observed in the males from day 14. A similar, yet insignificant, tendency was observed in the females. */**/***=males compared to baseline, #/##=females compared to baseline.\n\nThe relative bone density was significantly reduced compared to baseline measures in the males from day 14 and throughout the study (Figure 2D, Dataset 4: rawdata_xray). A similar, however insignificant, tendency was observed in the females (Figure 2D). No difference was found between males and females at any time point. Overall, the data suggest a less severe progression in females compared to males.\n\nAll animals had a detectable bioluminescent signal on days 7 and 10, hence demonstrating successful inoculation of living cancer cells (Figure 3, Dataset 5: rawdata_BLI, Dataset 6: rawdata_bioluminescence). From day 14 an increasing number of animals lost the bioluminescence signal, suggesting recovery in a subset of animals. Compared to females with consistent bioluminescence signal, a subset of females losing bioluminescence signal displayed a significant decrease in the signal from day 17 (Figure 3). A similar but later occurring tendency was observed in the males (Figure 3).\n\nThe signal increases in both males and females during the initial 14 days, and reaches a plateau for the remaining of the study. From day 14 an increasing numbers of animals lost the bioluminescence signal. *=females compared to males.\n\nLoss of bioluminescence signal was observed in 40% of the females, but only in 20% of the males (Figure 4A and B). A significant trend for loss of signal was observed in both females and males, p=0.0079 and p=0.0353 respectively. A similar pattern in recovery was observed in previous independent in-house experiments. In these experiments, lack of osteolysis (determined by x-ray analysis) was used as an exclusion criterion. In one study, 5 out of 16 female animals displayed a lack of osteolysis (Figure 4C)14. Three additional experiments using male animals demonstrated lack of osteolysis at the end of the experiment in 3 out of 21, 1 out of 10 and 1 out of 11 animals, respectively12 (Supplementary figure S1 and Supplementary figure S2). Pooling the data from the five experiments demonstrated a significant difference in the odds ratio between females and males, reflecting a significantly greater proportion of females clearing active cancer growth compared to males, as indicated by loss of bioluminescence signal and/or lack of cancer-induced osteolysis, p=0.029, (Figure 4D).\n\n(A and B) Cumulative numbers of females and males losing bioluminescence signal over time. (C) Numbers of animals with restored states in five independent in-house experiments. The recovery is based on either loss of bioluminescence signal or lack of osteolysis. (D) A significant difference is found between the odds ratio of recovering in females and males.##/###=females without signal compared to females with signal.\n\nDespite the presence of living cancer cells during the initial 10 days, neither pain behavior nor relative bone density was significantly changed in animals losing bioluminescence signal (Figure 5A–D). A transient but insignificant decrease in the 50% withdrawal threshold was observed in animals later losing bioluminescent signal (Figure 5A, rawdata_vonfrey). In addition, a slight decrease in limb use, weight-bearing and relative bone density was observed on day 14, however the decrease was insignificant and returned to baseline levels on the next measured day (Figure 5B–D, rawdata_limbuse, rawdata_weight-bearing, rawdata_xray). Overall this suggests initial normal disease progression, followed by subsequent recovery and hence normal relative bone density and lack of pain behavior. In addition, a strong correlation between bioluminescence signal and relative bone density was observed (Figure 6A) supporting the division of animals into two groups; animals with a clear bioluminescence signal and osteolysis, indicating active cancer growth (Figure 6A, B and C) and animals with no bioluminescence signal and no osteolysis, indicating recovery (Figure 6A, E and D).\n\n(A) A transient, yet insignificant, decrease was observed in the 50% threshold in females. (B, C) Limb use and weight-bearing deficit was not observed in either females or males during the experiment. (D) The relative bone density remained the same throughout the experiment in both males and females.\n\n(A) Animals without bioluminescence signal all had high relative bone density (blue), whereas animals with high bioluminescence signal demonstrated low relative bone density (black). Examples of osteolytic (C) and non-osteolytic bone (D). Examples of high bioluminescence signal (B) and lack of signal (E).\n\nExclusion of animals successfully recovering aligned the onset and severity of pain behavior in both sexes (Figure 7A–C). In addition, a similar effect was seen on the relative bone density, reflecting similar bone degradation in both females and males with active growing cancer (Figure 5D).\n\n(A, B and C) Both females and males demonstrate significant decreases in 50% withdrawal threshold, limb use score and weight-bearing ration. (D) Both sexes show a similar extent of bone degradation. **/***/****=males compared to baseline, #/##/####=females compared to baseline.\n\n\nDiscussion\n\nTo increase the translational potential of preclinical research, it is essential to continually focus on refining and optimizing the animal models used. Increased focus has been given to sex as a variable, initially driven by the contradictive issue that preclinical research has been biased towards use of male animals despite the fact the chronic pain is highly overrepresented in women8. Although females are in most cases likely the most intuitive sex for models of metastatic breast cancer, such as the MRMT-1 model of cancer-induced bone pain, our data suggest that care should be taken when interpreting the data. Our data demonstrates that the females have an increased capacity for recovering from the disease state, reflected by loss of bioluminescence signal accompanied by normal limb use and weight-bearing ratio as well as normal relative bone density throughout the study.\n\nThe increased odds ratio for recovery in the females introduces a potential bias in interpretation of the data. In order to get a reliable model truly reflecting the human disease, it is critical to exclude the animals capable of recovery, as these animals are not reflecting the pain state observed in patients. This means that it is highly important to include a parameter for discrimination between an “active disease state” and a “recovered from disease state”. Inclusion of animals with who have cleared the cancer could ultimately result in false negative or false positive results due to increased variation and/or shifting of readouts towards baseline.\n\nIn addition, our data demonstrates that bioluminescence signal is a reliable measure for the active disease state. All animals with continuous bioluminescence signal subsequently developed pain-related behavior and decreased relative bone density. In contrast, animals losing bioluminescence signal during the experiment did not develop pain-related behavior and demonstrated normal relative bone density.\n\nOverall, this study demonstrates an increased capacity for recovery from the experimental disease state in females compared to males, yet the specific underlying mechanisms are currently not known. However, these could possibly be linked to sex-dependent differences in the immune responses. It is now generally accepted that inherent properties and influence of sex hormones induce more potent immune and inflammatory reactions in females compared to males15,16. It could therefore be speculated that the females’ increased recovery rate is linked to an increased immune response to inoculation with the cancer cells. It should be noted that the capacity for recovering was seen regardless of whether the cancer cells expressed luciferase or not, and is thus not due to an immune reaction to the luciferase enzyme. In addition, recent work from Sorge et al. demonstrate that different immune cells mediate pain hypersensitivity in female and male mice17. They found that whereas microglia activity is required for mechanical pain hypersensitivity in males, this is not the case in females. Overall this suggests that sex-dependent immune-differences could affect cancer-induced bone pain at different levels. Another factor potentially affecting the sex-dependent effect might be the local microenvironment in the bone. The microenvironment around the tumor might be different due to nonspecific sex-differences, hence potentially affecting the growth of the tumor cells following inoculation18–20. Interestingly, the observed difference is likely species or cell line dependent. We have previously reported that in a similar model of cancer-induced bone pain, based on 4T1-luc2 mammary cancer cell inoculation in femur of BALB/cJ mice, females have a significantly greater initial bioluminescence signal compared to males. The females had, in addition, an earlier onset of pain behavior, but a similar bone degradation rate5. This suggests that in the mouse model, intrinsic sex-dependent variation favors more aggressive progression in females compared to males, whereas the rat model displays better odds ratio for recovery in the females.\n\n\nData availability\n\nF1000Research: Dataset 1. Rawdata_vonfrey, 10.5256/f1000research.6827.d9668721\n\nF1000Research: Dataset 2. Rawdata_limbuse, 10.5256/f1000research.6827.d9668822\n\nF1000Research: Dataset 3. Rawdata_weight-bearing, 10.5256/f1000research.6827.d9668923\n\nF1000Research: Dataset 4. Rawdata_xray, 10.5256/f1000research.6827.d9669024\n\nF1000Research: Dataset 5. Rawdata_BLI, 10.5256/f1000research.6827.d9669125\n\nF1000Research: Dataset 6. Rawdata_bioluminescence, 10.5256/f1000research.6827.d9669226",
"appendix": "Author contributions\n\n\n\nS.F. conceived and designed the study and carried out all experimental procedures. T.A-D. assisted during experimental procedures. L.M. generated the MRMT-1-Luc2 cell line. A-M.H. provided advice and facilities. S.F. wrote the manuscript that was edited and approved by all coauthors.\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\nSupplementary Material\n\nRepresentative images of animals demonstrating osteolysis (A–F) and lack of osteolysis (G,H).\n\nRepresentatives of animals demonstrating bioluminescence signal (A–C, E–F) and lack of bioluminescence signal (D).\n\n\nReferences\n\nBarrett JE: The pain of pain: challenges of animal behavior models. Eur J Pharmacol. 2015; 753: 183–90. PubMed Abstract | Publisher Full Text\n\nRuggeri BA, Camp F, Miknyoczki S: Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery. Biochem Pharmacol. 2014; 87(1): 150–61. PubMed Abstract | Publisher Full Text\n\nFalk S, Dickenson AH: Pain and nociception: mechanisms of cancer-induced bone pain. J Clin Oncol. 2014; 32(16): 1647–54. PubMed Abstract | Publisher Full Text\n\nCurrie GL, Delaney A, Bennett MI, et al.: Animal models of bone cancer pain: systematic review and meta-analyses. Pain. 2013; 154(6): 917–26. PubMed Abstract | Publisher Full Text\n\nFalk S, Uldall M, Appel C, et al.: Influence of sex differences on the progression of cancer-induced bone pain. Anticancer Res. 2013; 33(5): 1963–9. PubMed Abstract\n\nMogil JS, Bailey AL: Sex and gender differences in pain and analgesia. Prog Brain Res. 2010; 186: 141–57. PubMed Abstract | Publisher Full Text\n\nGreenspan JD, Craft RM, LeResche L, et al.: Studying sex and gender differences in pain and analgesia: a consensus report. Pain. 2007; 132(Suppl 1): S26–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMogil JS, Chanda ML: The case for the inclusion of female subjects in basic science studies of pain. Pain. 2005; 117(1–2): 1–5. PubMed Abstract | Publisher Full Text\n\nZimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983; 16(2): 109–10. PubMed Abstract | Publisher Full Text\n\nMezzanotte L, Aswendt M, Tennstaedt A, et al.: Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain. Contrast Media Mol Imaging. 2013; 8(6): 505–13. PubMed Abstract | Publisher Full Text\n\nMedhurst SJ, Walker K, Bowes M, et al.: A rat model of bone cancer pain. Pain. 2002; 96(1–2): 129–40. PubMed Abstract | Publisher Full Text\n\nFalk S, Schwab SD, Frøsig-Jørgensen M, et al.: P2X7 receptor-mediated analgesia in cancer-induced bone pain. Neuroscience. 2015; 291: 93–105. PubMed Abstract | Publisher Full Text\n\nDixon WJ: Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol. 1980; 20: 441–62. PubMed Abstract | Publisher Full Text\n\nFalk S, Ipsen DH, Appel CK, et al.: Randall Selitto pressure algometry for assessment of bone-related pain in rats. Eur J Pain. 2015; 19(3): 305–12. PubMed Abstract | Publisher Full Text\n\nChrousos GP: Stress and sex versus immunity and inflammation. Sci Signal. 2010; 3(143): pe36. PubMed Abstract | Publisher Full Text\n\nFurman D, Hejblum BP, Simon N, et al.: Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination. Proc Natl Acad Sci U S A. 2014; 111(2): 869–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSorge RE, Mapplebeck JC, Rosen S, et al.: Different immune cells mediate mechanical pain hypersensitivity in male and female mice. Nat Neurosci. 2015. PubMed Abstract | Publisher Full Text\n\nFukusato T, Aoyama H, Mori W: Age and sex differences in bone metastasis of hepatocellular carcinoma in Japanese autopsy cases. Gastroenterol Jpn. 1989; 24(2): 127–34. PubMed Abstract\n\nSakaguchi S, Goto H, Hanibuchi M, et al.: Gender difference in bone metastasis of human small cell lung cancer, SBC-5 cells in natural killer-cell depleted severe combined immunodeficient mice. Clin Exp Metastasis. 2010; 27(5): 351–9. PubMed Abstract | Publisher Full Text\n\nWright LE, Guise TA: The microenvironment matters: estrogen deficiency fuels cancer bone metastases. Clin Cancer Res. 2014; 20(11): 2817–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFalk S, Al-Dihaissy T, Mezzanotte L, et al.: Dataset 1 in: Sex-difference affects disease progression in the MRMT-1 model of cancer-induced bone pain. F1000Research. 2015. Data Source\n\nFalk S, Al-Dihaissy T, Mezzanotte L, et al.: Dataset 2 in: Sex-difference affects disease progression in the MRMT-1 model of cancer-induced bone pain. F1000Research. 2015. Data Source\n\nFalk S, Al-Dihaissy T, Mezzanotte L, et al.: Dataset 3 in: Sex-difference affects disease progression in the MRMT-1 model of cancer-induced bone pain. F1000Research. 2015. Data Source\n\nFalk S, Al-Dihaissy T, Mezzanotte L, et al.: Dataset 4 in: Sex-difference affects disease progression in the MRMT-1 model of cancer-induced bone pain. F1000Research. 2015. Data Source\n\nFalk S, Al-Dihaissy T, Mezzanotte L, et al.: Dataset 5 in: Sex-difference affects disease progression in the MRMT-1 model of cancer-induced bone pain. F1000Research. 2015. Data Source\n\nFalk S, Al-Dihaissy T, Mezzanotte L, et al.: Dataset 6 in: Sex-difference affects disease progression in the MRMT-1 model of cancer-induced bone pain. F1000Research. 2015. Data Source"
}
|
[
{
"id": "10035",
"date": "20 Aug 2015",
"name": "Juan M Jiménez-Andrade",
"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 very interesting and well presented study; however the data does not support the title and conclusions at all of the present study. For this reviewer it is unclear what is the functional implication of females having an increased capacity for recovering from cancer state, given that there are non-significant differences in terms of pain behaviors and bone degradation between females and male rats. I think the authors are overstating the results and the results are not properly interpreted.Additionally, the authors have not considered that the loss of bioluminescence signal over time may due to gender-related differences in terms of bioavailability and excretion of D-Luciferin. In fact, the authors are strongly recommended to present the in vivo quantification of bioluiminescence signal after i.p. injection (as presented in figure 1B) in both females and males.Finally, a histopathological analysis of the bone is strongly needed in order to suggest that females rats are recovering faster than male rats. EDIT 21/08/2015: This review was mistakenly published with a ‘Not Approved’ status, this has now been amended to an ‘Approved with Reservations’. The text has not been changed in any way.",
"responses": [
{
"c_id": "1592",
"date": "18 Sep 2015",
"name": "Sarah Falk",
"role": "Author Response",
"response": "Dear Professor Jiménez-Andrade Thank you for your very constructive review. We have taken your comments into carefully consideration, and made the following changes: We have changed the title to “Effect of sex in the MRMT-1 model of cancer-induced bone pain”, as we realize that the original title could be misleading. Regarding the functional implication of the study, we have extended the discussion to clarify that the main result of the study is the increased capacity for recovery from the tumor-induced disease in the female rats compared to the male rats. This can potentially affect the interpretation of data produced with the model.The last line in the abstract is in addition changed to “interpretation of data”, to emphasize that the increased capacity for recovery do not affect the overall progression in cancer-bearing animals, but that the recovery in some animals can mask the actual effect of the cancer, as the data from the recovered animals will shift the mean towards baseline. Sex-related differences in terms of bioavailability and excretion of D-Luciferin should not affect the result in the study. The bioluminescence emission is based on acute injection of D-Luciferin at a level that saturates the system. Bioluminescence signal is measure 10min past injection on each measure day, meaning that if there were sex-differences in bioavailability and excretion is should be seen on all measuring day, and not only in a subset of animals from day 17 and beyond. Also as can be seen in supplement figure S2 the loss of signal is seen as an all-or-non response; either there is a signal or no signal at all. If the loss of bioluminescence signal were caused by changes in bioavailability and excretion in a subset of animals a more gradient decrease in signal would be expected and not a complete loss of signal as seen in these animals. Additional histopathological analysis could be performed, however since loss of bioluminescence signal is accompanied by both lack of pain behavior and osteolysis it is unlikely that the animals should have living cancer cells in tibia. To emphasis this point, we have added a section in the discussion."
}
]
}
] | 1
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https://f1000research.com/articles/4-445
|
https://f1000research.com/articles/4-898/v1
|
24 Sep 15
|
{
"type": "Opinion Article",
"title": "Design for learning – a case study of blended learning in a science unit",
"authors": [
"Roslyn Gleadow",
"Barbara Macfarlan",
"Barbara Macfarlan"
],
"abstract": "Making material available through learning management systems is standard practice in most universities, but this is generally seen as an adjunct to the ‘real’ teaching, that takes place in face-to-face classes. Lecture attendance is poor, and it is becoming increasingly difficult to engage students, both in the material being taught and campus life. This paper describes the redevelopment of a large course in scientific practice and communication that is compulsory for all science students studying at our Melbourne and Malaysian campuses, or by distance education. Working with an educational designer, a blended learning methodology was developed, converting the environment provided by the learning management system into a teaching space, rather than a filing system. To ensure focus, topics are clustered into themes with a ‘question of the week’, a pre-class stimulus and follow up activities. The content of the course did not change, but by restructuring the delivery using educationally relevant design techniques, the content was contextualised resulting in an integrated learning experience. Students are more engaged intellectually, and lecture attendance has improved. The approach we describe here is a simple and effective approach to bringing this university’s teaching and learning into the 21st century.",
"keywords": [
"Blended learning",
"Moodle",
"educational design",
"science communication",
"on-line learning",
"higher education"
],
"content": "Introduction\n\nThe massive change in communication and information technology in the past ten years raises questions about how, or even whether, we should harness this to teach our students. As teaching and research academics in a research-intensive university we are keen to engage students in “discipline knowledge” (Breen, 1999), but what is an appropriate way to do that? Recording of lectures and ready availability of lecture notes and slides on-line is routine at most universities. This has led to heated debate about the role of presentation tools such as PowerPoint (Horvath & Lodge, 2015; Sørensen, 2015).\n\nThe reduction in the number of students attending lectures in person has given impetus to calls for lectures to be replaced with other forms of teaching, or for recordings to be abolished. However, these may not be effective in addressing the real issue of student engagement, since the proportion of students downloading slides and listening to lectures is often less than 20% in any one week. Lectures did not disappear after the invention of the printing press, but they did evolve from the reading of texts to compilations of learned material. Current changes in digital technology indicate that there is clearly a need for lectures to evolve further.\n\nThe current student cohort, sometimes referred to as “Millennials”, is often accused of being self-centred and lazy (Stein, 2013). This mean-spirited generalisation ignores the challenges these students face in juggling increasingly busy lives with the competing demands of work/life/study (Abbott, 2013). As a result their approach to study is more targeted and goal oriented. Students are used to sourcing information rapidly, with minimum financial and mental cost, and to be entertained in the process. Simple, well-organised websites such as Wikipedia are particularly useful for sourcing information in a targeted way. As educators, our role is to not just help students fulfil the minimum requirements to pass, but to inspire students to take control of their own learning, rather than just consume. Here we present a case study to serve as a model for organising and contextualising content and learning to better engage students, helping them develop effective learning strategies, and to show how attention to design principles can transform the student experience.\n\n\nRationale: Designing for learning\n\nLearning management systems are, too often, used as a file repository by busy academics that then gets rolled over from one year to the next, without change. We face the university-wide challenge of ensuring deep content that is both engaging and accessible. This can be addressed by creating interactive content that ensures students can readily access key information – for example, an interactive glossary lets students check their understanding of new keywords. The pedagogical challenge of clearly defining the purpose of learning and assessment activities is best addressed when it is clearly contextualised within unit objectives.\n\nLearning design can be described as the “the complex process of planning, decision making, design, and creativity in [the] facilitation of student learning” (Laurillard et al., 2013). It is different from planning for learning that must consider institutional constraints such as timetabling, mode (face-to-face, online, blended), class sizes, and sociocultural backgrounds of students. When re-designing this unit, we wanted to take the opportunity to place the student at the centre the process. The optimal situation is one where the students are directed through learning activities designed to deconstruct the concepts and make the relationships between them transparent (Laurillard, 2012).\n\nOur plan was to bring sound pedagogical theory of learning together with a smattering of instructional design to create a blended learning methodology that makes sense to busy academics in a university context. Dalziel (2009) refers to two determining factors to designing for learning: (1) the building of a learning pathway to sequence learning activities, and (2) the description and dissemination of practice. In this case study, we describe the steps and rationale taken in re-designing the learning pathway in a large undergraduate science unit, its impact on the students, and the plans for disseminating this model of good practice to other faculty members. Building a learning pathway requires a re-framing of the content and learning activities for the students and incorporates considerations of learning outcomes and resources that maximise the opportunities for learning through interaction with self (through reflection), and with peers as well as with the teacher (face-to-face and online channels). Thus the strategies for learning in the online environment can establish learning pathways that encourage students to explore and discover their own way through the content. (Sims et al., 2002). Figure 1 graphically represents Laurillard’s Conversation Framework with its emphasis on teacher-student interaction in the learning process and their shared responsibility when developing understanding. This interactivity is an essential component for the successful implementation of teaching and learning (Sims et al., 2002), and we suggest that all students will benefit from a course that is designed with the learner at its heart.\n\nBased on Laurillard's conversation model reflecting teacher/student interaction (BTBL Bytes, 2015).\n\nStudents learn best when they are actively engaged and can construct their own knowledge (Laurillard, 2012). Teachers and learners both play roles in this process (See Figure 1). It is the teacher’s responsibility to lower the barriers for learning by clearly outlining details of the assessments and the sequence of topics and related learning activities and ensuring that the content, learning activities and that assessments are aligned to the learning outcomes (Gleadow et al., 1993). Delivery strategies include pre-class, in-class and post-class teaching and learning activities that could include group project work, class presentations, excursions, guest speakers, and so on. How this learning takes place (e.g. face-to-face, blended, online, workplace or an internship) should be chosen on the basis of its effectiveness in the context. Students participate by engaging in these activities. This can be challenging and it is important to involve students in this process and to be clear about what is expected of them. Feedback is critical in the learning and teaching cycle if students are to improve and consolidate their learning. Students expect timely and detailed responses to their queries and qualitative feedback on their work (Hannon et al., 2002).\n\n\nImplementing learning design principles\n\nThe learning management system at our institution is Moodle, an open-source learning platform that is designed to give educators around the world a secure, integrated system through which to deliver learning. Academics have limited access to training in the effective use of Moodle and this combined with a busy teaching and research load means that we find, unsurprisingly, that Moodle is used in a very basic way. The default format is linear and without an understanding of learning theory to inform their decisions, academics use their online teaching space as a repository for their lecture slides and reading lists with a few forums thrown in. This was the situation with this core science unit when the learning designer started to work with the academic. Figure 2 is a very common Moodle page layout. This structure provides no clues for the learner as to where to find key materials such as assessments, lecture slides, resources and learning activities. These are hidden from the learner in this configuration.\n\n(a) Previous layout with week-by-week arrangement, typical of many courses; (b) Menu buttons and themes positioned on the front page.\n\nThe structure chosen for an online course should maximize the learning opportunities being presented: “Whatever medium is used for a text, its meaning is revealed through its structure” (Laurillard, 2012) and in the online space, the structure needs to be the one that makes it easiest for the learners to navigate through the key points and supporting material. We addressed this issue over the first few months of the project, discussing the interplay between the face-to-face components of the course - the lectures and workshops - and the online space with reference to established principles of effective learning design and the underpinning pedagogy.\n\nThe first thing that a learning designer can be expected to address when embarking on a unit evaluation process is a critical analysis of the learning outcomes and their alignment to the delivery and assessment strategies. This process encompasses all factors that can impact on the learning environment including learning outcomes, curriculum, assessment, and teaching and learning activities (Laurillard et al., 2013). It is at this stage that inconsistencies can be identified and a process of remediation to address anomalies can be established. This was not the case with SCI2010. There was a rich, engaging face-to-face learning environment that was well established with a team of highly motivated tutors to support the academic in the delivery of a varied and stimulating learning environment that incorporated authentic assessment activities.\n\nThe assessments and learning activities were aligned, and the content was in a constant state of renewal; so what was left for a learning designer to do? What value could be added? Consequently, it was the online learning environment on which we focused our attention and we set about re-designing Moodle as a teaching and learning space rather than a filing system. We decided to re-build the online course to represent best practice in learning design and integrate sound pedagogic online facilitation protocols. These were: structure and organisation; aesthetic design; contextualisation; clear learning pathway; and online facilitation. Here’s how this worked in practice.\n\nOur university is implementing a program for enhancing teaching that aims to ensure effectiveness through high quality design of learning outcomes and assessment regimes, multifaceted activities, and optimal delivery methods. The course chosen to spearhead a program for enhancing teaching across our university was SCI2010 Scientific Practice and Communication. The course deals with the nature and origins of science, ethical practice and science communication. This large, interdisciplinary unit is compulsory for all science students studying at the Melbourne and Malaysian campuses, or by distance education. There are approximately 1200 students (600 per semester) mostly in their second year of University and taking degrees in Physics, Biology, Chemistry, Biomedical sciences, Mathematics and Psychology. Students are, on average, 20.3 years old, with over one-third from homes where a language other than English is spoken (more at the international campus). A survey of graduates found that only one-fifth of students would have taken the subject if it was not compulsory, but in hindsight over two-thirds said they had learnt things not otherwise covered in their degree and that it should remain compulsory (unpublished data, The Faculty of Science, Monash University 2009). It is thus both challenging and rewarding to present the subject matter in a way that is intellectually engaging and relevant to students from a wide range of disciplines.\n\nThe course had always had a ‘Quick links’ section at the top of the page (as seen in Figure 2b). This guided the students to resources that they needed to access in a timely manner. These included Assessments, Lecture resources, Quizzes, and the highly rated, What’s on this week? that linked directly to the relevant page with the weekly learning activities. The buttons had been used for the past few years, and only required minimal modification to reflect the new navigation system. The unit Introduction (which was already in the Unit Guide) was brought into the online course front and centre, presenting this unit in context in the wider course and emphasising the learning outcomes as they applied in practice.\n\nThere was an obvious need to change the weekly headings to something that best described the topic and give the student more clues and support for their learning. As Al-Samarraie et al. (2013) propose, a well-designed structure underpinning the learning process will facilitate students’ understanding of the concepts leading to successful outcomes. The section headings were changed from Week 1, Week 2 to topic names such as Is science special? Can we afford self-deception? Can scientists be bad? Each topic was presented with a consistent weekly structure to create expectations of learning activity. This structure included an introduction to the topic, the learning outcomes, and a pre-class activity to activate thinking for this week’s concept, learning activities – Something to read, Something to do, Something to think about- a series of questions for reflection, and the link to the lecture slides.\n\nAs we worked through this makeover, it became obvious that by contextualising the topics with more supporting information and activity, we were actually accentuating the major themes of the unit. It must be emphasised here that at no point did the content change, but rather the way that the learning resources and activities were presented changed the focus from a list of resources to a more thematically contextualised, learner-centred structure. This caused a re-think in the delivery and a shift in the paradigm to where the online space was truly connected to the face-to-face interaction.\n\nAs a start, the design of the online space in Moodle was changed from collapsed topics to a more open setting. This different unit layout eschews the linear format, introduces images to guide the learner to different sections, whetting their appetite for further investigation. Each section in the new configuration has its own image and description to guide the students quickly to course material that they need (Figure 3). This design and clear learning pathway was implemented to make obvious to the student the actions required to achieve specified learning outcomes. The learner is guided through scaffolded activities, discussions, opportunities for reflection, self-test quizzes, and extension activities if needed or desired. We believe that an altered learning landscape motivates the learners to engage with the prescribed materials and activities at a deeper level and reflectively participate in the learning experience. In doing this they are learning to become pro-active participants in their environment, actively reshaping their landscape to support on-going learning (Goodyear, 2015).\n\nPictures from Creative Commons. Image of scientists: Mars-discovery-district; Image of globe: Kotomi.\n\nQuestion of the week - a Moodle polling activity. Students view a short video or image and are asked to participate in a poll. Video: A Brief History Of Science (2013).\n\nFor us, contextualising the learning means adding value to the materials and activities presented online; interacting with the learners through the instructions and guidelines; and being present in that space with them, similar to that described by Laurillard (2012). In order to get to the underlying purpose of each activity, the learning designer (BM) prompted the teacher (RG) by asking: “If you were to present this video/activity/article to read in a face-to-face class, what would you say by way of introduction?” This forced the teacher to really think about the context and that then shaped the writing surrounding the learning activities so that the purpose was crystal clear, and would make sense to the students, forestalling questions such as: Why am I doing this? What’s the point of this?\n\nThe learning pathway was designed to guide the learner through clearly sign-posted themes and topics in order to help the students understand the progressive and cumulative building of knowledge; and to help them synthesise and apply the key concepts (Figure 5). Students could, of course, access all the weekly resources through the quick links at the top of the page, and some did. However, our feedback and analytics of the hits per page suggest that most students took advantage of the learning opportunities presented in this format.\n\nA typical layout and introduction for each ‘lecture’ activity is shown in Figure 6. At the top there is a very short introduction, followed by a very short video and a Moodle Choice activity. This was designed to stimulate thinking on the topic, and by voting the students had to consider the issue and act, thus activating their thinking on the new concept and preparing them for the new learning. The next step for students is to access the Lecture resources. Again, the information was organised in a consistent format, designed to fit on a single screen of a computer, (see Figure 4) incorporating a short introduction, a list of learning outcomes, and a list of things to do post-lecture, called ‘Something to read’ (content and further reading), ‘Something to do’ (a related activity), ‘Something to think about’ (opportunity to reflect, extend and apply the learning.). The link to the lecture slides was at the bottom of the page.\n\nThe same structure every week to create expectations of learning activity. The students are directed to the video BBC: Science and Islam - Part 1 in “Something to Do”.\n\nIn order for this blended model to be effective, all the tutors on the course were encouraged to participate in the online activities in private discussion forums (on-line and face-to-face) and bring those discussions into the classroom. The guided online activities were referred to and discussed in class, reinforcing to the students that the online space was valued as much as the face-to-face interaction. This seamless interaction in the online and face-to-face spaces highlighted to the students that the teachers were active in both spaces and the each mode was an essential element of the course delivery.\n\n\nStudent evaluation and responses\n\nThere were 763 students enrolled in the unit: 633 at the Melbourne campus in Clayton, 109 at the campus in Kuala Lumpur, Malaysia (where students do the same program but have their own lecturers) and 21 students taking the unit by distance education. Data on student responses to the new layout and structure was collected in four ways. Firstly, we determined how many students were accessing the material and participating in the non-compulsory pre-lecture choice activities. Secondly, an online survey was conducted in Week 9 of the teaching program (see Supplementary material for wording of the questions). Thirdly, observations of attendance and engagement, and the type of questions that students were asking during the lead up to the final examinations were made.\n\nOn average, 159 students took part in each pre-lecture poll, ranging from 415 in week 1 to 92 in the final week (Table 1). Participation in the ten polls was completely optional, and attracted no marks. The weekly online revision quiz opened after the lecture and closed the evening before the lecture the following week. Students could make up to three attempts in this time. Students were rewarded with a ‘participating mark’ of 0.25% of the semester grade for attempting each quiz, but while they got feedback on their answers, no marks were given for getting the right answers. On average, there were over 1000 attempts at the quiz each week, which means that some students were attempting them multiple times.\n\nThere were 763 student enrolled in the unit across three campuses. Semesters run for 12 weeks (excluding the study period). There were no lectures in Weeks 4, 8 and 12.\n\nThere was the opportunity for students to provide open-ended responses (see Supplementary Data) as well as the ranking of specific aspects. Overall students were very positive about the changes:\n\n“Your Moodle site is awesome! I wish all our units were like that”.\n\n“…By far my favourite moodle page of any subject.”\n\nStudents were particularly positive to the questions about the navigation of the site, with 80.5% agreeing that the navigation was logical (Question 2), and the information easily accessible (Figure 7). Students singled out the Quick Link buttons at the top of the page with 60.3% strongly agreeing that they liked being able to navigate the site using the buttons on the home page, and 55.2% strongly agreeing that is was easy to find information about the assignments. This was particularly rewarding, as a recurring complaint in student evaluations in past years was that it was hard to find out what was required for the assignments.\n\nThe full text of the questions is in Supplementary Data. The complete dataset is available as a.csv file.\n\n“The start buttons made for excellent navigation…”\n\n“It [the quick link menu] makes it easy to find the quizzes, information for workshops and information from lectures. Moodle isn’t always easy to navigate so this has definitely helped.”\n\nOf course, not everyone liked the new layout. The comment (below) highlighted one of the problems that we anticipated: if information is only accessible via the themes, then more clicking is required. It is good web design to be able to access the same material in several different ways, and this was incorporated into the design, but clearly this was not immediately obvious to everyone:\n\n“I didn’t like the organisation of the course content on Moodle. I much prefer the normal unit layout with everything on one page under different subheadings. It was often quite annoying having to click through many different pages in order to find information.”\n\nThe major focus of the revisions was the lecture pages, while the workshop pages had only minimal information on them. Interestingly, this could be detected by the student responses. Over 55% of students strongly agreed and a further 30% somewhat agreed that “the lecture support pages helped me understand the lectures…” but only 29% strongly agreed with the statement that “that “the workshop support pages helped me to get more out of the workshops”. Other comments on the changes included:\n\n“Really liked the things to read, do and think about sections for every week. Really helped me keep on track with the subject and what is required.”\n\n“Workshop resources should be linked to the lectures rather than having a separate tab.”\n\n“I found the course content to flow logically from week to week.”\n\nThe Moodle book was developed in response to students wanting more specific information; something particularly important in this course as there isn’t a textbook. Reluctant to move from good design and communication practice (few words, lots of images), I transferred and edited existing written materials into the on-line Moodle Book format. For example, feedback from 2014 included the following:\n\n“Suggest you make the slides more text rich? The way they are you have to listen to the whole lecture to work out what it is about”\n\nNot everyone worked out that this type of information was now available via the Moodle Book, as one student commented in the present survey:\n\n“please put more explanation and sentences in the slides instead of a bunch of images”\n\nAs so often happens in education, students like what you’ve done and then want more of it. For example, the Moodle book is the first one of its kind in the Faculty of Science, but one student commented:\n\n“I like the moodle book however it should be supplied by a pdf.”\n\nIn a typical semester lecture attendance can fall below 20% by the end of semester. Although we did not collate the data formally, counts of students at the end of the implementation program showed a shift closer to 25%. As expectations rise, we expect attendance to further improve. Why does this matter? Lectures should be the place where we inspire, direct and interact with students. By contextualising lectures, and making them part of a blended learning program, students should be better equipped to engage intellectually, and not be passive recipients of information.\n\nStudents in this course actively engage in Discussion forums during semester and in the lead up to the examinations. This semester there were far fewer questions asking for clarification about the objectives of each topic, and more questions about the application, reflecting deeper learning. For example, comments along the lines of:\n\n“What are we supposed to know about the lectures on….?”\n\nWere replaced by comments such as:\n\n“Just wanted to clarify one of the examples on….given in the lecture...”\n\n\nDiscussion\n\nThe introduction of e-learning technology has been a game changer in education (Mor & Craft, 2012). The LMS with its collaborative affordances now competes with the teacher for attention as many students tune into their lectures online rather than turning up in person. It falls to the academic – who is not usually trained as a teacher and is allocated little time or support to develop the newly required skill-set – to design a learning pathway that incorporates meaningful interactivity between the learner and the teacher; the learner and the online content and activities; and the learner and other learners. This focused interaction is critical to the success of the learner experience and will ultimately influence the efficacy of the learning environment. On the other hand, the use of digital technologies is taken for granted by students who expect that their lectures and assessments will be available to them online; but to the academic, who is time poor, anything beyond the basic online presence can be seen as window dressing. The onus is on academics to determine whether the increased workload and upskilling required to develop new digital resources is a good use of their time (Sheey et al. 2006). However, even busy academics will find the time to integrate methodologies if they are convinced that the change in practice will make a difference. Teachers’ use of learning technologies will increase if they are convinced that the pedagogy is sound, and if they are inspired and enthused enough to implement these changes into their teaching practice (Macfarlan & Everett, 2010).\n\nManagement of cultural change requires simultaneous implementation from top-down and bottom-up (Brown, 2014; Fullan, 1999; Patria, 2012). Top-down incentives include training, mentoring, showcasing, and research. Teaching and learning are high on the agenda of the senior management of our university and there is a concerted effort from all stakeholders to challenge the current status quo of teaching and effect a cultural shift with a move towards interactive rather than didactic teaching approach using innovative, effective and efficient online and face-to-face teaching modalities, providing the opportunity for deeper and relevant learning to be realised. (Better Teaching Better Learning vision, OVPLT, 2014). The provision of Education Designers can be a highly effective way of fostering innovative approaches to teaching and learning and guide academics towards the development of a pedagogy that incorporates digital resources. For a successful change process to be enacted Fullan & Stiegelbauer (1991) suggest that the likelihood of success will be higher when the individual's personal goal align with the organization's goal. It then falls to management to communicate the need for change and clearly articulate the support offered to manage this intended change.\n\nBottom-up incentives come if academics see that the process will lead to improved student outcomes that are simple and time-efficient. Academics are more likely to engage with cultural change if it is manageable, supported, and endorsed by their peers (Patria, 2012). It takes the example of academics who act as change agents to implement this shift and undergo the rigors of detailed student feedback before others are prepared to follow. This was the case with this science unit; the positive student feedback was a motivating force for other academics who are now open to the discussion surrounding “unit enhancement” and accessing the support of the learning designer to discuss the possibilities. This development of consultation, support and modelling good practice involves the academics in “…a dynamic process that enacts participants [academics] to reflect about the values, processes and outcomes of an educational intervention.” (Ghislandi & Raffaghelli, 2015). The outcome is the development of a model for an iterative process and a culture of reflective practice that encourages experimentation with new tools and pedagogical approaches in learning design. Our model is expected to be further refined, guided by evaluation of students’ motivation and learning outcomes.\n\nThe changes described here focused on structure and design, with only relatively minor modifications to the actual course content. Nevertheless, change needs to be carefully managed, so that those affected are brought along with it, and not alienated. The initial hesitance in implementing the changes proposed by the model described in this paper arose from the concern that if the course looked different to other courses, then it might be less acceptable to students. Indeed, the organisation had been converted from a Lectures/Workshops/Assignments layout to the weekly list in 2012 in order to be consistent with the majority of other teaching units. Students do not always appreciate change, possibly because it means they cannot generalise from one task to the next. They are mostly interested in the assessments and passing an exam. Vygotsky’s (1978) social learning theory means little to students whose workload is increased by a teacher’s exhaustive use of the collaborative affordances of the LMS. We spent a short time in each lecture referring to the LMS and talking about the outcome of the pre-week polls, however as only a minority of students attend lectures this was a bit like preaching to the converted. In order to help students during this time of transition it is going to be necessary to spend time explaining the rationale of this altered design, giving them the opportunity to reflect on their own learning ability and extending their understanding of the part they play in the teaching and learning process (Ghislandi & Raffaghelli, 2015).\n\n\nFinal reflections\n\nThe design we have described here used a consistent thread as a strategy within which we could create learning opportunities for a diverse group of students. The underlying principle was: How can we help our learners to move from their current state of learning development to that “sweet spot” where what they know meets what they need to know. We aimed to create an environment where the learning tasks were not so easy that the learners became bored and switched off, but were sufficiently challenged and motivated to work through difficult tasks with support from teachers and peers. We liken this to Vygotsky’s Zone of Proximal Development (1978) that describes the distance between the actual development of a learner as determined by independent problem solving ability and the potential development as evidenced through collaboration with others.\n\nSuch change is inevitable. Our learners can now readily access up-to-date information anywhere, anytime, and this requires us to adapt our methodology to meet increasingly complex challenges. As a consequence, we need to move “from distributors of knowledge to designers of learning experiences.” While it still falls to the teacher to manage content and assessment, a student-centred learning paradigm necessitates a collaborative learning environment where learners explore, enquire, analyse, and engage in authentic learning activities. There is, however, little support for teachers in developing skills in design for learning and a paucity of culture to nurture such practices.\n\nGetting buy-in from other time-poor academics can be challenging. The pain is more in taking time to rethink what it is that you want to teach rather than the implementation. Making design explicit and shareable delivers consistency and makes implementation straightforward. The key lesson has been to set up in a step-wise manner with judicious use of time release so everything doesn’t have to be ready before the start of the teaching period. It is also possible to lower the hurdles by generating generic pages ready for the content to be added. Teaching academics should be encouraged to experiment with the technology available to them and, by reflective practice, work out what suits them. Institutions that support them through this process will help create a better learning environment for both lecturers and students.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw dataset for Gleadow et al., 2015 'Design for learning – a case study of blended learning in a science unit', 10.5256/f1000research.7032.d101855",
"appendix": "Author contributions\n\n\n\nRMG is coordinator and principle lecturer in the Unit “Scientific Practice and Communication. BM is an education designer employed by the Office of the Vice Chancellor (Teaching and Learning) and was assigned to work on this project as part of the University-wide unit enhancement program. Both authors agree to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThis program was funded by a Teaching initiative Grant from the Faculty of Science and the School of Biological Sciences, Monash University to RMG.\n\nI confirm that the 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 Melissa McKnight in implementing the changes to Moodle, building the templates and creating the weekly quizzes. We also thank Associate Lecturer Dr Bronwyn Isaac, for implementing and adapting to the changes and the tutors who embraced and implemented the changes, and gave us unfiltered feedback.\n\n\nSupplementary materials\n\nStudent survey.\n\nWe provided the opportunity in Week 9 for students to provide open-ended responses as well as the ranking of specific aspects of the program.\n\nClick here to access the data.\n\n\nReferences\n\nAl-Samarraie H, Teo T, Abbas M: Can structured representation enhance students’ thinking skills for better understanding of E-learning content? Comput Educ. 2013; 69: 463–473. Publisher Full Text\n\nBrown S: “You can't always get what you want”: change management in higher education. Campus-Wide Information Systems. 2014; 31(4): 208–216. Publisher Full Text\n\nDalziel J: Prospects for learning design research and LAMS. Teaching English with Technology. 2009; 9(2): i–iv. Reference Source\n\nFullan MG, Stiegelbauer S: The new meaning of educational change. (2nd ed), New York: Teachers College Press, 1991; 2(4): 336–343. Publisher Full Text\n\nFullan M: Change forces the sequel. London, Philadelphia: Open University Press, 1999. Reference Source\n\nGhislandi PMM, Raffaghelli JE: Forward-oriented designing for learning as a means to achieve educational quality. Brit J Educ Technol. 2015; 46(2): 280–299. Publisher Full Text\n\nGleadow RM, Ladiges PY, Handasyde K, et al.: Innovative teaching methods in Biology incorporating self-study and multimedia programs. In Promoting Teaching in Higher Education. Reports from the National Teaching Workshop. (eds. J Bain, E Lietzow and B Ross), 1993; 305–318. (Griffith University Press: Brisbane). Reference Source\n\nGleadow R, Macfarlan B: Dataset 1 in: Design for learning - a case study of blended learning in a science unit. F1000Research. 2015. Data Source\n\nGoodyear P: Teaching as design. In HERDSA Review of Higher Education. 2015; 2. : 27–50. Reference Source\n\nHannon P, Umble KE, Alexander L, et al.: Gagne and Laurillard’ s models of instruction applied to distance education: A theoretically driven evaluation of an online curriculum in public health. Int Rev Res Open Dist Learn. 2002; 3(2): 1–16. Reference Source\n\nLaurillard D: Teaching as a design science: building pedagogical patterns for learning and technology. New York: Routledge, 2012. Reference Source\n\nLaurillard D, Charlton P, Craft B, et al.: A constructionist learning environment for teachers to model learning designs. Blackwell Publishing Ltd. 2013; 29(1): 15–30. Publisher Full Text\n\nMacfarlan B, Everett R: E-Mentors: A Case Study In Effecting Cultural Change. In Donnelly R, Harvey J, and O’ Rourke K (eds) Critical Design and Effective Tools for E-Learning in Higher Education: Theory into Practice. New York, IGI Global, 2010; 244–261. Publisher Full Text\n\nMor Y, Craft B: Learning design: reflections upon the current landscape. Res Learn Technol. 2012; 20: 85–94. Publisher Full Text\n\nPatria B: Change Management in the Higher Education Context: A Case of Student-centred Learning Implementation. Int J Educ. 2012; 4(4): 176–191, ISSN 1948-5476. Publisher Full Text\n\nSheey P, Marcus G, Costa F, et al.: Implementing e-learning across a faculty: Factors that encourage uptake. Proceedings of the 23rd Annual Conference of the Australasian Society for Computers in Learning in Tertiary Education: Who’ s Learning? Whose Technology? 2006. Reference Source\n\nSims R, Dobbs G, Hand T: Enhancing quality in online learning: Scaffolding planning and design through proactive evaluation. Distance Education. 2002; 23(2): Carfax Pub. Publisher Full Text\n\nVygotsky LS: Mind in society: The development of higher psychological processes. Cambridge, MA: Harvard University Press, 1978. Reference Source"
}
|
[
{
"id": "10477",
"date": "07 Oct 2015",
"name": "Mary Williams",
"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 enjoyed reading this article and recommend it for indexation.I appreciate the authors’ understanding of today’s students, who are different from those of a generation ago particularly in their attitudes and expectations of their digital learning environment. As this article indicates, course instructors and designers can be more effective in their instruction by leveraging today’s learners’ skills and abilities. There are two important messages for educators here.Use Moodle more effectively. The authors present a before-and-after description of their redesign of a course Moodle page. It is common, as the authors say, for course organizers to use Moodle as an electronic filing system, but this usage does not particularly enhance a course. Rather, Moodle can be integrated into the course design to help students to navigate assignments and expectations. Furthermore, images and design elements are not difficult to incorporate into Moodle pages yet provide them with the contemporary feel our digital learners have come to expect. I would encourage the authors to disseminate their good ideas by sharing generic Moodle pages and template with others. The expression “Something to read, something to do, something to think about” provides a simple mnemonic device for instructors when designing a course and students when negotiating it that I think will enhance teaching and learning independently of the digital environment. As someone who has navigated several MOOCs, keeping track of assignments can be tricky; a schedule that requires certain tasks to be performed weekly will make the students’ job easier. I also like the way these three tasks span the spectrum of teaching methods: read this (student is relatively passive), do this (student must be more active), think about this (student must step back and grasp the big ideas that the first two tasks lead to). This framework will also help instructors make sure that their reading lists and assignments are more than just busy work but are truly relevant to the week’s objectives. Two small suggestions: I first read this as a printed PDF without hyperlinks, so I would have appreciated having all of the in-text references listed in the References section (e.g., Breen, 1999; Horvath & Loge, 2015 etc.).Also, I found Figure 7 confusing. There seems to be a formatting problem on the Y axis. Furthermore, although the legend says “..Where 1 is a negative and 4 is a positive” the legend in the graph suggests that the leftmost column (blue) means Agree, which confuses me. Somehow this graph needs to be clarified.",
"responses": []
},
{
"id": "10478",
"date": "09 Oct 2015",
"name": "Katharina Freund",
"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 provides constructive and clear advice for learning designers and academics looking to re-design courses for blended delivery, and to make more effective use of the LMS (in this case, Moodle). I appreciated the accessible writing style, and would recommend this article to academics to assist in their teaching.A few small comments:I have some reservations about generalising student cohorts based on generational distinctions such as \"Millenials\", as this does not necessarily address the diversity of the student body. I do agree with your overall point - that it's easy to source information online - though. There was a display error in Figure 7, where the numbers on the Y-axis of the graph displayed as signs (like #$). I'm glad of the discussion on culture change as an important part of the process towards more effective use of Moodle, as I think this is one of the most important elements. I am reminded of the work on innovation diffusion in higher education - perhaps this might be relevant for inclusion in any future revisions. (See for example McLaren and Kenny 2015 in Australian Universities' Review).I was curious as to the life of the project and workload involved in making these changes. How long did it take, and how many people worked on it? Obviously there was a positive effect on this course, and the new design was appreciated by students on the whole. How scalable was this initiative to more courses, and more institutions, and what sort of investment would be required?Thank you for the invitation to peer review; I found the article insightful.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-898
|
https://f1000research.com/articles/4-1250/v1
|
12 Nov 15
|
{
"type": "Case Report",
"title": "Case Report: ‘Z’ osteotomy - a novel technique of treatment in Blount’s disease",
"authors": [
"Raju Karuppal",
"Rahul Mohan",
"Anwar Marthya",
"Gopakumar TS",
"Sandhya S",
"Rahul Mohan",
"Anwar Marthya",
"Gopakumar TS",
"Sandhya S"
],
"abstract": "Blount’s disease is a progressive form of genu varum due to asymmetrical inhibition of the postero medial portion of the proximal tibial epiphysis. The surgical treatments involved in correction of Blount’s disease are often technically demanding, complicated procedures. These procedures can lead to prolonged recovery times and poor patient compliance. In such a context we are suggesting “fibulectomy with Z osteotomy” of the proximal tibia, a relatively simple and highly effective technique. This technique is based on correcting the mechanical axis of the lower limb thereby restoring growth from the medial physis of proximal tibia. We have used a new surgical technique, which includes fibulectomy followed by a Z-shaped osteotomy. We have used this simple technique in a 5 year-old boy with unilateral Blount’s disease. The femoro-tibial angle was corrected from 18.2° of varus to 4.2° of valgus. The angular correction obtained after operation was 22°. There were no postoperative complications. This technique has the advantages of correcting both angular and rotational deformities simultaneously. The purpose of this case study is to introduce a new surgical technique in the treatment of Blount’s disease.",
"keywords": [
"Infantile tibia vara",
"Blount’s disease",
"Z tibial osteotomy"
],
"content": "Case report\n\nA 5 year-old Indian male child presented in June 2013 with unilateral right sided genu varum (Figure 1) noted since January 2013. The deformity was gradually progressing in character. There was no significant history of trauma or infection. Clinical examination showed 18.2° of varus and 10° of intortion of tibia. Biochemical investigations were normal. X-ray showed depression of medial tibial plateau with beaking of posteromedial tibial metaphysis (Figure 2) MRI showed an irregular medial physeal line, postero-medial depression, thinning of medial epiphyseal cartilage and concomitant increase in the joint space (Figure 3). As the child is already 5 years old, to achieve a rapid complete correction surgical treatment was opted. Of the many surgical options like wedge osteotomy and ilizarov correction have its own many demerits. Hence we decided for Z osteotomy which will correct the angular and rotational deformities. It also has predictable result and potential for minimal complications.\n\n\nOperative technique of Z osteotomy\n\nPre-operative planning includes quantifying the tibio-femoral angle from standing antero-posterior X-ray of both lower limbs. The surgical aim is to achieve a correction of 5° to 7° of valgus. The rotational correction needed should also be assessed clinically. The patient should be in supine position. A tourniquet is applied and a fibular osteotomy is performed at the middle of the fibula. The proximal shaft of the tibia is then exposed through a 5 cm long incision just below the tibial tuberosity in the midline.\n\nFor the left tibial varus deformity, the upper horizontal limb of the Z osteotomy starts at the medial border of tibia, one finger breadth below the tibial tuberosity, to the anterior border of the tibia. The vertical limb descends down from this point along the anterior border for a same distance of the upper horizontal limb. The lower horizontal limb of the Z osteotomy starts from this point horizontally to the lateral border of the tibia. (For the right tibial deformity, the reverse Z osteotomy is made).\n\nA Z shape is marked on the proximal tibia by drill holes which are connected by an osteotome to complete the osteotomy. The width of the wedge to be removed is calculated as 1mm for each 1° of angle to be corrected. A wedge of bone is removed between the vertical and lateral horizontal limb of the Z osteotomy (Figure 4) and the distal tibia is derotated and angulated laterally then re-engaged in the corrected position. The osteotomy site is then stabilized with one or two k wires (Figure 5). The final alignment is confirmed by an X-ray image intensifier. The leg is then immobilized in a long-leg cast. In the presented case, this was worn for eight weeks, after which weight-bearing was allowed. The postoperative period was uneventful.\n\nWedge of bone is removed (Shown in blue shape) and the distal tibia is derotated and then re-engaged in the corrected position. The red line shown on the fibula is the fibular osteotomy.\n\na. Close-up view of correction of deformity by Z osteotomy and stabilization with 2 K wires, b. Varus deformity is corrected to normal alignment, c. Immediate post-operative X-ray anterior posterior view, d. Immediate post-operative X-ray lateral view.\n\nThe femoro-tibial angle was corrected from 18.2° of varus to 4.2° of valgus. The angular correction obtained after operation was 22°. There were no major complications or any neurological problems in our case. The result was favorably comparable with other reported surgical techniques. At the last follow-up in May 2015 child had maintained the correction of angular and rotational alignment (Figure 6).\n\na. Standing front view shows deformity is corrected to normal and comparable to the opposite side, b. Standing side view shows deformity is corrected, c. Front view of leg in knee flexed position, d. Side view of leg in knee flexed position, e & f. X-ray AP and lateral view shows consolidation of osteotomy site with normal tibial mechanical axis.\n\n\nDiscussion\n\nInfantile tibia vara or Blount’s disease was the first described by Erlacher in 19221. The three-dimensional complex deformity of Blount’s disease includes varus, internal rotation, and (sometimes) procurvatum2. The progressive varus deformity in Blount’s disease is thought to be due to repetitive, compressive injury of the proximal tibial growth plate medially with relative overgrowth of the lateral tibial physis3. The varus deformity may improve by the age of 4 years; hence operation should be delayed unless significant lateral thrust or other symptoms develop. The spontaneous resolution of the varus deformity in Blount’s disease is rare4.\n\nA rapid complete correction would generally be achieved by an osteotomy. It is not advisable to operate on physis to avoid growth disturbances. The commonly used osteotomies are closing and opening wedges at proximal tibia. Other alternatives are Dome and chevron-type osteotomies5. The results of closing-wedge, proximal tibial osteotomy was published by Laurencin et al.6. It has many disadvantages like fracture of the medial cortex, which would produce over correction and shortening of the limb. Martin et al.7 described the result of Opening-wedge-type osteotomy. The disadvantages of this technique include undercorrection of the internal tibial torsion and instability at the osteotomy site, which requires rigid internal fixation. The disadvantages of external fixation for stabilising osteotomies for tibia vara are longer associated consolidation times, unsightly scars and the need for expensive, complex devices8. It also has other demerits, like pin-track infections and postoperative neurapraxia.\n\nThe best way to obtain correction should be a simple procedure as near to the deformity (as high in the tibia as possible) to promote rapid union and quick remodeling9.\n\nThe Z osteotomy of tibia as it has many advantages. Biomechanically, it is more stable than the closing and opening wedge osteotomies because of the special geometry of the osteotomy. Rotational deformity can be simultaneously corrected without affecting the stability and contour of the bone. Bone healing is predicted to be better because of the larger surface area at the osteotomy site. In the surgeon’s perspective it is simple to learn and perform. Only one or two K wires for short term fixation are required because of the inherent stability of the Z shape of the osteotomy, hence a second surgery for the implant removal can be avoided as well. The correction achieved following Z osteotomy is based on the principle of correcting mechanical axis of lower limb thereby restoring growth from the medial tibial physis. As such this procedure does not have any contraindications or limitations similar to other corrective osteotomies.\n\n\nConclusion\n\nThe Z osteotomy of tibia is a useful simple technique for the correction of tibia vara in Blount’s disease, which has not been previously described in the literature. The Z osteotomy achieves angular and rotational correction of the deformity requiring minimal internal fixation. The amount of correction can be predetermined by appropriate wedge dimensions. It is an easy technique to learn and perform. It allows correction of the deformity while maintaining length, restoring joint alignment and mechanical axis of the limb.\n\n\nConsent\n\nWe have obtained informed consent for publication of clinical details and images from the parent of the child.",
"appendix": "Author contributions\n\n\n\n\n\n1) Dr.Raju Karuppal: Has been instrumental in the conception, design, acquisition and interpretation of data. Has done drafting the article and final approval of the version to be published.\n\n2) Dr.Rahul Mohan: Has contributed in the acquisition, analysis and interpretation of data. He has contributed in drafting the article and revising it critically for important intellectual content.\n\n3) Dr.Anwar Marthya: Has been instrumental in the conception, design, acquisition and interpretation of data. Has done drafting the article and final approval of the version to be published. He has been involved in the final approval of the version to be published.\n\n4) Dr.Gopakumar.T.S: Has involved in the conception and design of study. Has done drafting the article and final approval of the version to be published.\n\n5) Sandhya S: Has involved drafting the article or revising it critically for important intellectual content and final approval of the version to be published.\n\n\nCompeting interests\n\n\n\nThe authors declared 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\nBlount WP: Tibia vara Osteochondrosis deformans tibiae. J Bone Joint Surg (Am). 1937; 19(1): 1–29. Reference Source\n\nRab GT: Oblique tibial osteotomy revisited. J Child Orthop. 2010; 4(2): 169–172. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacnicol MF: Realignment osteotomy for knee deformity in childhood. Knee. 2002; 9(2): 113–120. PubMed Abstract | Publisher Full Text\n\nZayer M: Osteoarthritis following Blount’s disease. Int Orthop. 1980; 4(1): 63–6. PubMed Abstract | Publisher Full Text\n\nStaheli LT: The lower limb. In: Morrissy RT (Ed), Pediatric orthopedics. (3rd edn), Philadelphia: J.B. Lippincott, 1990; 741–66.\n\nLaurencin CT, Ferriter PJ, Millis MB: Oblique proximal tibial osteotomy for the correction of tibia vara in the young. Clin Orthop Relat Res. 1996; (327): 218–24. PubMed Abstract\n\nMartin SD, Moran MC, Martin TL, et al.: Proximal tibial osteotomy with compression plate fixation for tibia vara. J Pediatr Orthop. 1994; 14(5): 619–22. PubMed Abstract | Publisher Full Text\n\nPrice CT, Scott DS, Greenberg DA: Dynamic axial external fixation in the surgical treatment of tibia vara. J Pediatr Orthop. 1995; 15(2): 236–43. PubMed Abstract\n\nAly TA, El-Rosasy M, Saied MS: Upper Tibial Coronal Plane- Oblique Osteotomy for Deformity Correction in Blount’s Disease. Pan Arab J Orth Trauma. 2004; 8(2): 135–138. Reference Source"
}
|
[
{
"id": "14311",
"date": "14 Jun 2016",
"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 an interesting, nicely illustrated, article describing a surgical technique that can be applied for the management of Blount's disease. Although most surgeons use external fixation for the correction of such deformities, the current article addresses another possibility, namely acute correction and internal fixation. Some patients (especially small children) might not tolerate external fixation, thus this technique offers another tool in the surgeon's armamnetarium. Therefore it is a contribution to the literature if alternative techniques are presented. The postoperative result is also nicely illustrated.",
"responses": []
},
{
"id": "14967",
"date": "01 Aug 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 varus deformity in Blount’s disase. The case report and surgical steps are clearly described. The described method appears to be a simple and the clinical and radiological correction achieved by the described procedure looks good and hence could contribute as an additional technique in the management of Blount’s disease.\nSome of my queries and suggestions are as follows:\nCase report (line 5) – “Clinical examination showed 18.2 deg of varus”. It would be better if authors can round off the deformity to 18 deg, as assessing 0.2 deg of varus CLINICALLY would be really challenging.\n\nCase report (line 10) – Even though, X ray shows medial joint space widening, the MRI does not show concomitant increase in the joint space. Hence that sentence may be modified.\n\nWhile de-rotating and taking the distal fragment into valgus, the superomedial corner of the distal fragment is seen to be unduly prominent (on radiograph). Considering the fact that the medial border of tibia is subcutaneous, whether this undue prominence can lead to wound dehiscence is a cause of worry.\n\nSignificant gap (on radiograph) is noted at the proximal aspect of the osteotomy site after de-rotation and it would be nice to know if authors feel that the wedge of bone removed from the lateral side can be put back as a graft on the medial side if significant gap is noted.\n\nFigure 4 – The fibular osteotomy demonstrated in the figure 4 is a medial to lateral downward sloping short oblique osteotomy. However, in the antero-posterior radiograph of the index case the fibular osteotomy appear as a transverse cut. Readers would be interested to know about the best direction for the fibular osteotomy which would help in translation of the fibula during de-rotation.\n\nDiscussion – It would be informative if authors can discuss the merits and demerits of RAB’s osteotomy which seems to be simpler than ‘Z’ osteotomy. Also, post-operative correction can be adjusted if under or over correction is noted by wedging of the cast in RAB’s osteotomy.\n\nConclusion – I/we feel ‘Z’ osteotomy is not simpler than RAB’s osteotomy. The conclusion may be modified describing ‘Z’ osteotomy as another new technique to correct the mechanical axis.\n\nIt would be worth mentioning that a longer follow up is required to know the status of the proximal tibial medial physis and if the correction of the deformity is maintained till skeletal maturity.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1250
|
https://f1000research.com/articles/4-1244/v1
|
11 Nov 15
|
{
"type": "Opinion Article",
"title": "On the pitfalls of peer review",
"authors": [
"Wilfred van Gunsteren"
],
"abstract": "The review process of academic, scientific research and its basic tenets is considered, thereby distinguishing between (i) reviewing of manuscripts to be published in the scientific literature, (ii) reviewing of research proposals to be financed by funding agencies, (iii) reviewing of educational or research institutions with respect to their proper functioning, and (iv) reviewing of scientists with the aim of appointing or tenuring faculty.",
"keywords": [
"research manuscripts",
"research proposals",
"faculty appointment",
"faculty tenure",
"peers",
"performance indicators",
"institutional review",
"editorial process"
],
"content": "Introduction\n\nScientific research is primarily driven by curiosity, by the desire to determine relationships between observations and to develop models that can be used to understand and predict phenomena in the world in which we live. It is a continuous process of refinement and extension of knowledge and understanding. Every generation of researchers stands on the shoulders of those who have gone before. This is why reporting research results, be it positive or negative ones, in the scientific literature is of importance to the progress of our understanding and knowledge. The scientific literature constitutes the database of scientific knowledge. Because any research may be flawed due to erroneous observations, overseen correlations, incorrect assumptions, or just sloppy reasoning, any model or theory that is proposed must be justified and tested against data that are both tangible and publicly available. Publication of research results allows other scientists to check the validity of a proposed model or theory without having to repeat the original work, thereby facilitating scientific progress. Thus the integrity of published research is of fundamental value to the academic community of scholars.\n\nSince the 18th century quality control of research publications has been exerted by peer review: the judgement of scientific reports by academics with equivalent knowledge. Peer review can only function under the umbrella of the ethics of science1. This assumes an unbiased examination of the opinion or data based on logical and empirical criteria and places trust in the competence and honesty of the reviewers, be they a colleague or a competitor. However, with the expansion in both the size and number of research institutions over the past half century, the number of research publications has grown rapidly, reaching about 1.4 million per year in 2013. To properly evaluate such a large number of manuscripts is a challenge and puts severe strain on the peer review system. The increasing mass of submitted manuscripts of decreasing quality and relevance is slowly choking the review system and thus slowly corrupting the database of knowledge2. It also leads editors of journals to use odd arguments, “I regret to inform you that our editors have now carefully considered your manuscript and feel it unsuitable for publication as we have not been able to secure reviews.” A testimonium paupertatis.\n\nPeer review is also called upon by agencies funding research in the process of evaluating research proposals in regard to funding. This role of peer review adds a financial dimension to the review process: money that is allotted to the research of a competitor will generally not be spent on research of the reviewer. One only has to remind oneself of the steady stream of scandals in the banking industry to fear the impact of money upon the ethics of science.\n\nA third type of reviewing is the evaluation of educational or research institutions such as universities in regard to how they function compared to that envisaged by the boards of such institutions. This role of peer review adds yet further dimensions to the review process including the goals and outcomes in teaching of science and research, and the effectiveness of the corresponding organisational structure. Here peer review requires more than reading and judging manuscripts and research proposals.\n\nPeer review is also invoked by universities when recruiting faculty. Apart from the ability to offer inspiring teaching and original and high-quality research, aspects of a candidate’s personality are to be gauged: openness of mind, views on academic ethics, sensitivity to academic issues broader than research and teaching, for example. Here peer review requires ‘seeing through’ a person with respect to psychological, social, ethical and organisational abilities.\n\n\nReview of manuscripts to be published in the scientific literature\n\nIn view of the development of the internet and its storage capacity, one could consider the option to refrain from peer review and allow anyone to publish whatever she or he wishes. As the development of Wikipedia has shown, absence of any form of review may quickly lead to a loss of reliability of the material published. Thus some validation3 and selection of submitted manuscripts by peer review seems necessary to avoid too much corruption of the database of scientific knowledge. A reviewer should formulate an opinion on the quality of a manuscript:\n\n1. Clarity of text, tables and figures.\n\n2. Reproducibility of the results from the data specified.\n\n3. Sound connection between the results and the conclusions (no overstatements).\n\n4. Embedding of the results in the literature (proper referencing).\n\n5. Relation to other methods addressing the same problem.\n\n6. Novelty of the method or results.\n\n7. Relevance of the results to the scientific community.\n\nThe first six aspects can be addressed objectively, whereas judgement of the relevance of particular research is subjective. Reviewers should not prescribe what they think to be important and should be written or omitted from the manuscript, or prescribe particular references remotely related to the topic to be included4,5.\n\nThe quality of the reviewer’s report is to be evaluated by the editor who requested it:\n\n1. Apparent knowledge of a reviewer regarding the subject of the manuscript.\n\n2. Validity and consistency of the arguments of a reviewer. I have seen reviewers who initially judge the reported research to be “relevant, a novel approach and publishable”, but after the authors had refused to comply with an inappropriate request by the reviewer, then judge the manuscript as “not providing physical insight and inappropriate for publication in the journal”.\n\n3. Possible bias because of a vested interest of a reviewer, e.g. reflected in criticism of an Introduction or Discussion, without criticism of the results themselves.\n\nEditorial decisions should be consistent as function of time. I have seen an editor asking for the addition of data and, upon this request being honoured, rejecting the manuscript.\n\nOne way to avoid your work being reviewed and edited by persons with insufficient knowledge of the field is to submit to journals maintained by professional organisations such as the national chemical or physical societies. These suffer less from sensationalism and are less influenced by hypes when selecting manuscripts. As a colleague once confided when questioned about overstating his results: “Of course you tone down the wording of a manuscript after it has been accepted for publication by Science or Nature.” Or, as a former colleague at the ETH once said: “Why lose time arguing with incompetent reviewers or editors of a high-profile journal, if you can get your work competently reviewed and smoothly published in a quality journal such as Helvetica Chimica Acta? If the published science is of real, lasting importance, it will sooner or later be noticed, irrespective of the journal.”. A genuinely academic opinion.\n\n\nReview of research proposals\n\nFunding agencies also use peer review to select research proposals for funding6,7. Often particular research goals are set, such as relevance to society or to the development of methods, tools or materials of practical interest, e.g. for industry. Innovation is a much cherished, frequent request. This leads to scientists echoing these goals in the introduction of the proposal, e.g. claiming they will develop multiple drugs to treat wide-spread diseases such as AIDS, stroke or dementia, while the proposed research itself is at best only remotely related to achieving this goal. Such a discrepancy between claims and content in a proposal is at odds with the ethics of science and undermines the credibility of the scientist and as a result the chance of getting the proposal approved, because the credibility of the researcher must be considered by the reviewer when answering the question as to whether the proponent will be able to successfully carry through the proposed research. Obtaining funding for basic science and risky but well-thought-through projects with a long-term perspective becomes difficult if short-term relevance is requested by agencies8.\n\nResearch proposals should be judged in terms of (i) the attainability of the stated goals using the proposed means, (ii) the risk of failure versus the resources requested, and (iii) the ability of the proponent to carry through the proposed research. These must be considered while always remembering that the result of an exploration of uncharted territory cannot be planned, no matter how many milestones are requested. One can plan to put a man on the moon, but not to invent a new material.\n\n\nReview of educational research institutions\n\nUniversities regularly use peer review to evaluate the performance of their different departments regarding three major aspects of their activity: (i) effectivity and content of their teaching, (ii) quality and novelty of their research, and (iii) effectivity of their organisational structure. Such peer review also has its pitfalls. Not only are more aspects involved than only the quality of research, but also the sheer number of scientists and personnel to be evaluated makes this a nearly impossible task to execute based on a site visit lasting just a few days. Of course one could require the reviewing committee to spend more time at the institution, but this will reduce the willingness of good scientists to participate in reviews. I was once asked to chair a committee tasked with evaluating the performance of the chemistry departments of ten Dutch universities. The agency in charge estimated the time required would be 45 days. When I asked whether they thought the president of the ETH would appreciate me spending about nine weeks in The Netherlands, I did not get an answer from the agency. It is also almost impossible to obtain a reliable impression regarding the teaching abilities of staff during a visit of a few days. In addition, increased specialisation hampers institutional review: it is nearly impossible to cover all types of research performed in a large department by a review committee consisting of even 10 to 15 scientists. Most have much less.\n\nThe effectivity of an organisational structure can only be judged by reviewers who are familiar with the socio-cultural and political environment of the institution. For example, an organisational structure that functions well within the context of the US culture and research landscape based on funding through research agencies and foundations may be not appropriate in a European context where funding is primarily through government channels. Or, teaching to British high-school graduates may require an approach different from teaching to graduates from German or Swiss Gymnasiums.\n\nIn view of these odds, and because an academic institution generally changes rather slowly, it would be wise to limit institutional review to once in say 10 years, and to select a reviewing committee consisting of scientific peers with academic experience and a sense for the socio-cultural environment of the institution to be reviewed.\n\n\nReview of scientists with respect to appointment or tenure\n\nOne of the most important tasks of university management is recruiting of faculty. Any error made will have lasting detrimental effects due to the long residence times of faculty and its central role in teaching and research and when serving as peers. The procedure of nomination of faculty and the role of peers in it should strike a balance between the opinion of scientific peers in a selection committee who are knowledgeable in the particular field of research of an open faculty position and who may judge the quality and originality of the research of a candidate on the one hand, and the opinion of the other members of the committee who are knowledgeable in other fields of research on the other hand. The latter may judge clarity of presentation and the maturity of the personality of a candidate without possibly being biased by feelings of collegiality with persons working in their field.\n\nAt the ETH this is secured by a selection committee composed by the president of the ETH upon proposal by the department in which the faculty position is located, and in which members from other departments and from outside the ETH constitute a majority, with an independent chairman of the committee, chosen from a pool of such chairs, and a secretary from the staff of the president9. Since the peers that are members of the department are a minority, they must convince the outsider peers of the quality of a candidate, a barrier against co-optation within a field of research. Yet the majority will follow the minority of department members in two cases. If the department members of the committee express their minority wish (i) to invite a particular candidate for a research presentation and interview, and (ii) to veto a candidate favoured for the faculty position by the majority of the committee, because they expect not to be able to work with the selected person. For a committee of 10 to 15 peers from different departments and institutions, judgement of research and teaching abilities of candidates should not be too difficult. But, for a thorough evaluation of personality characteristics a single research presentation and an interview by the committee may not be sufficient. One would rather observe how a future colleague would function in the different roles of a faculty member of a university. The latter is much easier when evaluating candidates for tenure. The members of the department of a tenure candidate have the opportunity to observe the functioning of a tenure-track professor a few years before proposing tenure to the tenure committee, which then only has the duty to see to it that the quality standards in research and teaching are fulfilled.\n\nEssential for recruiting of faculty is the composition of a selection committee in regard to judging research, teaching and personality of candidates and its ability to conduct an open and honest discussion on real issues regarding candidates, i.e. not on scientifically rather irrelevant issues10 such as citations of recent research publications, i.e. short-term popularity, h-indices or grant money gathered. Being one of many co-authors of a paper in a high-profile journal such as Science or Nature is not to be considered to reflect scientific quality or long-time vision.\n\n\nThe use of indicators of performance\n\nThe time pressure on reviewers will inevitably induce them to rely on performance indicators rather than spending time to investigate in depth the research of a scientist. However, measurement results in numbers, and numbers reflect quantity, not quality. Quality cannot be caught in a number. It is also seductive to compare numbers10–12. In other words, numbers lead to rankings, and rankings lead to competition. Excessive competition undermines care and rigour, encouraging activities close to or, ultimately, beyond the boundaries set by the ethics of science2. The increasing pressure to violate academic principles is illustrated by the mounting number of cases of plagiarism and scientific fraud13. Focus on quantity as opposed to quality also leads to the aversion of risk: truly difficult and innovative research is shunned. A focus on competition will not enhance the quality of research. Quality measured by metrics alone is an illusion and the cost to society is growing inefficiency14.\n\nIndicators such as number of citations of publications, grant money gathered, number of successful students educated, or student satisfaction are only useful to detect extremes. A curriculum vitae with more than 1000 research publications must raise questions regarding the true involvement of the person in question in the research and the scope of the issues addressed. On the other hand, a lack of publication activity may indicate a lack of effort, the inability to finalise work, or reflect the difficulty of the research being executed. Student evaluations of courses are dependent on the difficulty of the topic, whether the course was logically structured, on the size of the class, whether the course was obligatory, how many credit points could be earned, the knowledge of the students, etc.. As a teacher I consistently received higher marks for an optional 3rd year course on algorithms and programming, a well-structured topic taught to a class of about 20, than for a general obligatory freshman course on computer science for about 200 chemists and biologists, for whom the topic was not their primary interest. High marks for teaching may reflect more the ability to entertain than to inspire and teach. Low marks may reflect a lack of interest by the students in the subject as much as genuine lack of clarity.\n\nOne of the most perverse consequences of the growing importance of rankings and competition between universities I have seen was a quarter-page advertisement in a daily newspaper in The Netherlands. In it, the University of Utrecht thanked five of its scientists for having obtained a European Research Council Consolidator Grant. My parents – although not being graduates of this university - would turn in their grave, they used to say “Science needs no applause”, an echo from times gone by?\n\n\nWhat’s to be done?\n\nScience lives from an open exchange of arguments and data. It is damaged by reviewers prescribing what kinds of arguments can or cannot be published. Research proposals should not be evaluated by reviewers easily impressed by hype or unjustified promise of utility in order that scientists who follow trends, promising much but delivering poorly, can be barred from funding. The larger the gap between proposals, publications and scientific reality, the greater the long-term damage to the academic community of scholars and its credibility will become. If the curriculum vitae of an applicant for a faculty position lists the number of citations or an h-index value or the amount of grant money gathered, this is to be regarded as a sign of superficiality and misunderstanding of the academic research endeavour, a basic flaw in academic attitude, or at best as a sign of bad taste2.\n\nTo maintain their credibility as impartial peers, reviewers should refrain from reviewing work by close collaborators and should be conscious of potential bias when reviewing work of colleagues. Networks of scientific friends that review the work of each other in an unjust manner undermine scientific integrity. They constitute a perversion of the ethics of science5. Yet, excluding from review all persons with whom a proponent of a research proposal has a joint publication may lead to equally perverse outcomes. As a former vice-president for research of the ETH discovered, this rule excluded more than 260 scientists familiar with a given field from reviewing an ETH research grant proposal.\n\nThe process of review by peers has its pitfalls. It needs to be handled with care and a sense of proportion. There is, however, no viable alternative. Using indicators, which primarily reflect quantity, not quality, leads to perverse incentives and should be avoided14.",
"appendix": "Competing interests\n\n\n\nThe author declared no competing interests.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI thank David Gugerli, Alan Mark, Frédéric Merkt, Sereina Riniker, Philippe Hünenberger, and Jožica Dolenc for helpful comments and suggestions.\n\n\nReferences\n\nMerton RK: On the Shoulders of Giants: A Shandean Postscript: The Post-Italianate Edition. (with Umberto Eco and Denis Donoghue), University of Chicago Press, Reprint Edition, ISBN 0-226-52086-2. Reference Source\n\nvan Gunsteren WF: The seven sins in academic behavior in the natural sciences. Angew Chem Int Ed Engl. 2013; 52(1): 118–122. PubMed Abstract | Publisher Full Text\n\nvan Gunsteren WF, Mark AE: Validation of molecular dynamics simulation. J Chem Phys. 1998; 108: 6109–6116. Publisher Full Text\n\nReedijk J: Citations and ethics. Angew Chem Int Ed Engl. 2012; 51(4): 828–830. PubMed Abstract | Publisher Full Text\n\nBarbour V: Perverse incentives and perverse publishing practices. Sci Bull. 2015; 60(14): 1225–1226. Publisher Full Text\n\nSchatz G: Jeff’s View, Networks, fretworks. FEBS Lett. 2003; 553(1–2): 1–2. Publisher Full Text\n\nQuack M: Myths, Challenges, Risks and Opportunities in Evaluating and Supporting Scientific Research. In Incentives and Performance. Welpe IM, et al. Eds, Springer Intl. Publishing Switzerland. 2015; 223–239. Publisher Full Text\n\nThomas JM: Intellectual freedom in academic scientific research under threat. Angew Chem Int Ed Engl. 2013; 52(22): 5654–5655. PubMed Abstract | Publisher Full Text\n\nEichenberger T: Faculty Handbook. Office of Faculty Affairs ETH Zürich. Zürich. 2012; 1–40. Reference Source\n\nMolinié A, Bodenhausen G: Bibliometrics as weapons of mass citation. Chimia (Aarau). 2010; 64(1–2): 78–89. PubMed Abstract\n\nErnst RR: The follies of citation indices and academic ranking lists. A brief commentary to 'Bibliometrics as Weapons of Mass Citation'. Chimia (Aarau). 2010; 64(1–2): 90. PubMed Abstract | Publisher Full Text\n\nGibb BC: Lies, damned lies and h-Indices. Nat Chem. 2012; 4(7): 513–514. PubMed Abstract | Publisher Full Text\n\nExtance A: Data Falsification hits Polymer Mechanochemistry Papers. Chemistry World. 2015; 3: 9–10. Reference Source\n\nBinswanger M: Sinnlose Wettbewerbe: Warum wir immer mehr Unsinn produzieren. Herder Verlag, Freiburg im Breisgau, 2010. Reference Source"
}
|
[
{
"id": "11210",
"date": "01 Dec 2015",
"name": "Xavier Daura",
"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\nPeer reviewing an article on the pitfalls of peer review reminds you of the responsibility of an act that, due to time pressure, we often do less proficiently than we should. In addition, if the author of the manuscript happens to be a friend, you get directly confronted with the essence of the topic. Can we be impartial about the work of friends and direct competitors? My own opinion is that we ought to if we want to be credible as a collective; but we need to be transparent about it. In more practical terms, as van Gunsteren puts it, finding peers in the same field of expertise both with no connection and in no competition with a given author could be an arduous task. If we talk about an opinion article it is even questionable that the reviewer should prescribe the author any changes (assuming the article is void of nonsense, excess of commonplace arguments or bad writing). Opinion articles are probably the most personal form of scientific writing, their only requisites being the ability to provoke thought and generate discussion on a topic of interest. The article by van Gunsteren fully complies with these principles. It also has a wider focus than recent articles on peer review, thus including the evaluation of publications, projects (ex-ante, but a similar discussion would be valid for ex-post evaluations), institutions and researchers. The discussion of the four levels of peer review is illustrated with examples, understandably centred on the author’s own experience. Some of the examples would seem anecdotal wouldn’t we all have similar experiences to tell. In summary, a concise, easy-reading article worth adding to recent discussions on an evaluation system that while far from ideal is still the best we have.",
"responses": []
},
{
"id": "11208",
"date": "03 Dec 2015",
"name": "Daan P. Geerke",
"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\nStarting from a systematic overview of the reviewing process, the current paper summarizes the true values and the possible pitfalls in scientific and academic refereeing. It can also be seen as a (very) useful guide for anyone involved in reviewing, including those who are making policy for the assessment of individual scientists, faculties, research schools and institutes, which is increasingly putting additional pressure to academic staff. The striking examples given by the author illustrate the strong need to keep (or even start?) discussing the mode of reviewing within scientific communities. In line with the previous referee report, I can also exemplify this need by personal experiences with peer reviewers judging the quality of grant applicants based on the number of papers in journals with impact factor > 10, or with universities bringing out press releases about their top researchers defined as grant awardees. Therefore the current paper is in my opinion not only a very good but clearly also a highly timely overview of pitfalls in peer reviewing.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1244
|
https://f1000research.com/articles/4-95/v1
|
21 Apr 15
|
{
"type": "Review",
"title": "I drink for my liver, Doc: emerging evidence that coffee prevents cirrhosis",
"authors": [
"Jordan J. Feld",
"Élise G. Lavoie",
"Michel Fausther",
"Jonathan A. Dranoff",
"Jordan J. Feld",
"Élise G. Lavoie",
"Michel Fausther"
],
"abstract": "Evidence demonstrating that regular ingestion of coffee has salutary effects on patients with chronic liver disease is accumulating rapidly. Specifically, it appears that coffee ingestion can slow the progression of liver fibrosis, preventing cirrhosis and hepatocellular carcinoma (HCC). This should excite clinicians and scientists alike, since these observations, if true, would create effective, testable hypotheses that should lead to improved understanding on fibrosis pathogenesis and thus may generate novel pharmacologic treatments of patients with chronic liver disease.This review is designed to examine the relevant clinical and epidemiological data in critical fashion and to examine the putative pharmacological effects of coffee relevant to the pathogenesis of liver fibrosis and cirrhosis. We hope that this will inspire relevant critical analyses, especially among “coffee skeptics”. Of note, one major assumption made by this review is that the bulk of the effects of coffee consumption are mediated by caffeine, rather than by other chemical constituents of coffee. Our rationales for this assumption are threefold: first, caffeine’s effects on adenosinergic signaling provide testable hypotheses; second, although there are myriad chemical constituents of coffee, they are present in very low concentrations, and perhaps more importantly, vary greatly between coffee products and production methods (it is important to note that we do not dismiss the “botanical” hypothesis here; rather, we do not emphasize it at present due to the limitations of the studies examined); lastly, some (but not all) observational studies have examined both coffee and non-coffee caffeine consumption and found consistent effects, and when examined, no benefit to decaffeinated coffee has been observed. Further, in the interval since we examined this phenomenon last, further evidence has accumulated supporting caffeine as the effector molecule for coffee’s salutary effects.",
"keywords": [
"adenosine receptor",
"coffee",
"liver fibrosis",
"cirrhosis"
],
"content": "Analysis of clinical and epidemiological data\n\nIt was recognized decades ago that caffeine is a vasoactive molecule; this led to concerns that coffee consumption may be associated with an increased risk of cardiovascular diseases, potentially leading to an increased risk of all-cause mortality. An early study by Klatsky and colleagues (1993) to address this issue using the Kaiser-Permanente database found that although very high coffee intake (> 4 cups per day) was associated with a slightly increased risk of myocardial infarction (relative risk 1.4, 95% CI 1.0–1.9), there was no overall effect on mortality, largely due to an unexpected finding of fewer deaths due to cirrhosis in coffee drinkers than non-drinkers. With each additional cup of coffee consumed per day, the risk of death from cirrhosis fell by 23% (RR 0.77, 95% CI 0.67–0.89)4. Subsequent studies have confirmed that coffee consumption is associated with improved outcomes on many parameters of liver disease ranging from liver enzyme levels and histological activity to rates of liver fibrosis progression and incidence of cirrhosis and hepatocellular carcinoma (HCC)1–3. The almost exclusively observational nature of the data has made it difficult to draw strong conclusions about causation and to identify the specific mechanisms involved; however, the consistency and magnitude of the findings certainly justify further investigations to clarify how coffee improves liver health.\n\nEarly studies from Europe and Japan found that regular coffee consumption was associated with lower gamma glutamyl transferase (GGT) and alanine aminotransferase (ALT) levels5–9. Ruhl and Everhart (2005) used data from the National Health and Nutrition Examination Survey (NHANES) to evaluate the association between coffee and caffeine intake and ALT elevation in American patients at increased risk for liver disease from alcohol, viral hepatitis, or obesity. They found a lower prevalence of ALT elevation with increasing coffee and particularly with increasing caffeine intake. After adjustment for confounders, individuals in the highest quintile of caffeine consumption had less than one third the risk of ALT elevation of those in the lowest quintile (odds ratio (OR) 0.31, 95% CI 0.16–0.61)10. To explore possible explanations for their findings, they evaluated whether lower insulin resistance in coffee drinkers could account for the reduced ALT levels. Although coffee consumption was inversely associated with fasting insulin levels, the relationship between coffee or caffeine intake and ALT was unaffected by inclusion of insulin levels in the model10. Thus, the possible effect of coffee on insulin/sugar balance was not a sufficient mechanism to explain the effects observed.\n\nMore recently, Molloy and colleagues (2012) evaluated the effect of coffee and caffeine consumption in patients with non-alcoholic fatty liver disease (NAFLD). They found a weak but statistically significant inverse correlation between caffeine consumption and ALT levels. Notably, caffeine and coffee intake were similar between patients without any evidence of NAFLD and those with established non-alcoholic steatohepatitis (NASH), whereas intake was lower in patients with NASH than in those with simple steatosis, suggesting that the protective effect of coffee and/or caffeine may be greatest in patients at risk for progressive liver disease11.\n\nInterestingly, in patients with chronic hepatitis C virus (HCV) infection, no relationship between coffee or caffeine consumption and ALT levels has been observed, despite the fact that increasing intake was found to be associated with reduced histological activity and fibrosis on liver biopsy11,12. This observation raises the possibility that coffee and/or caffeine consumption directly inhibit hepatic fibrosis independent of reducing hepatic inflammation, providing a distinct rationale for the study of coffee/caffeine on liver fibrogenic mechanisms.\n\nMore important than an effect on aminotransferase levels, increasing coffee and caffeine consumption has been found to be associated with reduced liver fibrosis, a finding that has been largely consistent across studies in HCV and fatty liver disease, whether related to NASH or alcohol.\n\nInitial studies from Italy found that patients with cirrhosis consumed less caffeine, and specifically less caffeine from coffee, than age and sex-matched controls13. Odds ratios for presence of cirrhosis in this study increased as a function of coffee consumption: 0.47 (95% CI 0.20–1.10) for patients consuming 1 cup of coffee per day and 0.16 (95% CI 0.05–0.50) for patients consuming 4 cups per day. Here the reference against which the above groups are compared is lifetime coffee abstainers. Caffeine intake from sources other than coffee was similar between cases and controls; however, it is critical to note that coffee accounted for the vast majority of caffeine consumption in both groups (likely reflecting the dietary habits of the Italian population studied). Similar results were seen in other studies using a case-control design14,15.\n\nModi and colleagues evaluated a cohort of patients with chronic liver diseases of various etiologies and found that patients with advanced fibrosis consumed less coffee and less caffeine than those with milder liver damage12. The effect size was greatest in patients with chronic HCV infection. They also found no relationship between caffeine from sources other than coffee or intake of decaffeinated coffee and the severity of liver fibrosis. Coffee, and caffeine specifically, is metabolized almost exclusively within the liver, which has raised the issue that individuals with more advanced liver fibrosis may reduce coffee intake because of a greater clinical effect of lower doses with progressive hepatic impairment. It is also possible that individuals with more advanced liver disease reduce coffee intake due to a perception that coffee is unhealthy. Modi and colleagues (2010) found that results from caffeine consumption questionnaires were consistent over time, and patients with more advanced fibrosis did not report reducing coffee or caffeine consumption as their disease progressed12.\n\nTo assess the clinical significance of fibrosis progression, Freedman and colleagues (2009) evaluated the effect of coffee consumption in the large HALT-C study, which included only patients with bridging fibrosis (F3) or cirrhosis (F4)16. They found that at baseline, increased coffee consumption was associated with milder liver disease; perhaps more importantly, during the 4-year study period, they found that patients who consumed more coffee had a lower risk of experiencing adverse clinical outcomes. Patients who consumed no coffee had a risk of hepatic decompensation or HCC of 11.1 per 100 patient-years compared to just 6.3 per 100 patient-years in those consuming ≥ 3 cups per day. Once again, no beneficial effect was seen with tea or other sources of caffeine. Interestingly, coffee consumption was also associated with better clinical responses to peginterferon and ribavirin therapy in this cohort17.\n\nCoffee has been shown to be associated with less severe fibrosis in patients with NASH as well. Interestingly, although coffee consumption was associated with less severe hepatic steatosis, the effect may not be limited to liver injury11. Increasing coffee consumption was found to be associated with a lower risk of metabolic syndrome in Japanese men, particularly in those drinking ≥ 4 cups per day (OR 0.61, 95% CI 0.39–0.95). The reduced rate of metabolic syndrome was due to an inverse association between coffee consumption and both blood pressure and triglyceride levels after controlling for other relevant factors18. Large population-based studies have also found that increasing coffee intake is associated with a lower incidence of diabetes19–21. The recent finding that coffee consumption was associated with a lower risk of insulin resistance and liver fibrosis in patients with HIV-HCV co-infection raises the possibility that the beneficial hepatic effects of coffee on the liver may relate to improved metabolic parameters, even in patients with diseases other than NAFLD22.\n\nOverall, observational data have consistently shown that patients with more advanced liver fibrosis consume less coffee than those with milder disease, particularly in patients with HCV and NAFLD. Although these data are certainly suggestive of a clinical benefit of coffee on fibrosis progression, caution must be taken before drawing direct causal inferences from these observational, non-interventional studies.\n\nThe initial observation that increased coffee consumption was associated with a lower incidence of HCC came from epidemiological studies from Italy and Greece14. This finding has been confirmed in multiple subsequent studies, including meta-analyses from other parts of the world23,24. Reassuringly, similar effects have been seen in case-control and cohort studies. The most recent meta-analysis including 16 studies with 3153 cases of HCC found that coffee consumption was associated with an overall relative risk of 0.60 (95% CI 0.50 to 0.71) for HCC compared to those who drink no coffee at all25. The results were consistent across studies after controlling for confounders and importantly showed that the apparent benefits of coffee seemed to increase with each additional cup consumed per day (RR of 0.80 per cup per day). Cirrhosis is the single most important risk factor for HCC. Whether coffee directly affects hepatic carcinogenesis or reduces HCC by slowing the progression of fibrosis and development of cirrhosis remains unclear.\n\nThere are as many as 1000 substances in coffee, any of which may have hepatoprotective or anti-fibrotic properties. Most studies have focused on caffeine, diterphenoic alcohols (cafestol and kawheol), as well as possible antioxidant properties of chlorogenic acid and tocopherols. To date, no studies have found an association between caffeine consumption from sources other than coffee and reduced liver injury. However, in almost all epidemiological studies to date, the vast majority of caffeine in the diet came from coffee consumption. To achieve equivalent levels of total caffeine intake, individuals must consume much more tea or caffeinated soda than coffee. Particularly if, as suggested in some studies12, there is a threshold of caffeine intake for a beneficial effect, it may be difficult to reach this level from non-coffee sources of caffeine (see Table 2).\n\nCoffee preparation affects the composition of the final product. Interestingly, the apparent benefits of coffee may be greatest with filtered coffee. Drip coffee reduces cafestol and kawheol, which have been associated with increasing LDL cholesterol and possibly with increased ALT levels26. This difference was borne out in a recent study that found that increasing filtered coffee consumption but not espresso consumption was associated with lesser degrees of liver fibrosis in obese European patients. In this study, espresso intake was associated with lower HDL cholesterol levels, higher triglyceride levels and a higher prevalence of metabolic syndrome27. In response to a recent report documenting an association of coffee consumption with reduced total and cause-specific mortality28, Aubin and Berlin noted that the benefits were largely seen in the era of filtered coffee consumption and may not extend to espresso and other unfiltered coffee, products which are increasing in use globally29. This is further compounded by the high degree of variability between coffee preparations, with up to 6-fold differences in caffeine content between different commercially available espresso products30. Clearly, before interventional studies can be seriously considered, it will be critical to clarify what in coffee has a hepatoprotective effect and what dose would be safe and effective.\n\nCollectively the epidemiological data showing a beneficial association between increasing coffee consumption and severity of liver disease are strong. The consistency of the findings across different parameters of liver injury and in different liver diseases is reassuring. Importantly, coffee consumption has been associated not only with reduced liver fibrosis but also with a lower incidence of liver cancer and hepatic decompensation, which are critically relevant clinical outcomes. However, it is important to recognize some important limitations to the existing literature.\n\nSpecifically, the data are almost exclusively observational, and most studies have been cross-sectional in nature. Presumably a beneficial effect of coffee on liver disease would require prolonged exposure from early in the disease state to prevent progression, unless coffee somehow promotes fibrosis regression. As a result, studies finding an association between current coffee consumption and the current degree of liver fibrosis are limited due to a lack of accurate data on prior coffee intake. Although some studies have assessed the consistency of coffee intake over time, recall bias is still a major potential confounder. In addition, the possibility that patients with more advanced liver disease reduce their coffee intake over time specifically because of the severity of their liver disease must be considered, at least in part because they are often encouraged to reduce alcohol and tobacco use, both of which are highly correlated with coffee intake.\n\nUnfortunately, it is difficult, if not impossible, to perform controlled trials of coffee use with hard clinical endpoints, most of which take years to occur. Cardin and colleagues (2013) recently performed a crossover-controlled trial of filtered coffee intake (4 cups per day) compared to none over a 30-day period in patients with chronic HCV infection. They found that during the period of coffee drinking, AST levels decreased, but GGT and HCV RNA levels rose. They also found that 8-dyrdoxydeoxyguanosine (8-OHDG) levels decreased, and telomere length increased, which they interpreted to suggest less oxidative DNA damage31. Although the authors should be commended for trying to perform a controlled trial of coffee in patients with any chronic liver disease, it is hard to interpret the results. Numerous comparisons were made, and even those that were statistically significant were of questionable clinical importance. In addition, the biological plausibility is somewhat questionable given the short duration of the study. Overall, this study highlights the challenge of conducting controlled trials of dietary interventions.\n\nAs suggested above, there are exciting data from patients to suggest that coffee and/or caffeine prevent liver fibrosis; however, the cellular mechanisms by which this effect may work are not fully understood. In an attempt to elucidate these potential mechanisms, we will first examine some of the animal studies in which coffee and caffeine have been used in experimental models.\n\nWhether caffeine or filtered coffee itself has been studied in rodent liver fibrosis/cirrhosis models (dimethylnitrosamine (DMN), carbon tetrachloride (CCl4) or thioacetamide (TAA)), fibrosis has been attenuated32–37. Interestingly, one trial examining Turkish-style coffee, which is unfiltered, demonstrated that liver fibrosis was not decreased and aminotransferase levels were increased in animals receiving CCl4 and Turkish coffee38. It is important to note, however, that detailed mechanistic studies for the potentially beneficial effects of coffee in animal models of liver fibrosis have not been performed.\n\nOne way that researchers have attempted to distinguish effects of coffee vs those of caffeine is through the use of trials in which decaffeinated coffee and/or non-coffee caffeine have been administered32,35,38. The effect of non-coffee caffeine was protective against experimental liver fibrosis in three trials35,38,39. However, two trials showed that decaffeinated coffee was also antifibrotic, albeit to a lower extent than caffeinated coffee32,38. We have interpreted these trials as part of a work in progress. Although the main effect of coffee as an antifibrotic in animals receiving experimental pro-fibrotic agents is largely mediated by caffeine, it is necessary for more, well-designed experiments to be performed.\n\nCaffeine has varied pharmacological effects, but one of its potent and best characterized effects is inhibition of adenosine receptors (AR)40. There are four G protein-coupled receptors for extracellular adenosine: A1AR, A2aAR, A2bAR, and A3AR, each of which has its own signal transduction mechanism and downstream physiologic effects41,42. In addition, affinity for each receptor for adenosine varies as well: the high affinity receptors A1AR, A2aAR and A3AR are activated by low concentrations (>10 nM) of extracellular adenosine, whereas the low affinity A2bAR requires adenosine concentrations likely activated only in the setting of cell injury or death (>1 µM)43.\n\nIn the liver, one of the most studied functions of adenosine is its protective role against ischemia/reperfusion, with potential implication of A1AR44 and A2aAR45,46. The receptor that seems to be mainly responsible for adenosine protection is A2bAR47,48. A1AR was also shown to have a protective effect against ethanol-induced hepatotoxicity49 and to protect against alpha-naphthylisothiocyanate-induced cholestatic liver injury induced by DPCPX (a specific A1AR antagonist) in A1AR deficient mice50. A2aAR is expressed by heptatic stellate cells, where it regulates fibrogenesis and contractility51,52. A1AR and A2aAR antagonists were also shown to inhibit the protective effect of caffeine on portal hypertension-related complications53. A3AR is overexpressed in hepatocellular carcinoma cells, and its activation is linked to apoptosis54. A3AR agonists were shown to have anti-cancer properties in vitro and in vivo in the rat55. These agents are currently studied in ongoing clinical trials56. The same agents were also shown to have a protective effect against liver inflammation due to concanavalin-A injection in rats55. Thus some adenosine receptor antagonists, like coffee/caffeine, may act against liver inflammation and fibrosis.\n\nThe data presented in this section support the concept that, in well-established models of liver fibrosis in animals (almost exclusively rodents), coffee provides a protective effect. Until a better hypothesis is tested, we may conclude that the protective effect occurs at the level of HSC A2aAR, with caffeine acting as an inhibitor. An alternative possibility is worth considering, however. Specifically, caffeine may be blocking inflammation rather than fibrosis directly, since adenosinergic signaling in inflammatory cells is well-established57,58. In addition, it is naïve to assume that rodent models of liver fibrosis/cirrhosis, such as CCl4, are effective analogues of human diseases, such as viral hepatitis and alcoholic liver disease. That said, these models are strong in vivo tests of liver myofibroblastic function, so they are essential steps in the testing of coffee and caffeine testing in cirrhosis pathogenesis.\n\n\nConclusion\n\nIt seems very likely that coffee, acting through caffeine, and probably through inhibition of adenosinergic signals, prevents complications of chronic liver disease – specifically cirrhosis. Two features of the evidence are of particular importance. First, the fact that the literature in patients supporting coffee’s anti-cirrhotic effect continues to accrue without opposing studies suggests that the initial epidemiological associations were real. Although this could be accounted for in part by publication bias favoring positive studies, that is not a fully convincing explanation. Second, the observation that the studies in human are supported by animal and cellular data suggest that there is a rationale to give the human trials greater consideration. At present, it is rational to encourage the use of moderate amounts of brewed coffee in patients with chronic liver disease.",
"appendix": "Author contributions\n\n\n\nAll four authors contributed to the text of this manuscript. JF, EL, and MF wrote initial drafts of particular sections, and JD edited and wrote the final draft.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by NIH/NIDDK R01 DK076735 to JAD and a Roger L. Jenkins American Liver Foundation Postdoctoral Research Fellowship Award to MF.\n\n\nAcknowledgements\n\nThe authors would like to thank Dr. Bruce Cronstein for encouraging us to contribute this manuscript to the body of scientific literature.\n\n\nReferences\n\nSaab S, Mallam D, Cox GA 2nd, et al.: Impact of coffee on liver diseases: a systematic review. Liver Int. 2014; 34(4): 495–504. PubMed Abstract | Publisher Full Text\n\nDranoff JA, Feld JJ, Lavoie EG, et al.: How does coffee prevent liver fibrosis? Biological plausibility for recent epidemiological observations. Hepatology. 2014; 60(2): 464–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhalaf N, White D, Kanwal F, et al.: Coffee and Caffeine are Associated with Decreased Risk of Advanced Hepatic Fibrosis Among Patients with Hepatitis C. Clin Gastroenterol Hepatol. 2015. PubMed Abstract | Publisher Full Text\n\nKlatsky AL, Armstrong MA, Friedman GD: Coffee, tea, and mortality. Ann Epidemiol. 1993; 3(4): 375–381. PubMed Abstract | Publisher Full Text\n\nTanaka K, Tokunaga S, Kono S, et al.: Coffee consumption and decreased serum gamma-glutamyltransferase and aminotransferase activities among male alcohol drinkers. Int J Epidemiol. 1998; 27(3): 438–443. PubMed Abstract | Publisher Full Text\n\nCasiglia E, Spolaore P, Ginocchio G, et al.: Unexpected effects of coffee consumption on liver enzymes. Eur J Epidemiol. 1993; 9(3): 293–297. 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PubMed Abstract | Publisher Full Text\n\nMolloy JW, Calcagno CJ, Williams CD, et al.: Association of coffee and caffeine consumption with fatty liver disease, nonalcoholic steatohepatitis, and degree of hepatic fibrosis. Hepatology. 2012; 55(2): 429–436. PubMed Abstract | Publisher Full Text\n\nModi AA, Feld JJ, Park Y, et al.: Increased caffeine consumption is associated with reduced hepatic fibrosis. Hepatology. 2010; 51(1): 201–209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCorrao G, Zambon A, Bagnardi V, et al.: Coffee, caffeine, and the risk of liver cirrhosis. Ann Epidemiol. 2001; 11(7): 458–465. PubMed Abstract | Publisher Full Text\n\nGallus S, Bertuzzi M, Tavani A, et al.: Does coffee protect against hepatocellular carcinoma? Br J Cancer. 2002; 87(9): 956–959. PubMed Abstract | Publisher Full Text\n\nCorrao G, Lepore AR, Torchio P, et al.: The effect of drinking coffee and smoking cigarettes on the risk of cirrhosis associated with alcohol consumption. A case-control study. Provincial Group for the Study of Chronic Liver Disease. Eur J Epidemiol. 1994; 10(6): 657–664. PubMed Abstract | Publisher Full Text\n\nFreedman ND, Everhart JE, Lindsay KL, et al.: Coffee intake is associated with lower rates of liver disease progression in chronic hepatitis C. Hepatology. 2009; 50(5): 1360–1369. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreedman ND, Curto TM, Lindsay KL, et al.: Coffee consumption is associated with response to peginterferon and ribavirin therapy in patients with chronic hepatitis C. Gastroenterology. 2011; 140(7): 1961–1969. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatsuura H, Mure K, Nishio N, et al.: Relationship between coffee consumption and prevalence of metabolic syndrome among Japanese civil servants. J Epidemiol. 2012; 22(2): 160–166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuxley R, Lee CM, Barzi F, et al.: Coffee, decaffeinated coffee, and tea consumption in relation to incident type 2 diabetes mellitus: a systematic review with meta-analysis. Arch Intern Med. 2009; 169(22): 2053–2063. PubMed Abstract | Publisher Full Text\n\nHamer M, Witte DR, Mosdol A, et al.: Prospective study of coffee and tea consumption in relation to risk of type 2 diabetes mellitus among men and women: the Whitehall II study. Br J Nutr. 2008; 100(5): 1046–1053. PubMed Abstract | Publisher Full Text\n\nBoggs DA, Rosenberg L, Ruiz-Narvaez EA, et al.: Coffee, tea, and alcohol intake in relation to risk of type 2 diabetes in African American women. Am J Clin Nutr. 2010; 92(4): 960–966. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarrieri, MP, Sogni P, Cohen J, et al.: Elevated coffee consumption and reduced risk of insulin resistance in HIV-HCV coinfected patients (HEPAVIH ANRS CO-13). Hepatology. 2012; 56(5): 2010. PubMed Abstract | Publisher Full Text\n\nLarsson SC, Wolk A: Coffee consumption and risk of liver cancer: a meta-analysis. Gastroenterology. 2007; 132(5): 1740–1745. PubMed Abstract | Publisher Full Text\n\nTanaka K, Hara M, Sakamoto T, et al.: Inverse association between coffee drinking and the risk of hepatocellular carcinoma: a case-control study in Japan. Cancer Sci. 2007; 98(2): 214–218. PubMed Abstract | Publisher Full Text\n\nBravi F, Bosetti C, Tavani A, et al.: Coffee reduces risk for hepatocellular carcinoma: an updated meta-analysis. Clin Gastroenterol Hepatol. 2013; 11(11): 1413–1421.e1. PubMed Abstract | Publisher Full Text\n\nUrgert R, Essed N, van der Weg G, et al.: Separate effects of the coffee diterpenes cafestol and kahweol on serum lipids and liver aminotransferases. Am J Clin Nutr. 1997; 65(2): 519–524. PubMed Abstract\n\nAnty R, Marjoux S, Iannelli A, et al.: Regular coffee but not espresso drinking is protective against fibrosis in a cohort mainly composed of morbidly obese European women with NAFLD undergoing bariatric surgery. J Hepatol. 2012; 57(5): 1090–1096. PubMed Abstract | Publisher Full Text\n\nFreedman ND, Park Y, Abnet CC, et al.: Association of coffee drinking with total and cause-specific mortality. N Engl J Med. 2012; 366(20): 1891–1904. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAubin HJ, Berlin I: Coffee drinking and mortality. N Engl J Med. 2012; 367(6): 576; author reply 576–577. PubMed Abstract | Publisher Full Text\n\nCrozier TW, Stalmach A, Lean ME, et al.: Espresso coffees, caffeine and chlorogenic acid intake: potential health implications. Food Funct. 2012; 3(1): 30–33. PubMed Abstract | Publisher Full Text\n\nCardin R, Piciocchi M, Martines D, et al.: Effects of coffee consumption in chronic hepatitis C: a randomized controlled trial. Dig Liver Dis. 2013; 45(6): 499–504. PubMed Abstract | Publisher Full Text\n\nArauz J, Moreno MG, Cortes-Reynosa P, et al.: Coffee attenuates fibrosis by decreasing the expression of TGF-β and CTGF in a murine model of liver damage. J Appl Toxicol. 2013; 33(9): 970–979. PubMed Abstract | Publisher Full Text\n\nFurtado KS, Prado MG, Aguiar ESMA, et al.: Coffee and caffeine protect against liver injury induced by thioacetamide in male Wistar rats. Basic Clin Pharmacol Toxicol. 2012; 111(5): 339–347. PubMed Abstract | Publisher Full Text\n\nMoreno MG, Chavez E, Aldaba-Muruato LR, et al.: Coffee prevents CCl4-induced liver cirrhosis in the rat. Hepatol Int. 2011; 5(3): 857–863. PubMed Abstract | Publisher Full Text\n\nShim SG, Jun DW, Kim EK, et al.: Caffeine attenuates liver fibrosis via defective adhesion of hepatic stellate cells in cirrhotic model. J Gastroenterol Hepatol. 2013; 28(12): 1877–84. PubMed Abstract | Publisher Full Text\n\nShin JW, Wang JH, Kang JK, et al.: Experimental evidence for the protective effects of coffee against liver fibrosis in SD rats. J Sci Food Agric. 2010; 90(3): 450–455. PubMed Abstract | Publisher Full Text\n\nShi H, Dong L, Zhang Y, et al.: Protective effect of a coffee preparation (Nescafe pure) against carbon tetrachloride-induced liver fibrosis in rats. Clin Nutr. 2010; 29(3): 399–405. PubMed Abstract | Publisher Full Text\n\nPoyrazoglu OK, Bahcecioglu IH, Ataseven H, et al.: Effect of unfiltered coffee on carbon tetrachloride-induced liver injury in rats. Inflammation. 2008; 31(6): 408–413. PubMed Abstract | Publisher Full Text\n\nChan ES, Montesinos MC, Fernandez P, et al.: Adenosine A2A receptors play a role in the pathogenesis of hepatic cirrhosis. Br J Pharmacol. 2006; 148(8): 1144–1155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaly JW: Caffeine analogs: biomedical impact. Cell Mol Life Sci. 2007; 64(16): 2153–2169. PubMed Abstract | Publisher Full Text\n\nChen, JF, Eltzschig HK, Fredholm BB: Adenosine receptors as drug targets--what are the challenges? Nat Rev Drug Discov. 2013; 12(4): 265–286. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFredholm BB, IJzerman AP, Jacobson KA, et al.: International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors--an update. Pharmacol Rev. 2011; 63(1): 1–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAherne CM, Kewley EM, Eltzschig HK: The resurgence of A2B adenosine receptor signaling. Biochim Biophys Acta. 2011; 1808(5): 1329–1339. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim J, Kim M, Song JH, et al.: Endogenous A1 adenosine receptors protect against hepatic ischemia reperfusion injury in mice. Liver Transpl. 2008; 14(6): 845–854. PubMed Abstract | Publisher Full Text\n\nCao Z, Yuan Y, Jeyabalan G, et al.: Preactivation of NKT cells with alpha-GalCer protects against hepatic ischemia-reperfusion injury in mouse by a mechanism involving IL-13 and adenosine A2A receptor. Am J Physiol Gastrointest Liver Physiol. 2009; 297(2): G249–258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMandili G, Alchera E, Merlin S, et al.: Mouse hepatocytes and LSEC proteome reveal novel mechanisms of ischemia/reperfusion damage and protection by A2aR stimulation. J Hepatol. 2015; 62(3): 573–580. PubMed Abstract | Publisher Full Text\n\nZimmerman MA, Grenz A, Tak E, et al.: Signaling through hepatocellular A2B adenosine receptors dampens ischemia and reperfusion injury of the liver. Proc Natl Acad Sci U S A. 2013; 110(29): 12012–12017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChouker A, Ohta A, Martignoni A, et al.: In vivo hypoxic preconditioning protects from warm liver ischemia-reperfusion injury through the adenosine A2B receptor. Transplantation. 2012; 94(9): 894–902. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang P, Wang Z, Zhan Y, et al.: Endogenous A1 adenosine receptor protects mice from acute ethanol-induced hepatotoxicity. Toxicology. 2013; 309: 100–106. PubMed Abstract | Publisher Full Text\n\nYang P, Chen P, Wang T, et al.: Loss of A1 adenosine receptor attenuates alpha-naphthylisothiocyanate-induced cholestatic liver injury in mice. Toxicol Sci. 2013; 131(1): 128–138. PubMed Abstract | Publisher Full Text\n\nHashmi AZ, Hakim W, Kruglov EA, et al.: Adenosine inhibits cytosolic calcium signals and chemotaxis in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol. 2007; 292(1): G395–401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSohail MA, Hashmi AZ, Hakim W, et al.: Adenosine induces loss of actin stress fibers and inhibits contraction in hepatic stellate cells via Rho inhibition. Hepatology. 2009; 49(1): 185–194. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHsu, SJ, Lee FY, Wang SS, et al.: Caffeine ameliorates hemodynamic derangements and portosystemic collaterals in cirrhotic rats. Hepatology. 2014. PubMed Abstract | Publisher Full Text\n\nBar-Yehuda S, Stemmer SM, Madi L, et al.: The A3 adenosine receptor agonist CF102 induces apoptosis of hepatocellular carcinoma via de-regulation of the Wnt and NF-kappaB signal transduction pathways. Int J Oncol. 2008; 33(2): 287–295. PubMed Abstract | Publisher Full Text\n\nCohen S, Stemmer SM, Zozulya G, et al.: CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver. J Cell Physiol. 2011; 226(9): 2438–2447. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Q, Dai X, Yang W, et al.: Caffeine protects against alcohol-induced liver fibrosis by dampening the cAMP/PKA/CREB pathway in rat hepatic stellate cells. Int Immunopharmacol. 2015; 25(2): 340–352. PubMed Abstract | Publisher Full Text\n\nHasko G, Linden J, Cronstein B, et al.: Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov. 2008; 7(9): 759–770. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilne GR, Palmer TM: Anti-inflammatory and immunosuppressive effects of the A2A adenosine receptor. ScientificWorldJournal. 2011; 11: 320–339. PubMed Abstract | Publisher Full Text\n\nMcCusker RR, Goldberger BA, Cone EJ: Caffeine content of specialty coffees. J Anal Toxicol. 2003; 27(7): 520–522. PubMed Abstract | Publisher Full Text\n\nChin JM, Merves ML, Goldberger BA, et al.: Caffeine content of brewed teas. J Anal Toxicol. 2008; 32(8): 702–704. PubMed Abstract | Publisher Full Text\n\nKhan K, Naeem M, Arshad MJ, et al.: Extraction and Chromatographic determination of caffeine contents in commercial beverages. J Appl Sci. 2006; 6(4): 832–834. Publisher Full Text"
}
|
[
{
"id": "8419",
"date": "22 Apr 2015",
"name": "Heather Francis",
"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 review regarding an area of some controversy, the beneficial effects of coffee/caffeine on liver disease. Feld, et al. have presented recent studies from both human populations as well as animal studies that provide more mechanistic data. The review is concise and highlights important work without bogging the reader down in too much detail. From this review, it's clear that more work needs to be done to fully understand the potential benefits of coffee and caffeine on liver fibrosis and other liver diseases.",
"responses": [
{
"c_id": "1316",
"date": "27 Apr 2015",
"name": "Jonathan A Dranoff",
"role": "Author Response",
"response": "We greatly appreciate your kind review. We hope that two things arise from such a review:Investigators are encouraged to engage in new research projects related to this work. This will serve as a firm starting-point for those hoping to critically review this topic."
}
]
},
{
"id": "8403",
"date": "27 Apr 2015",
"name": "Kinji Asahina",
"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 review covering recent findings on beneficial effects of coffee on liver cirrhosis. The authors reviewed the relevant clinical and epidemiological data and suggested an inhibitory role of caffeine on adenosinergic signaling in hepatic stellate cells. Caffeine inhibits adenosine receptors. Contrary to this notion, the authors mentioned that antagonists for the receptors inhibit the protective effect of caffeine on portal hypertension-related complications on page 6. Please check whether antagonists for adenosine receptors inhibit the beneficial effects of caffeine in the liver.\n\nAre there papers showing adenosine receptor signaling in hepatic stellate cells? Does the inhibition the receptor suppress activation of hepatic stellate cells or induce their cell death? Caffeine has been shown to induce autophagy in hepatocytes. Is it possible that activation of autophagy in hepatocytes indirectly mediates the activation state of hepatic stellate cells in the liver? Please indicate Table 1 in the text.",
"responses": [
{
"c_id": "1315",
"date": "27 Apr 2015",
"name": "Jonathan A Dranoff",
"role": "Author Response",
"response": "Please see my answers to the questions above.This is a reasonable question, but one that is currently unanswered. There are indeed good data to demonstrate the presence of functional adenosine receptors in myofibroblastic HSC. Please see our collaborations with Waj Mehal's group. This is certainly possible, but it has not been examined directly. Natalie Torok's work shows elegantly that HSC can endocytose apoptotic hepatocyte remnants, so this is worth examining. I am not sure that I understand - both tables are labeled and described.Thanks for the supportive review."
}
]
},
{
"id": "8510",
"date": "29 Apr 2015",
"name": "Vijay H. Shah",
"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 review about beneficial effects of coffee on fibrogenesis and liver injury encompassing both lab and clinical studies. A couple points warrant the authors’ attention:Caffeine also regulates phosphodiesterases. Could this be a contributory factor for beneficial effects? Carbonated beverages also have significant levels of caffeine. Not aware of their benefits for fibrosis though. Any literature about this in context of fibrosis to support or refute the concept of caffeine as the antifibrotic component of coffee?",
"responses": [
{
"c_id": "1324",
"date": "29 Apr 2015",
"name": "Jonathan A Dranoff",
"role": "Author Response",
"response": "We thank the reviewer for his kind comments.Caffeine has several biochemical effects, including phosphodiesterase (PDE) inhibition. If the primary PDE inhibited were a regulator of cAMP (as is generally accepted), our collaborative work with Waj Mehal's group would suggest that caffeine might hasten liver fibrosis progression, since adenosine and its downstream effector cAMP are profibrogenic in vitro. I am not aware of any beneficial effects of carbonated beverages; however, the relative caffeine content of carbonated beverages is quite low relative to drip coffee. Moreover, in the case of sugar-containing soft drinks, potential benefits of caffeine would be likely to be masked by the adverse metabolic effects of simple sugars. Note also that the relatively low caffeine content even of black and oolong teas (green and white teas have even less caffeine) probably accounts for the lack of epidemiological data supporting their beneficial effects in liver fibrosis progression, if caffeine is indeed the active ingredient."
}
]
}
] | 1
|
https://f1000research.com/articles/4-95
|
https://f1000research.com/articles/4-1222/v1
|
06 Nov 15
|
{
"type": "Research Article",
"title": "Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock",
"authors": [
"Esmeralda Vicedo",
"Zofia Gasik",
"Yu-An Dong",
"Tatyana Goldberg",
"Burkhard Rost",
"Zofia Gasik",
"Yu-An Dong",
"Tatyana Goldberg",
"Burkhard Rost"
],
"abstract": "Recent experiments established that a culture of Saccharomyces cerevisiae (baker’s yeast) survives sudden high temperatures by specifically duplicating the entire chromosome III and two chromosomal fragments (from IV and XII). Heat shock proteins (HSPs) are not significantly over-abundant in the duplication. In contrast, we suggest a simple algorithm to “postdict” the experimental results: Find a small enough chromosome with minimal protein disorder and duplicate this region. This algorithm largely explains all observed duplications. In particular, all regions duplicated in the experiment reduced the overall content of protein disorder. The differential analysis of the functional makeup of the duplication remained inconclusive. Gene Ontology (GO) enrichment suggested over-representation in processes related to reproduction and nutrient uptake. Analyzing the protein-protein interaction network (PPI) revealed that few network-central proteins were duplicated. The predictive hypothesis hinges upon the concept of reducing proteins with long regions of disorder in order to become less sensitive to heat shock attack.",
"keywords": [
"Saccharomyces cerevisiae",
"heat-shock stress",
"chromosomal duplication",
"GO terms",
"protein disorder",
"protein-protein interactions",
"heat shock proteins"
],
"content": "Introduction\n\nSaccharomyces cerevisiae (baker’s yeast; for simplicity we mostly use yeast) was the first completely sequenced eukaryote1. Being simple to handle and manipulate has rendered yeast a preferred model organism for genetics, biochemistry and systems biology2–4. It grows optimally within a narrow temperature range but tolerates moderate deviations, some of which impinge upon cell structure and function, often through rapid physiological adaptations. One such adaptation mechanism is the duplication of the whole genome or particular chromosomes (aneuploidy)5–7 that contain the genes necessary to rapidly cope with specific adverse conditions over the course of several generations of evolving yeast8–14. Such evolutionary adaptations imbalance the genome15, destabilize reactions or pathways16,17, and cost energy18,19. Aneuploidy, therefore, is a transient solution. Over many generations exposed to the same adverse conditions refined specific and less expensive solutions replace aneuploidy20. Yeast cells can adapt to high-temperature stress by repeatedly duplicating chromosome III along with two other fragments (from chrIV and chrXII)20. Why specifically copy these regions? Can particular biophysical features and/or functions of the proteins encoded in these regions explain the choice?\n\nOne simple biophysical feature is protein disorder: most proteins adopt well-defined three-dimensional (3D) structures21–24, i.e. will largely remain identical at different times. In contrast, disordered regions do not adopt well-defined 3D structures in isolation25, i.e. without binding substrates they will look very different at different time points. Proteins with long disordered regions encompass some unique biophysical characteristics26–34. Such regions are so difficult to characterize experimentally that there is no good experimental data set proxy for “all proteins with long regions of disorder in yeast”. In contrast, acceptably accurate computational predictions are available for entire proteomes30,35,36. Protein disorder seems one means for prokaryotes to adopt to extreme environments, e.g. halophiles have more proteins with long disorder than their closest phylogenetic relatives, while thermophiles tend to have fewer37. Here, we hypothesized a similar effect to govern the response to high temperature-related duplication in yeast, namely that chromosomal regions duplicated under high temperature are depleted of proteins with long disorder.\n\n\nMethods\n\nWe downloaded the yeast (S. cerevisiae) proteome from UniProt (proteome ID: UP000002311)38 as fasta files including only the reviewed proteins (UniProtKB/Swiss-Prot). Removal of duplicates applying the method Uniqueprot239 (with 100% pairwise sequence identity, keeping the longer sequence) left 5667 proteins (Table S1A). We considered the 16 nuclear chromosomes (matched through http://www.yeastgenome.org40, the numbers of proteins per chromosome are given in Figure S1B). The yeastgenome.org resource also provided the annotations of heat-shock response proteins (HSR). Proteins known to interact with HSR proteins augmented this set of HSRs in the following way.\n\nBioGRID (version 3.1.86) provided the data for experimental protein-protein interactions (PPIs) in yeast. After filtering out redundancy (a-b and b-a counted only once) and excluding self-interactions (a-a), we based all subsequent analysis on the single largest connected component of the network. We focused on the most basic network features that allow the comparison and characterization of complex networks. The most elementary characteristic of a node is its degree or connectivity, defined as the number of interactions for a node (here protein), i.e. the number of interactions one protein has with all others. Another important parameter is the betweenness, i.e. the fraction of shortest paths between all other nodes that has to go through a given node. Additionally, we monitored the average degree of neighbors, which depends on the number of nodes and links in the network. These three parameters measured the importance of each node within the network.\n\nWe applied methods capturing different “flavors” of protein disorder29,41,42. IUPred (version 1.0) is based on statistical contact potentials and exclusively uses single sequences28,43. MD (Meta-Disorder)42 combines different original prediction methods through machine learning (neural network) with evolutionary profiles and predictions of solvent accessibility and protein flexibility. To some extent disorder is a gradual phenomenon, i.e. proteins may have more or less disorder44. On the other hand, prediction methods distinguish between a 30-residue loop resembling “protein disorder” and another resembling a region with “regular structure”29. Thus, protein disorder seems more a binary feature (it is there or not, or present/absent) than a gradual one25. Unfortunately, no argument or data determines one single correct threshold for what constitutes present/absent for protein disorder. Typically, experts use a length threshold of the type: protein disorder is present when at least T consecutive residues in a protein are predicted to be disordered. If so, this protein is considered to contain a long region of disorder. More disorder in this model could imply, e.g. more than one region, or the entire protein. We analyzed many alternatives to choose the threshold for long disorder, and found most to be redundant. We included different views only if they provided relevant information. In particular, we largely focused on one threshold to define “long disorder”: %long30, is the percentage of proteins with at least one region of ≥30 consecutive residues predicted as disordered (alternatives were: %long50 and %long80, i.e. with length thresholds at ≥50 and ≥80, and completely disordered implying no region of 30 consecutive residues without any disorder).\n\nWe applied BINGO (Biological Networks Gene Ontology44, version 2.44) to identify the enrichment of GO (Gene Ontology)45 terms in subsets of experimentally annotated proteins. We focused on “biological process” and “molecular function”. For two sets of proteins with annotated biological functions (more precisely: GO numbers) BINGO estimates to which extent their annotations differ in a statistically significant way. We visualized BINGO results with Cytoscape46 platform (version 2.8). Our analysis focused on the hypergeometric test in BINGO, which accurately estimates p-values as it tests without replacement. Following the common, procedure for BINGO, we considered p-values >0.05 as significant46. Testing multiple hypotheses may give many false positives (Type I error: incorrect rejection of true null hypothesis47,48). Using BINGO, we corrected for these through the Benjamini and Hochberg correction which provides strong control over the False Discovery Rate (FDR, expected proportion of erroneous null hypothesis rejections among all rejections48).\n\n\nResults and discussion\n\nIn response to high temperature yeast (S. cerevisiae) duplicates the entire chromosome III (for brevity we use chrN to denote ‘yeast chromosome N’ with N as Roman numerals following convention) and fragments from chrIV and chrXII20. The size of the 16 yeast chromosomes varies over six-fold (Table S1). The average protein length is similar between the 16 chromosomes (Figure S1, Table S1). The duplicated chrIII is the 3rd smallest with 183 genes, of which 153 are mapped and 132 constitute “verified ORFs”. Fewer genes are encoded only by chrI with 90, and chrVI with 125 proteins (Table S1). The relatively small number of genes on chrIII was one reason for choosing it as the first fully synthesized functional yeast chromosome49. In contrast to protein length, the percentage of proteins with predicted long regions of disorder differed significantly between the 16 yeast chromosomes (Figure 1).\n\nThe composition of proteins with long regions of disorder (y-axes) differed significantly between the 16 chromosomes of S. cerevisiae (x-axes) and also for the set of HSPs. The three rightmost marks on the x-axes describe: HSPs and the disorder predictions for the HSR-related duplicated fragments on chromosome IV and chromosome XII (frag IV and frag XII). The differences were similar for two different prediction methods (MD in black, IUPred in light gray), and for different thresholds with respect to the minimal length of a disordered region (A: ≥30 consecutive residues predicted in disorder, B: ≥50, C: ≥80). Dashed horizontal lines mark the averages over all chromosomes. Error bars are too small to become visible on the scale chosen. The least disorder content was predicted for chromosome III and chromosome X. Overall, all duplications in response to heat shock treatment reduced the level of protein disorder in the offspring.\n\nThe least protein disorder was predicted for chrIII and chrX (Figure 1, Table S2). That means heat response duplicates one of the two chromosomes with the least disorder. In addition, the fragments of chrIV and chrXII that are duplicated along with the entire chrIII also clearly have less disorder than the chromosomes from which they were taken (Figure 1). This enhances the effect of protein disorder reduction in response to high temperatures.\n\nThe other low-disorder option is chrX: Why not duplicate chrX in response to high temperatures? ChrX is more than twice as large as chrIII (Figure S1). Thus duplicating chrX would “cost” twice as much. This might be prohibitive. ChrX might also not contain the cell activities important for coping with high temperature. Furthermore, as chrX and chrIII are similar in disorder content while chrX has twice the proteins of chrIII, the duplication of chrX would increase the overall level of proteins with disorder that might become unfolded and thereby “jam” cellular activity more than the duplication of chrIII.\n\nAssume a certain amount of tolerable duplication were tolerable and that number were about 153 proteins (as for chrIII): where in the genome do we find a continuous stretch (within a chromosome) that has 153 proteins with the least disorder? Our results underscored the special role of chrIII (Figure S2): only 3% of all continuous genome fragments with 153 proteins have as little disorder as chrIII (corresponding numbers for chrX: 5%; 29-protein fragment from chrIV: 52%; 64-protein fragment from chrXII: 10%). These figures demonstrate that the duplication of chrIII might be THE optimal choice for a simple way to duplicate 153 proteins with as little disorder as possible.\n\nOur results explained why duplicating 150–200 proteins from another chromosome might have been potentially more damaging than the duplication of chrIII in response to high temperatures. In other words, our model might suggest why the duplication of this particular region is better than other duplications. However, what is the selective benefit from the proteins on chrIII? We expected to find the answer to this question in proteins that actively help with coping with heat stress. The immediate suspects are heat-shock proteins (HSP) and the proteins known to interact with these HSPs (HSP-binders). The known HSPs and HSP-binders scatter over all 16 yeast chromosomes (Figure S3). All regions duplicated in response to heat shock contain only one known gene coding for HSPs (HSP30) and one known HSP-binder (TAH1). This implied that 1.3% of all known HSPs and HSP-binders were duplicated in an event that duplicated 0.5% of all genes, i.e. a 2.6-fold over-representation. This statistically insignificant the finding that fewer than 1 in 50 of all HSPs and HSP-binders are duplicated might still be scientifically significant if HSP30 and TAH1/HSP90 were outstandingly important proteins for the given conditions. However, this is not the case, at least not given what is currently known about HSP30. Furthermore, introducing an extra copy of HSP30 into wild-type cells did not increase the ability of the cells to cope with high temperature (Dahan & Pilpel, personal communication).\n\nThe set of known HSPs (Figure S3) slightly changed expression levels in response to heat stress during the fixation of the trisomy20 only slightly but almost all HSPs were significantly up-regulated (arrows in Figure S3) when the “refined descendants” replaced the trisomy20. This could imply that the duplicated genes are essential for survival under heat stress. Nevertheless, quite contrary to the naïve expectation, the HSPs and the HSP-binders by no means explained the heat-stress-specific duplications observed experimentally.\n\nIncidentally, HSPs appeared particularly abundant in disorder regions of 30–50 consecutive residues (Figure 1A, in particular for IUPred). It has previously been argued that such disorder is required for proper function of HSPs50. In contrast, HSPs are depleted of longer disorder (>50; Figure 1B,C).\n\nOverall, we argue that HSPs could have explained the duplication of many other chromosomes, possibly even better than that of chrIII. Therefore, this explanation is not specific. Thus, we conclude that the duplication of known HSPs and HSP-binding proteins did not explain why chrIII was specifically duplicated. Many HSPs and HSP-binders might remain unknown. However, we have no scientific ground to suspect that the fraction of the unknown HSPs differs between the chromosomes, i.e. that there are particular HSPs on chrIII that remain undiscovered.\n\nAre any other proteins on chrIII important for growth under high temperature? Simply scanning GO45 annotations is insufficient: the question is not whether proteins on chrIII have certain functions, but whether these are overrepresented enough to explain why chrIII and not the other two small chromosomes (chrVI or chrI) are duplicated in response to high temperatures. In order to address this question, we need a GO term enrichment analysis of the duplicated regions51.\n\nGrowth and reproduction might be considered as the most important cell activities in the sense that the organism must grow and proliferate (cells that fail are not observed) even under stress. The GO enrichment analysis seemed to confirm this expectation (Figure 2): the two most abundant GO terms in the heat stress-duplicated regions were those related to (i) sexual reproduction (Figure 2 and Figure S2; “conjugation with cellular fusion”, “reproductive cellular process” and “response to pheromone”) and to (ii) sugar transport (hexose transport process as well as mannose, fructose and glucose transmembrane transporter activity; Figure S4).\n\nThe tree gives the complete set of all experimentally annotated GO-terms (Gene Ontology45) for any of the proteins on chromosome III that describe biological process (left branch) and molecular function (right branch). The enrichment analysis51 describes how much chrIII GO-terms are enriched with respect to all other GO-terms from yeast: all terms marked by yellow circles are significantly enriched. Sexual reproduction (7 GO-terms on chrIII) and transport (carboxylic acid and organic anion 4 GO-terms on chrIII) mapped to most overrepresented GO terms on this chromosome.\n\nThe major energy source of yeast is sugar, in particular hexose monosaccharides (C6H12O6; e.g. glucose, fructose, mannose). These nutrient sugars are imported into the cell through hexose transporters, which are encoded by HXT genes52,53. The HXT yeast genes on the duplicated fragment of chromosome IV (HXT3, HXT6 and HXT7) are almost five-fold over-represented with respect to random (yeast has 5667 NgenY genes, 243 NgenD are duplicated, 15 NgenHXT genes are in yeast; in a region with 243 NgenD genes we would find by chance 0.64 HXT genes in the duplicated regions pchance=NgenHXT *[NgenD/NgenY]). Two HXT genes on the duplicated chrIV fragment (HXT6 and HXT7) appear to encode high-affinity transporters required for growth at very low glucose concentrations (~0.1%54), i.e. these two would become particularly important when yeast is cultured under glucose limitation54. Interestingly, several works have detected duplication of these two genes (HXT6 and HXT7) in yeast populations evolving under low nutrient availability8,55. These numbers suggest that heat stress also puts strain upon obtaining the energy needed for growth and reproduction.\n\nSexual reproduction also appeared crucial for the survival of yeast cultured under heat stress56,57. Seven of the ten molecular functions to be significantly overrepresented in the heat stress-duplicated chrIII (Table S3) by a standard GO-term enrichment analysis51 are involved in reproduction. Three of these seven molecular functions are related specifically to sexual reproduction; the others pertain to general reproductive processes (Figure 2). In particular, the reproduction-related processes involve cell fusion (FUS1 and FIG258–60), pheromone response (STE50 which is also required for optimal invasive growth and hyperosmotic stress signaling61,62 and NOT1 that is also involved in several RNA regulation levels63), nuclear fusion, chromosome disjunction, nuclear segregation after mating (BIK1 which is involved in microtubule function during mitosis64,65), fusion of haploid nuclei during mating; KAR4 or KARyogamy plays a critical role in the choreography of the mating response66), cytokenesis (division of cytoplasma and plasma membrane of a cell and its separation into two daughter cells which is also relevant for asexual mitotic growth: CDC1067), specification of the site where the daughter cell will form (relevant for budding and asexual growth, also referred to as axial bud selection) and in the developmental process in which the size of a cell is generated and organized (also referred to as morphogenesis: CDC1067–69). All these genes are also required for the correct localization of other proteins involved in cytokinesis and bud site selection67,70–73. Other important processes and activities overrepresented on chrIII are related to the avoidance of oxidative stress (e.g. carboxylic acid transport – Figure 2 - which may be important for the survival since during the vegetative asexual reproduction cells were exposed to oxidative stress) and NAD(P)H nitro-reductase activity (Figure 2). The only nitroreductase-related proteins in yeast – HBN1 and FRM274 - are only on chrIII (Table S4). The proteins involved in these two activities (carboxylic acid transport and NAD(P)H nitro-reductase activities) are also implicated in cellular detoxification75, which is another task relevant for survival under stress.\n\nAll these data supported the view that chrIII is important for sexual reproduction. A seemingly convincing story, until we learned that the laboratory strains of yeast survived through asexual reproduction20, i.e. apparently did not need what is so uniquely enriched in the heat stress duplication. The set of proteins known to be involved in reproduction on chrIII (Table S3) had more disorder than the average for chrIII (Figure S5). Some of these proteins with long disordered regions might not work correctly in heat.\n\nWhy duplicate proteins that fail? Not having found a convincing answer, we propose two conjectures: first, sexual reproduction might “frame” another cellular activity of the same protein that is more relevant to the growth conditions applied during the evolution in the laboratory experiment. For instance, CDC10 is also required to maintain cell polarity (GO: 0030011), BUD3 and BUD5 are involved in axial cellular bud-site selection (GO: 0007120), KCC4 a bud neck kinase involved in budding and cell bud growth (GO: 0007117) and BIK1, which is involved in microtubule function during mitosis. All of these activities are related to asexual reproduction. Our second proposition seems more far-fetched, namely that the set of proteins with the strongest GO-enrichment might have been duplicated coincidentally, i.e. the disorder-rich proteins related to sexual reproduction might have been duplicated because they happened to be on chrIII but not due their relevance for the survival in heat. If so, there must be something else we have not found yet on chrIII.\n\nSeveral other processes were slightly enriched in the duplicated fragments with some relevance for yeast survival in heat but none of those gave a clear explanation (Figure 2): (i) fatty acid elongase, (ii) rRNA (guanine) methyltransferase, and (iii) the importin-alpha export receptor activities. We analyzed these in detail. (i) Fatty acid elongase: currently, only three proteins are known to be involved in lengthening fatty acids; two of those (ELO2 and APA1; Table S3) are on chrIII. Fatty acid elongases are involved in sphingolipid biosynthesis. The sphingolipids are components of the cellular membrane and bioactive signaling molecules that contribute to heat tolerance as they are directly involved in organizational cellular structures (e.g. cell membrane)76. (ii) rRNA methyltransferases: three yeast proteins are known to be involved in rRNA (guanine) methyltransferase activity; two of those (BUD23 and SPB1) are on chrIII (Table S4). It is believed that the modification of ribonucleotides optimizes the rRNA structure and represents a way to expand the topological potentials of RNA molecules. It is possible that the loss of modification affects fine-tuning of ribosome function that could give rise to the pronounced cold-sensitivity77. (iii) Importin-alpha nuclear export: two yeast proteins contribute toward the importin-alpha export receptor activity; one of those (MSN5) is in the duplicated fragment of chromosome IV. MSN5 knockout mutants show a variety of phenotypes, including carbon-source utilization, defects and sensitivity to high concentrations of ions, severe heat shock, and high pH78. Moreover, these mutants are partially sterile78. Therefore, this protein appears necessary for cell survival, especially under extreme conditions.\n\nOnly one cellular activity related to tRNA synthase appeared overrepresented on the duplicated fragment of chrXII (DUS3 and DUS4 proteins; Table S7). In particular, to the tRNA dihydrouridine synthases, which are responsible for the reduction of the 5,6-double bond of a uridine residue on tRNA (one of the numerous modifications observed on tRNA cytoplasmatic79). However, this particular finding appeared less relevant since the corresponding fragment was only duplicated in one of four growth experiments in response to high temperatures20.\n\nOne crucial limitation for any functional enrichment study remains the incomplete experimental annotation even for an organism as intensively studied as yeast. It may be that all our speculation above missed the real causation because the functions of the proteins that are really relevant remain uncharacterized. Therefore, we complemented our analysis with one aspect of function for which we have a complete prediction, namely the prediction of sub-cellular localization of all yeast proteins. The experimental localization annotations for yeast are still cover at most 70% of all proteins80. However, today’s top prediction methods, such as LocTree3, are very reliable80 and can make crucial differences for comparing ‘complete’ data sets81. We found nuclear proteins to be clearly depleted on chrIII (-4.6 percentage points with respect to the entire proteome; Figure S7A). Other abundant proteins found on chrIII were secreted (extra-cellular) or annotated as endoplasmic reticulum (ER) membrane proteins (each 3.2 percentage points higher than in the full yeast proteome). We also observed significantly more disorder in nuclear proteins (nuclear 77% vs. <40% for non-nuclear; Figure S8). This might explain the depletion of nuclear proteins on chrIII. While these findings were clear, they did not suggest a simple interpretation. The abundance of secreted proteins on chrIII (about 3.2 percentage points more on chrIII than in entire yeast; Figure S7A) implies that in the response to heat shock, more proteins are secreted into the ‘hot’ environment. Given the correlation between habitat and disorder37, we expect that proteins are more likely to sustain high temperatures with less disorder. Unfortunately, a GO enrichment study of the secreted proteins also did not provide the answer we had been hoping for. However, the “secretome” alone could not explain the lower content of disordered proteins on chromosome III (disorder entire yeast-chrIII=50%-43%=7%>3% for secretome; Table 1 and Figure S7A).\n\nAs proteins cannot be understood without also considering their networks of interaction, we compared the network of experimentally characterized PPIs between the entire yeast and those fragments that are duplicated in heat evolving populations. As for the differential analysis of any experimental annotation, the limitation of such an approach lies in the incompleteness of the experimental data. In all 16 chromosomes, the degree (number of interactions per protein) was lowest for chrIII (average=16±2; Figure 3A). A similar trend was observed for betweenness (number of times that a protein acts as a bridge along the shortest path between two other proteins: average=1800±300; Figure 3B). Furthermore, chrIII is one of the chromosomes with the largest mean value for the average neighbor degree (average=380±40; Figure 3C). Our network analyses confirm chrIII as a good choice for a first line of defense against high temperature because the proteins encoded on this chromosome play less essential roles for the overall PPI network. However, once again, this portrays the duplication as a solution with least possible damage without positively suggesting causation.\n\nWe began with the entire network of all PPIs with experimental annotations in yeast (Methods), and then differentially analyzed major network features: (A) Degree: The number of PPIs per protein (degree) was minimal for the proteins from chrIII (box in red; lowest mean - black dot and lowest median - black line in the box. (B) Betweenness: betweenness (number of times that a node acts as a bridge along the shortest path between two other nodes) was also lowest for chrIII. (C) Average neighbor degree: plotting the average degree for all network neighbors of all proteins on chrIII (i.e. all those proteins in direct PPI with proteins on chrIII), we observed a much less differentiated view. For this network feature, the proteins from chrIII had one of the highest means (black dot), but one of the lowest medians. Clearly, the proteins from the HSR-duplicated chromosome appeared less involved in the yeast network than expected by chance.\n\n\nConclusions\n\nOrganisms can duplicate the whole genome or particular chromosomes (aneuploidy) in response to sudden dramatic changes in the environment. As such coarse-grained major changes are costly, aneuploidy tends to give way to more fine-tune focused solutions that require many generations to evolve. The entire chromosome III and two fragments from chromosomes IV and XII in a culture of budding yeast (S. cerevisiae) were duplicated as a “transient evolutionary solution” in response to high temperature - a “transition” that fostered the survival of between 400 and 2,000 generations. Here, we reported that while the proteins on all 16 main chromosomes from yeast have similar length, they differ substantially in the fraction of proteins with long regions predicted to contain protein disorder (≥30–80 consecutive residues predicted as disordered by IUPred and MD). We found the regions duplicated under heat stress depleted of predicted disorder. In fact, chromosome III was one of the two chromosomes with the least disorder (Figure 1). The other (chromosome X) is twice as large, i.e. would cost twice to duplicate. Decreasing the overall content in protein disorder is likely an important strategy to protect against heat stress. A detailed analysis of the experimentally characterized PPI network in yeast revealed the duplicated proteins to be connected less than average (Figure 3). The PPI analysis, therefore, added to the explanation that the duplication causes minimal damage. However, why did the duplication create an advantage under heat stress? Surprisingly, we found no sustained evidence for a significant over-representation of HSPs in the duplication i.e. of proteins that usually help out under such stress. Instead, a Gene Ontology (GO) enrichment analysis suggested that the duplicated regions were enriched in processes related to reproduction and to the import of nutrients (Figure 2). The enrichment was strongest for proteins related to sexual reproduction although the heat stress survival was maintained through budding, i.e. through asexual reproduction. Nevertheless, the set of GO enriched proteins appeared so important that they were duplicated although high in disorder. This might point to where the explanation for the duplication might be found. Overall, our data suggested a very simple algorithm: identify the region with lowest protein disorder that is large enough, yet not too large and duplicate it along with possibly other fragments that are also depleted of disorder in order to cope with heat stress.",
"appendix": "Author contributions\n\n\n\nEV and BR conceived the study and designed the experiments. EV, ZG, YD, TG and MJ carried out the research. YD and TG provided expertise in protein-protein interactions and protein localization prediction respectively. EV prepared the first draft of the manuscript. EV, ZG and TG contributed to the graphics 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\nThis work, and all authors were supported by the German Research Foundation (DFG) and the Technische Universität München within the funding program Open Access Publishing.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThanks to Tim Karl and Laszlo Kajan (TUM) for invaluable help with hardware and software; to Marlena Drabik and Inga Weise (TUM) for administrative support; to Tobias Hamp and Edda Kloppmann for helpful comments on the manuscript. A special thanks goes to the Pilpel-lab (Weizman Inst. Rehovot, Israel), in particular to Yitzhak Pilpel (Weizman) and Orna Dahan (Weizman) for the data and crucial help. Last, not least, thanks to all those who deposit their experimental data in public databases, and to those who maintain these databases.\n\n\nSupplementary material\n\nSupplementary material for Vicedo et al., 2015 'Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock'.\n\nTable of Contents for Supplementary Material\n\nFigure S1: Number of genes per chromosome in Saccharomyces cerevisiae (yeast).\n\nFigure S2: Fragments with less disorder than proteins duplicated during heat shock response (HSR).\n\nFigure S3: Distribution of Heat Shock proteins (HSP) along chromosomes.\n\nFigure S4: Complete set of known GO (Gene Ontology) terms for a fragment of the HSR-duplicated chromosome IV.\n\nFigure S5: Disordered proteins differentiate by chromosomes and roles.\n\nFigure S6: Disorder difference between chromosome III proteins and their paralogs.\n\nFigure S7: Distribution comparison of chromosome III proteins and the complete yeast proteome across localization classes.\n\nFigure S8: Distribution of disordered/ordered nuclear proteins and other yeast proteins.\n\nTable S1: General information about yeast chromosomes.\n\nTable S2: Disorder abundance content.\n\nTable S3: Overrepresented GO terms for molecular function analysis of chromosome III.\n\nTable S4: Overrepresented GO terms for biological process analysis of chromosome III.\n\nTable S5: Overrepresented GO terms for molecular function of the duplicated fragment of chromosome IV.\n\nTable S6: Overrepresented GO terms for biological process of the duplicated fragment of chromosome IV.\n\nTable S7: Overrepresented GO terms for biological process of the duplicated fragment of chromosome XII.\n\nTable S8: Heat shock proteins distribution on chromosomes.\n\nClick here to access the data.\n\n\nReferences\n\nGoffeau A, Barrell BG, Bussey H, et al.: Life with 6000 genes. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang M, Bennett D, Erdman SE: Maintenance of mating cell integrity requires the adhesin Fig2p. Eukaryot Cell. 2002; 1(5): 811–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErdman S, Lin L, Malczynski M, et al.: Pheromone-regulated genes required for yeast mating differentiation. J Cell Biol. 1998; 140(3): 461–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamezani-Rad M: The role of adaptor protein Ste50-dependent regulation of the MAPKKK Ste11 in multiple signalling pathways of yeast. Curr Genet. 2003; 43(3): 161–70. PubMed Abstract | Publisher Full Text\n\nTruckses DM, Bloomekatz JE, Thorner J: The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae. Mol Cell Biol. 2006; 26(3): 912–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSandler H, Kreth J, Timmers HT, et al.: Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin. Nucleic Acids Res. 2011; 39(10): 4373–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBerlin V, Styles CA, Fink GR: BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J Cell Biol. 1990; 111(6 Pt 1): 2573–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiller RK, Rose MD: Kar9p is a novel cortical protein required for cytoplasmic microtubule orientation in yeast. J Cell Biol. 1998; 140(2): 377–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLahav R, Gammie A, Tavazoie S, et al.: Role of transcription factor Kar4 in regulating downstream events in the Saccharomyces cerevisiae pheromone response pathway. Mol Cell Biol. 2007; 27(3): 818–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nField CM, Kellogg D: Septins: cytoskeletal polymers or signalling GTPases? Trends Cell Biol. 1999; 9(10): 387–94. PubMed Abstract | Publisher Full Text\n\nCid VJ, Adamíková L, Cenamor R, et al.: Cell integrity and morphogenesis in a budding yeast septin mutant. Microbiology. 1998; 144(Pt 12): 3463–74. PubMed Abstract | Publisher Full Text\n\nLongtine MS, DeMarini DJ, Valencik ML, et al.: The septins: roles in cytokinesis and other processes. Curr Opin Cell Biol. 1996; 8(1): 106–19. PubMed Abstract | Publisher Full Text\n\nMadden K, Snyder M: Cell polarity and morphogenesis in budding yeast. Annu Rev Microbiol. 1998; 52: 687–744. PubMed Abstract | Publisher Full Text\n\nCarroll CW, Altman R, Schieltz D, et al.: The septins are required for the mitosis-specific activation of the Gin4 kinase. J Cell Biol. 1998; 143(3): 709–17. 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}
|
[
{
"id": "11122",
"date": "17 Nov 2015",
"name": "Melchor Sanchez-Martinez",
"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 research article entitled 'Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock' by Vicedo, Rost and co-workers shows how Saccharomyces cerevisiae reduces protein disorder to survive heat shock. It constitutes an interesting example about the usage of bioinformatic techniques to analyze protein disorder and its implications at a whole proteome level. In the article, there is a comprehensive explanation of study design, methods and analysis. The conclusions are well explained and justified on the basis of the results.Consequently, the manuscript is recommended for approval. It is a good piece of science that meets the indexation requirements of F1000Research.However I have some comments that the authors may consider and/or answer.As far as I know except some rare exceptions the protein disorder is reduced as temperature increases, oppositely as happens with ordered proteins or protein regions. With increasing temperature, disordered proteins and regions tend to adopt a transitory structure. Commonly this transitory structure is necessary for proteins to perform its biological function. In other recent works that the authors have published have published (Reference 37 in the References section), they stated that \"protein disorder appeared as a possible building block to bring about evolutionary changes such as the adaptation to different habitats\" and in that sense seems that more disorder should imply a better response to heat shock.Thus is surprising for me that in response to heat there is a protein disorder reduction, whereas I expect a disorder increment. Why does it happens? Maybe the answer is so easy as that the disordered proteins do not help to \"fight\" against heat shock or as the authors said \"...Some of these proteins with long disordered regions might not work correctly in heat...\", but I am curious about that. Do you have any evidence or supported hypothesis to explain that? Regarding to authors statement \"...Some of these proteins with long disordered regions might not work correctly in heat...\", a plausible way to study that and obtain a more conclusive answer could be to perform a molecular simulation. Maybe a Replica Exchange Molecular Dynamics or Monte Carlo simulations could give a better understanding of what happens with these protein at high temperatures.",
"responses": []
},
{
"id": "11121",
"date": "17 Nov 2015",
"name": "Paul Pavlidis",
"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\nVicedo et al. report a computational analysis sparked by the interesting findings of Yona et al. (2012) of yeast duplication of chrIII having a selective advantage in the face of heat stress. Yona et al. did not fully mechanistically explain the reason chrIII aneuploidy is the one selected for, so Vicedo et al. have proposed a hypothesis: that chrIII has a substantially lower number of disordered proteins. They test this computationally, followed by some additional bioinformatics and “by hand” characterization of chrIII genes (and some other regions of interest following from Yona et al.).The difficulty here is assigning cause vs. “permissive”. As Vicedo et al. report, the disorder hypothesis has limited predictive value because chrX genes also have a low disorder (on average), so the size of the chromosome is posed as the other important variable. However, Vicedo et al. seem to be proposing that “low disorder” is good for heat resistance per se (I grant them this) – and that overexpression of low disorder proteins is even better. I have difficulty with this second step, because the way the experiment of Yona et al. was done, it could easily be that there are “heat resistance proteins” on chrIII and that the overall duplication of chrIII is tolerated in the context of the advantage of overexpression those genes. But if this was the end of the story it would be hard to make a determination of whether this is a viable hypothesis.However, there is an obvious problem: the work of Yona et al. identified 17 genes on chrIII that appear to be the main culprits for the heat resistance (at least most of them). I see no mention of these 17 in Vicedo et al. nor of the 22 control genes tested by Yona et al. If Vicedo et al. are right then there should be a difference in the disorder of these two sets of proteins. Otherwise, the observations might still be relevant, but that the orderedness of chrIII proteins might be permissive for overexpression of the actual heat-resistance genes via aneuploidy. In that case it might be the rest of the proteins on chrIII that have the orderedness properties, not the 17. (Note that I was not familiar with the Yona work before this review and I have not checked to see if Yona et al. or others have done any followup.)Given the omission of discussion of the 17, the sections of this paper on network analysis, GO and localization cannot be interpreted with confidence. While I have some quibbles about them I would rather wait to see the response to the comments above.",
"responses": []
},
{
"id": "11125",
"date": "20 Nov 2015",
"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 Vicedo and colleagues presents an interesting observation: the authors have examined chromosomal regions (all of chromosome III and fragments of chromosomes IV and XII) that are duplicated in Saccharomyces cerevisiae in response to sudden exposure to high temperature and find that these chromosomal sequences are significantly decreased for genes encoding proteins with long disordered regions. The authors further analyzed these duplicated regions and the encompassed genes for any enrichment in annotated GO terms, as well as for encoded protein positioning in interaction networks. The results do not indicate significant GO term enrichment and reveal that the encoded proteins exhibit a decreased number of interactions per protein. The biological advantage to this duplication remains unclear. Comments/suggestions: The main conclusion presented here is interesting, but as the authors themselves attest, this observation does not explain a biological advantage behind the duplication. On p. 4, the authors state that introducing an extra copy of HSP30 into wild-type yeast does not modify the ability of the cells to cope with high temperature. The inclusion of laboratory data considering the effect of adding an extra copy of genes or chromosomal regions corresponding to some of the duplicated sequences would strengthen the paper significantly. This seems to be the easiest way to address a biological effect from duplication of a given gene. In regards to the analysis, are the observed GO function annotations enriched with respect to other chromosomes/segments as opposed to being enriched against the genome as a whole? If the advantage to the cell centered on the functions associated with the genes in the duplicated regions, then these regions relative to other regions may be enriched for a function. If I’m thinking of this correctly, that would be slightly different than comparing a region for enrichment against the whole genome. Maybe the authors could compare enrichment in one chromosome versus another or utilize a sliding window corresponding to the size of a duplicated fragment to identify regions that would be most enriched for some potentially interesting functions. That might be a more sensitive means of identifying a functional enrichment for the duplicated regions. Typos/stylistic suggestions: on p.2, first line under Introduction: I think it would be sufficient to state “The baker’s yeast Saccharomyces cerevisiae” rather than the text in parentheses. on p. 3, fourth paragraph under “Duplications in response to high temperature reduce protein disorder”: the first sentence in this paragraph (“Assume a certain amount …”) needs to be reworded. on p. 4, first paragraph, line 16: delete “the” from “insignificant the finding”",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1222
|
https://f1000research.com/articles/4-155/v1
|
17 Jun 15
|
{
"type": "Research Article",
"title": "YeATS - a tool suite for analyzing RNA-seq derived transcriptome identifies a highly transcribed putative extensin in heartwood/sapwood transition zone in black walnut",
"authors": [
"Sandeep Chakraborty",
"Monica Britton",
"Jill Wegrzyn",
"Timothy Butterfield",
"Basuthkar J. Rao",
"Charles A. Leslie",
"Mallikarjuna Aradhaya",
"David Neale",
"Keith Woeste",
"Abhaya M. Dandekar",
"Monica Britton",
"Jill Wegrzyn",
"Timothy Butterfield",
"Basuthkar J. Rao",
"Charles A. Leslie",
"Mallikarjuna Aradhaya",
"David Neale",
"Keith Woeste",
"Abhaya M. Dandekar"
],
"abstract": "The transcriptome provides a functional footprint of the genome by enumerating the molecular components of cells and tissues. The field of transcript discovery has been revolutionized through high-throughput mRNA sequencing (RNA-seq). Here, we present a methodology that replicates and improves existing methodologies, and implements a workflow for error estimation and correction followed by genome annotation and transcript abundance estimation for RNA-seq derived transcriptome sequences (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). A unique feature of YeATS is the upfront determination of the errors in the sequencing or transcript assembly process by analyzing open reading frames of transcripts. YeATS identifies transcripts that have not been merged, result in broken open reading frames or contain long repeats as erroneous transcripts. We present the YeATS workflow using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut. A novel feature of the transcriptome that emerged from our analysis was the identification of a highly abundant transcript that had no known homologous genes (GenBank accession: KT023102). The amino acid composition of the longest open reading frame of this gene classifies this as a putative extensin. Also, we corroborated the transcriptional abundance of proline-rich proteins, dehydrins, senescence-associated proteins, and the DNAJ family of chaperone proteins. Thus, YeATS presents a workflow for analyzing RNA-seq data with several innovative features that differentiate it from existing software.",
"keywords": [
"RNA-seq",
"transcriptome",
"open reading frame",
"extensin",
"proline-rich proteins",
"dehydrins",
"senescence-associated proteins",
"Computational genomics",
"Juglans nigra",
"black walnut",
"heartwood/sapwood transition zone"
],
"content": "Introduction\n\nAnalysis of the complete set of RNA molecules in a cell, the transcriptome, is critical to understanding the functional aspects of the genome of an organism. Most transcripts get translated into proteins by the ribosome1. Non-translated transcripts (noncoding RNAs) may be alternatively spliced and/or broken into smaller RNAs, the importance of which have only recently been recognized2. Transcriptional levels vary significantly based on environmental cues3, and/or disease4. Quantifying transcriptional levels constitutes an important methodology in current biological research. Traditional methods like RNA:DNA hybridization5 and short sequence-based approaches6 have been supplanted recently by a high-throughput DNA sequencing method - RNA-seq7,8. Concomitant with the introduction of RNA-seq has been the development of a diverse set of computational methods for analyzing the resultant data9–21.\n\nIn the current work, we present a methodology for analyzing RNA-seq data that has been assembled into transcripts (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). The process of associating genomic open reading frames (ORF) to a set of transcripts (transcriptome) is the key step in YeATS, enabling identification and correction of specific errors arising from sequencing and/or assembly, a novel feature missing in most known tools. These errors include transcripts that have not been merged, a transcript having broken ORFs and transcripts containing long repeats. Also, YeATS identifies noncoding RNAs by comparison to compiled databases22, transcripts with multiple coding sequences and highly transcribed genes (based on simple normalization of raw counts followed by sorting).\n\nHere, the YeATS workflow is demonstrated using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut (Juglans nigra L.). We have identified transcripts that have sequencing and/or assembly errors (~5%). A novel feature that emerged from our analysis was the presence of a highly transcribed gene that had no known homologous counterpart in the entire BLAST database. The amino acid composition of the longest open reading frame of this gene consists of a high percentage of leucine, histidine and valine, and classifies this as a putative extensin23. Given the economic and ecological importance of black walnut timber, characterization of such genes will enhance our understanding of the mechanisms underlying the unique properties associated with the wood of these trees24. The significance of proline-rich proteins25, dehydrins26, senescence-associated proteins27 and DNAJ28 proteins to the formation of heartwood was established through their transcriptional abundance. Finally, based on transcripts that have no known homologs, we have identified noncoding RNAs by comparison with the noncoding RNA database for Arabidopsis22. Thus, in the current work, we present a workflow (YeATS) with several novel features absent in most currently available software.\n\n\nMethods\n\nThe input to YeATS is a set of post assembly transcripts as a fasta file (φTRS). The first step is to identify the set of genes (proteins) encoded by φTRS. This is done by associating a proper open reading frame (ORF) to each transcript. This involves a comprehensive automated BLAST run29.\n\nFor each transcript in φTRS, we generate the three longest ORFs (using the ‘getorf’ utility in the EMBOSS suite30) (Figure 1). These three ORFs are BLAST’ed to the full non-redundant protein sequences (‘nr’) database. For a given E-value cutoff (1E-12 in the current work), we create four sets\n\n1. Only one ORF is less than the cutoff - the transcript is uniquely annotated.\n\n2. None of the ORFs is less than the cutoff - the transcript has no known homologs.\n\n3. More than one ORF is less than the cutoff.\n\n(a) The ORFs map to the same protein, often in distinct regions - this points to an error in the sequencing or the assembly.\n\n(b) The ORFs map to different proteins - these are instances of a transcript having two valid ORFs. We duplicate the transcript, associating each one to a different protein sequence.\n\nFor each transcript, the three longest open reading frames (ORF) are obtained using the ‘getorf’, and these were BLAST’ed to the full non-redundant protein sequences (‘nr’) database. Based on the number of significant matches, the transcriptome is partitioned. Unique genes have only one significant match, erroneous transcripts have multiple ORFs matching the same gene, while duplicate genes have multiple distinct matches.\n\nTo produce the uniquely annotated set of genes, we ignored entries with the keywords chromosome, hypothetical, unnamed, unknown and uncharacterized, in order to have a functional characteristic in the annotation, provided the final annotated entry has low E-value. Also, apart from comparing E-values, we also compare the BLAST score, choosing an ORF as unique if its BLAST score was more than twice any other BLAST score, even if other scores satisfied the E-value criteria.\n\nAlgorithm 1 describes the process of merging transcripts (SI Figure 1). For a given length (which varies from 5 to 15 in this case), the 5’ and 3’ sequences and identifiers of each transcript are stored in new string databases: 3’=Begin; 5’=End. Repetitive strings (strings that have only two letters) are ignored, as it is difficult to ensure their uniqueness. For each string of n length in the Begin (3’) string database, we find whether: a) unique matches of n length (one-to-one mapping) are present in the End (5’) string database and b) that the prefixes (initial transcript identifiers) of the transcripts are the same.\n\nInput: φTRS ⇐ Set of transcripts\n\nOutput: φTRSMERGED: Pairs of transcripts that can be\n\nmerged\n\nbegin\n\nφTRSMERGED ← 0;\n\nwhile NewStatesAdded do\n\nforeach TRSi in φTRS do\n\nφBEGIN ← 0;\n\nφEND ← 0;\n\nforeach len:5..15 do\n\nAddBeginingofTRS(φBEGIN,TRSi,len);\n\nAddEndofTRS(φEND,TRSi,len);\n\nend\n\nforeach stringi in φBEGIN do\n\n/* ignore strings that have less than 3 letters, these are repetitive*/\n\nIgnoreRepeats(stringi);\n\nif(∃ only one stringj in φEND) such that prefixof(TRSi) == prefixof(TRSj))[\n\nφTRSMERGED ←\n\nAddtoMergeableSet(TRSi,TRSj);\n\n]\n\nend\n\nend\n\nend\n\nreturn φTRSMERGED;\n\nend\n\nAlgorithm 2 describes the iterative method for identifying homologous genes in the genome based on the transcriptome. First, the transcriptome is converted to a set of protein sequences by choosing the appropriate ORF (described above) as the representative protein sequence, and a BLAST database (TRSDB) is created. An input protein sequence (possibly from another organism) of a gene of interest is used to query TRSDB using BLAST29. This results in a set of significant transcript matches which is pruned based on a cutoff identity (40% in this case) and the criterion that the sequence length should not differ more than another parameterizable value (50 in this case). Both these transcripts are now potential genes, and the above mentioned process is repeated for each of them, until no new transcripts are added.\n\nInput: G ⇐ Amino acid sequence of gene\n\nInput: TRSDB ⇐ BLAST database of the protein sequences from each transcript, choosing the longest ORF as the representative protein sequence\n\nInput: identitycutoff ⇐ Ignore matches which are less than identitycutoff % identical to the sequence under consideration\n\nInput: lengthcutoff ⇐ Ignore matches where the sequence length differs by more than lengthcutoff % from the sequence under consideration\n\nOutput: φgenes\n\nbegin\n\nφgenes ← G;\n\nφprocessed ← 0;\n\nNewStatesAdded ← 1;\n\nwhile NewStatesAdded do\n\nNewStatesAdded ← 0;\n\nforeach Gi in φgenes such that Gi is not in\n\nφprocessed do\n\nφprocessed ← Gi;\n\nϕiBLAST = BLAST Gi on TRSDB;\n\nforeach TRSi in ϕiBLAST do\n\ndifflength ←\n\nlength(Gi) – length(TRSi) ;\n\nif(identity(TRSi, Gi) > identitycutoff ^\n\n(difflength < lengthcutoff)) [\n\nNewStatesAdded ← 1;\n\nφgenes ← TRSi;\n\n]\n\nend\n\nend\n\nend\n\n/* This is not a TRS, but an input - remove this from the set*/\n\nremove G from φgenes;\n\nreturn φgenes;\n\nend\n\nThe raw counts for each transcript is normalized according to Equation 1, assuming a read length of 100.\n\n\n\nThe sequence alignment was done using ClustalW31. The alignment images were generated using SeaView32.\n\nTotal RNA was isolated from the xylem region immediately external to the heartwood of a 16 year-old black walnut. The tree was felled in November, cross sections about 1 inch thick were taken from the base and dropped immediately into liquid nitrogen. After the sections were fully frozen they were transported to the lab on dry ice. The transition zone was then chiseled and the xylem was ground using a freezer mill. The RNA was extracted from 100g of ground wood using lithium chloride extraction buffer, and subsequently treated with DNAse (to remove genomic DNA) using an RNA/DNA Mini Kit (Qiagen, Valencia, CA) per the manufacturers protocol. Presence of RNA was confirmed by running an aliquot on an Experion Automated Electrophoresis System (Bio- Rad Laboratories, Hercules, CA). The cDNA libraries were constructed following the Illumina mRNA-sequencing sample preparation protocol (Illumina Inc., San Diego, CA). Final elution was performed with 16 μL RNase-free water. Each library was run as an independent lane on a Genome Analyzer II (Illumina, San Diego, CA) to generate paired end sequences of 85 bp in length from each cDNA library. The reads were trimmed to remove adapter contamination and low quality sequences using Scythe and Sickle (https://github.com/ucdavis-bioinformatics). Trimmed reads were aligned to the J. regia ‘Chandler’ transcriptome fasta assembled using Trinity (manuscript in preparation) with BWA’s short read aligner v.0.6.2 (‘bwa aln’) (http://bio-bwa.sourceforge.net/)33.\n\n\nResults\n\nThe input dataset to the YeATS tool was a set of transcripts, transcript identifiers and their corresponding raw counts (see Supporting information), obtained from the tissue at the heartwood/sapwood transition zone (TZ) in black walnut (Juglans nigra L.) (Figure 2). These raw counts were normalized (see Methods), and transcripts with zero counts were ignored (see rawcounts.normalized.TZ in Dataset 1). There were ~24K such transcripts (ϕtranscriptTZ).\n\nA cross section of a mature black walnut (Juglans nigra) stem showing the light-colored sapwood (Secondary xylem), darkly colored heartwood which contains no living cells. The transition zone (TZ) is immediately external to the heartwood highlighted by the yellow line in the red box. Cell death is actively occurring in this TZ tissue.\n\nIn order to associate a transcript to a specific open reading frame (ORF), the ORFs of ϕtranscriptTZ is obtained using ‘getorf’ from the Emboss suite30 (see ORFS.tgz in Supporting information) (Figure 1). The three longest ORFs for each transcript is BLAST’ed to the full non-redundant protein sequences (‘nr’) database, and the results were used to characterize the genes.\n\nThere were ~1200 transcripts that had possible sequencing or assembly errors, ~22K transcripts that had significant matches (E-value<E-12) in the ‘nr’ database, 113 transcripts that had lower matches (E-12<E-value<E-08) in the ‘nr’ database, ~700 transcripts that had no matches in the ‘nr’ database and about 200 transcripts that could be merged based on overlapping amino acid sequences. We describe these in detail below.\n\nWe observed transcripts that had multiple ORFs that matched to the same gene with high significance (E-value<E-10). The possibility that such an occurrence is not an experimental artifact is low. Transcript C15259_G1_I1 is one such example, having two ORFs - ORF_36 (length = 144) and ORF_9 (length = 122), both of which match to the mitochondrial ATP-dependent Clp protease proteolytic subunit 234 (GenBank: CAN64666.1) from Vitis vinifera with E-values of 6E-92 and 7E-45, respectively. Figure 3 shows the alignment of these two ORFs to the Vitis vinifera protein indicated the possible site of the sequencing error or transcript misassembly. This aspect of the YeATS methodology can be used to estimate the sequencing and transcript assembly error rate. For example, in the current transcriptome of the walnut TZ, we found a 5% (1200 out of 24,000) error rate.\n\nTranscript C15259_G1_I1 has two ORFs - 9 and 36 - both of which match to the mitochondrial ATP-dependent Clp protease proteolytic subunit 2, mitochondrial (GenBank: CAN64666.1) from Vitis vinifera with E-values of 6E-92 and 7E-45, respectively. It is likely that the error occurred near the amino acid sequence ‘SAG’ marked in the figure. The current transcriptome of the walnut TZ had a 5% (1200 out of 24,000) error rate for this class of error.\n\nA small number of transcripts had long repeats (on the reverse strand), as identified by transcripts that had multiple identical ORFs. For example, transcript C50369_G5_I2 has two ORFs (length = 143) that matched to an uncharacterized protein (Uniprot id: XP_009362671, E-value= 4e-13). These ORFs were located on the reverse strand, and were exactly the same (Figure 4). There were only 8 such cases.\n\nTranscript C50369_G5_I2 had an ORF (length = 143, Uniprot id: XP_009362671, uncharacterized protein), with an exact match on the reverse strand. There were only eight such cases, and they could be manually corrected.\n\nAbout ~200 transcripts have been merged using conservative metrics by YeATS (see Methods, list.merge in Supporting information). For example, transcripts C55368_G1_I3 and C55368_G2_I1 were merged based on a stretch of 12 amino acids (NFDENRGALNSH) (Figure 5). The indicated single nucleotide difference might be the reason for the failure of the assembly program to merge these two transcripts. Transcript C55368_G1_I3 had two exact repeats of this stretch, which is a likely assembly error.\n\n(a) Transcripts C55368_G1_I3 and C55368_G2_I1 could be merged based on a stretch of 12 amino acids (NFDENRGALNSH) obtained from their ORFs. (b) The partial nucleotide sequences of these transcripts shows the repeat with only a single nucleotide difference. The indicated single nucleotide difference may explain the failure of the assembly program to merge these two transcripts. Interestingly, the transcript C55368_G1_I3 had two exact repeats of this stretch at the end which may have contributed to the failure of the assembly program to merge these transcripts.\n\nThese ORFS belong to the same transcript, and have significant matches to different proteins. (a) Genes on the reverse strand, having no overlap - clathrin light chain (value=3E-126) and a leucine repeat rich receptor-like serine/threonine protein kinase (E-value=0). (b) Genes on the same strand, having no overlap - RING/U-box superfamily protein (E-value=7E-149) and a homeodomain-like superfamily protein isoform (E-value=0).\n\nSome transcripts were associated with multiple ORFs with distinct significant matches in the ‘nr’ database. We demonstrate this for the transcript C8909_G1_I1, which had two ORFs - ORF_104 (length = 331) and ORF_45 (length = 390) which matched to a clathrin light chain35 (Uniprot id:XP_006481016.1, E-value=3E-126) and a leucine repeat rich receptor-like serine/threonine protein kinase36 (Uniprot id: XP_007026739.1, E-value=0), respectively. These ORFs were on opposite strands, and did not overlap. It was not possible to ascertain which was the correct gene product, and it is a distinct possibility that both strands were transcribed37. A slightly different situation arose when both the ORFs were on the same strand38, as in the case of the transcript C54995_G6_I2. For example, in transcript C54995_G6_I2, there were two ORFs - ORF_157 (length = 464) and ORF_231 (length = 543) that matched to a RING/U-box superfamily protein39 (Uniprot id: XP_007042454.1, E-value=7E-149) and a homeodomain-like superfamily protein isoform40 (Uniprot id: XP_007030696.1, E-value=0), respectively. Both of these proteins were on the same (reverse) strand of the transcript. These transcripts are candidates for chimeric41 or fusion42 genes, since the ribosome is known to bypass small nucleotide stretches separating two ORFs43.\n\nTable 1 shows the transcripts with the highest counts. Interestingly, the most abundant transcript had no homologous counterpart in the full BLAST ‘nr’ or ‘nt’ database (GenBank accession: KT023102). A proline-rich protein (PRP), a part of the protein superfamily of cell wall proteins consisting of extensins and nodulins, was found to have the second most abundant transcript23,44. Proline comprises 19% of the amino acids in the ORF of this transcript. PRPs are found as structural proteins in wood, and it was hypothesized that these proteins occur in the xylem cell walls during ligniflication, and influence the properties of wood45. PRPs were associated with carrot storage root formation46, were wound and auxin inducible46 and implicated in cell elongation47. PRPs are also an integral component of saliva responsible for the precipitation of antinutritive and toxic polyphenols by forming complexes48. Two DNAJ/HSP40 chaperone proteins, which are involved in proper protein folding, transport and stress response, showed high transcriptional levels28. Two DNAJ/HSP40 chaperone homologs (GenBank accession id: BI677935 and BI642398) were shown to be differentially expressed during summer at the sapwood/heartwood TZ of black locust49. The transcription levels of dehydrin-related proteins were shown to be seasonally regulated in the wood of deciduous trees26,50. However, this dehydrin protein is homologous to a 24kDa dehydrin (Uniprot id: AGC51777) from Jatropha manihot, a drought resistant plant51, unlike the ~100kDa proteins investigated in26. Senescence-associated proteins, and the related tetraspanins, were also highly transcribed27. One highly expressed transcript was homologous to a protein that is yet to be characterized.\n\nThere are several highly transcribed genes in the representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone (TZ) in black walnut that did not have any significant homologs (NSL) in the complete ‘nr’ or ‘nt’ database. For the ‘nr’ database, we use the three longest ORFs as query. The significance of dehydrins, senescence-associated and DNAJ proteins can be observed through their transcription abundance.\n\nWe demonstrated the (iterative) gene finding methodology in YeATS on a transcription factor that has an AP2 DNA binding motif (RAP2.6L in Aradidopsis, At5g13330)52. This protein showed differential tissue specific expression, and is likely to be involved in plant developmental processes and stress response53. Recently, the sequence of a homolog of RAP2.6L was deduced (Uniprot id: C1KH72, JnRap2) from an EST sequence isolated from tissue at the heartwood/sapwood TZ in black walnut (Juglans nigra L.), and its role in the integration of ethylene and jasmonate signals in the xylem and other tissues was established54,55. Using the sequence of JnRap2, we probed for other RAP2 genes in the TZ of walnut. We found three possible genes (C38523_G2_I1, C53728_G7_I1 and C53728_G7_I2) (Figure 7). It was observed that C53728_G7_I2 was closest to the JnRap2 gene (97.4% identity, 98.2% similar), and is probably the same gene. C53728_G2_I1 was also significantly homologous to the JnRap2 gene (84.4% identity, 92.4% similar), and it appears to be an allelic or splice variant, a conflict that can be resolved after the publication of the complete walnut genome. Raw counts (see Supporting information) demonstrated that the transcript C38523_G2_I1 had negligible expression levels in TZ, corroborating the previous detection of only one RAP2 protein in 54.\n\nMultiple sequence alignment of possible genes for a transcription factor that had a AP2 DNA binding motif compared to JnRap2, which was deduced from an EST sequence obtained from tissue at the heartwood/sapwood transition zone in black walnut.\n\nThe top three ORFs of ~600 transcripts had no match in the BLAST ‘nr’ database. Although these may be unique genes, another possibility that must be considered is that these are non-coding RNA genes2. The nucleotide sequences of these 600 transcripts were BLAST’ed to the database of noncoding RNAs in Arabidopsis22. Three matches were identified: C52424_G5_I11, C52424_G5_I4 and C53565_G3_I1. Both C52424_G5_I11 and C52424_G5_I4 are homologous to CR20, a cytokinin-repressed gene in excised cotyledons of cucumber, hypothesized to be non-coding RNA56. Analogous to the current work, the CR20 gene had alternate splicing56. C53565_G3_I1 had a 100% match to the Aradidopsis locus ATMG01380, a mitochondrial 5S ribosomal RNA, which is a component of the 50S large subunit of mitochondrial ribosome57.\n\n\nDiscussion\n\nHigh-throughput mRNA sequencing (RNA-Seq) has revolutionized the field of transcript discovery, providing several advantages over traditional methods7,8. Following isolation and fragmentation of RNA and subsequent generation of cDNA libraries, a high-throughput sequencing platform is selected to generate short reads58. Reconstruction of transcripts from these short reads (assembly) may be performed using a reference genome or de novo algorithms15–18,21,59. Sequencing biases, variable coverage, sequencing errors, alternate splicing and repeat sequences are some of the challenges faced by these assemblers14,60.\n\nSeveral post assembly computational tools provide further curation of transcripts resulting from the assemblers. The curation step involves identifying redundancies19,20, finding coding regions61, annotating the transcripts (https://transdecoder.github.io/) and detecting inaccuracies by aligning the transcripts to the genome62. In the current work, we present an integrated workflow for RNA-seq analysis (YeATS). YeATS includes most features of the tools mentioned above. Additionally, YeATS delivers several capabilities absent in these tools. A comprehensive BLAST analysis of the top three open reading frames of each transcript enables the identification of erroneous transcripts arising out of sequencing or assembly errors. These erroneous transcripts can be classified as: a) transcripts that have not been merged, b) transcripts that result in broken ORFs and c) transcripts that have long improbable repeats. Finally, YeATS provides annotation of the genes, enumerates homologous genes based on a template sequence and specified similarity threshold and identifies transcripts with multiple ORFs. The ribosome is known to bypass small nucleotide stretches separating two ORFs43. These are rare events, however, and thus unlikely to apply to the ~1200 transcripts that have broken ORFs pointing to the same gene63. Transcripts having multiple ORFs on the same strand are good candidates for chimeric41 or fusion42 genes dependent on ribosome bypassing.\n\nThe current work reveals and corroborates several aspects of the biology of hardwood trees. Probably, the most interesting is the detection of a highly transcribed gene (C52369_G2_I1) with no known homologs in the complete protein and nucleotide BLAST database, or significant matches in a database of long non-coding RNA genes22. If indeed the longest ORF of this transcript encodes a protein, it is 143 amino acids long, and is leucine (18%), histidine (13%) and valine (10%) rich (Figure 8). Although it is likely that this is a protein with leucine rich repeats, these proteins are typically larger proteins64. The regulatory stimuli of extensins are different for monocots (which also have different amino acid composition) and dicots23. A significant presence of extensin-like proteins in the cell wall of both developing and mature xylem (wood) have been reported for pine45,65. The publication of the walnut genome will aid the characterization of these genes by elucidating its promoter sequences.\n\nC52369_G2_I1 has a high percentage of leucine, histidine and valine, and is a putative extensin. C51134_G2_I2 is proline and lysine rich, and is homologous to an extensin and nodulin.\n\nWell characterized proteins like proline-rich proteins25,45, dehydrins26, senescence-associated proteins27 and DNAJ/HSP40 chaperone49 proteins were also abundant in the transcriptome. While Arabidopsis supports secondary growth, it fails to accumulate wood; it is therefore interesting to identify highly transcribed genes that are missing in the Arabidopsis proteome (Table 2). The DNAJ/HSP40 chaperone, dehydrins and tetraspanin proteins are found in the Arabidopsis proteome (TAIR10_pep_2010121466), while the putative extensin, the proline-rich protein, a probable zinc transporter protein, an uncharacterized protein and senescence-associated protein appear to be unique to the walnut proteome.\n\nThe wood quality of walnut and Arabidopsis are quite different. It is informative to identify genes (proteins) that are absent in Aradidopsis, since they are likely to be responsible for the differences. The DNAJ/HSP40 chaperone, dehydrins and tetraspanin proteins are found in the Arabidopsis proteome, while the putative extensin, the proline-rich protein, a probable zinc transporter protein, an uncharacterized protein and senescence-associated protein appear to be unique to the walnut proteome.\n\nAlso, we corroborated the presence of a transcription factor that has a AP2 DNA binding motif52,54, and identify additional splice/allelic variants with similar transcriptional levels. Once again, the knowledge of the walnut genome would enable a more profound understanding of such genes.\n\n\nConclusions\n\nIn summary, the current work elucidates an integrated workflow for RNA-seq analysis with several innovative features for identifying and correcting erroneously assembled transcripts. We demonstrated this workflow by characterizing the transcriptome of the tissue at the heartwood /sapwood TZ in black walnut.\n\n\nData availability\n\nF1000Research: Dataset 1. YeATS Dataset, 10.5256/f1000research.6617.d4973067\n\n\nSoftware availability\n\nhttps://github.com/sanchak/YEATSCODE\n\nhttp://dx.doi.org/10.5281/zenodo.18382\n\nGNU General Public License version 3.0 (GPLv3)",
"appendix": "Author contributions\n\n\n\nThe YeATS tool suite was designed by Chakraborty, Britton and Wegrzyn did the analysis of the transcriptome, Woeste isolated the RNA, Butterfield was involved in the validation. The rest of the authors were involved in various aspects of the study design. Chakraborty wrote the first draft and the rest of the authors were involved in the editing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors wish to acknowledge support from the California Walnut Board and UC Discovery program.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful to Mary Lou Mendum for her inputs in preparing the manuscript.\n\n\nSupplementary figure\n\nThe contents of the ‘Begin’ and ‘End’ databases are represented along with the shared amino acid residues at the 3’ and 5’ ends, respectively. The ‘merge5’ command identified the String1 pair in the ‘Begin’ and ‘End’ databases due to the shared sequence ‘abcde’. The ‘merge5’ command failed to identify the String2 pair since six residues are shared. 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Publisher Full Text\n\nOkamuro JK, Caster B, Villarroel R, et al.: The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci U S A. 1997; 94(13): 7076–7081. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrishnaswamy S, Verma S, Rahman MH, et al.: Functional characterization of four APETALA2-family genes (RAP2.6, RAP2.6L, DREB19 and DREB26) in Arabidopsis. Plant Mol Biol. 2011; 75(1–2): 107–127. PubMed Abstract | Publisher Full Text\n\nHuang Z, Zhao P, Medina J, et al.: Roles of JnRAP2.6-like from the transition zone of black walnut in hormone signaling. PLoS One. 2013; 8(11): e75857. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang Z, Tsai CJ, Harding SA, et al.: A cross-species transcriptional profile analysis of heartwood formation in black walnut. Plant Mol Biol Report. 2010; 28(2): 222–230. Publisher Full Text\n\nTeramoto H, Toyama T, Takeba G, et al.: Noncoding RNA for CR20, a cytokinin-repressed gene of cucumber. Plant Mol Biol. 1996; 32(5): 797–808. PubMed Abstract | Publisher Full Text\n\nBarciszewska MZ, Szymanski M, Erdmann VA, et al.: Structure and functions of 5s rRNA. Acta Biochim Pol. 2001; 48(1): 191–198. PubMed Abstract\n\nMardis ER: The impact of next-generation sequencing technology on genetics. Trends Genet. 2008; 24(3): 133–141. PubMed Abstract | Publisher Full Text\n\nHaas BJ, Papanicolaou A, Yassour M, et al.: De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013; 8(8): 1494–1512. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoberts A, Trapnell C, Donaghey J, et al.: Improving RNA-seq expression estimates by correcting for fragment bias. Genome Biol. 2011; 12(3): R22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArrial RT, Togawa RC, Brigido Mde M: Screening non-coding RNAs in transcriptomes from neglected species using PORTRAIT: case study of the pathogenic fungus paracoccidioides brasiliensis. BMC Bioinformatics. 2009; 10: 239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao QY, Wang Y, Kong YM, et al.: Optimizing de novo transcriptome assembly from short-read RNA-seq data: a comparative study. BMC Bioinformatics. 2011; 12(Suppl 14): S2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHerr AJ, Gesteland RF, Atkins JF: One protein from two open reading frames: mechanism of a 50 nt translational bypass. EMBO J. 2000; 19(11): 2671–2680. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones DA, Jones JDG: The role of leucine-rich repeat proteins in plant defences. Advances in botanical research. 1997; 24: 89–167. Publisher Full Text\n\nAllona I, Quinn M, Shoop E, et al.: Analysis of xylem formation in pine by cDNA sequencing. Proc Natl Acad Sci U S A. 1998; 95(16): 9693–9698. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLamesch P, Berardini TZ, Li D, et al.: The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 2012; 40(Database issue): D1202–D1210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChakraborty S, Dandekar A, Rao BJ, et al.: Dataset 1 in: YeATS - a tool suite for analyzing RNA-seq derived transcriptome identifies a highly transcribed putative extensin in heartwood/sapwood transition zone in black walnut. F1000Research. 2015. Data Source"
}
|
[
{
"id": "10335",
"date": "05 Oct 2015",
"name": "Varodom Charoensawan",
"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\nChakraborty and coworkers proposed a new platform for analysing transcriptomic data from RNA-seq (YeATS -Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). The key feature of the tool highlighted by the authors is error estimation and correction of assembled transcripts, which is performed by analysing ORFs predicted in each transcript and merging of transcripts. This error-filtering step is supposedly missing in most other existing tools to date. In addition, YeATS is able to perform other common RNA-seq analytic tasks, such as transcript abundance estimation. From the point of view of a frequent user of NGS tools, rather than a developer, I can see that such a tool can be useful for improving transcript assembly and estimation, especially in organisms with no or poorly annotated genomes. However, there are a number of points that, to me, would improve the tool and the article, and it would be great if the authors could address/clarify. I would be happy to discuss this further if any of my comments are not clear.It would be nice to include a performance evaluation of this new platform against existing tools, or with vs’ without the transcript error correction step by YeATS. One way to do this might be to take an existing RNA-seq dataset from a well-annotated organism such as Arabidopsis as a gold standard, and perform transcript assembly-estimation with and without correction by YeATS, and compare this to the transcript estimation using genomic information (e.g. by mapping reads to annotated transcriptomes/genomes). Does YeATS indeed improve the coverage and specificity of transcript estimation, for instance? Along the same lines as the comment above, it would be useful if the authors could comment on the time and/or computing resources required to perform the correction step. Also, are the accuracy and computing resources dependent on the read lengths and/or sequencing platforms? (Or is it intended for Illumina reads as used in the example?). Both the source code of YeATS and the data set used to illustrate its usage have been deposited and described at the end of the article. However, to this reviewer’s understanding, there is a set of Perl scripts deposited to Github, but it is still not clear to me how the tool/workflow should be implemented. The README does not seem to describe this. Could the author point out if there is already a guideline or documentation on how to use or integrate YeATS into an existing NGS workflow, if that already exists? To my understanding, the input of YeATS is a set of assembled transcripts performed by other tools (e.g. Trinity). However, this step was not clearly described in the “in vitro methods” section on Page 5. Instead, it seems the trimmed reads were directly aligned to J. regia transcriptome, which is somewhat confusing. Could you please clarify these? The authors described the genes as highly “transcribed” in walnut (according to RNA-seq from this study?) that are not present in Arabiodopsis “proteome”. I found these to be slightly disconnected.\n\nMinor comments:Figure 1: Is the “no” label between the boxes “Choose longest ORF” to “Gene annotation” necessary?Page 3, 2nd column, line 12: modify the text to “often in distinct regions of the transcript..” for clarity?Page 5, 1st column: There were ~24K “of” such transcriptsFigure 6’s legend: These ORF”s”",
"responses": [
{
"c_id": "1653",
"date": "06 Nov 2015",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "We would like to thank you for taking the time to review this paper. Please find our responses below.Chakraborty and coworkers proposed a new platform for analysing transcriptomic data from RNA-seq (YeATS -Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). The key feature of the tool highlighted by the authors is error estimation and correction of assembled transcripts, which is performed by analysing ORFs predicted in each transcript and merging of transcripts. This error-filtering step is supposedly missing in most other existing tools to date. In addition, YeATS is able to perform other common RNA-seq analytic tasks, such as transcript abundance estimation. From the point of view of a frequent user of NGS tools, rather than a developer, I can see that such a tool can be useful for improving transcript assembly and estimation, especially in organisms with no or poorly annotated genomes.We appreciate your positive comments, and the possibility of value addition by the suggested methodology for existing NGS flows. We believe that this is the first attempt to associate the key information encoded by transcripts within ORFs to assess the accuracy of the assembly.However, there are a number of points that, to me, would improve the tool and the article, and it would be great if the authors could address/clarify. I would be happy to discuss this further if any of my comments are not clear. It would be nice to include a performance evaluation of this new platform against existing tools, or with vs without the transcript error correction step by YeATS. One way to do this might be to take an existing RNA-seq dataset from a well-annotated organism such as Arabidopsis as a gold standard, and perform transcript assembly-estimation with and without correction by YeATS, and compare this to the transcript estimation using genomic information (e.g. by mapping reads to annotated transcriptomes/genomes). Does YeATS indeed improve the coverage and specificity of transcript estimation, for instance?YeATS evaluates the accuracy of a transcriptome, but it is dependent on downstream tools (like MAKER) to use this for proper annotation of the genes. Thus, there are no existing tools that we could compare it with directly. A highly curated database like the Arabidopsis would not be a fair comparison, since it might have been annotated looking at several data points. However, we have extensively used the YeATS pipeline in processing the newly sequenced walnut genome (manuscript in review), and established erroneous assembly for several genes of interest. The transcriptome from several other tissues were included in the genome study. Interestingly, the 5% error estimate remained the same.Along the same lines as the comment above, it would be useful if the authors could comment on the time and/or computing resources required to perform the correction step. Also, are the accuracy and computing resources dependent on the read lengths and/or sequencing platforms? (Or is it intended for Illumina reads as used in the example?).The run times for most of the processing required in YeATS is a few hours on a 16 GB, 16-core machine, barring the search for homologies in the BLAST ’nr’ database, which can be time-intensive for a comprehensive search. This search can be significantly accelerated when the organism under investigation has well-annotated protein databases (as in the current case), much in lines of the newly introduced SMARTBLAST (http://blast.ncbi.nlm.nih.gov/smartblast/), to run times under a day. Run times are dependent on the number of transcripts only, since the input to YeATS is an assembled transcriptome from a tool like Trinity. We have included this information in the manuscript.Both the source code of YeATS and the data set used to illustrate its usage have been deposited and described at the end of the article. However, to this reviewers understanding, there is a set of Perl scripts deposited to Github, but it is still not clear to me how the tool/workflow should be implemented. The README does not seem to describe this. Could the author point out if there is already a guideline or documentation on how to use or integrate YeATS into an existing NGS workflow, if that already exists?We have provided a README that describes the step in the YeATS workflow. However, this is not a push-button methodology, and goes through several steps, each of which is dependent on the previous step. Also, we have used custom schedulers, and thus several steps need to be adjusted depending on available resources. For example, the number of parallel jobs and the time-step between each submission is controlled through a custom-script. Thus, we have provided the key algorithms in detail in the paper for any developer to easily replicate our results. Furthermore, we are enhancing several of the programs based on more sophisticated algorithms (like using kmers, compression of data, etc). A proper release of this software will require some more time, but this manuscript was not meant to be a software article.To my understanding, the input of YeATS is a set of assembled transcripts performed by other tools (e.g. Trinity). However, this step was not clearly described in the in vitro methods section on Page 5. Instead, it seems the trimmed reads were directly aligned to J. regia transcriptome, which is somewhat confusing. Could you please clarify these?The input of YeATS is indeed a set of assembled transcripts performed by other tools like Trinity. We have modified the methods section to clarify this.The authors described the genes as highly transcribed in walnut (according to RNA-seq from this study?) that are not present in Arabiodopsis proteome. I found these to be slightly disconnected.We agree that these results are slightly disconnected to the general narrative of this paper, which focuses on post-assembly methodologies to assess the accuracy of assembled transcripts. However, these are interesting results that emerge during the analysis of the transcriptome of the transition zone of walnut, which has been obtained for the first time. And thus, though this may be of interest to researchers in the field, there is too little data to spin-off another paper to publish these findings.Minor comments:Figure 1: Is the no label between the boxes Choose longest ORF to Gene annotation necessary?We have changed the label ’no’ to ’unannotated’. Long ORFs that do not have have significant matches are probably uncharacterized genes, and the genome could be annotated accordingly (although the annotation of novel genes is another problem not addressed in the current paper).Page 3, 2nd column, line 12: modify the text to often in distinct regions of the transcript.. for clarity?We have clarified this: ‘The ORFs map to different fragments of the same protein. This points to an error in the sequencing or the assembly, which breaks down the contiguous ORF into two fragments.’Page 5, 1st column: There were 24K of such transcripts Figure 6s legend: These ORFsWe have made these modifications. Once again, we are thankful for your insightful comments, and hope to have addressed your concerns."
}
]
}
] | 1
|
https://f1000research.com/articles/4-155
|
https://f1000research.com/articles/4-1215/v1
|
05 Nov 15
|
{
"type": "Research Article",
"title": "Integrated analysis of oral tongue squamous cell carcinoma identifies key variants and pathways linked to risk habits, HPV, clinical parameters and tumor recurrence",
"authors": [
"Neeraja M. Krishnan",
"Saurabh Gupta",
"Vinayak Palve",
"Linu Varghese",
"Swetansu Pattnaik",
"Prach Jain",
"Costerwell Khyriem",
"Arun Hariharan",
"Kunal Dhas",
"Jayalakshmi Nair",
"Manisha Pareek",
"Venkatesh Prasad",
"Gangotri Siddappa",
"Amritha Suresh",
"Vikram Kekatpure",
"Moni Kuriakose",
"Binay Panda",
"Neeraja M. Krishnan",
"Saurabh Gupta",
"Vinayak Palve",
"Linu Varghese",
"Swetansu Pattnaik",
"Prach Jain",
"Costerwell Khyriem",
"Arun Hariharan",
"Kunal Dhas",
"Jayalakshmi Nair",
"Manisha Pareek",
"Venkatesh Prasad",
"Gangotri Siddappa",
"Amritha Suresh",
"Vikram Kekatpure",
"Moni Kuriakose"
],
"abstract": "Oral tongue squamous cell carcinomas (OTSCC) are a homogeneous group of tumors characterized by aggressive behavior, early spread to lymph nodes and a higher rate of regional failure. Additionally, the incidence of OTSCC among younger population (<50yrs) is on the rise; many of whom lack the typical associated risk factors of alcohol and/or tobacco exposure. We present data on single nucleotide variations (SNVs), indels, regions with loss of heterozygosity (LOH), and copy number variations (CNVs) from fifty-paired oral tongue primary tumors and link the significant somatic variants with clinical parameters, epidemiological factors including human papilloma virus (HPV) infection and tumor recurrence. Apart from the frequent somatic variants harbored in TP53, CASP8, RASA1, NOTCH and CDKN2A genes, significant amplifications and/or deletions were detected in chromosomes 6-9, and 11 in the tumors. Variants in CASP8 and CDKN2A were mutually exclusive. CDKN2A, PIK3CA, RASA1 and DMD variants were exclusively linked to smoking, chewing, HPV infection and tumor stage. We also performed a whole-genome gene expression study that identified matrix metalloproteases to be highly expressed in tumors and linked pathways involving arachidonic acid and NF-k-B to habits and distant metastasis, respectively. Functional knockdown studies in cell lines demonstrated the role of CASP8 in a HPV-negative OTSCC cell line. Finally, we identified a 38-gene minimal signature that predicts tumor recurrence using an ensemble machine-learning method. Taken together, this study links molecular signatures to various clinical and epidemiological factors in a homogeneous tumor population with a relatively high HPV prevalence.",
"keywords": [
"oral tongue squamous cell carcinoma",
"somatic variants",
"gene expression",
"tumor recurrence",
"CASP8",
"TP53",
"RASA1",
"HPV"
],
"content": "Introduction\n\nSquamous cell carcinomas of the head and neck (HNSCC) are the sixth leading cause of cancer worldwide1. Tumors of the head and neck region are heterogeneous in nature with different incidences, mortalities and prognoses for different subsites and accounts for almost 30% of all cancer cases in India2. Oral cancer is the most common subtype of head and neck cancer in humans, with a worldwide incidence in >300,000 cases. The disease is an important cause of death and morbidity, with a 5-year survival of less than 50%1,2. Recent studies have identified various genetic changes in many subsites of the head and neck using high-throughput sequencing assays and computational methods3–7. Such multi-tiered approaches using the exomes, genomes, transcriptomes and methylomes from different squamous cell carcinomas have generated data on key variants and in some cases, their biological significance, aiding our understanding of disease progression. Some of the above sequencing studies have identified key somatic variants and linked them with patient stratification and prognostication. This, along with the associated epidemiology, enables one to look beyond the discovery of driver mutations, and identify predictive signatures in HNSCC.\n\nA previous study from the cancer genome atlas (TCGA) consortium with HNSCC patients (N = 279) identified somatic mutations in TP53, CDKN2A, FAT1, PIK3CA, NOTCH1, KMT2D and NSD1 at a frequency greater than 10%7. Additionally, the TCGA study identified loss of TRAF3 gene, amplification of E2F1 in human papilloma virus (HPV)-positive oropharyngeal tumors, along with mutations in PIK3CA, CASP8 and HRAS, and co-amplifications of the regions 11q13 (harboring CCND1, FADD and CTTN) and 11q22 (harboring BIRC2 and YAP1), in HPV-negative tumors, described to play an important role in pathogenesis and tumor development7. Chromosomal losses at 3p and 8p, and gains at 3q, 5p and 8q were also observed in HNSCC7. Tumors originating in the anterior/oral part of the tongue, or oral tongue squamous cell carcinoma (OTSCC) tend to be different from those at other subsites as oral tongue tumors are associated more with younger patients8 and spread early to lymph nodes9. Additionally oral tongue tumors have a higher regional failure compared to gingivo-buccal cases10 in oral cavity. Tobacco (both chewing and smoking) and alcohol are common risk factors for this group of tumors among older patients8. The role of HPV, both as an etiological agent and/or risk factor along with its role as a good prognostic marker in OTSCC, unlike in oropharyngeal tumors, is currently uncertain. It remains to be explored whether HPV acts as an etiological agent in the development of oral tongue tumors or simply represents a superinfection in patients. Additionally, HPV infection status currently does not influence disease management in OTSCC.\n\nHere, we present data towards a comprehensive molecular characterization of OTSCC. We performed exome sequencing, whole-genome gene expression, and genotyping arrays using fifty primary tumors along with their matched control samples, towards identification of somatic variants (mutations and indels), significantly up- and down-regulated genes, loss of heterozygosity (LOH) and copy number variations (CNVs). We integrated all the molecular data along with the clinical parameters and epidemiology such as tumor stage, nodal status, HPV infection, risk habits and tumor recurrence to interpret the effect of changes in the process of cancer development in oral tongue. We identified significant somatic variations in TP53 (38%), RASA1 (8%), CASP8 (8%), CDKN2A (6%), NOTCH1 (4%), NOTCH2 (4%), and PIK3CA (4%) from the exome sequencing study in OTSCC. The key variants were validated using an additional set of primary tumor samples. Variants in TP53 and NOTCH1 were found in mutually exclusive sets of tumors. Additionally, we found frequent aberrations in chromosomes 6–9, and 11 in tumor samples. We observed a strong association between somatic variations in some key genes with one or more risk habits; for example, CDKN2A and PIK3CA with smoking; CASP8 with consuming alcohol and chewing tobacco; RASA1 with chewing and tumor stage, and HPV infection, along with DMD and PIK3CA. From the gene expression analysis, we found matrix metalloproteases (MMPs) to be highly expressed in OTSCC. Pathway analysis identified Procaspase-8, Notch, Wnt, p53, extracellular matrix (ECM)-receptor interaction, JAK-STAT and PPAR to be some of the significantly altered pathways in OTSCC. We implemented an ensemble machine-learning method11 and identified a minimal gene signature set that distinguished a group of tumors with loco-regional recurrence from the non-recurrent set. Finally, we performed functional analysis of CASP8 gene in HPV-negative and HPV-positive OTSCC cell lines to establish its role in the process of tumor development.\n\n\nResults\n\nWe collected tumor and matched control (adjacent normal and/or lymphocytes) samples from 50 patients diagnosed with OTSCC, with informed consent. Data from patient habits, epidemiology and clinical parameters are presented in Figure 1A and Additional file 1A. About two-thirds of the patients (N = 31) included in our study were in the younger age group (≤50yrs), with 20% female patients in the total pool. Approximately, 70% of the patients were positive for at least one risk habit, namely, smoking, alcohol consumption or chewing tobacco (33% of patients smoked tobacco, 40% consumed alcohol and 42% chewed tobacco). HPV infection status in the primary tumors was established with type-specific qPCR or HPV16 digital PCR. Thirty-three percent of the patients were deceased at the time of completing the analysis. About 60% of the tumors were moderately differentiated, 25% well differentiated and the rest were poorly differentiated. Among the patients recruited, 60% were node-positive, 70% had no recurrence, 9% had distant metastasis and 24% had loco-regional recurrence at the time of completing the analysis. The mean and median follow-up durations for patients were nearly 30 months and 21 months, respectively. About 27% of the tumors were early stage tumors (T1N0M0 and T2N0M0) and the rest 73% were late stage tumors (tumors belonging to the rest of the TNM stage).\n\nA. The OTSCC samples are represented in color-codes with their corresponding status on; node (P: positive, N: negative); stage (E: early, L: late), recurrence (Y: loco-regionally recurrent, N: non-recurrent and M: distant metastatic); grade (WD: well-differentiated, MD: moderately-differentiated and PD: poorly-differentiated); disease-free survival or DFS (L: low/≤12mo, M: mid/12–24mo and H: high/>24mo); HPV (P: positive and N: negative); and habits (chewing, alcohol and smoking, Y: yes and N: no). B. Somatic mutation frequency per megabase (MB) is represented as scatterplot with the median point as a fine dotted line. C. Genes with significant somatic variants. D. Frequency histogram of nineteen cancer-associated genes bearing somatic missense and nonsense variants (mutations and indels). E. Columns representing mutually exclusive sets of genes. F. Significant copy number insertions and deletions (CNVs), alongside the chromosome cytobands (the numbers of cancer-associated genes within each cytoband are listed on the right).\n\nWe re-discovered variants, as described previously12 using whole-genome arrays, to validate the variant call accuracy as obtained from the exome sequencing data. We validated ~99% of the SNPs discovered from Illumina sequencing in both the tumor and matched control samples (Additional file 2). After filtering and annotation, we identified 19 cancer-associated genes bearing significantly altered somatic variants in OTSCC (Figure 1D). These were validated using Sanger sequencing in two sets of samples, one using the same tumor-control pairs used in the exome sequencing (the discovery set, Additional file 1A) and second, using an additional 36–60 primary tumors (validation set, Additional file 1B) for genes altered in ≥ 5% of the tumor samples. All the TP53 variants were validated in the discovery set. Three out of the four variants were validated for CASP8. The mutant alleles for the heterozygous variants in HLA-A, OBSCN, ING1, TTK and U2AF1 discovered by exome sequencing were difficult to interpret from the results of the validation using Sanger sequencing as they were present at a very low frequency (Additional file 3). Combining data from the validation set; the mutation frequencies for RASA1 and CDKN2A rose significantly to 10.71% and 16.47% in primary tumors respectively but those for TP53 and CASP8 remained largely unchanged (Additional file 3).\n\nThe somatic mutation frequency per megabase (MB) ranged from 10–45 with a median around 25 (Figure 1B). The median value for transition to transversion (ti/tv) ratio for both the tumor and its matched control samples was ~2.5 (Additional file 4). Overall, T->C changes were most frequent, followed by G->A and then T->G. Habits (smoking and alcohol consumption), nodal status, HPV infection, tumor grade and stage had no significant impact on the distribution of these nucleotides (Additional file 5). We used the workflow described in the Methods section to identify somatic mutations and indels in tumor samples following which we used three functional tools, IntOGen19, MutSigCV21 and MuSiC222 for variant interpretations (Additional file 6). In order to identify genes harboring significant variants, we used the intersection of these tools, following the criteria that the somatic variants be callable in the matched control sample and present in a single sequencing read in the control sample. This resulted in a final list of 19 cancer-associated genes (Figure 1C), which were divided into three categories with varying mutation frequencies (Figure 1D). The three frequency tiers were ≥ 30% (TP53), 6–30% (RASA1, CASP8 and CDKN2A) and 2–5% (NOTCH1, NOTCH2, DMD and PIK3CA were prominent among them).\n\nNext, we looked for mutual exclusivity of finding somatic variants in the genes and found that many of these genes harbor variants in a mutually exclusive manner across samples (Figure 1E), suggesting the possibility that there might be some common pathway(s) involved in the development of OTSCC. We observed mutual exclusivity among somatic variants in NOTCH1 and NOTCH2 genes, and expanded this finding to identifying 15 such mutually exclusive sets (Figure 1E). Among them, CDKN2A, HLA-A and TTK form a mutually exclusive set with TP53; RASA1, OBSCN, HLA-A, AJUBA and TTK are mutually exclusive with either NOTCH1 alone, or NOTCH2 and ANK3 together; NOTCH1, NOTCH2, HLA-A, AJUBA, ANK3, TTK, MLL2, ING1 or KEAP1, are mutually exclusive with CASP8 alone, or FAT1 and DMD together; FAT1, HLA-A, AJUBA, ANK3, TTK, MLL2, ING1 or KEAP1, are mutually exclusive with PIK3CA or DMD or NOTCH1 and OBSCN, or CDKN2A and OBSCN; U2AF1, MLL2 and TTK form a small mutually exclusive set. We juxtaposed the positions of the somatic variants from final list of all 19 genes (Additional file 7) detected in OTSCC against those found in the TCGA data using the cBioPortal. We found that the somatic variants in OTSCC were in the same domains where mutations were observed earlier in many of the genes (Additional file 7).\n\nCNV analyses using data from the whole-genome single nucleotide polymorphism (SNP) genotyping arrays revealed a large chunk of chromosome 9, bearing cancer-associated genes like CDKN2A, NF1 and MRPL4, to be affected in about 17% of the tumors (Figure 1F and Additional file 8). We found several CNVs of short stretches (in low kb range) within chromosomes 6–8, 11, 17 and X in many tumors.\n\nWe further classified the 19 cancer-associated genes from the previous analyses and linked those with habits, clinical parameters and HPV infection. Among the genes harboring significant somatic variants, we found CDKN2A to be mutated only in the never-smokers and past smokers, PIK3CA to be mutated only in the smokers, and TP53 to be mutated at a 20% greater frequency in the smokers, CASP8 mutation has a 12% greater frequency in those that consumed alcohol or chewed tobacco. RASA1 was exclusively mutated only in the non-chewers (Figure 2A). HPV-negative patients harbored somatic variants in DMD and PIK3CA, while HPV-positive patients alone had somatic variants in RASA1. Only the moderate- and well-differentiated tumor samples harbored variants in CASP8, while NOTCH1 was mutated largely in the poorly-differentiated tumors. Node-positive tumors had a 19% greater occurrence of TP53 variants. Somatic variants in RASA1 occurred exclusively in the late stage tumors (Figure 2A). We further studied the association of affected cancer-related signaling pathways with habits and clinical parameters, and found that recurrence and HPV infection had the highest impact (Figure 2B). The Procaspase-8 activation, Notch, p53 and Wnt signaling pathways were linked most with many of the clinical parameters, HPV infection and habits (Figure 2B).\n\nA. Histograms showing relationship between genes with significant somatic variants and various clinical and epidemiological parameters. For genes solely mutated in one of the clinical or epidemiological categories, or those mutated at a >= 5% frequency between two categories. B. Stack net charts of relative patient fraction (%) for each of the eight cancer-associated signaling pathways and their relationship with various clinical and epidemiological parameters.\n\nSignificant (q val ≤ 0.05) differentially expressed genes with a fold change of at least 1.5 revealed a consistent pattern of differential expression across the tumor samples (21 up- and 23 down-regulated genes, Figure 3A and Additional file 9). Genes involved in peroxisome proliferator-activated receptor (PPAR) signaling- (e.g., MMP1) and ECM-receptor interaction pathways (LAMC2 and SPP1) were up-regulated and CRNN, APOD, SCARA5 and RERGL were down-regulated in a majority of tumors (Figure 3A). Next, we studied the pathways involving genes with aberrant expression and their link with HPV infection and other clinical parameters. Genes in the arachidonic acid metabolism and Toll-like receptors were differentially expressed in patients with no smoking history (never smokers or past smokers) and alcohol habits (Figure 3B). SERPINE1 (a gene in HIF-1 signaling pathway) was differentially expressed in patients that are habits-negative. The NF-κ-B signaling pathway was differentially expressed only in metastasized tumors.\n\nA. Expression changes (green – up- and red – down-regulation) representing significantly differentially expressed genes in tumors. B. Stacked histograms representing relative patient fraction (%) for each of the 19 cancer-associated pathways and their relationship with clinical and epidemiological parameters.\n\nCASP8 is mutated in a significant number of oral tongue tumors [this study, 5, 7]. Caspase-8 is an important and versatile protein that plays a role in both apoptotic (extrinsic or death receptor-mediated) and non-apoptotic processes13,14. We studied the functional consequences of CASP8 knockdown through a siRNA-mediated method in an HPV-positive UM:SCC-4715 and an HPV-negative UPCI:SCC04016 OTSCC cell lines. Prior to the functional assay, the concentration of siRNA required for silencing, extent of CASP8 knockdown and cisplatin sensitivity (IC50) in both these cell lines was tested (Additional file 10). The invasion of cells was greater in both UM:SCC-47 and UPCI:SCC040 cell lines when CASP8 was knocked down (Figure 4A). To analyze the effect of caspase-8 on the migration property of cells, scratches were made on the confluent monolayer of cells and the wound closure area was measured at different time points (0hr, 15hr, 23hr & 42hr, Figure 4B). The wound closure was faster in CASP8 knockdown HPV-negative cells compared to the HPV-positive cells. At 15hr, 23hr and 48hrs, about 65%, 90% and 100% of the wound got closed respectively in the HPV-negative cell line compared to 50%, 70% and 85% respectively during the same time period in the HPV-positive cell lines (Figure 4B). siRNA knockdown of CASP8 rescued the chemo-sensitivity caused by cisplatin treatment as evident by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survival assay (Figure 4C). Interestingly, we found that the extent of rescue is greater in the HPV-negative cell line compared to the HPV16-positive one.\n\nResults from A. Matrigel cell invasion assay (plotted with respect to the control cells), B. Wound healing assay, and C. MTT cell survival assay (plotted with respect to the control cells) in UPCI:SCC040 (HPV-negative) and UM:SCC-47 (HPV-positive) cell lines.\n\nAfter cataloging the significantly altered genes in OTSCC, we wanted to see whether there is a relationship between the altered genes and loco-regional recurrence of tumors and metastasis. In order to do this, we used an ensemble machine-learning method implemented by variable elimination using random forests11 (Figure 5). We used multiple testing correction and the 0.632 bootstrapping method17 to estimate false positives. We discovered a 38-gene minimal signature that discriminated between the non-recurring, loco-regionally recurring and distant metastatic tumors (Figure 5). The .632+ bootstrap errors, indicative of prediction specificity, varied across non-recurrent, recurrent and distant metastatic tumors. The median error was low (0.03) and intermediate (0.3) for the non-recurrent and the loco-regionally recurrent categories respectively but was relatively higher (1.0) for the metastatic tumors. The errors were proportional to the number of representative samples within each category.\n\nGenes harboring somatic variants (in color) that are a part of the minimal signature set for tumor recurrence derived from random forest analyses are used.\n\nWe looked at significant pathways altered in OTSCC, taking into account all the molecular changes in tumors and found apoptosis, HIF, Notch, mTOR, p53, PI3K/Akt, Wnt and Ras to be some of the key signaling pathways affected in OTSCC (Figure 6). In addition, histone methylation, cell cycle/immunity and mRNA splicing processes were also affected. The complete list of pathways is provided in Additional file 11.\n\nGenes harboring significant somatic variants and with expression changes in tumors were used in Cytoscape to derive a set of important signaling pathways implicated in OTSCC.\n\n\nDiscussion\n\nSquamous cell carcinomas of the oral tongue are an aggressive group of tumors with a higher incidence in the younger population (≤50yrs), which spread early to lymph nodes and have a higher regional failure compared to gingivo-buccal cases8–10. Previous sequencing studies3–5,7 grouped oral tongue tumors with tumors from the oral cavity, but a rise in the incidence of oral tongue tumors, especially among younger people who never smoked, consumed alcohol or chewed tobacco warrants further investigation of this subgroup of oral tumors. Additionally, the role of HPV in oral tongue tumors, unlike in oropharyngeal cases18–20, is not well understood both in terms of incidence and prognosis. A meta-analysis of HPV-positive HNSCC tumors from multiple studies conducted at multiple locations concluded that HPV-positive patients, especially in oropharynx, have improved overall and disease-specific survival21. A past study has presented data that the HPV incidence in the oral tongue is low22 and some argue against any link between HPV infection and aggressive oral tongue tumors23. Although there is no consensus on rate of HPV incidence among oral tongue tumor patients, it is generally believed that it is low compared to oropharyngeal tumors. However, some studies in the past24, albeit from a different geography, established a much higher rate of HPV infection in oral tongue tumors.\n\nWe applied stringent filtering steps and used multiple annotation tools to come up with a list of 19 cancer-associated genes that harbored somatic variants in OTSCC. Most of these genes were also found in other studies, including the recent TCGA HNSCC study7, with some notable differences. A comparison of somatic variants discovered in all HSNCC studies, including the current study, is provided in Additional file 12. The frequency for somatic changes in CASP8, NOTCH1, CDKN2A and FAT1 genes in previous studies3–7 were, 4–34%, 13–18%, 2–16% and 13–50%, respectively. This is different from what we found in the current study (8%, 4%, 6% and 2% respectively for the same genes). This may partly be attributed to the total number of tumor samples used in different studies but may also be due to a unique pattern of mutations specific to the oral tongue subsite. It appears from our study that the latter is the case. For example, in one of the earlier studies6 involving similar number of patients as in the current study, CASP8 and FAT1 were mutated in 34% and 50% of the patients but we find the frequency to be 8% and 2%, respectively. In some earlier studies, it was not possible to categorize and identify oral tongue-specific variants as the sites were classified under oral cavity3,4.\n\nAlthough the somatic variants discovered from our study appear to be distributed uniformly across the genome, the significant copy number variation events are more concentrated in chromosomes 6–9 and 11 (Figure 1F and Additional file 8). One of the most important genes harboring somatic mutations discovered in our study is CASP8, the product for which derived from the precursor Procaspase-8. Caspase-8 is an important protein implicated in both apoptotic and non-apoptotic pathways14. Recent analysis from the TCGA study7 suggests that mutations in CASP8 co-occur with mutations in HRAS, and are mutually exclusive with amplifications in the FADD gene. In our functional studies, the most important observation was that caspase-8 shows different effects in HPV-positive and HPV-negative cells, the effect being more pronounced in HPV-negative cells (Figure 4). Therefore, it is possible that HPV-negative tumors activate a completely different set(s) of pathways and/or may have different chemosensitivity towards drugs than the HPV-positive tumors. It was shown previously that HPV-positive HNSCC cell lines are resistant to TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and treatment of cells with the proteasome inhibitor bortezomib sensitizes HPV-positive cells towards TRAIL-induced cell death mediated by caspase-825. The E6 protein of HPV interacts with the DED domain of caspase-8 and induces its activation by recruiting it to the nucleus26. Our observation on the role of caspase-8-mediated apoptosis being more pronounced in the HPV-negative OTSCC cell line is similar to the observation on the role of CASP8 in HPV-negative patients made earlier in TCGA study7. Taken together, genes including CASP8 regulate key pathways (Figure 6) that might play important role in the development of tumors in oral tongue.\n\nIn the past, several large sequencing studies have been undertaken in HNSCC3–5,7 that contained very few HPV-positive oral tongue patients. Our study is based on a unique patient cohort and attempts to link molecular signature with different clinical and epidemiological parameters. The prevalence of HPV is very high in oral tongue tumors from India, including in our cohort, compared with studies using cohorts elsewhere. Currently we are completing a larger study on HPV prevalence in different head and neck subsites and we don’t see the same high prevalence of HPV in non-oral tongue tumors in the oral cavity, for example in buccal tumors, in one of our studies (Palve et al., unpublished observation from upcoming publication). The exact reason for this high prevalence is not know. Additionally, our observation that some HPV-positive patients harbored TP53 mutations is counter-intuitive, owing to the fact that E6 is known to block p53. Although we don’t know the reason behind this, there is a possibility that HPV-positive tumors harboring TP53 mutations represent a unique class of tumors and it will be interesting to see if those tumors recur early or late compared to the HPV-positive tumors that have wild type p53 function. Therefore, this study is unique in that respect.\n\nIdentifying a signature for tumor recurrence prospectively in primary tumors may add significant advantage to disease management. In order to do this, we used a machine-learning method using the molecular changes identified in this study, in three batches of primary tumors; non-recurring, loco-regionally recurring and tumors with distant metastasis. We identified a 38-gene signature to be significantly distinguishing the three groups. The bootstrapping error for the non-recurring and the loco-regionally recurring groups were low (N = 34, .632 error = 0.03 and N = 10, .632 error = 0.3 respectively) but not in the metastatic tumor group (N = 4, .632 error = 1). This was due to the small sample numbers (N = 4) in the metastatic category, justifying the need for a larger sample set to validate the signature. The 38 gene signature identified in our study, however, needs to be validated in a much larger cohort in the future to achieve its true potential as a prognostic panel in OTSCC.\n\nFinally, we were keen to see if the current study leads to finding novel drug candidates in OTSCC. We based our assumption on the fact that genome-wide somatic variant discovery in tumors may give rise to possibilities of finding novel drug targets/candidates or may led us to use existing drugs prescribed for other indications. In an attempt to identify if any of the significantly altered genes found in the current study could potentially act as drug targets, we screened for available drugs against them. We found drugs against three targets out of which two have undergone at least one clinical trial (Additional file 13).\n\n\nMethods\n\nInformed consent was obtained voluntarily from each patient enrolled in the study. Ethical approval (NHH/MEC-CL/2014/197) was obtained from the Institutional Ethics Committees of the Mazumdar Shaw Medical Centre. Matched control (blood and/or adjacent normal tissue) and tumor specimens were collected and used in the study. Patients diagnosed and treated at the cancer clinic of the Mazumdar Shaw Medical Centre for oral tongue tumors were subjected to a screening procedure before being enrolled in the study. Only those patients, where the histological sections confirmed the presence of squamous cell carcinoma with at least 70% tumor cells in the specimen, were used in the current study. At the time of admission, patients were asked about the habits (chewing, smoking and/or alcohol consumption). Fifty treatment-naïve patients who underwent staging according to AJCC criteria, and curative intent treatment as per NCCN guideline involving surgery with or without post-operative adjuvant radiation or chemo-radiation at the Mazumdar Shaw Medical Centre were accrued for the study (Additional file 1). Post-treatment surveillance was carried out by clinical and radiographic examinations as per the NCCN guidelines.\n\nHPV was detected by using q-PCR (Applied Biosystems 9700) using HPV16- and HPV18-specific TaqMan probes and primers, and digital PCR (BioRad QX100) using TaqMan probes and primers to detect HPV in primary tumor samples. The primers, probes and cycling conditions for q-PCR and ddPCR were as follows. For q-PCR: 5’ GCA CAG AGC TGC AAA CAA CT 3’; 3’ GCA TAA ATC CCG AAA AGC AA 5’; probe-ATTAGAATGTGTGTACTGCAAGCA-FAM-BHQ and 5’ TGA CAC TGT GCC TCA ATC CT 3’; 3’ AGA GCC ACT TGG AGA GGG AG 5’; Probe-TGCCTGCTTCACCTGGCAGC-VIC-BHQ for HPV16 and HPV18 respectively. The cycling conditions for q-PCR were: 95°C : 3 min, 95°C : 30 sec, 55°C for HPV16 and 60°C for HPV18 : 30 sec, 72°C : 30 sec for 40 cycles. For ddPCR: 5’ ACT GTC AAA AGC CAC TGT GT 3’; 3’ GCT GGG TTT CTC TAC GTG TT 5’ and Probe-AGGGGTCGGTGGACCGGTCGATGT-FAM-BHQ for HPV16. The cycling conditions for ddPCR were: 95°C: 10 min, 95°C : 315 sec, 55°C : 20 sec for 40 cycles.\n\nExome libraries were prepared using Agilent SureSelect, Illumina TruSeq and Nextera exome capture kits (Additional file 14) following manufacturers’ specifications. Paired end sequencing was performed using HiSeq 2500 or GAIIx and raw reads were generated using standard Illumina base caller (HCS 2.0). Read pairs were filtered using in house scripts (Additional file 15 and Additional file 16) and only those reads having ≥75% bases with ≥ 20 phred score and ≤ 15 Ns were used for sequence alignment against human hg19 reference genome using NovoAlign (v3.00.05)27. The aligned files (*.sam) were processed using Samtools (v0.1.12a)28 and only uniquely mapped reads from NovoAlign were considered for variant calling. The alignments were pre-processed using GATK (v1.2-62)29 in three steps before variant calling. First, the indels were realigned using the known indels from 1000G (phase1) data. Second, duplicates were removed using Picard (v1.39). Third, base quality recalibration was done using CountCovariates and TableRecalibration from GATK (v1.2-62), taking into account known SNPs and indels from dbSNP (build 138). Finally, UnifiedGenotyper from GATK (v2.5-2) was used for variant calling, using known SNPs and indels from dbSNP (build 138). Raw variants from GATK were filtered to only include the PASS variants (standard call confidence ≥ 50) within the merged exomic bait boundaries. Two out of 50 tumor samples did not confirm to the QC standards, therefore excluded from all further analyses. Therefore, all the downstream analyses were restricted to 48 primary tumors. The variants were further flagged as novel or present in either dbSNP138 or COSMIC (v67) databases, based on their overlap. In addition to GATK, we also used Dindel30 to call indels. Both GATK and Dindel calls were filtered for microsatellite repeats (flagged as STR). The raw variant calls were used to estimate frequencies of nucleotide changes and transition:transversion (ti/tv) ratios. Exome-filtered PASS variants specific to the tumor samples, with respect to both location and actual call, were retained as somatic variants, which were further filtered to exclude variants where the region bearing the variant was not callable in the matched control sample, and those where the matched control sample had even one read covering the variant allele.\n\nScripts used to perform various filtering steps are provided in Additional file 16. The numbers of raw reads, after QC, alignment statistics, numbers of variants pre- and post-filters are provided in Additional file 2.\n\nWe estimated cross-contamination using ContEst (June 2013)31 in the tumor samples (Additional file 16). Locus-wise and gene-wise driver scores were estimated by CRAVAT32 using the head and neck cancer database with the CHASM33 analysis option. Genes with a CHASM score of at least 0.35 were considered significant for comparison with other functional analyses (Additional file 16). Somatic mutations were normalized with respect to the exome bait size (MB) to calculate the somatic mutation frequency per MB.\n\nAnnotation and functional analyses of somatic variants was performed using IntoGen (web version 2.4)34,35, MutSigCV (v1.3.01)36,37 and MuSiC2 (v0.1)37. Somatic variants, filtered to contain only those callable in the matched normal but not covered by any read in the control samples (VCF), were used for IntoGen with the 'cohort analyses' option. We also ran MutsigCV1.3 with these variants using coverage from un-filtered variants of all tumor samples (Additional file 16). Pooled alignments for all normal and tumor samples (BAM), each, along with pooled variants for all normal samples (MAF) were analyzed using MuSiC2 to calculate the background mutation rates (bmrs) for all genes, and identify a list of significantly mutated genes (p-value of convolution test ≤ 0.05; Additional file 16). A condensed list of 19 genes, common between at least two analyses was compiled (Figure 1D).\n\nHigh quality DNA (200ng), quantified by Qubit 2.0 (Invitrogen), was used as the starting material for whole-genome genotyping experiments following the manufacturer’s specifications. Briefly, the genomic DNA was denatured at room temperature (RT) for 10 mins using 0.1N NaOH, neutralized and used for whole genome amplification (WGA) under isothermal conditions, at 37°C for 20 hrs. Post-WGA, the DNA was enzymatically fragmented at 37°C for 1hr. The fragmented DNA was precipitated with isopropanol at 4°C and resuspended in hybridization buffer. The samples were then denatured at 95°C for 20 mins, cooled at RT for 30 mins and 35µl of each sample was loaded onto the Illumina HumanOmni 2.5-8 beadchip for hybridization (20hrs at 48°C) in a hybridization chamber. The unhybridized probes were washed away and the Chips (HumanOmni 2.5-8 v1.0 and v1.1, Additional file 2) were prepared for staining, single base extension and scanning using Illumina’s HiScan system.\n\nWe filtered the SNP locations to retain only those, called without any error, contained within the exome boundaries as per the sequencing baits, and which were callable (covered by at least five sequencing reads). At these locations, we estimated the overlap for individual SNP calls, i.e., chr/pos/ref/alt and for no calls; i.e., chr/pos/ref/ref; between sequencing and array platforms (Additional file 16).\n\nCNVs and LOHs were identified using cnvPartition 3.1.6 plugin in Illumina GenomeStudio v2011.1, with default settings except for a minimum coverage of at least 10 probes per CNV/LOH with a confidence score threshhold of at least 100 (Additional file 17). Somatic CNVs and LOHs were extracted by filtering out any region common to CNVs and LOHs detected in its matched control. Somatic CNVs and LOHs were further filtered with respect to common and disease-related CNVs and LOHs using CNVAnnotator38. Overlaps with common CNVs and LOHs were discarded, reporting only the overlaps with disease-related, and novel CNVs and LOHs. We categorized the CNVs and LOHs within each cytoband and reported those with an occurrence in at least 10% of the patient samples.\n\nGene expression profiling was carried out using Illumina HumanHT-12 v4 expression BeadChip (Illumina, San Diego, CA) in tumor and matched normal tissues (Additional file 9) following manufacturer’s specifications. Total RNA was extracted from 20mg of tissue using PureLink RNA (Invitrogen) and RNeasy (Qiagen) Mini kits. RNA quality was checked using Agilent Bioanalyzer 2100 using RNA Nano6000 chip. Samples with poor RNA integrity numbers (RIN) (<7), indicating partial degradation of RNA, were processed using Illumina WGDASL assay as per manufacturer's recommendations. The RNA samples with no degradation were labelled using Illumina TotalPrep RNA Amplification kit (Ambion) and processed according to the array manufacturer’s recommendations. Gene expression data was collected using Illumina’s HiScan and analyzed with the GenomeStudio (v2011.1 Gene Expression module 1.9.0) and all assay controls were checked to ensure quality of the assay and chip scanning. Raw signal intensities were exported from GenomeStudio for pre-processing and analyzed using R further.\n\nGene-wise expression intensities for tumor and matched control samples from GenomeStudio were transformed and normalized using VST (Variance Stabilizing Transformation) and LOESS methods, respectively, using the R package lumi39. The data was further batch-corrected using ComBat40 (Additional file 16). The pre-processed intensities for tumor and matched control samples were subjected to differential expression analyses using the R package, limma41 (Additional file 16). Genes with significant expression changes (adjusted P value <= 0.05) and fold change of at least 1.5 were followed up with further functional analyses.\n\nWe used presence or absence of somatic mutations/indels data in the entire set of genes for all the OTSCC patients, along with their recurrence patterns as training set for the random forests11 analyses using the varSelRF package in R. This method performs both backward elimination of variables and selection based on their importance spectrum, and predicts recurrence patterns in the same set by iteratively eliminating 2% of the least important predictive variables until the current OOB (out-of-bag) error rate becomes larger than the initial or previous OOB error rates. In order to understand the specificity of the best minimalistic predictors of tumor recurrence, we estimated the 0.632+ error rate17 over 50 bootstrap replicates. We used the varSelRFBoot function from the varSelRF Bioconductor package to perform bootstrapping. The .632+ method is described by the following formula:\n\nErr0.632′=Err0.632+(Err1−err)(.368⋅.632⋅R′)1−.368⋅R′\n\nwhere Err(.632'), Err(.632), Err(1) and err are errors estimated by the .632+ method, the original .632 method, leave-one-out bootstrap method and err represents the error. R’ represents a value between 0 and 1. Another popular error correction method used is leave-one-out bootstrap method. The .632+ method was designed to correct the upward bias in the leave-one-out and the downward bias in the original .632 bootstrap methods.\n\nFor all iterations of all random forest analyses, we confirmed that the variable importance remained the same before and after correcting for multiple hypotheses comparisons using pre- and post- Benjamin-Hochberg FDR-corrected P values. R commands for variable elimination using random forests, 0.632+ bootstrapping and re-computing importance values after multiple comparisons testing are provided in Additional file 16.\n\nConsensus list of genes from analysis, filtering and annotation of variant calls and from differential expression analysis using whole genome micro-arrays, were mapped to pathways using the web version of Graphite Web42 employing KEGG and Reactome databases. The network of interactions between genes was drawn originally using CytoScape (v3.1.1)43 using the .sif file created by Graphite Web (Additional file 16).\n\nWe used Circos (v0.66)44 (Additional file 18 and Additional file 19) for multi-dimensional data visualization. Additionally, we used the cbioportal protal (http://www.cbioportal.org/) to visualize variants within the 19 genes harboring significant variants. All of the mandatory fields accepted by Mutation Mapper were provided for select genes from our study to create structural representations for each gene including domains. Such diagrams from our study, the HNSCC study and all cancer studies from TCGA were collated using the image-editing tool, GIMP (v2.8.0) (www.gimp.org). SNPs and indels were visualized for each individual tumor sample using IGV (v1.5.54)45, along with the reads supporting variants (Additional file 20).\n\nPrimers were designed using the NCBI primer designing tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and used in Sanger sequencing for validation. The sequences of all primers (IDT) used for validation is provided in Additional file 21. We tested the specificity of the designed primers using UCSC's tool, In Silico PCR. The variant-bearing region was amplified by using specific primers and used in Sanger sequencing (Additional file 14). The somatic variants were confirmed by sequencing in the entire tumor and matched control DNA set used for the exome sequencing followed by further validation in 60 additional tumor samples (Additional file 1B).\n\nThe human OTSCC cell lines UPCI:SCC040 (gift from Dr. Susan Gollin, University of Pittsburgh, PA, USA)16 and UM-SCC47 (gift from Dr. Thomas Carey, University of Michigan, MI, USA)15 were used in the study. All the cells were maintained in Dulbecco’s Modified Eagles’ Media (DMEM) supplemented with 10% FBS, 1% MEM nonessential amino acids solution & 1% penicillin/streptomycin mixture (Gibco) at 37°C with 5% CO2 incubator.\n\nWe performed the siRNA-based knockdown using UPCI:SCC040 and UM:SCC47 cell lines for CASP8 gene. The expression of Caspase-8 was transiently knocked down using ON-TARGETplus Human CASP8 smart pool siRNA (L-003466-00-0010; Dharmacon) along with an ON-TARGETplus Non-targeting siRNA (D-001810-01-20; Dharmacon). The transfection efficiency for the two cell lines (UPCI:SCC040 and UM:SCC47) were optimized using siGLO Red Transfection Indicator (D-001630; Dharmacon). The siRNA duplexes were transfected using Lipofectamine-2000 according to the manufacturer’s instructions (Invitrogen). The siRNA-oligo complexes medium was changed 8 hrs post transfection. The efficiency of transfection along with the mRNA expression was analyzed at 24 and 48 hrs post transfection by qRT-PCR. The specific down-regulation of CASP8 was confirmed by three independent experiments.\n\nRNA was extracted from cell pellets and tissues using RNeasy Mini kit spin columns (Qiagen) following manufacturer’s protocol. Genomic DNA contamination was removed by RNase-Free DNase Set (Qiagen) and the total RNA was eluted in nuclease free water (Ambion). The RNA samples were estimated using Qubit 2.0 fluorometer (Invitrogen) and the integrity was checked by gel electrophoresis. The RNA samples were stored at -80°C until further used. The cDNA was synthesized with 400ng total RNA, using a SuperScript-III first strand cDNA synthesis kit, and following the manufacturer’s instructions (Invitrogen). The cDNA was then subjected for quantitative real-time PCR (q-RT-PCR) using KAPA SYBR FAST qPCR Master Mix (KK4601, KAPA). The primer pairs used for testing the expression of caspase-8 in q-RT-PCR were, forward 5'-ATGATGACATGAACCTGCTGGA-3' and reverse 5’-CAGGCTCTTGTTGATTTGGGC-3'. The amplification was done on Stratagene MX300P real time machine. The cycling conditions were: step-1 95°C-3min, step-2 95°C-3sec, step-3 60°C-60sec then repeat steps 2–3 for 40 cycles following dissociation curve at 60°C-60sec, 95°C-1min, 60°C-60sec.\n\nTo normalize inter-sample variation in RNA input, the expression values were normalized with GAPDH. All amplification reactions were done in triplicates, using nuclease free water as negative controls. The differential gene expression was calculated by using the comparative CT method of relative quantification46.\n\nMTT cell proliferation assay was performed as per manufacturer’s instructions (Sigma) to assess cell viability. Briefly, cells were seeded on 96-well plates containing DMEM with 10% FBS & incubated overnight. After treatment with 0.1% DMSO (vehicle control), or Cisplatin for 48 hrs, medium was changed and 100 µl of MTT solution (1mg/ml) was added to each well. The cells were further incubated for 4hrs at 37°C. The formazan crystals in viable cells were dissolved by adding 100µl of dimethyl sulfoxide (DMSO) (Merck). The absorbance was recorded at 540 nm using reference wavelength of 690 nm on micro plate reader (Tecan Systems). Data were normalized to vehicle treatment, and the cell viability was calculated using GraphPad Prism software (version 4.03; La Jolla, CA). All the experiments were performed in triplicates.\n\nCells were cultured up to 80% confluency in 12 well plates; serum-starved for 24 hrs and then wounded using a 200µl pipette tip. The wound was washed with 1× PBS and the cells were grown in DMEM containing 10% FBS. Cells were imaged at 10× magnification at 0 hr, 15 hrs, 23 hrs and 42 hrs. For each well, three wounds were made and the migration distance was photographed and measured using Carl Zeiss software (Zeiss). Each experiment was performed in triplicates.\n\nThe ECM gel (E1270, Sigma) was thawed overnight at 4°C and plated at requisite concentrations (for UPCI:SCC040: 1.5mg/ml and UMSCC047: 2mg/ml) onto the transwell inserts and incubated overnight in the CO2 incubator at 37°C with 5% CO2. Cells were serum-starved for overnight, harvested, counted and seeded (UPCI:SCC040: 50,000 cells and UMSCC047: 20,000 cells per well) on top of the matrigel transwell-inserts (2 mg/ml) in serum-free medium as per manufacturer’s specifications (Sigma). D-MEM containing 10% FBS and 1% NEAA was added to the lower chamber. The 24-well plates containing matrigel inserts with cells were incubated in 37°C incubator for 48 hrs. At the end of incubation time, cells in the upper chamber were removed with cotton swabs and cells that invaded the Matrigel to the lower surface of the insert were fixed with 4% paraformaldehyde (Merk Milipore), permeabilized with 100% methanol, stained with Giemsa (Sigma), mounted on glass slides with DPX mounting agent and counted under a light microscope (Zeiss). Each experiment was performed in triplicates.\n\n\nConclusions\n\nWe have catalogued genetic variants (somatic mutations, indels, CNVs and LOHs) and transcriptomic (significantly up- and down-regulated genes) changes in oral tongue squamous cell carcinoma (OTSCC) and used those in an integrated approach linking genes harboring somatic variants with common risk factors like tobacco and alcohol; clinical, epidemiological factors like tumor grade and HPV; and tumor recurrence. We found CASP8 gene to be significantly altered and play an important role in apoptosis-mediated cell death in an HPV-negative OTSCC cell line. Finally, we present data towards a minimal gene signature that can predict tumor recurrence.",
"appendix": "Author contributions\n\n\n\nBP: conceived, designed and supervised the study, wrote the manuscript; NMK: analyzed the data and wrote the manuscript; SG: analyzed the data and critically read the manuscript; SP, PJ, CK, VKP: analyzed the data; VP, GS, AS: produced data on CASP8 functional analysis; LV, AKH, MP: produced sequencing data; KD and JN: produced array data; GS, AS, VK and MAK: provided clinical data, clinical input and associated clinical information. All authors have seen and agreed to the final content of this manuscript.\n\n\nCompeting interests\n\n\n\nNone.\n\n\nGrant information\n\nResearch presented in this article is funded by Department of Electronics and Information Technology, Government of India (Ref No:18(4)/2010-E-Infra., 31-03-2010) and Department of IT, BT and ST, Government of Karnataka, India (Ref No:3451-00-090-2-22).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAdditional files\n\nAdditional files for Krishnan et al., 2015 'Integrated analysis of oral tongue squamous cell carcinoma identifies key variants and pathways linked to risk habits, HPV, clinical parameters and tumor recurrence'.\n\nAll the additional files can be downloaded from Figshare (http://figshare.com/s/c928faa66f2a11e586d506ec4b8d1f61).\n\nAdditional file 1. Patient details used in the study.\n\nAdditional file 2. Sequencing, read QC, alignment and variant calls, OMNI SNP genotyping array validation.\n\nAdditional file 3. Validation using capillary gel electrophoresis based on Sanger sequencing in A. discovery set and B. validation set.\n\nAdditional file 4. The ratio of transitions to transversions (ti/tv) was estimated using the exome-filtered GATK PASS variants for tumor and matched control samples. The dotted lines depict the respective median ti/tv ratios.\n\nAdditional file 5. Effect of habits, clinical parameters and HPV infection on individual nucleotide change.\n\nAdditional file 6. Functional annotation of somatic variants using IntOGen, MuSiC2, MutSigCV.\n\nAdditional file 7. Position and frequency of somatic variants in protein domains found in this study, TCGA HNSCC, and in studies involving all cancer types using mutation mapper in the cBioPortal.\n\nAdditional file 8. Cytoband-wise representation of CNVs found in all 48 samples along with clinical parameters and patient epidemiology.\n\nAdditional file 9. Transformed, normalized and batch-corrected intensities following expression assay and results from differential expression analyses.\n\nAdditional file 10. Functional validation for the role of CASP8 in OTSCC cell lines.\n\nAdditional file 11. List of all pathways affected by somatic mutations/indels, copy number variations and expression changes (log2FC≥=0.6).\n\nAdditional file 12. Comparative sample frequency of important variants found in this and other HNSCC studies.\n\nAdditional file 13. Drug candidates and their targets in head and neck cancer.\n\nAdditional file 14. Supplementary Methods.\n\nAdditional file 15. Read QC filter scripts’ executable.\n\nAdditional file 16. Scripts used in the study.\n\nAdditional file 17. GenomeStudio output of all LOHs and CNVs found using cnvPartition plugin in GenomeStudio.\n\nAdditional file 18. Circos data and config files.\n\nAdditional file 19. Circular genomic representation using Circos (v0.66) of LOHs, somatic variants, CNVs with >= 10% frequency of patients bearing them, and genes with significant expression changes (|log2FC|>=0.6).\n\nAdditional file 20. IGV snapshots of all significantly mutated somatic variants in this study.\n\nAdditional file 21. Primer sequences used in the Sanger validation study.\n\nClick here to access the data.\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\nMishra A, Meherotra R: Head and neck cancer: global burden and regional trends in India. Asian Pac J Cancer Prev. 2014; 15(2): 537–550. PubMed Abstract | Publisher Full Text\n\nStransky N, Egloff AM, Tward AD, et al.: The mutational landscape of head and neck squamous cell carcinoma. Science. 2011; 333(6046): 1157–1160. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgrawal N, Frederick MJ, Pickering CR, et al.: Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science. 2011; 333(6046): 1154–1157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPickering CR, Zhang J, Yoo SY, et al.: Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers. Cancer Discov. 2013; 3(7): 770–781. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIndia Project Team of the International Cancer Genome Consortium: Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups. Nat Commun. 2013; 4: 2873. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCancer Genome Atlas Network: Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015; 517(7536): 576–582. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLlewellyn CD, Johnson NW, Warnakulasuriya KA: Risk factors for squamous cell carcinoma of the oral cavity in young people--a comprehensive literature review. Oral Oncol. 2001; 37(5): 401–418. PubMed Abstract | Publisher Full Text\n\nKuriakose M, Sankaranarayanan M, Nair MK, et al.: Comparison of oral squamous cell carcinoma in younger and older patients in India. Eur J Cancer B Oral Oncol. 1992; 28B(2): 113–120. PubMed Abstract | Publisher Full Text\n\nPathak KA, Das AK, Agarwal R, et al.: Selective neck dissection (I-III) for node negative and node positive necks. Oral Oncol. 2006; 42(8): 837–841. PubMed Abstract | Publisher Full Text\n\nBreiman L: Random Forests. The Netherlands: Kluwer Academic Publishers; 2001; 45(1): 5–32. Publisher Full Text\n\nPattnaik S, Vaidyanathan S, Pooja DG, et al.: Customisation of the exome data analysis pipeline using a combinatorial approach. PLoS One. 2012; 7(1): e30080. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOberst A, Green DR: It cuts both ways: reconciling the dual roles of caspase 8 in cell death and survival. Nat Rev Mol Cell Biol. 2011; 12(11): 757–763. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFulda S: Caspase-8 in cancer biology and therapy. Cancer Lett. 2009; 281(2): 128–133. PubMed Abstract | Publisher Full Text\n\nLansford CD, Grenman R, Bier H, et al.: Head and neck cancers. New York: Kluwer Academic Publishers, 2002. Publisher Full Text\n\nTelmer CA, An J, Malehorn DE, et al.: Detection and assignment of TP53 mutations in tumor DNA using peptide mass signature genotyping. Hum Mutat. 2003; 22(2): 158–165. PubMed Abstract | Publisher Full Text\n\nTibshirani R, Efron B: Improvements on Cross-Validation: The .632+ Bootstrap Method. J Am Stat Assoc. 1997; 92(438): 548–560. Publisher Full Text\n\nKumar B, Cordell KG, Lee JS, et al.: Response to therapy and outcomes in oropharyngeal cancer are associated with biomarkers including human papillomavirus, epidermal growth factor receptor, gender, and smoking. Int J Radiat Oncol Biol Phys. 2007; 69(2 Suppl): S109–111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorden FP, Kumar B, Lee JS, et al.: Chemoselection as a strategy for organ preservation in advanced oropharynx cancer: response and survival positively associated with HPV16 copy number. J Clin Oncol. 2008; 26(19): 3138–3146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFakhry C, Zhang Q, Nguyen-Tan PF, et al.: Human papillomavirus and overall survival after progression of oropharyngeal squamous cell carcinoma. J Clin Oncol. 2014; 32(30): 3365–3373. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDayyani F, Etzel CJ, Liu M, et al.: Meta-analysis of the impact of human papillomavirus (HPV) on cancer risk and overall survival in head and neck squamous cell carcinomas (HNSCC). Head Neck Oncol. 2010; 2: 15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDahlgren L, Dahlstrand HM, Lindquist D, et al.: Human papillomavirus is more common in base of tongue than in mobile tongue cancer and is a favorable prognostic factor in base of tongue cancer patients. Int J Cancer. 2004; 112(6): 1015–1019. PubMed Abstract | Publisher Full Text\n\nSalem A: Dismissing links between HPV and aggressive tongue cancer in young patients. Ann Oncol. 2010; 21(1): 13–17. PubMed Abstract | Publisher Full Text\n\nElango KJ, Suresh A, Erode EM, et al.: Role of human papilloma virus in oral tongue squamous cell carcinoma. Asian Pac J Cancer Prev. 2011; 12(4): 889–896. PubMed Abstract\n\nBullenkamp J, Raulf N, Ayaz B, et al.: Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent, E6-independent mechanism. Cell Death Dis. 2014; 5: e1489. PubMed Abstract | Publisher Full Text\n\nManzo-Merino J, Massimi P, Lizano M, et al.: The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8. Virology. 2014; 450–451: 146–152. PubMed Abstract | Publisher Full Text\n\nNovocraft: Novoalign. 2011. Reference Source\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. 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): 1297–1303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlbers CA, Lunter G, MacArthur DG, et al.: Dindel: accurate indel calls from short-read data. Genome Res. 2011; 21(6): 961–973. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCibulskis K, McKenna A, Fennell T, et al.: ContEst: estimating cross-contamination of human samples in next-generation sequencing data. Bioinformatics. 2011; 27(18): 2601–2602. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarter H, Chen S, Isik L, et al.: Cancer-specific high-throughput annotation of somatic mutations: computational prediction of driver missense mutations. Cancer Res. 2009; 69(16): 6660–6667. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDouville C, Carter H, Kim R, et al.: CRAVAT: cancer-related analysis of variants toolkit. Bioinformatics. 2013; 29(5): 647–648. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGundem G, Perez-Llamas C, Jene-Sanz A, et al.: IntOGen: integration and data mining of multidimensional oncogenomic data. Nat Methods. 2010; 7(2): 92–93. PubMed Abstract | Publisher Full Text\n\nSchroeder MP, Gonzalez-Perez A, Lopez-Bigas N: Visualizing multidimensional cancer genomics data. Genome Med. 2013; 5(1): 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLawrence MS, Stojanov P, Mermel CH, et al.: Discovery and saturation analysis of cancer genes across 21 tumour types. Nature. 2014; 505(7484): 495–501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDees ND: MuSiC2. 2015.\n\nZhao M, Zhao Z: CNVannotator: a comprehensive annotation server for copy number variation in the human genome. PLoS One. 2013; 8(11): e80170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDu P, Kibbe WA, Lin SM: lumi: a pipeline for processing Illumina microarray. Bioinformatics. 2008; 24(13): 1547–1548. PubMed Abstract | Publisher Full Text\n\nJohnson WE, Li C, Rabinovic A: Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostat. 2007; 8(1): 118–127. PubMed Abstract | Publisher Full Text\n\nRitchie ME, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSales G, Calura E, Martini P, et al.: Graphite Web: Web tool for gene set analysis exploiting pathway topology. Nucleic Acids Res. 2013; 41(Web Server issue): W89–97. 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\nKrzywinski M, Schein J, Birol I, et al.: Circos: an information aesthetic for comparative genomics. Genome Res. 2009; 19(9): 1639–1645. PubMed Abstract | Publisher Full Text | Free 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\nSchmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008; 3(6): 1101–1108. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11114",
"date": "09 Nov 2015",
"name": "Nishant Agrawal",
"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 an integrated genetic, epigenetic, and expression analysis from 50 oral tongue SCC. The findings were confirmed using a data set from TCGA. The manuscript presents surprising data which is interesting and has potential clinical implications. It is surprising that 23 of 50 patients with oral tongue SCC are HPV/p16 positive. This is not entirely consistent with previously published literature. Although the authors comment on this, this finding is rather “unique.” It is very possible that the improved survival is due to HPV status and not mutation status as these results maybe confounded. Although 50 patients is a relatively large sample size for such a study, a much larger study is necessary to really have immediate clinical impact.",
"responses": []
},
{
"id": "11116",
"date": "17 Dec 2015",
"name": "Thomas Carey",
"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 reports the the genetic analysis of 50 oral tongue primary tumor cancers treated at a single center, listed as paired sets. The study appears to be well done and thorough with carefully performed informatics. In the abstract it is stated that 50 paired primary oral tongue cancers were studied, but doesn't state whether the tumors were paired to normal blood or other tissue.The analysis includes single nucleotide variations, copy number variations, indels, regions with loss of heterozygosity. These somatic variations are linked to clinical parameters. In addition HPV was assessed by q-PCR and found to have a very high rate of positivity, which is surprising given the reported site of oral tongue. It was not clear if all HPV positive cases also had p16 expression. It would be valuable for the reader to know what portions of the HPV transcriptome was assessed. Did the authors examine E6 and E7 oncogene expression? It is questionable whether the HPV is a driver in 22 of 50 tumors. It would be important to verify the activity of HPV by assessing viral oncogene expression and level of expression. In tumors with HPV and mutant p53 it is unlikely that those are driven by HPV, given that p53 mutations in other HPV positive head and neck tumors is extremely rare. It would be helpful to have a table that shows how often mutant p53 was found in the HPV positive tumors and whether those patients had excessive use of carcinogenic substances. There are some novel findings in this study and some very surprising correlations, for example, CDKN2A mutations were found only in non-smokers. This is highly surprising given that in the US abnormalities of this locus is common in head and neck cancers in smokers. Thus, grouping never smokers and former smokers may not be a fair grouping. What does former smoker mean in this population? Only smoking and oral tobacco and alcohol use are discussed as etiologic factors. Are there no other factors in this part of India? Is betel nut or oral tobacco mixed with other substances included in the oral tobacco use category? Since these are all oral tongue cancers the information about etiology should be made more clear. The mutual exclusivity of several genetic variants is interesting but needs validation.The followup period is fairly short for the clinical correlates and relatively few patients recurred.Inclusion of a table of the clinical characteristics showing a breakdown of the T-class, N-class stage and outcome would be helpful to evaluate the clinical outcome and the association with the minimal gene signature. However, the non-recurrent tumors have minimal genetic changes, whereas the recurrent and metastatic tumors are far more complex. So the low genetic complexity alone may be a marker of good outcome rather than the minimal gene signature described for poor outcome. Addition of the primary tumor size, nodal status and stage would be informative on figure 5, which shows the minimal signature set for tumor recurrence.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1215
|
https://f1000research.com/articles/4-1212/v1
|
04 Nov 15
|
{
"type": "Review",
"title": "Recent advances in understanding of chronic kidney disease",
"authors": [
"Junna Yamaguchi",
"Tetsuhiro Tanaka",
"Masaomi Nangaku",
"Junna Yamaguchi",
"Tetsuhiro Tanaka"
],
"abstract": "Chronic kidney disease (CKD) is defined as any condition that causes reduced kidney function over a period of time. Fibrosis, tubular atrophy and interstitial inflammation are the hallmark of pathological features in CKD. Regardless of initial insult, CKD has some common pathways leading CKD to end-stage kidney disease, including hypoxia in the tubulointerstitium and proteinuria. Recent advances in genome editing technologies and stem cell research give great insights to understand the pathogenesis of CKD, including identifications of the origins of renal myofibroblasts and tubular epithelial cells upon injury. Environmental factors such as hypoxia, oxidative stress, and epigenetic factors in relation to CKD are also discussed.",
"keywords": [
"Chronic Kidney Disease",
"Hypoxia",
"Fibrosis",
"Tubular Atrophy",
"Pathogenesis",
"Nephrogenesis"
],
"content": "Introduction\n\nChronic kidney disease (CKD) is a growing health burden with an increasing incidence and prevalence worldwide. An estimated 13% of adults in the US and Japan have CKD, and the proportion of affected individuals increases each year because of an aging population and increases in diabetes and hypertension, the most common causes of CKD1,2. CKD is a risk factor for end-stage kidney disease (ESKD), cardiovascular disease, and overall mortality3. In the US, the economic costs of CKD and ESKD in patients over age 65 are $60 billion, representing 24% of total Medicare expenditures in 20114. Currently, the predominant problem is that therapeutic options for CKD are limited and often ineffective, meaning that there is essentially no cure for CKD. Therefore, translating our understanding of CKD pathogenesis into treatments is a high priority in the field.\n\nCKD is defined as any condition that causes abnormalities of kidney structure or function for a duration of more than 3 months with notable implications for patient health5,6 (Table 1). Regardless of initial etiology, fibrosis, tubular atrophy, and interstitial inflammation are common pathological features of CKD. Careful histological observations have demonstrated that functional impairment of the kidney is more highly correlated with tubulointerstitial damage than with glomerular injury, which is often associated with the loss of peritubular capillaries (PTCs)7. In addition, hypoxia is now accepted to be the final common mechanism underlying the progression of CKD to ESKD, which we discuss later in this article8,9.\n\naEither of the criteria below should be present for more than 3 months. Data are from the KDIGO (Kidney Disease: Improving Global Outcomes) 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. ACR, albumin-to-creatinine ratio; AER, albumin excretion rate; GFR, glomerular filtration rate.\n\nThe current understanding of CKD is based on a broad range of studies focused on the genetic risk factors for the development and progression of CKD, the pathogenesis of renal fibrosis (e.g., the origin and activation of renal myofibroblasts, fibrogenic mediators and signaling, crosstalk with tubular cells, vasculature, and inflammatory cells), tubular injury and repair, mediators and dynamics of renal inflammation, and cellular adaptations to the microenvironment such as hypoxia and oxidative stress. This article reviews some of the recent advances in our understanding of CKD from two vantages: cellular regeneration and hypoxia. A better understanding of CKD pathogenesis will hopefully provide insights leading to better management of CKD in the future.\n\n\nKidney development and regeneration\n\nNephrogenesis requires precise sequential and reciprocal interactions between renal progenitor cells and their integration with vasculature. In mammals, the metanephric kidney develops through interactions between the metanephric mesenchyme (MM) and uretic bud10. MM nephron progenitors give rise to Six2+ cap mesenchyme progenitor cells (which later differentiate into nephron epithelia, including proximal and distal tubular cells, the loop of Henle, and podocytes) and Foxd1+ cortical stromal progenitor cells (which later differentiate into cortical and medullary interstitial cells, mesangial cells, and pericytes)11–14. Nephrogenesis ceases at approximately the third post-natal day in mice15 and 36 weeks of gestation in humans16. Low nephron number is associated with a risk of renal disease and hypertension17, and low birth weight and prematurity are the most robust clinical surrogates for low nephron number18. The molecular event that governs the end of nephron formation is unknown and is an ongoing topic of research10,19. The regenerative capacity of glomeruli is limited after birth, and many studies have focused on the source of regenerated tubular cells following acute kidney injury (AKI) and the origin of myofibroblasts in CKD.\n\nA proliferative burst of tubular cells occurs during kidney injury. Sophisticated lineage-tracing studies have excluded the possibility of extrarenal cells contributing to tubular regeneration20. Recent studies further support the self-proliferation of existing differentiated tubular cells rather than the contribution of stem-like cells to epithelial proliferation after AKI21–23.\n\nWhereas the origin of the repairing tubule is becoming clearer, less is known regarding the signals that regulate epithelial dedifferentiation, proliferation, and polarization. One signal is known to derive from inflammatory cells. Cellular stress in tubules induces the activation of innate immunity through the production of cytokines and chemokines, which exacerbate tubular injury by recruiting macrophages, neutrophils, and proinflammatory lymphocytes24. One study demonstrates that a lack of interleukin-1 receptor-associated kinase-M leads to persistent proinflammatory macrophage infiltration with higher tubular phagocytosis activity and thus limited tubular re-epithelialization25. This effect was reversed by tumor necrosis factor-alpha blockade, indicating that cytokine-induced tubular attack overwhelms tubular repair capacity. Advances in genetic manipulation at the desired time point, together with a better understanding of myofibroblasts, now allow the study of signaling from fibroblasts or myofibroblasts to tubular cells in vivo.\n\nHowever, whether tubular regenerative capacity is itself limited in CKD is unknown. The intrinsic limit of tubular regenerative capacity may be related to disturbances in metabolism, endoplasmic reticulum stress, cell cycle arrest, or DNA damage26. In addition, the first direct reprogramming of renal epithelial cells to Six2+ nephron progenitor cells was accomplished by the addition of a combination of six transcription factors, including SIX2 and OSR127. These reprogrammed cells differentiated into epithelial cells in a re-aggregation assay, providing another strategy for replacing the epithelial layer if correct integration into nephrons can be achieved. In parallel, several groups have succeeded in the induction of cells of renal lineage, including intermediate mesoderm as well as individual differentiated cells such as proximal tubular cells or podocytes from embryonic stem or induced pluripotent stem cells11,28,29. Similar to the maintenance of nephron progenitor potency in the stromal-epithelial niche during kidney development, sophisticated programs may be required to maintain this potency30.\n\nMyofibroblasts are extracellular matrix-producing cells that drive fibrogenesis. The origin of renal myofibroblasts has been another area of major debate. Currently, FoxD1-Cre-labelled pericytes31, P0 (myelin protein 0)-Cre-labelled resident fibroblasts32, and renal erythropoietin-producing (REP) cells33 are reported as the origins of myofibroblasts. The absence of permanent specific markers and a shared developmental program makes it difficult to determine their precise origin. Their similar localization—near CD31+ endothelial cells in the interstitium—and gene expression patterns (PDGFRβ (platelet derived growth factor receptor beta) and CD73) suggest that they represent an overlapping cell population. A recent study reported that Gli1+PDGFRβ+CD73− cells, a small fraction of the total PDGFRβ population, are the major cellular origin of myofibroblasts in multiple organs, including kidney, heart, and liver34. Unified theories require further investigation.\n\nTriggers of the transdifferentiation of resident fibroblasts, REPs, or pericytes to alpha-smooth muscle actin-producing myofibroblasts also remain unclear. Factors produced by injured tubular and inflammatory cells, including vascular endothelial growth factors (VEGFs), platelet-derived growth factors (PDGFs), fibroblast growth factors, and transforming growth factor-beta, activate pericytes and induce their detachment from capillaries and their transdifferentiation to myofibroblasts33,35. In a typical inflammatory fibrogenic model known as unilateral ureteric obstruction (UUO), this transdifferentiation was found to be partially reversible in REPs after removal of the insult33. Recently, a comprehensive DNA microarray analysis of pericyte-to-myofibroblast transition was performed by using translational ribosome affinity purification in UUO, which may yield clues to help characterize these cells36.\n\n\nMediators of chronic kidney disease progression\n\nProteinuria is an established mediator of CKD pathogenesis, and lowering proteinuria retards CKD progression37–40. Protein overload exacerbates tubulointerstitial injury in a number of ways: direct tubular injury, including lysosomal rupture and energy depletion; activation of intratubular complement components, which leads to tubular cell activation or injury; and stimulation of inflammatory and fibrogenic mediators41–43.\n\nThe fact that nonproteinuric CKD is common and that renin-angiotensin-aldosterone inhibitors have renoprotective effects beyond lowering blood pressure and reducing proteinuria suggests that there are other key mediators of CKD pathogenesis. Chronic hypoxia of the tubulointerstitium is now widely accepted as the final common pathway in CKD progression8,9 (Figure 1). Once PTC rarefaction occurs, hypoxia in the affected region triggers phenotypic changes in tubular cells (e.g., proliferation rate and apoptosis), which in turn serve as a source of mediators involved in inflammatory cell infiltration and fibrosis. Fibrosis further impairs local oxygenation, while hypoxia induces sterile inflammation. Hypoxic responses are also induced by inflammatory transcription factors44. Thus, hypoxia is intricately linked to inflammation and oxidative stress, causing a vicious cycle leading to CKD pathogenesis.\n\nTubulointerstitial hypoxia, inflammation, and oxidative stress form a vicious cycle in chronic kidney disease (CKD) progression. Glomerular injury results in a decrease in peritubular capillary (PTC) blood flow and subsequent tubulointerstitial hypoxia. Hypoxia and proteinuria cause tubular injury, which in turn triggers the production of cytokines and chemokines and promotes inflammatory cell infiltration into the tubulointerstitium. Damaged PTC also facilitates inflammatory cell infiltration. Hypoxia, inflammation, and oxidative stress promote the transdifferentiation of resident fibroblasts, renal erythropoietin-producing cells, or pericytes to extracellular matrix (ECM)-producing myofibroblasts. Direct interactions between the injured tubular cells and myofibroblasts also play a role. Fibrosis further impairs local oxygenation.\n\nHypoxia-inducible factors (HIFs) are transcription factors that function as master regulators of biological adaptive responses to hypoxia45. HIFs consist of an alpha subunit (HIF-1α, HIF-2α, and HIF-3α) and a common beta subunit. Under normoxic conditions, HIF-α is hydroxylated by prolyl hydroxylase (PHD) and undergoes proteasomal degradation. HIFs regulate the expression of more than 150 genes, including those involved in anaerobic metabolism (e.g., glucose transporter-1), hematopoiesis (erythropoietin, or EPO), and angiogenesis (e.g., VEGF and angiopoietins). In response to hypoxia in kidney, HIF-1α is expressed in tubular cells, whereas HIF-2α is expressed mainly in endothelial cells and interstitial fibroblasts46.\n\nIn kidney disease, despite the hypoxic milieu, HIF activation is considered to be suboptimal. In the early phase of UUO (day 2), induction of HIF-1α and its target genes was disrupted, although pronounced hypoxia was confirmed by a hypoxia-detecting probe33. In another study using a rat CKD model, indoxyl sulphate, a representative uremic toxin, impeded the recruitment of transcriptional coactivators to HIF-1α, causing insufficient upregulation of HIF-1 target genes while leaving HIF-1α protein level unaffected47. This was reversed by an oral adsorbent for CKD, AST-120, that is currently in clinical use. Indeed, genetic and pharmacological modulation of HIFs in the kidney has been a subject of great interest, not only for investigating the roles of HIFs but also as a potential therapeutic tool. The renoprotective effects of HIF activation have been demonstrated in various AKI models, whereas those in CKD models have variable outcomes48. Pepck-Cre-mediated conditional knockout of HIF-1α in proximal tubules ameliorated fibrosis in UUO49, whereas global HIF activation by Vhl knockout ameliorated inflammation and fibrosis in the same model50. Global HIF activation by PHD inhibition reduced the tubulointerstitial injury associated with reduced tubular injury and capillary rarefaction in CKD rats51 and improved oxygen metabolism in diabetic rats52. HIF-1 in tubular cells exhibits both autocrine (e.g., cell cycle regulation and metabolic regulation) and paracrine (e.g., angiogenic and fibrogenic factors) signaling, which may result in different long-term renal outcomes. Additional cell type-specific and time-dependent manipulations of HIF activity may yield further insight for the development of future kidney therapies.\n\nRenal anemia is a frequent complication of CKD. The pathogenesis of renal anemia includes chronic inflammation, iron deficiency, shortened erythrocyte half-life, and, most importantly, EPO deficiency. One explanation for the observed EPO deficiency is the accumulated indoxyl sulphate observed in CKD. Indoxyl sulphate is reported to suppress EPO production in a HIF-dependent manner53. The identification of REPs also provided insight into the causes of EPO deficiency. REPs were repressed of EPO producing potential upon transdifferentiation to myofibroblasts in UUO through the activation of nuclear factor-kappa-B (NF-κB) signals33. REP-specific PHD2 knockout mice recovered EPO production in UUO and lipopolysaccharide-treated mice via HIF-2 activation54. This finding is in accordance with the observation that the pharmacological activation of HIFs by PHD inhibitors augmented EPO production in patients with ESKD55. Notably, PHD2 knockout-mediated HIF activation in REPs did not affect the inflammatory or fibrotic pathology of UUO; REP plasticity seems to be regulated by multiple signals at multiple levels.\n\nWhat causes angiogenesis insufficiency in CKD? Hypoxia signals generally promote angiogenesis56, and PTC development is thought to be regulated by angiogenic factors (e.g., VEGF, fibroblast growth factors, angiopoietins, and PDGF) secreted from tubular cells as well as endothelial and mesenchymal precursors. Doxycycline-regulated tubular-specific VEGF-A deletion during development led to a marked reduction of PTC, whereas deletion of VEGF-A post-natally between days 21 and 42 did not result in pronounced PTC rarefaction57. This suggests a difference in tubulovascular crosstalk in the developing and adult kidney. Another study that focused on pericyte-endothelial crosstalk in the adult kidney58 showed that PDGFβ and VEGF receptor signaling induced pericyte detachment from PTC and their transdifferentiation to myofibroblasts in UUO. These unusual behaviors by angiogenic factors may in part explain the insufficient angiogenesis in adult kidneys, including CKD kidneys.\n\nOxidative stress, another type of oxygen disturbance, is inevitably present in CKD and inseparably linked to hypoxia and inflammation59 (Figure 1). Oxidative stress is caused by increased reactive oxygen species (ROS) production or impaired antioxidant capacity, or both. Factors such as proteinuria, uremic toxin, hyperglycemia, and increased activity in the intra-renal angiotensin system contribute to increased oxidative stress in CKD. The Keap1-Nrf2 (Kelch-like ECH-associated protein 1-nuclear factor-erythroid-2-related factor 2) system is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by ROS. Impaired Nrf2 activity is observed in various animal CKD models, and the activation of Nrf2 ameliorates antioxidant defense and inflammation. Pharmacological activation of the Nrf2 pathway has been challenged with synthetic triterpenoid bardoxolone methyl in type 2 diabetic CKD patients. A phase 2 BEAM (52-Week Bardoxolone Methyl Treatment: Renal Function in CKD/Type 2 Diabetes) trial showed promise for the use of bardoxolone methyl to increase estimated glomerular filtration rate (eGFR) compared with a placebo (mean change of 8.2 to 11.4 ml/min per 1.73 m2, depending on the dose group) in moderate-to-severe diabetic CKD patients60 (eGFR 20 to 45 ml/min per 1.73 m2). Notably, increased albuminuria was observed in the bardoxolone methyl group, despite significantly improved kidney function. A study in cynomolgus monkeys suggests that bardoxolone methyl decreases the expression of megalin, which is primarily responsible for albumin reabsorption in proximal tubules, resulting in increased albuminuria61. Whether and how Nrf2 is related to reduced megalin expression remain unknown. The subsequent phase 3 BEACON (Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes Mellitus: the Occurrence of Renal Events) trial in diabetic CKD stage 4 patients (eGFR of 15 to less than 30 ml/min per 1.73 m2) was terminated because of a higher rate of cardiovascular events in the bardoxolone methyl group than in the placebo group62. Controversies exist as to the cause of increased cardiovascular events during bardoxolone methyl treatment and as to the appropriate selection of a target patient population for this therapy63,64. Interventions designed to prevent oxidative stress remain important therapeutic options for CKD.\n\n\nNew technology-driven advances in understanding of chronic kidney disease\n\n‘-Omics’ approaches have rapidly expanded our understanding of CKD. Genome-wide association studies have identified multiple genetic loci associated with kidney function-related traits65–68. The shared loci among multiple ethnic groups include the UMOD locus, which encodes the abundant urinary protein uromodulin produced by the epithelial cells of the thick ascending limb of the loop of Henle (TAL). Further animal studies have demonstrated the causal role of UMOD risk variants in hypertension and CKD by modulating salt handling in the TAL69. An example for a specific ethnic group is APOL1. The higher incidence of ESKD in African Americans compared with European Americans led to the identification of APOL1 variants as risk factors for the development and progression of CKD among African Americans in the general population70–72.\n\nEpigenetic regulation in CKD is emerging as an important topic. As proposed in the “metabolic memory” theory of diabetic nephropathy, hypoxia may be remembered via epigenetic changes to play a crucial role in the pathogenesis of CKD. Epigenetic modifications include cytosine DNA methylation, noncoding RNA, and histone post-translational modification73. Differentially methylated regions were observed in the cortical tubules of CKD patients and controls, especially in enhancer regions of key fibrotic genes74. Microarray approaches have identified a number of potential microRNAs responsive in CKD animal models75. MicroRNA-21 was shown to promote fibrosis by repressing peroxisome proliferator-activated receptor-alpha, by either germline deletion of miR21 or oligonucleotide administration of anti-miR21 in UUO76. Hypoxia is also reported to alter the chromatin conformational structure dynamically and cause histone modifications in human umbilical vein endothelial cells, which result in transcriptional changes of HIF-1 target genes77. Prolonged ischemic-reperfusion injury has caused histone modifications at proinflammatory and profibrotic genes prior to fibrosis, which may be related to CKD pathogenesis78. Interventional studies for these epigenetic modifications are anticipated.\n\n\nPerspectives\n\nTechnological developments in genome editing, genome-wide analysis, and dynamic multiplex four-dimensional measurement, as well as advances in the fields of stem cell and regenerative biology are considerable. It is now possible to investigate the contextual, environmental, and interdependent coordination between multiple players in the kidney79. Needless to say, translating the results of basic research in animal models to the bedside will require a number of additional studies. One example is the lack of animal models that mimic human CKD pathophysiology. To overcome these issues, research using samples from patients with CKD is under way. Overall, this is an exciting time for CKD research, as a fuller understanding of its pathogenesis lays the foundation for pathogenesis-based kidney therapy.\n\n\nAbbreviations\n\nAKI, acute kidney injury; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; EPO, erythropoietin; ESKD, end-stage kidney disease; HIF, hypoxia-inducible factor; MM, metanephric mesenchyme; Nrf2, Nuclear factor-erythroid-2-related factor 2; PDGF, platelet-derived growth factor; PDGFRβ, platelet-derived growth factor receptor beta; PHD, prolyl hydroxylase; PTC, peritubular capillary; REP, renal erythropoietin-producing; ROS, reactive oxygen species; TAL, thick ascending limb of the loop of Henle; UUO, unilateral ureteric obstruction; VEGF, vascular endothelial growth factor.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe work of the authors is supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (26111003 to MN) and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (15H04835 to MN and 26461215 to TT).\n\n\nReferences\n\nCenters for Disease Control and Prevention: Chronic Kidney Disease Surveillance System—United States. Reference Source\n\nJapanese Society of Nephrology: CKD Practice Guide 2012.\n\nSarnak MJ: Cardiovascular complications in chronic kidney disease. Am J Kidney Dis. 2003; 41(5 Suppl): 11–7. PubMed Abstract | Publisher Full Text\n\nU.S. Renal Data System, USRDS 2013 Annual Data Report: Atlas of Chronic Kidney Disease and End-Stage Renal Disease in the United States. 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Clin Exp Pharmacol Physiol. 2013; 40(2): 148–57. PubMed Abstract | Publisher Full Text\n\nHiggins DF, Kimura K, Bernhardt WM, et al.: Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition. J Clin Invest. 2007; 117(12): 3810–20. PubMed Abstract | Free Full Text\n\nKobayashi H, Gilbert V, Liu Q, et al.: Myeloid cell-derived hypoxia-inducible factor attenuates inflammation in unilateral ureteral obstruction-induced kidney injury. J Immunol. 2012; 188(10): 5106–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTanaka T, Kojima I, Ohse T, et al.: Cobalt promotes angiogenesis via hypoxia-inducible factor and protects tubulointerstitium in the remnant kidney model. Lab Invest. 2005; 85(10): 1292–307. PubMed Abstract | Publisher Full Text\n\nNordquist L, Friederich-Persson M, Fasching A, et al.: Activation of hypoxia-inducible factors prevents diabetic nephropathy. J Am Soc Nephrol. 2015; 26(2): 328–38. 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}
|
[
{
"id": "11085",
"date": "04 Nov 2015",
"name": "Motoko Yanagita",
"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",
"responses": []
},
{
"id": "11084",
"date": "04 Nov 2015",
"name": "William Couser",
"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",
"responses": []
},
{
"id": "11086",
"date": "04 Nov 2015",
"name": "Takashi Yokoo",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1212
|
https://f1000research.com/articles/3-308/v1
|
17 Dec 14
|
{
"type": "Research Article",
"title": "Subdivisions of the adult zebrafish pallium based on molecular marker analysis",
"authors": [
"Julia Ganz",
"Volker Kroehne",
"Dorian Freudenreich",
"Anja Machate",
"Michaela Geffarth",
"Ingo Braasch",
"Jan Kaslin",
"Michael Brand",
"Volker Kroehne",
"Dorian Freudenreich",
"Anja Machate",
"Michaela Geffarth",
"Ingo Braasch",
"Jan Kaslin"
],
"abstract": "Background: The telencephalon shows a remarkable structural diversity among vertebrates. In particular, the everted telencephalon of ray-finned fishes has a markedly different morphology compared to the evaginated telencephalon of all other vertebrates. This difference in development has hampered the comparison between different areas of the pallium of ray-finned fishes and the pallial nuclei of all other vertebrates. Various models of homology between pallial subdivisions in ray-finned fishes and the pallial nuclei in tetrapods have been proposed based on connectional, neurochemical, gene expression and functional data. However, no consensus has been reached so far. In recent years, the analysis of conserved developmental marker genes has assisted the identification of homologies for different parts of the telencephalon among several tetrapod species.Results: We have investigated the gene expression pattern of conserved marker genes in the adult zebrafish (Danio rerio) pallium to identify pallial subdivisions and their homology to pallial nuclei in tetrapods. Combinatorial expression analysis of ascl1a, eomesa, emx1, emx2, emx3, and Prox1 identifies four main divisions in the adult zebrafish pallium. Within these subdivisions, we propose that Dm is homologous to the pallial amygdala in tetrapods and that the dorsal subdivision of Dl is homologous to part of the hippocampal formation in mouse. We have complemented this analysis be examining the gene expression of emx1, emx2 and emx3 in the zebrafish larval brain.Conclusions: Based on our gene expression data, we propose a new model of subdivisions in the adult zebrafish pallium and their putative homologies to pallial nuclei in tetrapods. Pallial nuclei control sensory, motor, and cognitive functions, like memory, learning and emotion. The identification of pallial subdivisions in the adult zebrafish and their homologies to pallial nuclei in tetrapods will contribute to the use of the zebrafish system as a model for neurobiological research and human neurodegenerative diseases.",
"keywords": [
"telencephalon",
"teleost",
"Actinopterygii",
"amygdala",
"hippocampus",
"neuroanatomy",
"vertebrate brain",
"homology",
"evolution",
"neurogenesis"
],
"content": "Background\n\nThe functions of the different parts of the telencephalon encompass control of sensory and motor, autonomic and endocrine functions, as well as cognitive tasks like memory, learning and emotion. The structures in the telencephalon can be assigned either to its dorsal part, the pallium, or its ventral part, the subpallium1–3. The telencephalon of most vertebrates forms by an evagination of the neural tube, where the central lumen of the neural tube expands to form the two paired telencephalic vesicles1,3,4. However, in ray-finned fishes the rostral neural tube is thought to bend outward resulting in two telencephalic hemispheres separated by an unpaired ventricle and covered by a thin roof plate, thus referred to as “everted”1,3,4. In the rayfin teleost zebrafish, it has been shown that the morphogenetic movement that creates this different layout of the telencephalon does not result from a simple lateral outward bending of the telencephalic walls5. Instead, telencephalon morphogenesis comprises first the generation of a ventricular outfolding between telencephalon and diencephalon, followed by an enlargement of the pallial territory rostrally5. The different development of the telencephalon results in an unpaired ventricle and a different arrangement of the parts in the pallium compared to all other vertebrates1,4,6. Hence, due to its everted nature, a comparison between the parts of the pallium of rayfin fishes and all other vertebrates has been difficult. The correct determination of homologous pallial areas between teleosts and tetrapods is critical for the usage of teleost fish as neurobiological models as well as models for human neurological diseases. In recent years, a variety of different studies have demonstrated that using the expression pattern of conserved developmental regulatory genes as landmarks is a useful approach to identify homologous subdivisions of brain regions between divergent vertebrate species. The advantage of using the expression of conserved developmental genes lies in the uncoupling of anatomical and developmental differences of the brain part of interest between divergent species. This approach has been especially valuable for the telencephalon with its great variability in morphology between vertebrate species and has led to the clarification of the homology within subdivisions of the telencephalon between different vertebrate species, such as the domestic mouse, the chicken and the African clawed frog (e.g.7–22). For example, the gene expression of Tbr1 and Eomes (Tbr2) has been successfully used to identify the extent of the pallium in tetrapod embryos9,21,23. In addition, absence of Emx1 expression and presence of Tbr1 expression delineate the ventral pallium in tetrapod embryos9,10,12,13,21.\n\nThe pallium in teleosts has generally been subdivided in a medial (Dm), dorsal (Dd), central (Dc), lateral (Dl) and posterior (Dp) part4. The pallium shows a notable structural variety and different subdivisions of these broad divisions have been described in different teleost species3,24–29. The subdivisions of the pallium and their homologies to nuclei in other vertebrate species have not been resolved, but different models based on neurochemical and connectional data have been suggested3,29–38. Ablation experiments combined with behavioral experiments suggest that the lateral nucleus of the pallium shows a similarity in function to the hippocampus and the medial nucleus of the pallium to the amygdala of amniotes39–43. Based on the expression of nicotine adenine dinucleotide phosphate diphorase (NADPHd) and Parvalbumin, a new model of four subdivisions (Dm, Dc, Dl, and Dp) has been proposed for the adult zebrafish pallium32. However, a comprehensive study of pallial subdivisions based on different conserved molecular markers is still missing in the adult zebrafish.\n\nThe object of this study was to analyze expression of conserved marker genes to identify subdivisions within the adult zebrafish pallium. Here, we investigated the expression patterns of the molecular marker genes emx1, emx2, emx3 to identify a ventral pallial subdivision both in the larval and adult zebrafish pallium. The expression of Prox1 in a dorsal subdivision of Dl caudally suggests that it is homologous to the dentate gyrus in mouse. Combinatorial expression of ascl1a, emx1, emx2, emx3, and eomesa shows four main divisions in the pallium, Dm, Dc, Dl, and Dp. The combinatorial expression pattern also suggests a subdivision of Dl in a dorsal and ventral subdivision (which we have named Dld and Dlv, respectively).\n\n\nMaterial and methods\n\nFish were kept under standard conditions at a 14 hours light/10 hours dark cycle as previously described44,45. All procedures were in accordance with the live animal handling and research regulations of the local Animal Care and Use Committee, the Regierungspräsidium Dresden (permit AZ 24D-9168.11-1/2008-1 and -4). Wildtype experimental animals (Biotechnology Center Dresden) were adult fish from the gol-b1 line in the AB genetic background46. Adult fish were 6–8 months old and had a 24mm–32mm body length, zebrafish larvae were 7dpf old.\n\nBrains (either dissected or within the skull) were fixed at 4°C overnight in 2–4% paraformaldehyde/0.1M phosphate buffer (PB), pH 7.5. They were washed 1 × 10 minutes and then up to 1h in 0.1M PB and subsequently transferred for decalcification and cryoprotection to 20% sucrose/20% EDTA in 0.1M PB, pH7.5. Brains were frozen in 7.5% gelatine/20% sucrose and sectioned into 14–16 µm cryosections. Sections were stored at -20°C.\n\nCompared to tetrapods, teleost fish have undergone an additional whole genome duplication: the teleost genome duplication (TGD) (reviewed in47). Thus, there is the possibility of two co-orthologous genes in zebrafish compared to the single tetrapod gene.\n\nWe analyzed if there are two co-orthologous genes compared to the tetrapod gene using Ensembl73 gene trees (http://www.ensembl.org) and synteny analysis with the Synteny Database (http://syntenydb.uoregon.edu;48). This is clearly the case, e.g., for human EOMES with two TGD co-orthologs in zebrafish, eomesa and eomesb (Figure S1A,B).\n\nTwo genes are currently termed ascl1 in zebrafish. Zebrafish ascl1a is clearly orthologous by phylogeny and conserved synteny to Ascl1 in lobefins (tetrapods and coelacanth). Zebrafish ascl1b not only has a separate ortholog in coelacanth but also shows conserved synteny to tetrapod Ascl2, suggesting that ascl1b is in fact the missing teleost ascl2 gene.\n\nThere are two prox1 genes described in zebrafish, yet while prox1a is clearly orthologous to tetrapod Prox1, teleost prox1b shows no conserved synteny to teleost prox1a or tetrapod Prox1. This suggests that teleost prox1b represent a more distant prox paralog and is not a TGD paralog of prox1a. The phylogeny of the emx genes in zebrafish has previously been determined in49.\n\nRNA in situ hybridization on sections and on whole-mount brains and RNA probe generation was essentially performed as previously described7,50. Briefly, after defrosting at room temperature (RT), sections were rehydrated for 15 minutes in PBS with 0.3% TritonX (PBSTx) and incubated with the probe overnight at 62–65°C. Information on the antisense in situ riboprobes can be found in7 for ascl1a (NM_131219), emx1 (NM_198144), emx2 (NM_131280), emx3 (NM_131279), eomesa (NM_131679). The in situ probe eomesb (NM_001083575) was cloned from zebrafish embryonic cDNA with the following primers (eomesb-F, TTTCCAAAACGAAAAGCGTA, eomesb-R, GAGCCAGAACTGGATCCTTCT). The eomesb probe was tested on 3dpf embryos and showed specific staining only in the midbrain at 3dpf (Figure S2, Dataset 1). The sections were washed at 60–65°C in washing solution (1 × SSC, 50% deionized formamide) for 1 × 15 minutes and 2 × 30 minutes followed by 2 × 30 minutes MAB with 0.1% Tween-20 (MABT) washes. Sections were incubated for 1h at RT in 2% DIG-blocking reagent (Roche) and incubated with anti-DIG antibody (Roche Diagnostics, sheep, polyclonal, Fab fragments conjugated to alkaline phosphatase, #11093274910) diluted 1:4000 in 2% DIG-blocking reagent overnight at 4°C. Subsequently, sections were washed 4 × 20 minutes in MABT, equilibrated with staining buffer and stained with the substrate NBT/BCIP. The staining was controlled using a stereomicroscope. Finally, sections were washed 2 × 5 minutes in PBS, postfixed with 4% PFA for 20–30 minutes, washed again 2 × 10 minutes in PBS and mounted with 70% glycerol in PBS. All washing steps were performed on a shaker, all incubation steps in a humid chamber. To test for nonspecific binding of the antibodies that detect digoxigenin, which are not endogenous to vertebrate tissue, we performed control experiments in which the labeled RNA was omitted from the hybridization mix. No signal was detected in the absence of the riboprobe, demonstrating that the antibody reacts specifically with the synthetic RNA (Dataset 2).\n\nImmunohistochemistry on cryosections was performed as previously described51. Briefly, to retrieve the antigens of Prox1, sections were pre-incubated in 50 mM Tris-buffer (pH 8.0) at 99°C for 5 minutes, cooled down to RT over 15 minutes and washed for 5 minutes in PBS and twice for 10 minutes in PBSTx. The sections were then incubated in primary and secondary antibodies in PBSTx. The primary antibody Prox1 (AB5475, 1:2000, Millipore) was incubated overnight at 4°C and secondary antibodies for 1h at room temperature. The slides were washed in PBSTx and mounted. The secondary antibody (dilution 1:750) was Alexa 488-Fluor conjugated (A-11034, Invitrogen, Karlsruhe).\n\nConfocal images were acquired with Leica TCS-SP5 confocal microscope. Brightfield images were acquired with Zeiss Axio Imager Z1. The images were processed using ImageJ v. 1.4.3.67 and Adobe Photoshop CS2. Composites were assembled using Adobe Photoshop CS2 and Adobe Illustrator CS2.\n\nWe primarily followed the nomenclature proposed in32 with modifications based on our combinatorial expression pattern analysis, which suggest a subdivision of Dl in a dorsal and ventral subdivision (which we have named Dld and Dlv, respectively). In the figures we employ a two-color code to separate subdivisions that can be made based on the current marker (red dashed line) from those that we make based on other markers (white dashed line). The black dashed line indicates the boundary between D and V. The distinction of ventricular zone versus neuronal layer was based on cellular morphology. The ventricular zone is the region where cells are directly facing the ventricle. The neuronal layer is characterized by cells with a round shape.\n\n\nResults\n\nIn tetrapod embryos, Eomes (Tbr2) expression is found in the ventricular zone and mantle layer in all parts of the pallium at embryonic stages9,21,23. In the zebrafish embryo, eomesa expression is present throughout the dorsal telencephalon52,53. Its TGD paralog, eomesb is not present in the embryonic or adult telencephalon (Figure S2, Dataset 1) and thus was not further taken into account. In the adult zebrafish, eomesa positive cells are scattered in a salt-and-pepper pattern along the ventricular zone of Dm along the rostro-caudal axis (Figure 1A–D, arrowheads). Further, eomesa expression is present in the ventricular zone and neuronal layer of Dc, Dlv and Dld in the rostral telencephalon and at mid-telencephalic levels and in the ventricular zone and neuronal layer of Dld and Dp at the anterior commissure (Cant; Figure 1A–C). Caudal to Cant, eomesa is expressed in the ventricular zone and neuronal layer of Dp, sporadically in Dld and in bed nucleus of the stria medullaris (BNSM, Figure 1D). In summary, eomesa is differentially expressed in the pallium along the rostro-caudal axis (Figure 1E–H, Dataset 3; Table 1). The most abundant parenchymal eomesa expression is detected in parts of the central and lateral dorsal telencephalic nuclei.\n\nA. In the rostral telencephalic, eomesa expression is found in the ventricular zone (vz) of Dm (arrowheads) and in the vz and neuronal layer (nl) of Dc and Dlv. B. At mid-telencephalic levels, eomesa expression is present in the vz of Dm (arrowheads), in the nl of Dc and in the vz and nl of Dld and Dlv. C. At the anterior commissure (Cant), eomesa expression is present in the vz of Dm (arrowhead) and in the vz and nl of Dld and Dp. Inset shows close-up of vz of Dm D. Caudal to Cant, eomesa expression is present in the vz of Dm (arrowhead), in the vz and nl of Dp and in BNSM. Inset shows close-up of vz of Dm E.–H. Summary of the expression pattern of eomesa at rostral (E.), mid-telencephalic (F.), commissural (G.) and postcommissural levels (H.). A.–D. Brightfield images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The black dashed line indicates the boundary between D and V. Scale bars = 50µm in A–D.\n\n0 expression not detected, 1 weak expression, 2 moderate expression, 3 strong expression, vz ventricular zone, nl neuronal layer,\n\na scattered cells in the ventricular zone; b no expression from mid-telencephalic levels; c part of Dc posterior to Cant; d scattered cells in the neuronal layer; e no expression posterior to Cant; f expression at mid-telencephalic level shortly anterior to Cant, shortly caudal to Cant, only scattered Prox1+ cells are present; g expression present in the rostralmost telencephalon in the vz, moving caudally expression in vz and nl.\n\nIn tetrapod embryos, the ventral pallial subdivision is characterized by absence of Emx1 and presence of Tbr19,10,12,13,21. In embryonic and adult zebrafish, tbr1 is expressed throughout the pallium7,53,54. Thus, we investigated the expression of emx1, emx2 and emx3 to identify a ventral pallial subdivision in the zebrafish pallium. In zebrafish, the three emx genes are expressed at 1 day post fertilization (dpf) in the embryo throughout the dorsal telencephalon49,55,56. In 7 day-old larva, the expression of emx1 and emx2 is restricted to a small caudo-lateral area in the dorsal telencephalon (Figure S3A,B,D, Dataset 4). The expression of emx3 is found throughout the dorsal telencephalon (Figure S3C,E, Dataset 4). In the adult zebrafish pallium, emx1 and emx2 are expressed in a very restricted fashion. The expression of emx1 only starts in the mid-telencephalon shortly rostral to Cant, where it is restricted to the ventricular zone and neuronal layer of Dp. This expression pattern continues to Cant (Figure 2A). Furthermore, emx1 expression is present in Dp and in EN caudal to Cant (Figure 2B). The expression of emx2 is only found caudal to Cant in an area in Dc lateral to Vp (Figure 2C). Similar to the larvae, emx3 shows the broadest expression of the emx genes. In the rostral telencephalon, the expression of emx3 is found in the ventricular zone and neuronal layer of Dm and weakly in scattered cells in the neuronal layer of Dc (Figure 2G). Additionally, the expression of emx3 is weakly present rostrally in the ventricular zone of Dlv (Figure 2G, arrowheads). At mid-telencephalic levels, emx3 expression is found in the ventricular zone and neuronal layer of Dm, in scattered cells in Dc and in the ventricular zone and neuronal layer of Dlv (Figure 2H). At Cant, expression of emx3 is found in the ventricular zone and neuronal layer of Dm, in scattered cells in Dc and in the ventricular zone and neuronal layer of Dp (Figure 2I). Caudal to Cant, the expression gets weaker in Dm, Dc and Dp (data not shown). In summary, emx1 and emx2 show a very restricted expression pattern in the adult zebrafish pallium (Figure 2D–F, Dataset 5; Table 1). Expression of emx3 is present in Dm, Dc and Dlv/Dp (Figure 2J–L, Dataset 5; Table 1).\n\nA. At the anterior commissure (Cant), emx1 expression is found in neuronal layer (nl) and ventricular zone (vz) of Dp. B. Caudal to Cant, emx1 expression is present in the vz and nl of Dp and in EN. C. Caudal to Cant, emx2 expression is present in part of Dc. D.–F. Summary of the expression pattern of emx1 and emx2 at commissural (D.) and postcommissural levels (E.,F.). G. In the rostral telencephalon, emx3 expression is present in the vz and nl of Dm, weakly in scattered cells of the nl od Dc and in the vz of Dlv (arrowheads). H. At mid-telencephalic levels, emx3 expression is found in the vz and nl of Dm, in scattered cells in Dc, and in the vz and nl of Dlv. I. At Cant, emx3 expression is present in the vz and nl of Dm, in scattered cells in Dc and in the vz and nl of Dp. J.–L. Summary of the expression pattern of emx3 at rostral (J.), mid-telencephalic (K.) and commissural levels (L.). A.–F. Brightfield images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The black dashed line indicates the boundary between D and V. Scale bars = 50µm in A–D.\n\nDuring mouse development, Prox1 expression is found in the amygdala, dentate gyrus and in the neocortex57. At adult stages, however, strong Prox1 expression is restricted to the dentate gyrus of the hippocampus and is commonly used as a specific marker for granule cells of the hippocampus57–60. In the adult zebrafish pallium, Prox1+ cells only are present in the midtelencephalon shortly rostral to Cant in the neuronal layer of Dld (Figure 3A). This expression pattern continues to the anterior commissure (Figure 3B). Shortly caudal to Cant, only scattered Prox1+ cells are present in Dld, more caudally no Prox1+ cells can be found (data not shown). In summary, Prox1 staining is present in Dld starting at mid-telencephalic levels until shortly caudal to Cant (Figure 3A–D, Dataset 6; Table 1).\n\nA. At mid-telencephalic levels, Prox1 positive cells are found in the neuronal layer (nl) of Dld shortly before the anterior commissure (Cant). B. At Cant, Prox1 positive cells are found in the nl of Dld. C.–D. Summary of expression pattern of Prox1. A.–B. Confocal images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The yellow dashed line indicates the boundary between D and V. Scale bars = 50µm in A–B.\n\nIn tetrapod embryos, Ascl1 is expressed a subpopulation of progenitors in the dorsal telencephalon61,62. In the zebrafish embryo, ascl1a expression is present in the caudomedial ventricular zone of the dorsal telencephalon63. In the adult zebrafish pallium, ascl1a is expressed in scattered cells in the ventricular zone of Dm and in scattered cells in the ventricular zone of Dlv (Figure 4A–D, arrowheads, Figure 4I,J, Dataset 7; Table 1). At Cant and posterior to Cant ascl1a is present in scattered cells in Dm and Dp (Figure 4E–H, arrowheads, Figure 4K,L, Dataset 7; Table 1).\n\n\n\nA.–B. In the rostral telencephalon, ascl1a expression is present in scattered cells in the ventricular zone (vz) of Dm (A, arrowheads) and Dlv (B, arrowheads). C.–D. At mid-telencephalic levels, ascl1a expression is present in scattered cells in the vz of Dm (C, arrowheads) and Dlv (D, arrowheads). E.–F. At Cant, ascl1a expression is present in scattered cells in the vz of Dm (E, arrowheads) and Dp (F, arrowheads). G.–H. Posterior to Cant, ascl1a expression is present in scattered cells in the vz of Dm (G, arrowheads) and Dp (H, arrowheads). I.–L. Summary of the expression pattern of ascl1a rostral (I.), mid-telencephalic (J.), commissural (K.) and postcommissural levels (L.). A.-H. Brightfield images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The black dashed line indicates the boundary between D and V. Scale bars = 50µm in A–C.\n\n\nDiscussion\n\nDue to the different development, the pallium of ray-finned fishes has a markedly different morphology compared to all other vertebrates, which makes the comparison between the areas of the pallium of ray-finned fishes to pallial nuclei of other vertebrates particularly challenging. Yet, the correct assignment of homologous pallial areas between teleosts and tetrapods is essential for usage of the teleost fish model in neurobiological research. We have analyzed several conserved molecular marker genes that are found in specific areas of the pallium in the domestic mouse, chicken and the African clawed frog. Based on the expression analysis we identify four main subdivisions of the pallium (Dm, Dl, Dc and Dp) and propose that Dl is subdivided in a dorsal (Dld) and ventral part (Dlv). Based on our data we also suggest putative homologies to pallial nuclei in tetrapods. We suggest that Dm is homologous to the ventral or ventral/lateral pallium, Dc to the dorsal pallium, Dl to the medial pallium, and suggest that Dp comprises a specialized part of Dl (Figure 5). Additional marker analysis, lineage tracing experiments and functional analyses will be necessary to substantiate the proposed pallial subdivisions and their homology to pallial nuclei in tetrapods. As our study is based solely on comparative gene expression data, we have discussed our results in the framework of gene expression data of other organisms and subsequently compared them to connectional, neurochemical, and functional data in teleosts. For clarity, we discuss our results separately for each pallial subdivision.\n\nA.–D. Cross-sections at the levels indicated through the telencephalon. E. Schematic diagram of a cross section through the mouse telencephalon for comparison (modified after14), note that the amygdaloid complex (AC, brown) is derived both from subpallium (grey) and pallium. Light grey areas (Th/Hyp) are part of the diencephalon. Indicated is also a model of the putative homology of the subdivisions in the adult zebrafish pallium to regions in the tetrapod pallium taking the data presented in this paper into account. Additional marker analysis, lineage tracing experiments and functional analyses are necessary to substantiate the proposed homology to pallial nuclei in tetrapods. * in scattered cells, ** in Dlv in the ventricular zone, moving caudally expression in the neuronal layer and ventricular zone, note that Prox1 positive cells are present in Dld shortly before the anterior commissure, n/a = not applicable. The black dashed line indicates the boundary between D and V. The red dashed lines indicate the boundaries between different nuclei in D. AC amygdaloid complex, ACo anterior cortical amygdalar area, BC basal amygdalar complex, BNSM bed nucleus of the stria medullaris, Ce central amygdala, CP Caudateputamen, DG dentate gyrus, EN entopeduncular nucleus, HF hippocampal formation, Hyp hypothalamus, Me medial amygdala, NCx neocortex, pCx piriform cortex, Po preoptic region, Th thalamus, V area ventralis telencephali, Vp postcommissural nucleus of the area ventralis telencephali, Vs supracommisural nucleus of the area ventralis telencephali.\n\nIn the African clawed frog, the chicken, and the domestic mouse, four pallial divisions have been identified in the embryo, the ventral pallium (VP), the lateral pallium (LP), the dorsal pallium (DP) and the medial pallium (MP)9,10,13,21. In the African clawed frog, chicken, and domestic mouse, the ventral pallial subdivision is characterized in the embryo by the absence of Emx1 expression and presence of the pallial marker Tbr19,10,13,21. In embryonic and adult zebrafish, tbr1 is expressed throughout the pallium7,53,54. In adult ray-finned fishes, Dm has been proposed to be homologous to the ventral pallium (pallial amygdala) based on topological, connectional and functional data29–31,64–66. In contrast, Nieuwenhuys (2009) proposed that Dm is homologous to the lateral pallium based on topology and Yamamoto et al. (2007) suggest that Dm together with Dd and Dld is homologous to the dorsal pallium. Thus, we analyzed the expression of the emx genes in the larval and adult zebrafish to determine if the absence of these markers identifies a ventral pallial subdivision in the zebrafish pallium. In zebrafish, the emx genes show a dynamic expression pattern both in the embryo and the adult. At 1dpf, the three emx genes are expressed at throughout the dorsal telencephalon49,55,56. We found that expression of emx1 is restricted to a caudolateral expression domain in the zebrafish larvae at 7dpf and to Dp in the adult (Figure 2D,E, Figure S3). Similarly, emx2 also shows a restricted expression pattern to a caudolateral domain both in the zebrafish larvae at 7dpf and in the adult zebrafish pallium (Figure 2F, Figure S3). The expression of emx3 is present in the entire pallium in the zebrafish embryo and larvae (49, Figure S3C). In the adult, emx3 is present in the rostral telencephalon in Dm, Dc and in the ventricular zone of Dlv (Figure 2J). Moving caudally, emx3 is present in Dm, in scattered cells in Dc and in the ventricular zone and neuronal layer of Dlv and Dp (Figure 2K,L). As it has been previously suggested that zebrafish emx3 supplies the functions provided by Emx1 and Emx2 in mouse49, we took the combined expression pattern of the three emx genes into account for our analysis. The lack of an area in Dm that is tbr1 positive and emx gene negative suggests that zebrafish might not have a distinct ventral pallial subdivision and that Dm is either homologous to the lateral pallium, or that Dm comprises a combined ventral and lateral pallium (Figure 5A–D). Emx1 alone might not be an adequate marker for the ventral pallium in the domestic mouse and chicken, as they have lost the Emx3 gene49.\n\nBoth the ventral pallium and the lateral pallium contribute together with subpallial derivatives to the amygdaloid complex in tetrapods10,67,68. In rodents, the Cannabinoid Receptor (CB1) is expressed in a subset of neurons in the basolateral amygdala69,70. In the weakly electric fish Apteronotus leptorhynchus and zebrafish, cb1 is expressed in Dm71–73, thus supporting the model that Dm is homologous to the pallial amygdala. Based on gene expression analysis, we have suggested previously that the supracommissural nucleus of the area ventralis telencephali (Vs) is homologous to the dorsal and ventral part of the central amygdala and the bed nucleus of the stria terminalis (BST) and that the postcommissural nucleus of the area ventralis telencephali (Vp) is homologous to the dorsal part of the central amygdala and the BST7. Thus, it is plausible that Vs and Vp (subpallial amygdala) and Dm (pallial amygdala) form the amygdaloid complex in the adult zebrafish (Figure 5A–E).\n\nBased on topological and gene expression data it has been proposed that the entire Dl4 or Dl excluding Dlv or Dp is homologous to the medial pallium in other vertebrates24,30,32,64. In contrast, Wullimann and Mueller (2004) considered only Dlv to be homologous to the medial pallium and Yamamoto et al. (2007) proposed that only the dorsal part of Dl is homologous to the medial pallium. Functional ablation experiments suggested that Dl is equivalent to the hippocampus of tetrapods65. Our combinatorial expression data suggests that Dl is subdivided in Dld and Dlv rostrally and at Cant and posterior to Cant in Dld and Dp (Figure 5A–D). During mouse development, Prox1 expression is found in the amygdala, dentate gyrus and in the neocortex57. At adult stages, however, strong Prox1 expression is restricted to the dentate gyrus of the hippocampus and is commonly used as a specific marker for granule cells of the hippocampus57–59. In the adult zebrafish, Prox1 positive cells are exclusively present in the caudal part of Dld. In summary, our data suggests that Dl (excluding Dp, see below) may be homologous to the medial pallium and hippocampus and the caudal part of Dld homologous to the dentate gyrus in the domestic mouse (Figure 5A–E). We will discuss the possible homology of Dp in the subsequent part.\n\nIt has previously been shown that Dp receives olfactory input and has been on this basis homologized to the lateral pallium in amphibians and all other gnathostomes with evaginated forebrains4. Being homologous to the lateral pallium, it should be located next to Dm in the embryo, the presumptive ventral pallium. The discrepancy between the position of Dp in the adult and an expected location next to Dm has led to different models to explain the different position of Dp in the adult pallium: “the partial pallial eversion model” by Wullimann and Mueller31,32,52,74, the “eversion-rearrangement theory” by Northcutt and Braford33,64,75, and the “new eversion model” by Yamamoto and colleagues37. In the “partial pallial eversion model”31,32,52,74, based on connectional and gene expression data, it has been proposed that the homolog of the lateral pallium does not participate in the eversion. Wullimann and Mueller put forward that neuroblasts generated by the ventricular zone of the uneverted, medially located lateral pallium homolog migrate laterally to give rise to the submeningeally located nucleus Dp31,74. Our gene expression analysis does not support this part of their modified partial eversion model. Based on differential gene expression we can identify Dp, which is not located submeningeally, but has its own ventricular zone even in the adult4,64,76. New neurons are still generated in the zebrafish pallium in the adult77,78. Thus, a small area of ventricular zone should be still present next to Dm to generate neurons for Dp. However, we have not observed neuroblasts migrating laterally across the pallium to reach Dp in the adult by BrdU pulse chase studies or genetic lineage tracing51,76,77. The “eversion-rearrangement theory” by Northcutt and Braford suggests that differential expansion of the ventricular surface of some pallial zones and differential proliferation and migration of neuroblasts from the different ventricular zones might result in displacement or shifting of the different pallial subdivisions33,64,75. Similar to the “partial pallial eversion model”, they propose that a small stretch of Dp is still located between Dm and Dl. Our gene expression data does not support this aspect of both models, as we do not identify a small area of ventricular zone and neuronal layer sandwiched between Dm and Dc rostrally and Dm and Dld more caudally that matches the gene expression profile found in Dp.\n\nIn the “new eversion model”, the eversion was suggested to occur in a caudolateral direction leading to a shift of the arrangement of the different pallial subdivisions37. Yamamoto and colleagues propose that ventral pallial and lateral pallial homologs are not present in the rostral but only in the caudal pallium. In their model they suggest that Dp is homologous to the lateral pallium37. In contrast, we find the putative ventral or ventral/lateral pallial homolog Dm contiguously from rostral to caudal. However, we also identify a separate Dp nucleus only in the posterior part of the pallium.\n\nIn contrast to the models discussed above, Nieuwenhuys has put forward that the lateral olfactory tract in vertebrates with everted telencephala is not homologous to the olfactory tract in vertebrates with evaginated telencephala4. He bases this on data that showed considerable variation in the pattern of secondary olfactory projections among different groups of ray-finned fishes and showed secondary olfactory projections both to Dm and Dp4,29,75. He suggests that the olfactory input has increased overall in Dl, with a subsequent confinement to Dp and has decreased in Dm during ray-fin fish evolution. He considers the lateral olfactory tract as an apomorphy of actinopterygian pallia4. Even though our gene expression data does not support the models that suggest a migration or displacement of Dp from a position equivalent to the lateral pallium to its caudolateral position, we can identify Dp as a subdivision of Dl separate from Dld. Furthermore, Neuropeptide Y and Parvalbumin immunohistochemistry clearly distinguishes Dl from Dp79. Furthermore, as discussed above, our gene expression data suggest that Dm might be homologous to the lateral pallium or comprise a combined ventral and lateral pallium, which might suggest that Dp is not homologous to the lateral pallium. Our gene expression data is consistent with Nieuwenhuys’ model that Dp comprises a specialized part of Dl that receives olfactory input, even though the other parts of Dl might be homologous to the medial pallium and hippocampus in tetrapods (Figure 5A–D).\n\nIn the “modified partial pallial eversion model”32, based on expression of Parvalbumin and nicotine adenine dinucleotide phosphate diphorase (NADPHd) in Dl, it was suggested that Dc is a true histogenetic unit that has its own germinative zone rostrally, but is caudally displaced to a more central location by differential growth of Dm and Dl. Mueller and colleagues suggest that Dc is homologous to the dorsal pallium in other vertebrates and that Dd is absent in zebrafish32. This model is consistent with connectional and gene expression data in Apteronotus leptorhynchus24. In addition, it has been noted that Dc shows no significant immunoreactivity to Calretinin, Neuropeptide Y or Thyrosine hydroxylase separating it from the surrounding areas of the pallium79. Other models based mainly on topology suggest that Dm, Dd and Dld are homologous to the dorsal pallium37, whereas others have homologized Dd with the dorsal pallium4,29,30,33,64 and have suggested that Dc is not a separate unit, but represents the deeper zone of the periventricular areas of Dm, Dd and Dl. Our gene expression data is consistent with the second part of the “modified pallial eversion model” of Mueller and colleagues. We have adapted the nomenclature of Mueller et al. (2011) to call the displaced nucleus Dc, even though our data do not rule out the possibility that Dc is rather a displaced Dd nucleus. Accordingly, Dc does not have its own germinative zone caudally, but only in the rostral part of the pallium. However, the morphogenesis of the telencephalon has been analyzed between 1dpf and 5dpf and no such displacement of Dc has been described5, so if it is the case, then it has to happen after 5dpf. To conclusively evaluate the displacement of Dc, detailed lineage analysis using Cre/loxP technology should be performed from embryonic stages till adulthood.\n\nIn the adult zebrafish, proliferating cells are found scattered in the ventricular zone of the pallium76–78. In the adult zebrafish pallium, ascl1a is found in scattered cells in the ventricular zone of Dm and in Dlv/Dp. In addition, eomesa is present in scattered cells in the ventricular zone of Dm and in the ventricular zone of Dld and Dlv/Dp. Emx1 is also present in the ventricular zone of Dp and emx3 is found in the ventricular zone of Dm, Dlv and Dp (Figure 5A–D).\n\nIn mouse, Ascl1 is expressed in a subpopulation of progenitors in the dorsal telencephalon61,62. Eomes (Tbr2) is present in intermediate progenitors in the cortex in the embryo and in the dentate gyrus both in the embryo and the adult and is part of the glutamatergic differentiation cascade that also contains NeuroD and Tbr180. In the adult zebrafish, neurod and tbr1 as well as the vesicular glutamate transporters vglut1/2.1/2.2 marking glutamatergic neurons are expressed throughout the pallium, suggesting that the glutamatergic differentiation cascade may still be present in the adult zebrafish telencephalon7,72. The expression in scattered cells in the ventricular zone suggests that eomesa and ascl1a may label progenitor subpopulations in the pallium and eomesa could play a role in differentiation of glutamatergic neurons in the adult zebrafish brain. In addition, emx1 and emx3 might be involved in regulating neuronal differentiation processes in the pallium. It will be interesting to perform comprehensive lineage analysis using Cre/loxP technology to follow the progeny of these different markers in the adult zebrafish.\n\n\nConclusions\n\nIn this study, we present a new model of the subdivisions in the adult zebrafish pallium based on conserved marker gene expression and propose putative homologies to pallial nuclei in tetrapods. Additional marker analysis, lineage tracing experiments and functional analyses are necessary to substantiate the proposed pallial subdivisions and their homology to pallial nuclei in tetrapods. It is important to identify pallial areas in adult zebrafish and their homologies to pallial nuclei in tetrapods to improve our knowledge about the zebrafish brain, in order to implement the zebrafish system as an ideal model for neurobiological research and as a model for human neurodegenerative diseases.\n\n\nList of abbreviations\n\nBNSM\n\nbed nucleus of the stria medullaris\n\nBST\n\nbed nucleus of the stria terminalis\n\nCant\n\nanterior commissure\n\nD\n\narea dorsalis telencephali\n\nDc\n\ncentral part of the area dorsalis telencephali\n\nDd\n\ndorsal part of the area dorsalis telencephali\n\nDl\n\nlateral part of the area dorsalis telencephali\n\nDld\n\ndorsal part of Dl\n\nDlv\n\nventral part of Dl\n\nDm\n\nmedial part of the area dorsalis telencephali\n\nDp\n\nposterior part of the area dorsalis telencephali\n\ndpf\n\ndays post fertilization\n\nEN\n\nentopeduncular nucleus\n\nnl\n\nneuronal layer\n\nOB\n\nolfactory bulb\n\nPo\n\npreoptic region\n\nV\n\narea ventralis telencephali\n\nVp\n\npostcommissural nucleus of the area ventralis telencephali\n\nVs\n\nsupracommisural nucleus of the area ventralis telencephali\n\nvz\n\nventricular zone\n\n\nData availability\n\nFigshare: Gene expression analysis in the adult zebrafish pallium. Doi: http://dx.doi.org/10.6084/m9.figshare.126619481",
"appendix": "Author contributions\n\n\n\nJG and MB conceived of the study and the experimental design, analyzed the data and co-wrote the manuscript. JG carried out most of the experiments, MB coordinated and supervised the study. VK performed the Prox1 immunohistochemistry. DF performed the ascl1a in situ hybridization. AM and MG performed the in situ hybridizations on larval brains. IB performed phylogenetic and synteny analyses. JK participated in the design of the study and helped analyze the data. 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\nThis work was supported by grants to MB from the Deutsche Forschungsgemeinschaft (SFB 655 A3), European Union (ZF-Health) and the Center for Regenerative Therapies Dresden (CRTD).\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 the Didier Stainier lab, the Monte Westerfield lab and the Steve Wilson lab for sharing plasmids. We are thankful to Anja Menge, Sabine Hübner, Julia Ludwig and Paul Morath for technical support. We thank Salvador Martinez and Stefano Suzzi for comments on the manuscript.\n\n\nSupplementary figures\n\nA. The topology of the Ensembl Gene Tree suggest that the teleost eomes co-orthologs, eomesa and eomesb, were duplicated at the base of the teleost lineage and thus most likely during the teleost genome duplication (TGD). B. Dotplot from the Synteny Database shows that the human EOMES gene region on chromosome Hsa3 shows extensive double conserved synteny to zebrafish chromosomes Dre19 and Dre16 which contain eomesa and eomesb, respectively, providing strong evidence for the TGD origin of teleost eomes co-orthologs.\n\nA. eomesb expression is not found in the telencephalon (T), but present in the midbrain (M), arrowhead, shown is a lateral view. Di Diencephalon, H Hindbrain.\n\nA. emx1 expression is present in the lateral part of the dorsal telencephalon (D, arrowhead), shown is a ventral view. B. emx2 expression is present in the lateral part of D (arrowhead), shown is a parasaggital view. C. emx3 expression is present throughout D, shown is a parasaggital view. D. Summary of the distribution of emx1 and emx2 expression in D in a sagittal view. E. Summary of the distribution of emx3 expression in D in a sagittal view. A.–C. Brightfield whole-mount images, A. ventral, B.–C. sagittal views. Scale bars = 50µm A–C.\n\n\nReferences\n\nNorthcutt RG: The forebrain of gnathostomes: in search of a morphotype. Brain Behav Evol. 1995; 46(4–5): 275–318. PubMed Abstract | Publisher Full Text\n\nNorthcutt RG: Evolution of the telencephalon in nonmammals. Annu Rev Neurosci. 1981; 4: 301–350. PubMed Abstract | Publisher Full Text\n\nNieuwenhuys R, Meek J: The Telencephalon of Actinopterygian Fishes. Cereb Cortex. 1990; 8A: 31–73. Publisher Full Text\n\nNieuwenhuys R: The forebrain of actinopterygians revisited. Brain Behav Evol. 2009; 73(4): 229–252. PubMed Abstract | Publisher Full Text\n\nFolgueira M, Bayley P, Navratilova P, et al.: Morphogenesis underlying the development of the everted teleost telencephalon. Neural Dev. 2012; 7: 32. 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{
"id": "7078",
"date": "08 Jan 2015",
"name": "Monica Folgueira",
"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\nMorphological differences between evaginated and everted telencephali hampers comparisons between brain morphotypes and complicates establishing homologies. This manuscript presents a comparative analysis of the expression pattern of a few transcription factors in the everted pallium of the zebrafish. These data will help us understand the organization of the zebrafish pallium and compare it with the pallium of other vertebrates. The manuscript will be of interest for readers working on comparative neuroanatomy and development, among other fields.The manuscript is generally clear, well written and appropriate. The analysis of data is generally well performed, but maybe authors could clarify the following points:Based on their expression analysis, authors identify five subdivisions of the pallium that they name as Dm, Dlv, Dld, Dc and Dp after adapting nomenclature from Mueller et al. (2011). I think it could be clearer in the text how data supports the division between “Dc” and “Dld” at rostral levels. In addition, as authors show that “Dld” represents a different histogenic unit to “Dlv”, I wonder if “Dld” could be equivalent to “Dd/Dld” of other teleosts. Maybe authors could comment on this in the manuscript. eomesa and Prox1 seem to show differences between anterior and posterior regions of Dld. Yet, authors consider Dld as a continuous unit. Dc also seems to show differences from rostral to caudal levels (e.g. emx2+ is only expressed in a caudal portion of Dc, while eomesa is only expressed in a rostral portion). Thus I wonder if more subnuclei could be identified in the zebrafish pallium, thus the organization of the pallium being even more complex. Maybe authors could comment on these observations (if correct). Authors discuss the divisions of the zebrafish pallium as corresponding to histogenic units of other vertebrates (ventral, lateral, dorsal and medial pallium). In addition, they propose certain homologies for specific nuclei, such as “Dm” being homologous to the “pallial amygdala” or “caudal part of Dld” homologous to the “dentate gyrus”. In my opinion, it is right to propose and discuss about such putative homologies in the manuscript. However, in my opinion, they are too speculative to be included in a summary figure, as this could mislead readers. I suggest removing these homologies from diagrams A-D in Figure 5, just referring to putative homologies for histogenic units.Minor comments:Discussion: authors state that ‘Our gene expression data is consistent with the second part of the “modified pallial eversion model” of Mueller and colleagues’ To me, it is not clear what to authors mean by “second part of the modified pallial eversion model”. Figure 1C-D: ventricular expression is not clear in the original data sets. Figure 2C: Authors state that red lines indicate subdivisions based on the current marker. However, emx2 only seems to be labelling a central area at this telenphalic level (Dc). So, it does not seem to support the limits between Dm, Dl and Dp. Figure S3: stages of development of the larvae are missing. Image C: in my opinion, OB limits are not accurately marked. Although the stage of development of the larva is not shown, the OB is certainly bigger than labelled between 3-5dpf (it seems correctly marked but not labelled in E-D). I find the abbreviation TGD unnecessary.",
"responses": [
{
"c_id": "1670",
"date": "04 Nov 2015",
"name": "Julia Ganz",
"role": "Author Response",
"response": "We thank Monica Folgueira for her helpful comments and suggestions.Based on their expression analysis, authors identify five subdivisions of the pallium that they name as Dm, Dlv, Dld, Dc and Dp after adapting nomenclature from Mueller et al. (2011). I think it could be clearer in the text how data supports the division between “Dc” and “Dld” at rostral levels.The difference between Dc and Dld is the absence of emx3 positive cells in Dld. We have included this point in the discussion to make it clearer and now write:“Dld is different from Dc based on absence of emx3 positive cells in Dld.”In addition, as authors show that “Dld” represents a different histogenic unit to “Dlv”, I wonder if “Dld” could be equivalent to “Dd/Dld” of other teleosts. Maybe authors could comment on this in the manuscript.The unit ‘Dld” could be an equivalent to “Dd/Dld” of other teleosts (such as in trout). As there are, however, to our knowledge, no good markers that have been used to identify “Dd/Dld”, we do not want to introduce another subdivision that has not been used so far in zebrafish. It would be really interesting though to test if our proposed subdivision “Dld” is equivalent to “Dd/Dld”.eomesa and Prox1 seem to show differences between anterior and posterior regions of Dld. Yet, authors consider Dld as a continuous unit. Dc also seems to show differences from rostral to caudal levels (e.g. emx2+ is only expressed in a caudal portion of Dc, while eomesa is only expressed in a rostral portion). Thus I wonder if more subnuclei could be identified in the zebrafish pallium, thus the organization of the pallium being even more complex. Maybe authors could comment on these observations (if correct).We have included different color coding in our summary figure to reflect these changes in gene expression (light green – medium green – dark green for Dc, and light yellow – dark yellow for Dld). We have also added an explanation to this color code in the figure legend and write:“The different shades of green (Dc) and yellow (Dld) indicate gene expression changes along the rostro-caudal axis.”At this point, we have avoided adding additional subdivision acronyms as they might be potentially confusing especially when comparing the subdivisions in zebrafish to other teleost species.Authors discuss the divisions of the zebrafish pallium as corresponding to histogenic units of other vertebrates (ventral, lateral, dorsal and medial pallium). In addition, they propose certain homologies for specific nuclei, such as “Dm” being homologous to the “pallial amygdala” or “caudal part of Dld” homologous to the “dentate gyrus”. In my opinion, it is right to propose and discuss about such putative homologies in the manuscript. However, in my opinion, they are too speculative to be included in a summary figure, as this could mislead readers. I suggest removing these homologies from diagrams A-D in Figure 5, just referring to putative homologies for histogenic units.We have adopted the suggestion from the reviewer Steve Wilson to add a question mark to figure 5 (“Putative homologous region in the tetrapod pallium (?)”) to indicate that the putative homologies that we propose based on our work will need additional studies. We also point this out both in the figure legend of figure 5 and at the beginning of the discussion.Minor comments:Discussion: authors state that ‘Our gene expression data is consistent with the second part of the “modified pallial eversion model” of Mueller and colleagues’ To me, it is not clear what to authors mean by “second part of the modified pallial eversion model”.We have clarified the text and now write:“Our gene expression data is consistent with the “modified partial pallial eversion model” of Mueller and colleagues with regard to Dc.” Figure 1C-D: ventricular expression is not clear in the original data sets.The cells at caudal levels of the telencephalon are scattered and a bit weaker labeled than at rostral levels, but there are scattered eomesa positive cells present at the ventricular zone of Dm at all levels.Figure 2C: Authors state that red lines indicate subdivisions based on the current marker. However, emx2 only seems to be labelling a central area at this telencephalic level (Dc). So, it does not seem to support the limits between Dm, Dl and Dp.We have modified this and changed the red lines in the figure. Figure S3: stages of development of the larvae are missing. Image C: in my opinion, OB limits are not accurately marked. Although the stage of development of the larva is not shown, the OB is certainly bigger than labelled between 3-5dpf (it seems correctly marked but not labelled in E-D).We have included the stages of development in the figure legend. We have included the OB label in S3E,D. We have changed the OB limits in the figure S3C accordingly.I find the abbreviation TGD unnecessary.The abbreviation has been removed."
}
]
},
{
"id": "7076",
"date": "29 Jan 2015",
"name": "Perrti Panula",
"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 title accurately describes the contents of the paper, and the abstract describes the findings and main conclusions well.The analysis of the selected markers in adult zebrafish telencephalon, particularly the pallium as is done here, is important for any functional studies on the zf brain.The study thus forms a good basis for further studies on adult fish brain. I have some comments on the controls below. Whereas the adult part is of high quality and will be very helpful in further analysis of e.g. circuits (which are essential to understand the functional significance of the results) and behavior, the developmental part could be improved.Some detailed comments:It needs to be indicated how the probe specificity has been verified. No probe-controls cannot replace a non-reacting probe control such as sense probe or partly scrambled probe controls, because occasionally probes bind non-specifically to e.g. proteins. From the point of view of the conclusions regarding correlation to telencephalic areas of other species, the developmental part in the study could be improved. Although mentioned in the abstract, only a limited dataset is shown at 7 dpf (on p. 4, last chapter), and even here the data is difficult to find because the title indicates only analysis of adults.The age/stage of the samples is not indicated in Fig. S3, and the samples look earlier than 7 dpf. It would be particularly useful to see a series a developmental series in a set of images in which the brain domains could be easily identified. In this respect Fig.S3 is of limited value. The resolution is not very good, and clearly better figures could be provided. At least from 5 dpf on the brains are easily prepared from the skull and penetration is excellent. Adding data on the developmental expression series would significantly improve the paper. Whole brain ish can be easily done on even 1-month-old fish. The data during the whole first week, or (even better) two weeks would be useful.In summary, the adult data is very useful and only a note on controls should be added. The developmental data could be improved to the same high level as the adult data.",
"responses": []
},
{
"id": "7077",
"date": "20 Feb 2015",
"name": "Stephen W. Wilson",
"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 teleosts, the pallial telencephalon undergoes eversion during its development rather than the more common evagination that occurs in other vertebrates. These differing modes of morphogenesis have made it difficult to resolve homologies between pallial domains in teleosts and other vertebrates. This paper describes the pallial expression of zebrafish orthologues of genes that have helped to define pallial sub-domains in other vertebrates.\n\nThe methods are clearly described, the data are of high quality and the discussion of results are balanced and informative. The authors acknowledge the limitations of using gene expression patterns and suggest additional experimental approaches that could be used to address the validity of hypotheses raised in the discussion of the data.\n\nThere is a great deal of information of use for comparative neuroanatomy studies and some observations challenge current models of teleost telencephalic organisation. The expression analyses suggest that pallial sub-divisions show considerable variation from rostral to caudal telencephalon and it will be interesting to study how such organisation arises during development. I have no revisions that I consider to be essential. I think that Monica Flogueira's concern about the summary figure potentially being misleading could most simply be addressed by adding a question mark after the suggested homology term.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-308
|
https://f1000research.com/articles/4-479/v1
|
05 Aug 15
|
{
"type": "Software Tool Article",
"title": "NetMatchStar: an enhanced Cytoscape network querying app",
"authors": [
"Fabio Rinnone",
"Giovanni Micale",
"Vincenzo Bonnici",
"Gary D. Bader",
"Dennis Shasha",
"Alfredo Ferro",
"Alfredo Pulvirenti",
"Rosalba Giugno",
"Fabio Rinnone",
"Giovanni Micale",
"Vincenzo Bonnici",
"Gary D. Bader",
"Dennis Shasha",
"Alfredo Ferro",
"Alfredo Pulvirenti"
],
"abstract": "We present NetMatchStar, a Cytoscape app to find all the occurrences of a query graph in a network and check for its significance as a motif with respect to seven different random models. The query can be uploaded or built from scratch using Cytoscape facilities. The app significantly enhances the previous NetMatch in style, performance and functionality. Notably NetMatchStar allows queries with wildcards.",
"keywords": [
"network querying",
"exact graph matching",
"approximate graph matching",
"cytoscape app",
"statistical significance",
"background network models",
"randomization",
"biological network motifs"
],
"content": "Introduction\n\nBiological networks such as protein-protein interaction, transcription regulatory, gene regulatory, and metabolic networks are often referred to as complex systems1. The term complex relates to the existence of non-trivial substructures contained within them. The study of complex systems involves the analysis of the way in which their elements interact rather than only their individual roles. Computationally, such a study entails the ability to query networks to find specific patterns of interactions.\n\nPossible queries might include the identification of positive and negative autoregulation, coherent and incoherent feed forward loops, single-input modules and dense overlapping regulons2 in a given target network N. Sub-networks that occur surprisingly often in a network may be preferred by evolution. For that reason, NetMatchStar offers the ability to compute a p-value against null models from seven distinct randomizing methods and suggests the one that shares the network properties of N in terms of degree distribution, cluster coefficient and assortativity.\n\nThe availability of computational tools for the analysis of biological networks has been helpful in providing novel biological insights on the function of many previously uncharacterized proteins. Several different methods have been developed for this purpose: (i) Network Motif fiding3–12, network querying13–15 and network alignment16–21 algorithms.\n\nMost of the approaches dealing with this kind of graph analysis entail subgraph matching. Such a problem has been widely studied and several methods and systems have been proposed. The approaches can be categorized according to the methodology they use. The first category is the tree search based algorithm. Those methods look for a solution of the problem in a state space by making use of a depth-first approach. Algorithms using such approach include Ullmann22, VF223 and the recently introduced RI24. The second category consists of algorithms using Constrained Programming techniques. Such methods aim at filter pairs of nodes which will not be in a matching solution. Many algorithms exploit such approaches25–27. The last category uses a database approach by exploiting the virtues of indexing. Such algorithms extract a set of features which define an index of the query that will be used for searching through the target one. The goal is to identify candidate subgraphs in the target one which are possibly isomorphic to the query. Algorithms using such a technique include28–31. NetMatchStar works on Cytoscape 3.2+ and is based on the NetMatch software in13. It deals with both exact queries and approximate ones, in which wildcards are used to match unspecified number of elements.\n\nNetMatchStar integrates the RI algorithm proposed for biological real networks which outperforms other existing algorithms24. For illustration purposes, NetMatchStar has been tested on a biological dataset24,32 and an overview of its performance concludes the paper.\n\n\nMethods\n\nA graph G is a pair (V, E), where V is the set of nodes and E ⊆ (V × V) is the set of edges.\n\nUsing a graph Q to query a target network graph N means to perform a subgraph isomorphism, which entails finding an injective function that maps each node of Q to a unique node of N such that nodes and edges labels are preserved. Assessing the statistical significance of Q implies a simulation process, where first a set R of r random graphs are generated according to a specific model. Then the number of occurrences of Q in each random graph is counted and a p-value is computed which is defined as the fraction of the r graphs where Q occurs at least as often as in N. The lower the p-value is, the more significant Q is as a motif. The significance of Q can also be evaluated through the z-score, which is defined as the difference between the number of occurrences of Q in N and the average number of occurrences of Q in the r random graphs, divided by the standard deviation of the frequencies of Q in R. A strongly positive value of the z-score means that Q is significant as a motif.\n\nA simple enumeration algorithm to find Q in N generates all possible maps between the nodes of the two graphs and checks whether any generated map is a subgraph isomorphism. The common aim of existing algorithms is to discover unsuccessful mappings as early as possible and to filter them away22. NetMatchStar uses the algorithm RI proposed in24, whose efficiency is mainly due to the choice of a search strategy, i.e. the ordering with which query nodes are mapped. For example, a variable ordering may begin with a query node having the highest degree or having the most uncommon label in the target graph. The variable ordering of RI is based only on the query graph topology. Roughly, the chosen order creates constraints as early as possible in the matching phase. The nodes having high valence and that are highly connected with nodes previously present in the ordering tend to come early in the variable-ordering. The aim of RI is to avoid costly pruning techniques by finding a static search strategy such that the number of constraints that are verifiable from a partial solution are maximized.\n\nApproximate queries are graphs containing wildcard structures. They may contain nodes and edges which can match any value of node or edge labels in the network and approximate paths constrained in length to be less than or greater than m, where m is a positive integer. NetMatchStar first matches all the specified subparts of the queries exactly and then joins the matches by network traversal. The network traversal phase checks that all traversed paths satisfy the query path constraints.\n\nIn NetMatchStar, the user can choose among seven different generative models to compute the statistical significance of a motif. In all cases, except for the shuffling model, the simulation starts with the generation of a network with |V| nodes having the same labels as the target network N and no edges. Then, new edges between existing nodes are added until we obtain a network with |V| nodes and |E| edges, just like N. In the following, we briefly describe each random model.\n\nShuffling model. In the shuffling model33 an existing network is \"rewired\" by repeatedly swapping the destinations of two randomly chosen edges, where possible. The result is a graph with the same degree distribution of the original network.\n\nErdos-Renyi model. The Erdos-Renyi (ER) model34 corresponds to a graph where two nodes connect each other randomly and independently. There are two variants of the ER model. In the G(|V|, |E|) model the algorithm randomly creates a network uniformly over all networks that have |V| nodes and |E| edges. In the G(|V|, p) model, edges between nodes are independently created with a user-defined probability p. NetMatchStar implements the G(|V|, p) variant of the ER model.\n\nWatts-Strogatz model. The Watts–Strogatz model35 produces graphs characterized by the small-world property, where most nodes can be reached from every other by a small number of hops, when there is no direct link between them. The model works in two phases. In the first one a lattice of |V| nodes is created where each edge is connected to d neighbors on its left and d neighbors on its right. Then, edges are randomly shuffled with rewiring probability β. Low values of β produce a quasi-regular graphs, where nodes have approximately the same degree, while high values of β produce networks which are very close to the ER model.\n\nBarabasi-Albert model. Also known as the preferential attachment model, this model36 creates graphs where the more connected a node is, the more likely it creates new links. Graphs generated with BA model are scale-free, meaning that the degree distribution follows a power law, with a few high-degree nodes and many low-degree nodes. The BA model starts with the creation of a complete initial seed network of k nodes. The remaining |V| – k nodes are added one at a time. Each new node is attached to d existing nodes, such that the probability of selecting an existing node u is proportional to the degree of u.\n\nGeometric model. The geometric model37 describes graphs in which the information about the location of nodes in the space determines the topology and might be useful to represent spatially oriented networks (e.g. transportation and neuronal networks). In the geometric model each node is represented as a point in a d-space. An edge between two nodes exists if the distance between corresponding points is within a threshold r.\n\nForest-Fire model. In the Forest-Fire (FF) model38, a new node v attaches to the network by iteratively exploring existing edges starting from one or more anchor nodes, called ambassadors, which are chosen randomly. At each step of the exploration, v creates out-links with newly discovered nodes with a forward probability p and in-links with a backward probability r, and continues exploration from those nodes. The FF model describes time-evolving networks where the number of edges grows super-linearly in the number of nodes and the distance between nodes shrinks as new nodes arrives.\n\nDuplication model. In the duplication model39 the duplication of the information is considered as a dominant evolutionary force for the growth of a network, such as in many biological networks. At each step of the duplication model a random node u is selected. Then, a new node v is created and connected to neighboring nodes of u with probability p. The lower is p, the more divergent is v as a copy of u.\n\nThe NetMatchStar Cytoscape App has been developed in Java 7 on top of the Cytoscape 3.2 API. The software is composed by a core module, which implements basic algorithms and data structures, plus a user interface module that integrates the analyses into the Cytoscape interface. The core module provides data representations, graph analysis (i.e. graph matching and motif searching) and two different types of attribute comparator that differentiate in exact and approximate comparison. The CyNetworks are converted into graph structures to optimize the graph traversal procedures. The user interface is designed by following the Model-View-Controller architectural pattern. The Model component adds up result data representations to the functionality provided by the software’s core module. The View component implements the graphical panels of the interface. The main panel of the app adds up, as a further tab, to the Control Panel of the Cytoscape interface. This integrates the graphical panels where the user can select the networks to be processed, the parameters of the analysis, and the results. The Control component ensures the communication between the Model and the View by implementing the set of tasks performed by NetMatchStar. This component is developed by following the Cytoscape 3.1 app guidelines, such that every task is implemented as a Cytoscape Task Java class.\n\nThe main frame of NetMatchStar contains three tabbed panels:\n\n\"Matching\" panel (Figure 1), to specify the target and the query graphs and run the matching task;\n\n“Significance” panel (Figure 2), for the statistical significance of the query as a motif according to a specific random model;\n\n\"Motif library\" panel (Figure 3), which contains a set of predefined queries for the matching task.\n\nIn this example, the network of Figure 4 has been provided as query, while the Mus musculus network provided in 24 has been chosen as target graph.\n\nIn the following subsections, we will describe all the required steps for the matching and motif verification of a query graph in a target network.\n\nQuery and network graphs can be uploaded in NetMatch-Star, by clicking on the folder icon in the toolbar of \"Matching\" panel (Figure 1). Each uploaded network will be added to the Network list of Cytoscape. In the drop-down lists of \"Network Properties\" and \"Query Properties\" section, the user can select one of the uploaded networks as a query or target network for the matching and statistical significance tasks. Likewise, the user may upload node and edge labels as Cytoscape attributes and link them to the nodes and edges of the target network and query graph.\n\nInstead of loading an existing network, the user can create a query from scratch or by starting from a pre-defined set of queries.\n\nTo create a new query, the user must click on the \"plus\" icon of \"Matching panel\" (Figure 1). A new panel for the creation of a new network will be opened (Figure 4). A right click on the panel will open the standard Cytoscape menu to add, edit or remove elements of the graph. Such a menu also includes the \"NetMatchStar\" menu item, which lets the user change the label of a node or edge and set a path between two nodes. By default, newly added nodes and edges will be labeled with the wildcard \"?\", corresponding to a node or a direct link between nodes with unspecified label. Any other character will be associated to a specific label. Paths between two nodes i and j are defined as special attributes for the edge (i, j). The length of a path is specified by an expression of the form aopb, where a and b are two integers (or the wildcard \"?\") and op is one of <, ≤, ≥, >, =. The \"?\" character is used to leave the minimum or maximum length of the path unspecified. For instance, the expression ”? ≤ 2” means that the corresponding path must have at most length 2, while ”? > 3” corresponds to a path of length greater than 3. A query with a \"?\" character in at least a node and/or edge is an approximate query for NetMatchStar.\n\nIn this example, an approximate query with 3 nodes and 3 edges has been created, where 2 nodes have a specific label and one edge represents an approximate path of length at most 2 (’?<=2’). The remaining elements of the graph have an unspecified label (’?’). By selecting an edge and right-clicking, a menu will be shown for changing its label or set the approximate path.\n\nBy clicking on \"Save\" button on panel, the user can store the query graph created from scratch on disk as text files in a .SIF format with nodes and edges attribute files with extensions respectively .NA and .EA.\n\nThe pre-defined set of queries includes small topologies which have been identified as motifs in many real networks2, such as feed-forward loops, diamonds, single-input modules and dense overlapping regulons. Figure 3 shows all the pre-defined queries that can be selected from the \"Motifs library\" tabbed panel. They are drown as directed graphs but can be used to query both directed and undirected networks. By clicking on one of these topologies, the user can visualize the query and modify it, as previously described, i.e. adding new nodes/edges, changing node/edge labels and setting paths between nodes. Modifying the pre-defined query does not change the original “library” entry, but only a copy of it.\n\nThe “Significance” panel (Figure 2) contains all the parameters for the evaluation of the statistical significance of a motif subnetwork. It consists of three subpanels. In the top subpanel the user can choose the number of random graphs to generate for the statistical test (between 0 and 100) and the seed for generating pseudorandom numbers. In the middle subpanel the user can compute a set of metrics for the target graph and sample random graphs, one for each model. Metrics include the average degree, the average clustering coefficient and the assortativity index. At the end of the computation, the resulting values are shown in a separate window. Values of these metrics can suggest to the user which random model best describes the features of the input network and should be used.\n\nThe bottom subpanel let the user choose a random model and set its parameters (if any). In “Shuffling” model, “Lab shuffling” option can be selected for enabling shuffling also on node and edge labels (if present), while “sw/edg” denotes the number of successful swaps per edges. The “Erdos-Renyi” model has no parameters. In “Watts-Strogatz”, “Rew prob” is the probability of rewiring β. The \"Barabasi-Albert” model defines “Init nodes”, the number of initial nodes in the complete seed network. The “Duplication” model has two parameters: “Init nodes”, the number of nodes in the initial seed network, and “Edg prob”, the edge duplication probability. In the “Geometric” model, parameter “Dim” denotes the dimension of the space where points are placed. Finally, “Forest-fire” contains parameter “Ambass”, that is the number of ambassadors nodes. For each model, all the remaining parameters are estimated based on the number of nodes and edges of the target network.\n\nOnce a target network and a query has been provided in the \"Matching\" panel (Figure 1), the user can either look for all occurrences of the query within the input graph or check if the query is a motif or not.\n\nIn the first case, the user must click on the \"Match\" button in the \"Matching\" panel (Figure 1). Once the matching task has been completed, a table with all the occurrences of the query in the target will be shown as a tabbed panel in the \"Result Panel\" of Cytoscape (Figure 5) and the input graph will be visualized. For each occurrence, NetMatch-Star reports its nodes and an image depicting its topology. By selecting a row in the table, the user can visualize the corresponding occurrence in the target network. If the option \"Create a new child network\" is disabled, nodes of the occurrence will be highlighted in yellow within the input network, otherwise the occurrence will be visualized in a separate window. By clicking on \"Save\" button on result panel, the user can store the results as text file.\n\nRecalling that the nodes of the network are not uniquely labeled and thus the query may have different matches, to check if a query is a motif, the user must click on one of the \"Start\" buttons of the “Significance” panel (Figure 2), depending on the random model that has been chosen to perform the significance test. When the simulation ends, a new window will appear with the following measures: the number of query occurrences in the real network, the mean and the standard deviation of the number of query occurrences in the random networks, the p-value and the z-score. The statistics of the test will be also reported on the “Log” panel located at the bottom of the “Matching” panel (Figure 1) and they can be consulted anytime.\n\n\nResults\n\nWe evaluated the performance of NetMatchStar on the biological networks provided in24 and compared it to the original NetMatch, developed for Cytoscape 2.8.\n\nIn Cytoscape others software are available for network motifs search. CytoKavosk40 is based on counting all k-size sub-graphs of a given network graph, while GraMoFoNe41 emulates the interface of NetMatchStar by allowing users to define a query and finding all occurrences similar to the query, with respect to node and edge deletions and node similarities. NetMatchStar contains predefined motif structures, checks the significance of a motif with respect to seven different random models and allows user to draw queries containing wildcards and manage the approximation they need.\n\nFigure 6 depicts the evaluation of NetMatchStar on three protein-protein interaction networks: Mus musculus, Homo sapiens and Danio rerio. They are large dense graphs. We randomly labeled networks with 32, 64, 128, 512 and 2048 synthetic labels and with 43 real labels corresponding to the Gene Ontology (GO) classes of the proteins (i.e. the nodes in the network). We used queries extracted from the networks by varying the number of nodes from 4, 8, 16, 32, and 64 and density from low to high (up the 90% of edges among nodes are present).\n\nFigure 7 evaluates NetMatchStar on protein back-bones graphs. They are large sparse graphs. The original labels are maintained since they are not unique (i.e., atoms names).\n\nFigure 8 evaluates NetMatchStar on contact map graphs. They are dense medium graphs. The original labels are maintained since they are not unique (i.e., amino acids).\n\nFigure 9 reports the querying performance of feed forward loop topology on Mus musculus with 512 labels. Queries are run exactly and approximated by unspecifying one, two and all node labels and replacing one edge with an approximate path constrained to less than 3 and 7 edges.\n\nFinally, for those queries we verified their statistical significance by using all random models (Figure 10) and we measured the average time required for generating random networks and searching the queries (Figure 11).\n\n\nSummary\n\nThis paper presented the biological network querying system NetMatchStar for Cytoscape 3.2.1. NetMatchStar improves upon its predecessor NetMatch in usability and performances. Moreover, it allows a comprehensive evaluation of statistical query significance. Future work includes semantic and ontological similarity search.\n\n\nSoftware availability\n\nThis section will be generated by the Editorial Office before publication. Authors are asked to provide some initial information to assist the Editorial Office, as detailed below.\n\nhttp://apps.cytoscape.org/apps/netmatchstar,\n\nhttp://alpha.dmi.unict.it/netmatchstar/\n\nhttps://github.com/fabiorinnone/NetMatchStar/tree/v3.1\n\nhttp://dx.doi.org/10.5281/zenodo.1904542\n\nCreative Commons Attribution-NonCommercial-ShareAlike 3.0 License.",
"appendix": "Author contributions\n\n\n\nRG conceived the project. FR, GM and VB enabled the porting of the code to the new versions of Cytoscape (from version 3.0 onward) and extended the functionality of the application. In particular, GM has worked on the statistical significance and VB on the graph matching algorithm. RG, GM, and AP wrote the main parts of the paper. All authors have tested, validated, improved the software and the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nRG, AP and AF have been founded by ProgrammaOperativoFondoEuropeo per lo SviluppoRegionale (PO-FESR 2007-2013), Linea di intervento 4.1.1.2. Grant number: CUP G23F11000840004.\n\n\nAcknowledgments\n\nWe would like to thank A. Cannella and D. Garofalo who worked in a preliminary porting on the software for the current version of Cytoscape; D. Skripin who developed NetMatch, M. Mongiovì who implemented, in the old version of the system, the motif verification based on shuffling model, and G. Pigola who worked in the old version in query output visualization and optimization. We also are grateful to all users of NetMatch for their contributions and suggestions.\n\n\nReferences\n\nAlbert R, Barabási AL: Statistical mechanics of complex networks. Rev Mod Phys. 2002; 74(1): 47. Publisher Full Text\n\nMilo R, Shen-Orr S, Itzkovitz S, et al.: Network motifs: simple building blocks of complex networks. Science. 2002; 298(5594): 824–827. PubMed Abstract | Publisher Full Text\n\nMete M, Tang F, Xu X, et al.: A structural approach for finding functional modules from large biological networks. BMC Bioinformatics. 2008; 9(Suppl 9): S19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRhrissorrakrai K, Gunsalus KC: MINE: Module identification in networks. BMC Bioinformatics. 2011; 12: 192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdamcsek B, Palla G, Farkas IJ, et al.: CFinder: locating cliques and overlapping modules in biological networks. Bioinformatics. 2006; 22(8): 1021–1023. PubMed Abstract | Publisher Full Text\n\nWernicke S, Rasche F: FANMOD: a tool for fast network motif detection. Bioinformatics. 2006; 22(9): 1152–1153. PubMed Abstract | Publisher Full Text\n\nWernicke S: Efficient detection of network motifs. IEEE/ACM Trans Comput Biol Bioinform. 2006; 3(4): 347–359. PubMed Abstract | Publisher Full Text\n\nAlon U: Network motifs: theory and experimental approaches. Nat Rev Genet. 2007; 8(6): 450–461. PubMed Abstract | Publisher Full Text\n\nBader GD, Hogue CWV: An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics. 2003; 4: 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrochow JA, Kellis M: Network motif discovery using subgraph enumeration and symmetry-breaking. In Research in Computational Molecular Biology. Springer, 2007; 4453: 92–106. Publisher Full Text\n\nRibeiro P, Silva F: Discovering colored network motifs. In Complex Networks V, Springer, 2014; 549: 107–118. Publisher Full Text\n\nRibeiro P, Silva F: G-Tries: a data structure for storing and finding subgraphs. Data Min Knowl Discov. 2014; 28(2): 337–377. Publisher Full Text\n\nFerro A, Giugno R, Pigola G, et al.: NetMatch: a Cytoscape plugin for searching biological networks. Bioinformatics. 2007; 23(7): 910–912. PubMed Abstract | Publisher Full Text\n\nBanks E, Nabieva E, Peterson R, et al.: NetGrep: fast network schema searches in interactomes. Genome Biol. 2008; 9(9): R138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBruckner S, Huffner F, Karp RM, et al.: Topology-free querying of protein interaction networks. J Comput Biol. 2010; 17(3): 237–252. PubMed Abstract | Publisher Full Text\n\nMicale G, Pulvirenti A, Giugno R, et al.: GASOLINE: a Greedy And Stochastic algorithm for optimal Local multiple alignment of Interaction NEtworks. PLoS One. 2014; 9(6): e98750. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMicale G, Continella A, Ferro A, et al.: GASOLINE: a Cytoscape app for multiple local alignment of PPI networks. [v2; ref status: indexed, http://f1000r.es/4f7] F1000Res. 2014; 3: 140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKalaev M, Bafna V, Sharan R: Fast and accurate alignment of multiple protein networks. J Comput Biol. 2009; 16(8): 989–999. PubMed Abstract | Publisher Full Text\n\nSahraeian SME, Yoon BJ: SMETANA: accurate and scalable algorithm for probabilistic alignment of large-scale biological networks. PLoS One. 2013; 8(7): e67995. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlannick J, Novak A, Srinivasan BS, et al.: Graemlin: general and robust alignment of multiple large interaction networks. Genome Res. 2006; 16(9): 1169–1181. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiao C, Lu K, Baym M, et al.: IsoRankN: spectral methods for global alignment of multiple protein networks. Bioinformatics. 2009; 25(12): i253–258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUllmann JR: An algorithm for subgraph isomorphism. J ACM. 1976; 23(1): 31–42. Publisher Full Text\n\nCordella LP, Foggia P, Sansone C, et al.: A (sub)graph isomorphism algorithm for matching large graphs. IEEE Trans Pattern Anal Mach Intell. 2004; 26(10): 1367–1372. PubMed Abstract | Publisher Full Text\n\nBonnici V, Giugno R, Pulvirenti A, et al.: A subgraph isomorphism algorithm and its application to biochemical data. BMC Bioinformatics. 2013; 14(Suppl 7): S13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSolnon C: Alldifferent-based filtering for subgraph isomorphism. Artif Intell. 2010; 174(12–13): 850–864. Publisher Full Text\n\nZampelli S, Deville Y, Solnon C: Solving subgraph isomorphism problems with constraint programming. Constraints. 2010; 15(3): 327–353. Publisher Full Text\n\nUllmann JR: Bit-vector algorithms for binary constraint satisfaction and subgraph isomorphism. J Experimental Algorithmics (JEA). 2010; 15: 1.6. Publisher Full Text\n\nHan WS, Lee J, Lee JH: Turboiso: towards ultrafast and robust subgraph isomorphism search in large graph databases. In Proceedings of the 2013 ACM SIGMOD International Conference on Management of Data. ACM, 2013; 337–348. Publisher Full Text\n\nShang H, Zhang Y, Lin X, et al.: Taming verification hardness: an efficient algorithm for testing subgraph isomorphism. Proceedings of the VLDB Endowment. 2008; 1(1): 364–375. Publisher Full Text\n\nZhang S, Li S, Yang J: GADDI: distance index based subgraph matching in biological networks. In Proceedings of the 12th International Conference on Extending Database Technology: Advances in Database Technology. ACM, 2009; 192–203. Publisher Full Text\n\nZhao P, Han J: On graph query optimization in large networks. Proceedings of the VLDB Endowment. 2010; 3(1–2): 340–351. Publisher Full Text\n\nSzklarczyk D, Franceschini A, Kuhn M, et al.: The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011; 39(Database issue): D561–D568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilo R, Kashtan N, Itzkovitz S, et al.: On the uniform generation of random graphs with prescribed degree sequences. Condensed Matter. 2004; 2: 1–4. Reference Source\n\nErdos P, Renyi A: On random graphs i. Publicationes Mathematicae. 1959; 6: 290–297. Reference Source\n\nWatts DJ, Strogatz SH: Collective dynamics of 'small-world' networks. Nature. 1998; 393(6684): 440–442. PubMed Abstract | Publisher Full Text\n\nBarabasi AL, Albert R: Emergence of scaling in random networks. Science. 1999; 286(5439): 509–512. PubMed Abstract | Publisher Full Text\n\nPenrose M: Random Geometric Graphs. Oxford Studies in Probability 5. Oxford University Press, 2003. Reference Source\n\nLeskovec J, Kleinberg J, Faloutsos C: Graphs over time: Densification laws, shrinking diameters and possible explanations. In Proceedings of the Eleventh ACM SIGKDD International Conference on Knowledge Discovery in Data Mining. KDD ’05, New York, NY, USA, ACM. 2005; 177–187. Publisher Full Text\n\nChung F, Lu L, Dewey TG, et al.: Duplication models for biological networks. J Comput Biol. 2003; 10(5): 677–687. PubMed Abstract | Publisher Full Text\n\nMasoudi-Nejad A, Ansariola M, Kashani ZR, et al.: CytoKavosh: a Cytoscape plug-in for finding network motifs in large biological networks. PLoS One. 2012; 7(8): e43287. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlin G, Sikora F, Vialette S: GraMoFoNe: a Cytoscape plugin for querying motifs without topology in protein-protein interactions networks. In Hisham Al-Mubaid, editor, Bioinformatics and Computational Biology (BICoB’ 10). International Society for Computers and their Applications (ISCA), 2010; 38–43. Reference Source\n\nRinnone F, Micale G, Bonnici V, et al.: NetMatch-Star: v3.1. Zenodo. 2015. Data Source"
}
|
[
{
"id": "10314",
"date": "15 Sep 2015",
"name": "Xifeng Yan",
"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\nNetMatchStar improves the previous NetMatch tool, a Cytoscape app developed by the authors. It has more functions (e.g., supporting approximate querying with wild cards) and runs faster. It shall be interesting to users who are looking for tools that are able to find all the occurrences of a query graph in a network and check for its significance under different null models. The manuscript has detailed description and evaluation results.",
"responses": []
},
{
"id": "10336",
"date": "21 Sep 2015",
"name": "Shaillay Dogra",
"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\nAlgorithmically, is a useful utility that has been implemented by the authors. Bringing out its multiple use-case scenarios in biological setting with interpretations for example will help novice users adapt this more widely. Biological explanations of network interpretations will strengthen understanding and usage. Also, there are 7 different algorithms implemented. In which kind of data or user-problem which algorithm is recommended, according to authors or empirical observations?Minor points:\"Algorithms using such a technique include..\" -- kindly mention here itself \"the RI algorithm proposed for biological real networks which outperforms other existing algorithms..\" -- please elaborate on this RI algorithm \"Values of these metrics can suggest to the user which random model\" - can the authors recommend some approximation of good/bad values?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-479
|
https://f1000research.com/articles/4-1207/v1
|
03 Nov 15
|
{
"type": "Correspondence",
"title": "The dorsal posterior insula is not an island in pain but subserves a fundamental role - Response to: “Evidence against pain specificity in the dorsal posterior insula” by Davis et al.",
"authors": [
"Andrew R. Segerdahl",
"Melvin Mezue",
"Thomas W. Okell",
"John T. Farrar",
"Irene Tracey",
"Thomas W. Okell",
"John T. Farrar",
"Irene Tracey"
],
"abstract": "An interesting and valuable discussion has arisen from our recent article (Segerdahl, Mezue et al., 2015) and we are pleased here to have the opportunity to expand on the various points we made. Equally important, we wish to correct several important misunderstandings that were made by Davis and colleagues that possibly contributed to their concerns about power when assessing our paper (e.g. actual subject numbers used in control experiment and the reality of the signal-to-noise and sampling of the multi-TI technique we employed). Here, we clarify the methods and analysis plus discuss how we interpret the data in the Brief Communication noting that the extrapolation and inferences made by Davis and colleagues are not consistent with our report or necessarily, in our opinion, what the data supports. We trust this reassures the F1000Research readership regarding the robustness of our results and what we actually concluded in the paper regarding their possible meaning. We are pleased, though, that Davis and colleagues have used our article to raise an important discussion around pain perception, and here offer some further insights towards that broader discussion.",
"keywords": [
"Pain",
"insula",
"brain imaging",
"ASL"
],
"content": "\n\nWe actually agree with a main premise of Davis and colleagues’ comments; namely, that the hunt for a single pain region is redundant and relying on 'single pain region' inference-based logic is flawed. The main finding presented in our study is that the contralateral dorsal posterior insula (dpIns) was the only region that was observed to track pain intensity; one of the many fundamental variables that are integrated by a number of other key brain regions into a complete tonic pain experience. At no point in the paper do we say that by identifying this ‘fundamental role for the dpIns’ in tracking pain intensity does this mean we’re “promoting the concept of a single spot”, as Davis and colleagues have themselves interpreted from our data. Nor do we ever suggest that the results presented as a Brief Communication should be used to regress back to an expired ‘one region fits all’ pedagogy of where ‘pain is in the brain’. Indeed, that view would be completely contrary to the view and concept about ‘pain representation in the brain’ that we’ve long held and have written extensively about via original studies, reviews and editorials over the past 16 years. We have even explicitly discussed the issue surrounding the concept of an ‘N1’ or ‘P1’ for pain and stated that there is ‘no P1 for pain like V1 for vision’.\n\nThe concept we’ve long held (and still do) is that due to pain being multifactorial and highly variable (even in response to the same nociceptive input) - contingent upon the context, cognitive and emotional state of an individual, it must be reflected in a malleable, flexibly accessible set of brain regions that variably activate in concert (i.e. network connectivity is key to perceptions) - (for examples, see: Berna et al., 2010; Denk et al., 2014; Lee & Tracey, 2013; Leknes et al., 2013; Ploghaus et al., 1999; Ploner et al., 2010; Ploner et al., 2011; Wiech et al., 2008; Wiech et al., 2010; Tracey, 2005a; Tracey, 2005b; Tracey, 2008; Tracey, 2011; Tracey & Johns, 2010; Tracey & Mantyh, 2007; Wiech & Tracey, 2009). Pain is not a unitary thing. Most recently, we have drawn upon computational approaches to show even that some pain experiences can be influenced by priors in a Bayesian brain - over-riding the information from nociceptive inputs to ultimately decide the pain experience (e.g. Wiech et al., 2014a). Further, we have shown that even in sleeping babies an extensive network of activity subserves nociceptive processing (Goksan et al., 2015). As we’ve explicitly written many times, pain – like all perceptions - requires a network of brain activity for it to emerge. However, to identify and dissect within that complex set of brain regions (using imaging or electrophysiological methods, neurophysiological or anatomical measures) what roles different regions or signals relate to in terms of the multifactorial pain experience, as well as to identify potentially nociceptive, pain intensity or other specific features of the pain experience is what many animal and human pain researchers have done for many years. The same aim held for this study, as set out, we thought clearly, in the introduction and references quoted. For the avoidance of doubt and in the interests of absolute clarity: we hold that if a region is found to have a specific role that appears quite fundamental and important to the pain experience – whether from animal or human studies – this does not mean it’s the sole region responsible for that complex pain experience, as Davis and colleagues have concluded and suggest our data implies. That is why we used throughout the paper and in the title the expression: “subserves a fundamental role in pain” – which doesn’t mean ‘it’s the only region involved’ in pain, which we agree would be naïve bordering on incredulous. Possibly the format of a brief communication didn’t help make this position clear.\n\nOur study explored what brain regions over a long period of time continuously activate and track a specific feature of the pain experience (in this case intensity) in response to a controlled manipulation of the nociceptive input using a novel imaging technique that allows such exploration. This is a very different approach for examining the neural correlates of painful experiences – and not one whereby the normal framework for thinking about the data can be assumed by applying what we know from conventional imaging/electrophysiological approaches and paradigms used to date (even the single-TI arterial spin labeling (ASL) studies). Perhaps this is why the highly significant result we found was so intriguing and challenging for all of us researchers to interpret, including ourselves.\n\nObviously no brain region, especially the insula, works in isolation as an island. Indeed, we had just showed precisely that in a publication prior to our Brief Communication; using functional and structural imaging we identified how extensively different divisions of the insula (including posterior) connect and communicate to other brain regions – even at rest (Wiech et al., 2014b). Yet, it is a fact that these other important brain regions were not continuously activated and involved in tracking a changing pain experience in the same fashion as we found was the case for the dpIns. We believe this was not for reasons of being underpowered (see below for correction to misunderstandings). In fact, even when lowering the statistical threshold dramatically, as we reported in the supplementary material of the paper but perhaps was missed, we did not find other areas significantly tracking the pain intensity ratings over the nearly two hour experiment. However, that result does not in itself nullify the relevance of these other brain regions for being necessary to bring about a complete painful experience, as we’ve shown many times (see refs above as examples). Indeed, we even showed that fact in the paper itself, as many regions were observed to have significant changes in cerebral blood flow (CBF) during the comparison of ‘peak pain’ to ‘rest’ [see: Supplementary Figure 2 in (Segerdahl et al., 2015)]. Perhaps Davis and colleagues wanted to see these supplementary data interrogated further – to see how the various regions we observed to be active during peak pain relative to baseline relate to the dpIns result? That withstanding, we’re left with how to best interpret and semantically describe our highly significant and robust result regarding the dpIns; a discussion that this platform provides a forum for.\n\nLet us now take each criticism in turn:\n\n1) Experimental method\n\nUnfortunately, Davis and colleagues provide an incorrect assessment of the imaging modality used, the analysis tools applied and the details that are actually reported in the manuscript. The following offers important corrections and clarifications to their review:\n\na. Multi-TI pCASL FMRI & absolute CBF quantification\n\nAdvances in ASL methods over recent years have been tremendous and are actually affording widespread penetration for clinical benefit (e.g. Alsop et al., 2014; Chappell et al., 2012; Kelly et al., 2013; Mezue et al., 2014; Okell et al., 2013). Davis and colleagues blur distinct, well described MRI methods and PET together into one way of 'measuring regional CBF'. Unfortunately, this ignores really important and different features of the various methods available, including the one used in our study, and the highly flexible paradigm designs that ASL based methods now afford neuroscience (compared to positron-emission tomography (PET)) to probe brain activity in very different ways and with varying degrees of improved signal-to-noise (SNR).\n\nAs we described in the paper, relative CBF (rCBF) volumes were actually collected approximately every 8s (not at 45s intervals as Davis and colleagues state). This is directly analogous to the ASL methods cited by Davis and colleagues. Further, the authors’ assertion that single-TI (or PET) is better than multi-TI ASL methods used is incorrect and unsupported when it comes to robustness of the measured blood flow and hence SNR. Rather, and as referenced in our paper, a key recognized benefit of the multi-TI approach is that it enables one to calculate absolute CBF (absCBF) at each voxel of the whole brain volume by incorporating each rCBF volume collected (i.e. at every 8s, at each TI) for the nearly two hour pain experience (Chappell et al., 2009; Mezue et al., 2014; Okell et al., 2013). By collecting each rCBF volume at different TIs, we are additionally able to incorporate information about changes in the time at which arterial signal arrives (i.e. arrival time = AAT) at a given voxel: a feature that is known to vary across space (i.e. all regions of the brain are not perfused instantaneously) and time (i.e. the dynamics of how perfusion changes over time during slowly-fluctuating pain is not known). This is a problem in the single-TI ASL studies quoted and accounting for this variability has been shown to maximize the robustness and reliability of measuring CBF over time within- and between-subjects (e.g. Mezue et al., 2014). In short, the multi-TI ASL method we used does not suffer from reduced temporal SNR efficiency compared to single-TI ASL (or worse, PET). In fact quite the opposite; using a multi-TI approach allows the ASL signal to be sampled around its peak, improving the SNR efficiency compared to single-TI ASL methods where a long TI must be used to reduce sensitivity to arterial transit time at which point the ASL signal has decayed considerably (Alsop et al., 2014).\n\nTo illustrate the robustness of our ASL data further, consider the temporal resolution of the data collection in our study versus the study by Owen et al. (2010) (cited by Davis and colleagues). While our behavioural data were collected at regular intervals comparable to what was used by Owen et al. (2010) [i.e. 21 behavioural data points in our study vs. 25 in Owens et al., 2010], in our study each ASL data point plotted in Figure 2 of the manuscript is far more rigorously sampled with more than double the number of rCBF time points being used to quantify CBF over the course of the full experimental paradigm. When Davis and colleagues assert that the study is significantly underpowered because it is only sparsely sampling brain activity once every 45s - this reflects a significant misunderstanding of the methods used in the study and the SNR benefits of our approach.\n\nIt is true that the temporal resolution of this method is longer than single-TI approaches. Any fast changes in CBF during this period will not be accurately represented in the output data. But that is not relevant here, as our experimental design was to observe brain activity linked to slowly changing sensory states that evolve over nearly two hours and were controlled by us – so the parameters of our measurement are appropriate for (indeed far faster than) the changing behavior being investigated (behavioural ratings were collected every 2.5 minutes, remember). Any variations that are occurring faster than the temporal resolution are attributed as noise in the fitting process, are represented in the variance maps that are utilized in subsequent levels of analyses and are therefore unlikely to bias the results in any significant way. We propagated all uncertainty in the CBF estimates through each stage of the analysis such that it was incorporated into the group level effects reported in the paper. In no way are we failing to observe potentially meaningful changes in CBF nor are we merely disregarding potential sources of variation that may be occurring on this time scale.\n\nWe did detail all these aspects in the relevant method sections and we were careful to highlight specific references to recent work that further details how the method is used and what the significant benefits are for such neuroscience applications. Please refer to the following references for further insight into these developments (Chappell et al., 2009; Mezue et al., 2014; Okell et al., 2013).\n\nb. FMRIB Software Library\n\nAll FMRIB Software Library (FSL) tools used in the paper are well-validated, publically available, ubiquitously employed across a range of experimental applications in over 1000 laboratories worldwide and are highly cited and cross-referenced. All brain activity is reported as significant using standardized and accepted criteria (e.g. voxels with supra-threshold statistics registered to a Montreal Neurological Institute (MNI)-standardized brain using standard space coordinates in FSL). The challenge of localizing group mean statistical maps is common to all fMRI studies. In the case of imaging healthy adults this is easily mitigated by employing FSL tools like Boundary-Based Registration (BBR) and FMRIBs Nonlinear Image Registration Tool (FNIRT) and defining activation clusters using Mixed Effects (z>2.3, p<0.01 cluster corrected) as Segerdahl et al. (2015) did. It is difficult to address Davis and colleagues’ criticisms of these approaches and the validity of our result that used globally accepted criteria without discrediting decades of fMRI imaging development, analysis optimization, and of course, the various publications by many pain imaging authors who use these very same FSL tools and criteria in their own publications. We confirmed the anatomical location of our results using the Harvard-Oxford cortical atlas in FSL that reported the greatest probability of voxels within the identified cluster of activity as residing in the insula; an observation that Davis and colleagues counter by simply referencing a different probabilistic-based atlas (Juelich). We selected the Harvard-Oxford atlas because it more comprehensively and accurately maps the entire insular cortex compared to the Juelich Atlas and therefore provides consistency with mapping of activity in other regions of the insula (i.e. the mid and anterior subdivisions).\n\nOur finding was further validated by cross-referencing with other reported studies exploring somatotopy to nociceptive inputs within this insula region and other studies in humans performing direct cortical stimulation in this region (Figure 3 in Segerdahl et al., 2015). This approach is valid and arguably, we think, goes beyond a comparison with studies that define activity within gross anatomical structures (e.g. ‘insula’, ‘posterior insula’ ‘cingulate’, ‘frontal cortex’, etc.), as is the case for the references (2,3,4,5,6,12) cited within the paper by Davis and colleagues. Excellent and relevant though these studies are, it is interesting to note that the reported coordinate of peak-activities from all these studies don’t overlap with our peak coordinate and is found to be proximal to the dpIns cluster when painful stimulation is used. We should also say though that even if they did overlap it could be argued that distinct specificity might still reside within that one cluster region considering the hundreds of thousands of neurons present – this vexing problem is common to many functional imaging studies and of course was one of the original motivations for employing multivariate pattern analyses, so that specificity to different tasks within ‘one blob’ of cluster activity could be better identified (e.g. Haynes, 2015). Also, it should probably be noted that earlier work of ours comparing the spatial specificity of neurally-derived functional activation as determined from BOLD data versus ASL highlighted ASL’s pre-eminence over BOLD – the latter being biased by local draining veins in some instances (Tjandra et al., 2005).\n\nHowever, Davis and colleagues do highlight a very meaningful topic of discussion about how best to map function across controversial boundary zones where two anatomically distinct regions align, as in the case of SII and dpIns. Although it was not possible to detail this important issue within the very strict word and reference limit of a Brief Communication, we did include references to discussions about the posterior insula medial operculum region (PIMO) in order to contextualize our results within the discussion regarding the possibility for nociceptive coding within a sub-region of the posterior insula (see: Craig, 2014; Evrard et al., 2014; Garcia-Larrea, 2012).\n\n2) Control experiment\n\nThe control experiment was completed on 12 participants (not seven as stated by Davis and colleagues), and this misreading forms another basis for their comments about the statistical power of the study. For clarity, the pain paradigm was done on 17 subjects. None of the subjects reported the vibration as painful and the group mean saliency rating for the vibration was 3.12 (s.e.m = 0.265) out of 10 [see: Supplementary Figure 1 in (Segerdahl et al., 2015)].). Power calculations suggest that a minimum of 12 subjects were necessary for group level statistical tests of absCBF data collected during a continuous sensory-motor task. This was clearly defined in both the Methods Checklist and the relevant methods sections of the paper.\n\nHowever, Davis and colleagues list a few worthy points about the control experiment that we would like to discuss in more detail here. Unfortunately, there was not adequate space to fully report the extensive preliminary work we conducted in preparation for this study that explores these very issues. First, the control experiment should be a stimulus that is as relevant, captivating and as engaging as the pain but without ever being painful (or unpleasant/pleasurable) for approximately 2hrs. Obviously, the suggestion to use an innocuous warm stimulus is sensible. Unfortunately, in practice it fails to fulfill these criteria (i.e. subjects report that it is not salient and not readily perceptible after a short period of time [unpublished pilot data; Psychophysical study currently ongoing]). In this regard, vibration improves upon these limitations, as it has the salience Davis and colleagues recognize as important and is perceptible, so long as the duration of the constant stimulation is limited to the timeframe we scanned in the study. We note, even the work by Owen et al. (2010) suffers from having the 'control' infusion induce pain and therefore not being completely innocuous.\n\nTherefore, it was not immediately evident how to compare pain and vibrotactile related effects directly given the nature of the experimental paradigms used.\n\nInstead, the design was established to compare the anatomical locations of CBF changes triggered by innocuous versus noxious tonic input where saliency is closely matched. However, as Davis et al. correctly point out, a direct comparison of the two conditions is a standard statistical approach used in other imaging modalities and paradigms, so we agree it could be informative for this discussion. Therefore, we’ve performed a comparison of the group level effects of the correlation between CBF and intensity ratings during pain versus the correlation between CBF and intensity ratings during vibration - where all experimental variables are closely matched (i.e. 12 subjects in each group (randomly selected from the pain cohort), 14 minutes of absolute CBF data included, group mean intensity is 3 out of 10 for both modalities).\n\nAt the group level, the unpaired t-test across conditions shows that the correlation between pain and intensity ratings is localised to the contralateral dpIns (compared to the vibration-related effects); whilst a conjunction analysis confirms that there is no significant overlap between the group mean effects of either condition here (Randomise: voxelwise, p<0.05). The authors’ logic dictates that increasing the N or lengthening the vibration task to boost SNR would potentially ‘reveal’ activity related to vibration within the dpIns region. However, this logic might reversibly also predict that the subthreshold SII activity seen in the vibration results would be now shown in the pain intensity tracking result (to a suprathreshold level perhaps)- yet this does not occur even with 17 subjects and including all data points. Therefore, while we acknowledge that thermosensation might well be represented within the dpIns – ongoing work in the laboratory that we look forward to sharing (accepting the saliency issue is still a problem here) – we hope this has helped clarify the parameters used for the control task and issues surrounding its use within these more complex paradigm designs. As an aside, Davis and colleagues' reference to early PET studies of vibration (refs: 4,5) aren't precisely relevant to the current discussion as the aim of our study was to investigate unique correlates of perception not to report what the main effects of different stimulus conditions relative to baseline are.\n\n3) Interpretation of the data\n\nDavis and colleagues criticized the discussion of our findings in the light of other literature regarding insula activity, saying that we missed important references and broader discussions. Alas, we would have very much liked to include that extensive and informative literature. However, we remind Davis and colleagues (and the readership) that this was not possible within the very strict requirements for a Brief Communication where we’re only allowed 20 references and 1200 words. Further, as Davis and colleagues are aware, much of our own published work has (ironically) focused on characterizing and dissecting the roles that different insula divisions play in the multifactorial experience that is acute and chronic pain (e.g. Baumgärtner et al., 2010; Brooks et al., 2005; Brooks & Tracey, 2007; Duff et al., 2015; Fairhurst et al., 2012; Ploghaus et al., 1999; Ploner et al., 2010; Ploner et al., 2011; Schweinhardt et al., 2006; Wanigasekera et al., 2012; Wiech et al., 2010; Wiech et al., 2014a; Wiech et al., 2014b; Wise et al., 2002) alongside the somatotopic studies quoted in the original paper) – nearly all of these references we couldn’t discuss or quote either due to space constraints despite their absolute relevance. Therefore, with full awareness of the literature cited alongside our own substantial contributions in understanding the complex role that the insula plays in pain mechanisms, we believe that we carefully interpreted our results while drawing upon that corpus of knowledge in the limited words allowed. We don’t believe our data undermines Craig's novel and important theories about interoception and the 'sentient self' - mechanisms that are unlikely to be anchored solely to the posterior insula (as asserted) and instead necessitate dynamic interaction with other regions such as the anterior insula, as Craig has stated (Craig, 2015).\n\nWe agree that a more insightful discussion centers on interesting recent work that attempts to rigorously interrogate the pain specificity of statistics maps by using multivariate pattern analysis (MVPA), as we and others have been doing (see: Brodersen et al., 2012; Brown et al., 2011; Duff et al., 2015; Marquand et al., 2010; Wager, 2015; Wager et al., 2013). Recent work by Woo et al. (2014) employed this approach to derive unique patterns of activation in regions like the dpIns that are specific to physical but not emotional pain (previously and wrongly inferred to be the ‘same’ because of overlapping BOLD statistics maps, as discussed above). Interestingly, their data support the interpretation that a region like the dpIns (see Figure 3 of Woo et al., 2014) has an identifiable role in coding attributes about nociceptive driven painful experiences.\n\nIn keeping with basic principles of good experimental design, we formulated our hypothesis to test one theory about which brain regions track the intensity of a slowly varying experience of pain in response to a carefully controlled change in peripheral nociceptive input. Understanding which brain regions are coding this information is of interest, we believe. The affective coding of that stimulus is highly relevant too - processing that is likely to engage higher-level pain network processing secondary to the more initial intensity coding of the input, as nicely described in a recent review (Garcia-Larrea & Peyron, 2013).\n\n\nConclusion\n\nIn closing, we would like to note that imaging tonic, slowly-fluctuating and spontaneous pain states in healthy controls (and in patients) is a small, emerging field within the pain neuroimaging community with very few laboratories to date using the method. In part, this is due to the difficulty in imaging the brain during pain experiences that are analogous to what patients are suffering from, alongside limitations in accessing state-of-the-art acquisition methods for measuring blood flow using ASL. It is difficult to induce a tonic pain state in volunteers that is robust, reliable, reproducible, easily controlled and is not invasive or risks permanent skin damage or infection. A number of important ASL/pain studies have been done by colleagues and us that have laid an excellent basis for the future (e.g. Howard et al., 2011; Howard et al., 2012; Liu et al., 2013; Owen et al., 2008; Owen et al., 2010; Owen et al., 2012; Maleki et al., 2013; Stagg et al., 2013; Segerdahl et al., 2012; Tracey & Johns, 2010; Tjandra et al., 2005 – alongside others quoted in the paper and embedded methods references). Neither should we ignore the foundational imaging physics work done in developing quantitative cerebral perfusion imaging (i.e. ASL) over the past several decades that have provided such opportunities for clinical and basic neuroscience (e.g. Detre & Aslop, 1999; Davies & Jezzard, 2003; Figueiredo et al., 2005; Guo & Wong, 2015; MacIntosh et al., 2008; Mutsaerts et al., 2015; Teeuwisse et al., 2014; Wang et al., 2003; Wong et al., 2006; Wong, 2007).\n\nWe hope our response informs the F1000Research readership about exciting developments in the field of novel ASL applications, underscores the robustness of our paradigm and reliability of the analyses used to quantify CBF dynamics; and lastly highlights the importance of evolving conceptually away from antiquated pain imaging dogmas (derived mostly from reverse inference-based statistical testing) about single “pain spots”. In that regard, we believe Davis and colleagues agree with us. We hold that if a brain region is continuously and metabolically active in response to a slowly varying peripheral nociceptive input and further that its degree of activity correlates with a concomitant changing subjective experience of pain intensity lasting over several hours then it must be important. We can argue semantics, but it seems to us that describing it as “subserving a fundamental role in pain” (does not mean only region) and potentially being an area that might be responsive only to nociceptive inputs that produce painful experiences – i.e. nociceptive/pain specific (alongside other areas to be yet possibly identified) best describes the data. We welcome alternative interpretations or descriptions of our results, having defended their reliability. We are of course eager to interrogate and explore the meaning of this result further within ongoing preclinical and clinical studies currently underway. We look forward to sharing these results with the wider community in due course.",
"appendix": "Author contributions\n\n\n\nAS, MM and IT prepared the first draft of the manuscript. All authors (AS, MM, TK, JF and IT) 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 no grants were involved in supporting this work.\n\n\nReferences\n\nAlsop DC, Detre JA, Golay X, et al.: Recommended implementation of arterial spin-labeled perfusion MRI for clinical applications: A consensus of the ISMRM perfusion study group and the European consortium for ASL in dementia. Magn Reson Med. 2014; 73(1): 102–116. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaumgärtner U, Iannetti GD, Zambreanu L, et al.: Multiple somatotopic representations of heat and mechanical pain in the operculo-insular cortex: a high-resolution fMRI study. J Neurophysiol. 2010; 104(5): 2863–72. 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}
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[
{
"id": "11308",
"date": "25 Nov 2015",
"name": "Arthur Craig",
"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\nSegerdahl and colleagues have replied clearly and thoughtfully to the issues raised by Davis et al. regarding the short paper in Nature Neuroscience. Their response seems collegial in tone, granting trans-atlantic cultural differences (e.g., the imperative “remember” might be received less positively in America than in Britain). Further, they recognize the benefits of a constructive discussion that is open to all, rather than admonishing the commentators for overlooking key statements and details (one of which was buried near the end of the legend of Supplementary Figure 2). On the whole, though, the discussion between these two camps resembles recurring disagreements between “splitters” and “lumpers” in pain research (e.g., see Berkley, 1980) as well as, more generally, the persistent controversy about functional localization in brain research. Nevertheless, a major difference between these two sides is that one group incorporates certain knowledge from work in the monkey, which to my mind is fundamentally significant.Davis and colleagues took issue mainly with the impression that Segerdahl et al. had proposed that the dorsal posterior insula (dpIns) is a “pain-specific” locus in human. That assertion is intimated in the initial portions of the paper, however the reply makes clear that the central issue is rather, why did the Multi-TI pCASL method reveal such “highly significant and robust” activation only in the dpIns after whole-brain correlation with subjective ratings of tonic “pain intensity” elicited by thermal modulation of post-capsaicin cutaneous hyperalgesia. This result is surprising because considerable prior evidence predicts that subjective feelings are associated with strong activation in the anterior insula and other sites (for review, see Ch. 7 in Craig 20152); apparently, that’s the reason that Segerdahl et al. submitted the short report instead of the full-length manuscript that they could (and perhaps should) have written.Davis et al. suggested that such a singular result could have resulted from faulty methodology, such as inadequate temporal sampling or underpowered statistics, but Segerdahl et al. convincingly rule out those two possibilities. Davis et al. rightfully point out that the “control” vibration results are inadequate to support a claim of pain specificity, although the “robust vibrotactile [PET] activation” they cite actually occurred in the mid-insula (see Ch. 6 in Craig 20152). In response, Segerdahl et al. claim that an innocuous thermal control stimulus, though desirable, would not maintain salience; however, imaging evidence suggests that an oscillating thermal stimulus would be salient and motivating (Hua et al. 2005; see Ch. 2 in Craig 20152). Davis et al. suggested also that inadequate anatomical registration of the ensemble of brain images could have obscured the expected activation in the anterior insula that had been observed in similar experiments by Henderson et al. (2007) and Owen et al. (2010). But, Segerdahl et al. rectified the misunderstanding of their anatomical protocol and explained the co-localization of the activation they observed in dpIns with the comparable results from several prior studies that had used different techniques (see Fig. 3 of the original paper). Indeed, the very precise localization of activation in the most dorsal extent of the posterior insula matches exactly the location of the terminus of ascending lamina I nociceptive-specific activity, which was demonstrated in the fundus of the superior limiting sulcus in the macaque monkey by the paper they cite (Craig 20146; see Ch. 5 in Craig 20152). Further, the antero-posterior somatotopic gradient identified in the monkey fits with the topographic order reported in the studies that Segerdahl et al. collated in Fig.3 and also with the posterior location of the “very specific ‘spot’” that they found.Lastly, Davis et al. challenged the interpretation of any activation locus as “pain-specific” and suggested that “the dpIns likely is involved in pain but overall is a non-specific perceptual way-station,” thereby ignoring the evidence in the monkey. Segerdahl et al. naturally replied that the evidence regarding multifactorial pain-related activation includes their “own substantial contributions,” meanwhile acknowledging the new conceptual contributions by the Multi-Voxel Pattern Analysis (MVPA) study of Wager et al. (2013) and by the correlative functional connectivity patterns found in individual subjects by Cheng et al. (2015) (from Davis’ lab). Yet, on one hand they cite attributions of higher-level cognitive and affective coding to the anterior insula in reviews by Garcia-Larrea & Peyron (2013) and Craig (20152), and on the other they point out that the new MVPA findings “support the interpretation that a region like the dpIns (see Figure 3 of Woo et al. 2014) has an identifiable role in coding attributes about nociceptive driven painful experiences.” Hmm. Does that mean modality-specific? noci-specific? algosity (see Greenspan’s comments)? a “pain switch”? or perhaps, an embedded cluster of neurons? They cannot say. Instead, Segerdahl et al. reiterate and expand the carefully worded concluding statements of their original paper, saying: “A growing body of literature suggests that a subsection of the posterior insula is both anatomically and functionally well suited to serve a primary and fundamental role in pain processing.” And “Using … a newly developed procedure and analysis, we were able to identify the dpIns as subserving a fundamental role in pain and the likely human homolog of the nociceptive region identified from animal studies. Future work targeting dpIns activity might provide a window to explore fundamental mechanisms related to how pain emerges from nociception as well.” These ideas and words remind me of Chapters 5 and 6 of my book and I must certainly agree.OK, so far, so good. Yet, the central issue remains: why did their rigorous quantitative analysis demonstrate robust activation only in the contralateral dpIns? Now, I will make a suggestion.The published evidence in the monkey tells us that a somatotopically-organized region of posterolateral thalamus that contains only nociceptive neurons projects topographically to the dpIns (Craig, 20152; 20146). The thalamic region includes both nociceptive-specific (NS) and polymodal nociceptive (HPC for heat, pinch, and cold) neurons that receive monosynaptic input from lamina I spinothalamic neurons; an adjacent sub-region of thalamus similarly relays thermoreceptive-specific (COOL) activity to an adjacent sub-region of the dpIns (as depicted in Fig. 12 in Craig 20152). Both classes of nociceptive neurons in the monkey respond to topical capsaicin and become sensitized, with trajectories that parallel human pain reports (unpublished obs.). The HPC neurons constitute the only sensory channel that can quantitatively explain human reports of temporally summating second (burning) pain in response to repeated brief-contact heat stimulation (Craig, 2004) as well as pain reports in response to the thermal grill (Ch. 3 in Craig, 20152); we can posit that they are directly responsible for the pain reports in the study of Segerdahl et al. Thus, the terminus of the HPC sensory channel in the dpIns could alone explain the activation focus they observed. Particular components of the parallel NS (first, sharp pain) sensory channel most likely would also be involved.However, the anomalous innocuous cold sensitivity of HPC neurons and the evidence for the involvement of some HPC neurons in signaling muscle work, vascular distension, and tissue immune and metabolic conditions all indicate that this sensory channel is not a binary, pain-specific signal but rather has a much broader role in interoception (Craig 20152). Along with sensory channels representing many other specific and non-specific bodily sensations, such as cool, warm, itch, sensual touch, taste, hunger, and thirst, the dpIns is thus proposed to contain interoceptive cortex. Both Davis et al. and Segerdahl et al. alluded to this perspective and also cited the evidence for thermoreceptive-specific activity in the dpIns, but Davis et al. seemingly regarded this knowledge as support for the assertion that the dpIns does not have a specific role in pain, which is a misrepresentation.Yet, these considerations do not resolve the central issue. For instance, the HPC channel is also relayed to area 3a in the primary sensorimotor area (Vierck et al., 2013; Craig, 20152; 20146), but that region was not activated. More importantly, neither the mid-insula nor the anterior insula displayed activation, and both are normally associated with subjective reports of immediate feelings of pain.To my mind, the key lies in the instructions to the individuals who participated in the experiment of Segerdahl et al., which can suggest what their brains were doing. Unfortunately, the exact instructions were not reported; we’re told only that they were asked to report the “pain intensity” at certain times and otherwise to remain focused on a fixation cross. We can surmise that, in order to perform as requested (and presumably earn a monetary reward), the participants (1) concentrated their attention on the intensity of sensation that originated from the stimulus site, but otherwise (2) ignored the ongoing feeling of unpleasantness as much as possible. Our brains are rather good at both tasks; first, we regard pain as a discriminative sensory capacity because we can focus our attention on the specific characteristics and the specific origin of the sensation, and second, as a homeostatic emotion that is crucial for survival, like hunger, pain has top motivational priority most of the time but can be inhibited when other emotional needs or goals are more urgent. Based on the available evidence for the neural correlates of focused attention and emotional feelings, that suggests (1) that focal activity at the somatotopically appropriate site in the HPC map within interoceptive cortex was enhanced by the center-surround (sombrero-like) modulation of endogenous attentional processes and (2) that activity that normally engenders the feeling of unpleasantness or pain was suppressed in interoceptive integration mechanisms in the mid-insula and anterior insula (and in related regions, such as area 3a and area 24c in the anterior mid-cingulate).To my mind, the mechanisms I propose offer a potential explanation for the singular observations of Segerdahl et al. They are incomplete – for instance, focal activation was not reported in the posterolateral thalamus and activity supporting the behavior of reporting the discriminative readout is missing – nonetheless, they provide practical inroads for future experiments. Most importantly, they incorporate knowledge about the crucial and specific role of dpIns in nociception and pain, and they build on the significant advance that the work of Segerdahl et al. has provided.",
"responses": []
},
{
"id": "11550",
"date": "15 Dec 2015",
"name": "Luis Garcia-Larrea",
"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 ‘response paper’, Segerdahl and colleagues endeavour to correct what they consider “important misunderstandings” made by Davis et al.1 in their target article. Most of the points raised by Davis and colleagues are of technical nature, and question the validity of the methods used by Segerdahl et al. to reach their conclusions. Here Segerdahl et al. thoroughly describe the methods employed, correct misunderstandings, and on this basis judge that the extrapolation and inferences made by Davis and colleagues are not consistent with their target report2. Also, they take the opportunity to expand on a number of conceptual points that were made.The response is broadly divided in two parts, respectively technical and interpretative. From the technical point of view, they rebut a number of points advanced in the Davis et al.’s paper, in particular their critique of the multi-delay ASL technique, the excessively long sampling intervals and the small number of control subjects, all of them contributing to a supposed underpower of the experiment. Segerdahl et al. vigorously correct several facts that seem to have been misunderstood in the Davis et al.’s comment, in particular that brain MRI volumes were acquired every 8 seconds –and not 45–, and that the control experiment was completed on 12 participants –not 7 as implied in Davis et al. These are important particulars that could have undermined the reliability of the MRI data under question; the clarification appears appropriate, and lends support to the reliability of Segerdahl et al.’s original results. I am unable to enter the highly technical discussion on the relative merits of uni- or multi-TI ASL methods. While the arguments advanced by Segerdahl to defend the superiority of multi-TI ASL appear solid, I must deplore that they are mostly based on their own productions, which limits generalisation. Reproducibility of functional imaging (or rather the lack of it) is a crucial problem3,4 and quoting external sources on multi-TI procedures would have strengthened the authors’ argument.I support and share one of the main notions in the response of Segerdahl et al., namely that they never claimed to have determined a sole ‘pain centre’, and that being fundamental for pain experience, as they wrote, does not equate being the sole region responsible for such experience. It seems clear to me that identifying a role for dorsal posterior insula (dpIns) in tracking pain intensity does not automatically promote the concept of a ‘single pain spot’. I would just add a nuance: while we have abundant experimental data of the dpIns coding properties under physiological conditions, this may not hold for neuropathic states where the dorsal insula is injured of highly deafferented, in which case the perceived intensity of pain may be coded by other areas –possibly including the insular cortex ipsilateral to the pain, which becomes disinhibited5. Also, and probably because of extensive cortical plasticity, long-standing destruction of both posterior insulae has been shown to be compatible with the ability to continuously rate the moment-to-moment level of pain6. Thus, the ‘fundamental’ role of the postero-dorsal insula tagged by Segerdahl et al. may be transferred to other networks in pathological conditions. The authors acknowledge the importance of one of the criticisms from Davis et al.’s comments, namely the lack of direct statistical comparison between pain and innocuous stimulation. Indeed, showing that one region exhibits significant activity changes in one condition (pain) and not in other (vibration) does not allow affirming that the activity of both regions would have differed significantly had they been compared directly. The error of comparing significance levels without directly testing their difference is said to be especially common in the neuroimaging literature7,8. To respond to this criticism, now Segerdahl and colleagues performed a comparison of the correlation between CBF and intensity ratings during pain versus the correlation CBF / intensity ratings during vibration. This approach seems fair to me, although the formulation of the results remains ambiguous, and I remain unsure whether there was a significant difference between the two correlations, or a region X condition significant interaction –the straightforward result I should have expected. This could have been clarified further.The notion of a gradient in the processing of noxious inputs from the lateral operculum (SII) to its medial portion (OP2) and posterior dorsal insula (dpIns) appears well backed by the literature. For instance, the sites where electrical stimulation induce non-noxious thermal sensations in humans predominate in the inner operculum (OP2) while those evoking clear pain tend to concentrate in the posterior insula9. Also, while medial opercular sites responded to barely perceptive thermal increases, the posterior insular response only emerged clearly at noxious levels10. Such a gradient is difficult to explain on a simple afferent connectivity basis, as the primate’s spinothalamic system projects in similar proportions to the granular insula and its adjacent operculum11. Labelled lines entailing a significant predominance of nociceptive-specific afferents in the posterior insula might contribute an explanation12, but this is difficult to reconcile with classical data showing the predominance of low threshold units in this same area13'14. Alternatively, functional differences between dpIns and operculum might reflect network properties, partially independent from those of isolated neurons. For instance, the posterior insula has a more extended connectivity pattern than the opercular region and S2 proper; the massive amount of afferent input to the insula may entail a greater background activity than in the operculum, hence obstructing the precise encoding of low-energy stimuli barely emerging from background noise. This might explain why posterior insula networks are biased toward encoding nociception despite the fact that approximately 70% of primate insular neurons can respond to non-noxious somatic inputs too13, and that intracranial recordings demonstrate posterior insular responses to non-noxious stimuli in humans15. Such a bias, however, reflects a progressive gradient, and not a clearcut functional cleavage, and failure to acknowledge this may lead us toward dangerous neo-phrenology. The suprasylvian operculum can also encode stimulus intensity in the painful ranges10, and the dpIns can also respond to non-noxious stimuli16.In conclusion, I believe the authors make clear their case that any notion of a ‘single pain centre’ in the human brain is absent from their line of thinking –and absent from the conclusions in their target paper. They also point out various methodological points that seem to have been misunderstood in the Davis et al.’s comment, while accepting the importance of others, such as the lack of direct statistical comparison between conditions. The important role we can ascribe to the dpIns should not be confounded with an absolute specificity of this area –especially when it comes to neuropathic lesions. And importantly, Segerdahl et al. acknowledge that the comments from Davis and her colleagues have triggered a most interesting, valuable and enlightening discussion, which should help many readers to get insight into current controversies on pain perception. To me, this is exactly how science advances.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1207
|
https://f1000research.com/articles/4-1200/v1
|
02 Nov 15
|
{
"type": "Review",
"title": "Advances in the understanding of delayed cerebral ischaemia after aneurysmal subarachnoid haemorrhage",
"authors": [
"Liam Flynn",
"Peter Andrews",
"Peter Andrews"
],
"abstract": "Delayed cerebral ischaemia has been described as the single most important cause of morbidity and mortality in patients who survive the initial aneurysmal subarachnoid haemorrhage. Our understanding of the pathophysiology of delayed cerebral ischaemia is meagre at best and the calcium channel blocker nimodipine remains the only intervention to consistently improve functional outcome after aneurysmal subarachnoid haemorrhage. There is substantial evidence to support cerebral vessel narrowing as a causative factor in delayed cerebral ischaemia, but contemporary research demonstrating improvements in vessel narrowing has failed to show improved functional outcomes. This has encouraged researchers to investigate other potential causes of delayed cerebral ischaemia, such as early brain injury, microthrombosis, and cortical spreading depolarisation. Adherence to a common definition of delayed cerebral ischaemia is needed in order to allow easier assessment of studies using multiple different terms. Furthermore, improved recognition of delayed cerebral ischaemia would not only allow for faster treatment but also better assessment of interventions. Finally, understanding nimodipine’s mechanism of action may allow us to develop similar agents with improved efficacy.",
"keywords": [
"Cerebral ischaemia",
"subarachnoid",
"haemorrhage"
],
"content": "Introduction\n\nAneurysmal subarachnoid haemorrhage (aSAH) has an incidence of 6–11 per 100,000 people per year and accounts for only 5% of all strokes1–4. Despite this, aSAH is the cause of one third of all stroke-related years of potential life lost before the age of 655. Approximately 70% of all people with aSAH will either die or require help with activities of daily living at six months after the initial injury5. The mean age of onset of aSAH is 55 years and, when combined with its poor morbidity and mortality, it causes an enormous socioeconomic burden6,7. The significant morbidity attached to aSAH can be attributed to rebleeding, delayed cerebral ischaemia (DCI), hydrocephalus, and other medical complications, despite successful treatment of the ruptured aneurysm. Of these complications, DCI is the most important cause of morbidity and mortality in patients who survive the ruptured aneurysm5,8,9. Between days 3 and 10 after the initial aSAH, 30–40% of patients will suffer DCI and half of these will have a poor outcome5,10,11.\n\nOur understanding of DCI is meagre at best. Conventionally, DCI was thought of as a neurological deficit observed at least three days after aSAH with radiological confirmation of large vessel narrowing and was often termed “vasospasm”. However, more contemporary articles question whether the relationship between angiographic cerebral vessel narrowing and neurological outcome is associative rather than causative and have highlighted the possibility of a multifactorial aetiology12–15. One of the problems with the disease and research surrounding DCI is the terminology applied. Terms include DCI, delayed ischaemic neurological deficit (DIND), delayed neurological deficit, secondary cerebral ischaemia, and vasospasm. In 2010, a consensus statement was issued defining DCI as a focal neurological impairment or decrease of ≥2 points on the Glasgow Coma Scale which lasts for ≥1 hour, is not apparent immediately after aneurysm occlusion, and cannot be attributed to other causes by means of clinical assessment, blood tests, or imaging16. The Neurocritical Care Society’s consensus definition was similar for DCI and also defined vasospasm as radiological evidence of cerebral vessel narrowing with corresponding neurology17.\n\n\nCerebral artery narrowing\n\nOver six decades ago, cerebral vessel narrowing was demonstrated by angiography after aSAH18. A decade later, a link was found between cerebral vessel narrowing and focal neurology19. Then in the late 1970s, it appeared that vessel narrowing was not only localised to the vascular territory of the aneurysmal bleed but also proportional to blood load and occurred between days 3 and 12 after the aSAH20,21. More contemporary authors found the onset of vessel narrowing started on day 3, was maximal by days 6–10, and lasted for up to two weeks22–24. The density, duration and volume of subarachnoid blood are key predictors of vessel narrowing21,25. Narrowing of cerebral arteries may cause a reduction in cerebral blood flow distal to the constricted vessel and contribute to secondary ischaemia26. The cause of vessel narrowing after aSAH is unclear but is thought to involve oxyhaemoglobin release, an inflammatory-mediated response, decreased nitric oxide levels, and an increased concentration of endothelin-1 (ET-1)14.\n\n\nOxyhaemoglobin\n\nOxyhaemaglobin induces cerebral artery vasoconstriction in vitro and in vivo in primates, which is not seen with methaemoglobin or bilirubin27–29. It is thought that oxyhaemoglobin decreases the production of prostacyclin and increases prostaglandin E2 in vessel walls, thereby causing vasoconstriction. It can also inhibit endothelial-dependent relaxation. The oxidation of oxyhaemoglobin to methaemoglobin, which occurs spontaneously, causes lipid peroxidation and vasoconstriction30. It is plausible that oxyhaemoglobin causes vasoconstriction by some or all of these mechanisms but attempts at modulating them have not completely reversed vessel narrowing or, importantly, improved outcomes.\n\n\nNitric oxide\n\nNitric oxide, which is responsible for the relaxation of vascular smooth muscle cells, appears to be depleted after aSAH. This may be due to a number of reasons, one of which is that nitric oxide is scavenged by haemoglobin, released during the breakdown of subarachnoid blood, due to nitric oxide’s high affinity for haemoglobin31,32. In addition to this, the production of nitric oxide may also be decreased due to the down-regulation of endothelial and neuronal nitric oxide synthase, which occurs in spastic arteries after aSAH33–35. Both of these mechanisms will lead to a decrease in the bioavailability of nitric oxide, which is then unable to counteract the effects of the vasoconstrictor ET-136. Furthermore, exogenous donors of nitric oxide, such as sodium nitroprusside and nitroglycerin, although associated with systemic side effects, have been shown to ameliorate cerebral artery narrowing37,38. In addition to the hypotension seen with these exogenous donors, there is also a concern that exposing nitric oxide to oxyhaemoglobin and deoxyhaemoglobin will lead to the formation of methaemoglobin, S-nitrosohaemoglobin and ferrous-nitrosyl-haemoglobin33. Interestingly, Kida et al. note in their comprehensive review that inhaled nitric oxide acts as a selective pulmonary vasodilator and avoids the hypotension seen with intravenous administration. Animal studies have demonstrated a reduction in ischaemia-reperfusion injuries after nitric oxide inhalation in extrapulmonary organs after cardiac injury. These have also been supported by proof-of-concept human trials39. The research discussed is used to support post-cardiac arrest ischaemia but Garry et al. also encourage further investigation of nitric oxide as a treatment of secondary brain injury in their review with reference to aSAH40.\n\n\nEndothelin\n\nEndothelin is key to maintaining the vascular tone of blood vessels, with ET-1 being the most potent endogenous activator of vasoconstriction. The amount of ET-1 in serum and plasma increases within minutes after the aSAH and peaks around days 3–4, the time at which DCI starts to occur. There also appears to be an excessive release of ET-1 by astrocytes around the time of onset of ischaemic symptoms41,42. ET-1 concentrations appear consistently elevated in patients with DCI. However, there are conflicting reports of ET-1 concentrations within the normal range in patients with radiological evidence of cerebral artery narrowing who do not have DCI43–45. Authors have questioned whether increased ET-1 marks ischaemic damage rather than arterial vessel narrowing in DCI14. Therefore, there are a number of different mechanisms that could be contributing to the arterial narrowing commonly seen after aSAH.\n\n\nAlpha calcitonin gene-related peptide\n\nAlpha calcitonin gene-related peptide (CGRP) is an endogenous neuropeptide and a potent vasodilator. CGRP exhibits its vasodilating properties by two mechanisms: one is nitric oxide and endothelium-dependent and the other is cyclic adenosine monophosphate mediated and is endothelium-independent46. Endogenous CGRP appears to be released, and is subsequently depleted, after aSAH to combat cerebral vasoconstriction which has led to the theory that exogenous CGRP may be beneficial in managing DCI47–49. Because CGRP can act independently of endothelial cells, which are morphologically damaged after aSAH, it may be successful in treating DCI. A number of animal studies and three human trials have investigated the effect of CGRP on cerebral arteries after aSAH. All animal studies appear to show either a reversal or improvement in cerebral artery narrowing46. The largest human trial, the European CGRP in aSAH study, demonstrated little improvement in morbidity or mortality from intravenous administration but noted that systemic side effects, such as hypotension, were limiting and suggested that intrathecal administration may be more beneficial, as endogenous CGRP acts on the abluminal side of vessel walls50. A trial investigating the effect of CGRP after intrathecal administration is still awaited.\n\n\nRadiological evidence\n\nAn often-cited argument against cerebral vasoconstriction being a causative factor of DCI is that, whilst up to 70% of patients demonstrate cerebral vessel narrowing on angiography, only 40% of these will manifest neurological deficits and only 30% develop DCI51–54. However, it must be acknowledged that even the consensus definition of DCI provided in the introduction has its limitations16. Patients with poor grade aSAH (World Federation of Neurosurgical Societies Grades IV and V), the group of patients most likely to develop DCI, are often sedated and mechanically ventilated and are particularly difficult to assess clinically55. Therefore, it is likely that we are under-diagnosing and under-treating DCI in this group of patients. Furthermore, it may be that the degree of large cerebral vessel narrowing does not correlate well with symptom severity26.\n\nFollowing a review of current tests available for the diagnosis of delayed cerebral ischaemia, Rodriguez et al. advise clinical examination and transcranial Doppler (TCD) in the screening and diagnosis of “vasospasm”. The authors reserve multi-modal magnetic resonance imaging (MRI) and computed tomography (CT) for specific situations, and acknowledge digital subtraction angiography (DSA) as the gold standard for diagnosis (Figure 1)56. Rabinstein et al. found that TCD and angiogram demonstrating cerebral vessel narrowing (termed vasospasm) only had a positive predictive value of 67% for cerebral infarction on CT8. We would expect this to be higher if cerebral vessel narrowing was the primary cause of DCI. Rates of cerebral infarction in patients with evidence of cerebral vessel narrowing range between 24 and 35% using CT57,58, but have been found to be as high as 81% in some studies using MRI59. In addition to this poor correlation between cerebral vessel narrowing and infarction, there is clinical evidence that up to 25% of delayed infarcts on CT are not in the same territory as the vessel narrowing, or are found in patients that did not demonstrate vessel narrowing at all60–62. Rabinstein et al. note that TCD and angiogram only agreed on the diagnosis of “vasospasm” in 73% of cases and so it could be that vessel narrowing simply wasn’t identified in patients who were later found to have evidence of infarcts on CT8. Despite these conflicting messages, clinical studies do report that those patients with radiological evidence of cerebral vessel narrowing are at greater risk of DCI62,63.\n\nNon-contrast CT scan of brain showing subarachnoid haemorrhage in classical “star sign” distribution with blood distributed along basal vessels.\n\nHerz et al. directly visualised pial artery constriction after application of blood or microtrauma to pial arteries in animal studies64. Further in vitro research has suggested that constriction of intraparenchymal arterioles occurs after aSAH and may contribute to DCI65. Maximal luminal narrowing has been seen between days 3 and 7 and repeated in vivo in mouse studies. The correlation between decreased regional cerebral blood flow and microvascular constriction appears stronger than that seen with large vessel narrowing65–67. Uhl et al. identified constriction of small vessels in surgical patients within the first 72 hours after aSAH by spectral imaging, and Pennings et al. later directly observed cerebral arterioles contracting after aSAH68,69. Therefore, it may be that vessel narrowing is consistently occurring with DCI but that we are not visualising it because it is microvascular and not readily visible on catheter angiography or TCD56.\n\nCT perfusion scanning (CTP) may provide haemodynamic evidence to support the diagnosis of DCI. Dankbaar et al. evaluated the diagnostic value of CTP for DCI and reported 84% sensitivity, 79% specificity, and 88% positive predictive values70. Sanelli et al. found that more CTP deficits occurred in patients with DCI than in those without71. Dankbaar et al. later suggested that patients with DCI exhibit worse cerebral perfusion (measured on CTP) than patients without DCI even before focal signs occurred. Encouragingly, they demonstrated partial recovery in areas of poor perfusion, suggesting that DCI could be partly reversible72. However, Killeen et al. concluded from their retrospective comparative study that CTP and DSA had similar test characteristics for identifying DCI in aSAH patients73.\n\n\nEndothelin-antagonists\n\nA shift in theory from cerebral vessel narrowing to a multifactorial aetiology occurred after the CONSCIOUS trials and a recent meta-analysis of pharmacological treatments for delayed cerebral ischaemia74–76. The meta-analysis demonstrated that, despite a reduction of cerebral vessel narrowing, no statistically significant effect on poor outcome was observed74. However, the authors note that the dissociation between a reduction in cerebral vessel narrowing but not poor outcomes could result from methodological problems, sample size, and insensitivity of outcome measures, in addition to a multifactorial aetiology of DCI. The CONSCIOUS trials were multicentre randomised controlled trials (RCT) investigating the effect of clazosentan, an endothelin-A (ET-A) antagonist, on “vasospasm” after aSAH. The first of these trials, CONSCIOUS-1, demonstrated that, despite a significant reduction in angiographic cerebral vessel narrowing, there was little evidence to support its use to improve morbidity and mortality and it was associated with increased rates of pulmonary complications, hypotension and anaemia76. CONSCIOUS-2 demonstrated no benefit from clazosentan in patients treated with surgical clipping, which led to the early termination of the trial75. Laban et al. recently published a review of animal studies investigating endothelin receptor antagonists after experimental aSAH and found no improvement in functional outcomes77. Perhaps more importantly, the review described insufficient animal data supporting endothelin receptor antagonists to warrant progression to a human trial. The authors also suggest that cerebral artery diameter, or “vasospasm”, is not a clinically relevant outcome measure in experimental aSAH studies77.\n\nThe example of clazosentan appears to provide evidence that cerebral artery narrowing is not the sole cause of DCI. However, there is conflicting evidence as more invasive methods of reducing vessel narrowing can improve outcomes (Figure 2). Kimball et al. reviewed 49 articles relating to interventional techniques to treat “vasospasm”. A total of 24 of the 27 publications (1,028 patients) reporting the use of transluminal balloon angioplasty noted an improvement in vessel diameter and neurological deficits. Twelve case series reported good angiographic and clinical results for patients who received papaverine (a vasodilator) administered approximate to the site of vessel narrowing78. Both techniques were associated with significant side effects and the quality of the studies was reported as very low to moderate (based upon the GRADE classification system)79. Nevertheless, the review does provide evidence that cerebral artery narrowing is likely to be strongly involved in the pathology of DCI.\n\nA and B: Anteroposterior (A) and lateral (B) angiograms of the left internal carotid artery demonstrate vessel narrowing at the level of the carotid siphon, the terminal internal carotid artery, the A1 segment of the anterior cerebral artery and the middle cerebral artery. C and D: Anteroposterior (C) and lateral (D) angiograms obtained after intra-arterial injection of nimodipine.\n\n\nNimodopine\n\nThe calcium channel antagonist nimodipine is the only proven intervention to reduce the incidence of DCI and improve outcomes after aSAH. Nimodopine was initially investigated as a vasodilator in the hope that it would aid post-ischaemic reperfusion, as it was thought that an increase in calcium in vascular smooth muscle cells led to “vasospasm”80,81. In 1989, the British Aneurysm Nimodipine Trial subsequently demonstrated a significant reduction in cerebral infarction rates and improved neurological outcomes at three months after aSAH82. A Cochrane review in 2007 concurred with these findings but noted that the supporting evidence was based mainly on one large study. This led to oral nimodipine becoming standard care for patients after aSAH83. Interestingly, the review found no statistically significant results to support the use of other calcium antagonists, magnesium sulphate, or intravenous administration of nimodipine.\n\nMagnesium sulphate is a non-competitive inhibitor of calcium channels and has vasodilatory and neuroprotective properties, similar to nimodipine. Hypomagnesaemia is common in patients after aSAH, appears to be proportional to the severity of the bleed, and is predictive of DCI84. Magnesium sulphate has also been shown to reduce cerebral artery narrowing and the size of ischaemic lesions after aSAH in animal models85. However, the Neurocritical Care Society guidelines advise against the routine administration of magnesium in patients with aSAH17. This is supported by data from the intravenous magnesium sulphate for aneurysmal subarachnoid haemorrhage (IMASH) and MASH-2 trials and a recent meta-analysis demonstrating no beneficial effect of magnesium in this group of patients86–88. A post hoc analysis of the IMASH trial reported an association between high plasma levels of magnesium and worse clinical outcomes89.\n\nIn summary, one calcium channel antagonist, nimodipine, has been shown to be effective in the prevention and treatment of DCI after aSAH whilst other calcium channel antagonists and a non-competitive inhibitor of calcium channels appear to have little effect on, or worsen outcomes.\n\nIt remains unclear how nimodipine exerts its neuroprotective effects but its action seems independent of any effect on large vessel narrowing90,91. It was thought that nimodipine may exert its effect by stopping calcium influx at a neuronal level, but no beneficial effect has been seen from administration in patients after ischaemic stroke or traumatic brain injury92–94. In addition to this, a recent systematic review found no benefit from nimodipine after traumatic SAH, suggesting that the mechanism of action of nimodipine is unique to aSAH95. Nimodipine has two properties that it does not share with other calcium channel antagonists. Firstly, it increases endogenous fibrinolytic activity, which may reduce the incidence of microthrombosis96. Secondly, it antagonises cortical spreading ischaemia in rats, which may be one of the culprits in DCI and is discussed in further detail below97.\n\n\nContemporary hypotheses\n\nEarly brain injury (EBI) refers to damage to the brain in the first 72 hours after the haemorrhage. There are a number of pathophysiological events in this time period that could influence later complications, such as DCI, and much of our understanding is derived from experimental data. One of these changes is a severe rise in intracranial pressure leading to decreased cerebral perfusion pressure, cessation of cerebral blood flow and ultimately global ischaemia and oedema98–100. The intracranial hypertension at ictus is often greater than systolic blood pressure, and the rate of increase and peak intracranial pressure appears to be proportional to the amount of arterial blood extravasating into the subarachnoid spaces from the aneurysm101–103. Cerebral spinal fluid outflow obstruction, in addition to hydrocephalus, further exacerbates intracranial hypertension104,105. However, the increase in intracranial pressure is not uniform and there are two distinct groups of patients in terms of their intracranial hypertension. The first, more common, scenario is an increase in intracranial pressure to the arterial diastolic pressure which then decreases to just above the patient’s baseline intracranial pressure102. These patients typically have a small volume haemorrhage with cerebral oedema. The second type of increased intracranial pressure is sustained due to either a progressive haematoma or acute hydrocephalus104,105.\n\nThe cerebral oedema seen after aSAH is often present on admission CT scans and becomes more common, being present in up to 20% of patients by day 698. Cerebral oedema is itself a poor prognostic factor after aSAH98,106,107. The global cerebral ischaemia that occurs during the initial aSAH may lead to the disruption of the blood-brain barrier, and initiate cell death mechanisms and inflammatory responses which all contribute to cerebral oedema. Regulated and unregulated neuronal cell death appears to occur within 24 hours after aSAH and as early as 40 minutes after the initial injury108–110. Serum and cerebrospinal fluid (CSF) levels of pro-inflammatory cytokines and vasoactive factors, such as tumour necrosis factor-α, interleukin-6, and interleukin-1 receptor antagonist, correlate with DCI and poor outcomes111,112.\n\nIn addition to these inflammatory responses, blood degradation products are thought to contribute to DCI and perhaps removing blood from the subarachnoid space may improve outcomes30,113. Continuous cisternal drainage and intrathecal administration of thrombolytics have been trailed with reports of success, and results of the EARLYDRAIN trial comparing continuous lumbar-CSF drainage with standard treatment are awaited114,115. A meta-analysis of the use of intrathecal thrombolytics suggested a reduction in the incidence of DCI but these findings were not statistically significant after excluding one study, which included intrathecal nimodipine in addition to thrombolytic therapy116.\n\nCerebral autoregulation, the ability of blood vessels to maintain constant cerebral blood flow (CBF) with arterial blood pressures between ~60 and 150 mmHg, is impaired after the aneurysm rupture117–119. Once impaired, autoregulation starts to rely on cerebral perfusion pressure and blood viscosity. Because of this, any change in intracranial pressure or systemic arterial pressure can potentially worsen oedema and ischaemia.\n\nA limitation to many of these theories is that the majority of data comes from animal studies of experimental aSAH models. Some authors have questioned whether we can reliably translate data derived from this model to human studies120,121. We await the results of a systematic review and meta-analysis of intracranial in vivo animal studies of EBI and delayed cerebral arterial vessel narrowing after aSAH122. The review aims to analyse aSAH models and define standard experimental parameters and endpoints for the study of EBI after aSAH and aSAH models of delayed cerebral arterial vessel narrowing.\n\nCortical spreading depolarisation (CSD), also termed cortical spreading depression, reflects a wave of depolarisation that spreads across grey matter at 2–5 mm/min. CSD is not a new theory, nor is it limited to aSAH, and has been implicated in brain injuries and migraine123. It occurs when a cation influx across cellular membranes exceeds the Na+ and Ca2+ pump action and is followed by water and shrinkage of the extracellular space by ~70% causing depression of EEG (electroencephalography) activity124,125. Because the Na+ and Ca2+ pump is ATP-dependent, to counteract the passive influx of cations across the membrane energy consumption increases, which leads to increased regional blood flow requirements. When there is a dysfunction of the vasculature in the region, as occurs after aSAH, severe microvascular spasm can occur, rather than vasodilation, causing “cortical spreading ischaemia”125. There is evidence that CSD occurs after the initial aneurysm rupture from both animal and human studies, and it is thought that after each depolarisation hypoperfusion of the cortex occurs due to vasoconstriction126. Furthermore, up to 75% of all CSD episodes occur between days 5 and 7 after the aSAH, which matches DCI chronology127. Another link between CSD and DCI comes from the CoOperative Study on Brain Injury Depolarisations (COSBID), which demonstrated that repeated CSD preceded DCI with little evidence of “vasospasm” on digital subtraction angiography (DSA), albeit in a small sample (thirteen patients)128.\n\nIncreased levels of procoagulants have been seen prior to DCI, specifically an increased von Willebrand factor 72 hours after aSAH and increased platelet-activating factors on day 4129–132. Microthrombi have also been identified at the autopsy of patients after aSAH, suggesting that they are involved in aSAH pathology132. The rate of rebleeding following aSAH has been significantly reduced following tranexamic acid administration. However, it may have led to an increased incidence of DCI separate from large vessel narrowing, possibly because the antifibrinolytic therapy caused microthrombosis and promoted DCI133–136. Unfortunately, the results of studies investigating antiplatelet agents in the treatment of microthrombosis after aSAH have been largely negative, including those investigating prophylactic low-molecular-weight heparin137,138.\n\n\nTherapies\n\nIntrathecal administration of nicardipine, a dihydropyridine calcium channel blocker, has been demonstrated in a number of clinical studies with varying results. Susuki et al. examined a series of 177 patients with Fisher grade III aSAH undergoing aneurysmal clipping and cisternal drainage within 48 hours of the aSAH139. Patients received 4 mg intrathecal therapy nicardipine every 12 hours on days 3–14 postoperatively. Of these patients, 11.3% had radiographic evidence of vessel narrowing and 5.7% had clinical signs of DCI. The authors note a significant reduction in “vasospasm” but also recognise that 18.6% of patients required a shunt operation. Shibuya et al. demonstrated a decreased incidence of DCI and angiographic vessel constriction by 20 and 26% respectively after prophylactic administration of 2 mg intrathecal therapy nicardipine via a cisternal drain when compared with control patients140. More recent trials also report positive findings, but are limited to cases of refractory “vasospasm” and have very small sample sizes141,142. However, nicardipine is associated with probable vasodilation-associated headaches, intracranial infections and hydrocephalus, and positive long-term outcomes from large RCTs are lacking. The NEWTON trial is a phase I/IIa multicentre RCT administering intrathecal nimodipine in patients with aSAH143. The trial uses EG-1962, a sustained delivery system of nimodipine in microparticles. These will be injected into the ventricles through an external ventricular catheter in patients undergoing coiling or clipping of ruptured aneurysms. It is thought that systemic effects are less likely to occur as nimodipine concentrations are much lower in the plasma than CSF144. We await the results of this trial and subsequent progressive trials with interest.\n\nStatins have been investigated as a potential treatment for DCI due to their multiple effects, although a recent meta-analysis of the four single-centre RCTs demonstrated no benefit from statins after aSAH145. Despite evidence that statins can reduce the duration of impaired autoregulation after aSAH, two more recent multicentre RCTs found no benefit from statin administration after aSAH146–148.\n\nAnother potential agent in the treatment of DCI is cilostazol, a phosphodiesterase 3 inhibitor and platelet aggregation inhibitor that affects smooth muscle cells. A meta-analysis of two RCTs and two quasi-RCTs demonstrated amelioration of cerebral vessel narrowing and a benefit on outcome at discharge, even after excluding the lower quality studies149–151. A subsequent trial has echoed these findings, but only one study has reported long-term outcomes and did not demonstrate improved outcomes with cilostazol151,152.\n\n\nConclusion\n\nIn summary, cerebral vessel narrowing is consistently seen after aSAH, but its location and severity is not predictably linked to DCI. There is no conclusive evidence to support the treatment of vessel narrowing in the management of DCI, despite some studies reporting improved outcomes, specifically after more invasive techniques. Nimodipine is the only effective treatment for DCI but we still do not understand how nimodopine exerts its neuroprotective effect, although it does not seem to work by reversing cerebral artery narrowing, at least not in large vessels. It is possible that we are not detecting microvascular vasoconstriction or ischaemia on CT and TCD and so our understanding of the pathology is limited. Furthermore, improved recognition of DCI clinically, from imaging and/or biochemical markers would not only allow for quicker treatment but also better assessment of interventions. DCI almost certainly has a multifactorial aetiology and it may be that only by combining interventions will we see improved outcomes, but first we must understand the aetiology. Understanding how nimodipine, the only drug with proven efficacy, exerts its effect may be the key to creating new interventions with improved efficacy. There remains a large amount of work to be done in understanding DCI and investigating future potential treatments.",
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PubMed Abstract | Publisher Full Text\n\nDorhout Mees SM, van den Bergh WM, Algra A, et al.: Antiplatelet therapy for aneurysmal subarachnoid haemorrhage. Cochrane Database Syst Rev. 2007; (4): CD006184. PubMed Abstract | Publisher Full Text\n\nSiironen J, Juvela S, Varis J, et al.: No effect of enoxaparin on outcome of aneurysmal subarachnoid hemorrhage: a randomized, double-blind, placebo-controlled clinical trial. J Neurosurg. 2003; 99(6): 953–9. PubMed Abstract | Publisher Full Text\n\nSuzuki M, Doi M, Otawara Y, et al.: Intrathecal administration of nicardipine hydrochloride to prevent vasospasm in patients with subarachnoid hemorrhage. Neurosurg Rev. 2001; 24(4): 180–4. PubMed Abstract | Publisher Full Text\n\nShibuya M, Suzuki Y, Enomoto H, et al.: Effects of prophylactic intrathecal administrations of nicardipine on vasospasm in patients with severe aneurysmal subarachnoid haemorrhage. Acta Neurochir (Wien). 1994; 131(1–2): 19–25. PubMed Abstract | Publisher Full Text\n\nGoodson K, Lapointe M, Monroe T, et al.: Intraventricular nicardipine for refractory cerebral vasospasm after subarachnoid hemorrhage. Neurocrit Care. 2008; 8(2): 247–52. PubMed Abstract | Publisher Full Text\n\nEhtisham A, Taylor S, Bayless L, et al.: Use of intrathecal nicardipine for aneurysmal subarachnoid hemorrhage-induced cerebral vasospasm. South Med J. 2009; 102(2): 150–3. PubMed Abstract | Publisher Full Text\n\nEtminan N, Macdonald RL, Davis C, et al.: Intrathecal application of the nimodipine slow-release microparticle system eg-1962 for prevention of delayed cerebral ischemia and improvement of outcome after aneurysmal subarachnoid hemorrhage. Acta Neurochir Suppl. 2015; 120: 281–6. PubMed Abstract\n\nHänggi D, Etminan N, Macdonald RL, et al.: NEWTON: Nimodipine Microparticles to Enhance Recovery While Reducing Toxicity After Subarachnoid Hemorrhage. Neurocrit Care. 2015; 23(2): 274–84. PubMed Abstract | Publisher Full Text\n\nVergouwen MD, de Haan RJ, Vermeulen M, et al.: Effect of statin treatment on vasospasm, delayed cerebral ischemia, and functional outcome in patients with aneurysmal subarachnoid hemorrhage: a systematic review and meta-analysis update. Stroke. 2010; 41(1): e47–52. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWong GK, Chan DY, Siu DY, et al.: High-dose simvastatin for aneurysmal subarachnoid hemorrhage: multicenter randomized controlled double-blinded clinical trial. Stroke. 2015; 46(2): 382–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKirkpatrick PJ, Turner CL, Smith C, et al.: Simvastatin in aneurysmal subarachnoid haemorrhage (STASH): a multicentre randomised phase 3 trial. Lancet Neurol. 2014; 13(7): 666–75. 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}
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"id": "11011",
"date": "02 Nov 2015",
"name": "Kyle Pattinson",
"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",
"responses": []
},
{
"id": "11012",
"date": "02 Nov 2015",
"name": "Stephan Mayer",
"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",
"responses": []
},
{
"id": "11013",
"date": "02 Nov 2015",
"name": "Michael N. Diringer",
"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",
"responses": []
},
{
"id": "11014",
"date": "02 Nov 2015",
"name": "Nikolaus Plesnila",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1200
|
https://f1000research.com/articles/4-36/v1
|
04 Feb 15
|
{
"type": "Research Article",
"title": "Tetranucleotide usage highlights genomic heterogeneity among mycobacteriophages",
"authors": [
"Benjamin Siranosian",
"Sudheesha Perera",
"Edward Williams",
"Chen Ye",
"Christopher de Graffenried",
"Peter Shank",
"Sudheesha Perera",
"Edward Williams",
"Chen Ye",
"Christopher de Graffenried",
"Peter Shank"
],
"abstract": "BackgroundThe genomic sequences of mycobacteriophages, phages infecting mycobacterial hosts, are diverse and mosaic. Mycobacteriophages often share little nucleotide similarity, but most of them have been grouped into lettered clusters and further into subclusters. Traditionally, mycobacteriophage genomes are analyzed based on sequence alignment or knowledge of gene content. However, these approaches are computationally expensive and can be ineffective for significantly diverged sequences. As an alternative to alignment-based genome analysis, we evaluated tetranucleotide usage in mycobacteriophage genomes. These methods make it easier to characterize features of the mycobacteriophage population at many scales.DescriptionWe computed tetranucleotide usage deviation (TUD), the ratio of observed counts of 4-mers in a genome to the expected count under a null model. TUD values are comparable between members of a phage subcluster and distinct between subclusters. With few exceptions, neighbor joining phylogenetic trees and hierarchical clustering dendrograms constructed using TUD values place phages in a monophyletic clade with members of the same subcluster. Regions in a genome with exceptional TUD values can point to interesting features of genomic architecture. Finally, we found that subcluster B3 mycobacteriophages contain significantly overrepresented 4-mers and 6-mers that are atypical of phage genomes.ConclusionsStatistics based on tetranucleotide usage support established clustering of mycobacteriophages and can uncover interesting relationships within and between sequenced phage genomes. These methods are efficient to compute and do not require sequence alignment or knowledge of gene content. The code to download mycobacteriophage genome sequences and reproduce our analysis is freely available at https://github.com/bsiranosian/tango_final.",
"keywords": [
"mycobacteriophages",
"computed",
"tetranucleotide",
"usage",
"deviation",
"genome",
"sequences"
],
"content": "Introduction\n\nMycobacteriophages, phages infecting mycobacterial hosts, are a subset of the estimated 1031 phage particles present globally. Mycobacteriophages infect a number of bacterial hosts from the genus Mycobacterium, and they are broadly classified into Siphoviridae and Myoviridae. Mycobacteriophages are present in both land and aquatic environments and play a large ecological role in the turnover and evolution of bacteria (Bohannan & Lenski, 2000, Chibani-Chennoufi et al., 2004, Hendrix, 2002). The recent rise of antimicrobial-resistant pathogenic bacteria has renewed interest in mycobacteriophages and the potential for phage therapy of Mycobacterium tuberculosis infections. Although in vivo experiments have not yet yielded promising clinical results, mycobacteriophages are still powerful diagnostic tools for the investigation of mycobacterial pathogenesis (Danelishvili, 2006, Hatfull, 2014, McNerney, 1999).\n\nThe genomic sequences of mycobacteriophages are mosaic and diverse. As of April 2014, 663 distinct mycobacteriophage genomes were available on the database PhagesDB.org; most were isolated on Mycobacterium smegmatis MC2155. Global Guanine + Cytosine (GC) content ranges from 50.3% to 70% (mean of 63.9%), and genome lengths range from 41kb to 165kb (mean of 67kb). In total, more than 50,000 distinct genes are found within the population. The majority of these genes are of unknown function and do not have homologs in other types of phages or bacteria (Hatfull et al., 2010). However, many genes are shared between closely related mycobacteriophages. Similar genes have been grouped into almost 4,000 phamilies (or phams, a play on gene families) based on shared amino acid sequence. Phams have been used to investigate horizontal gene transfer within the mycobacteriophage population and to create phylogenetic trees.\n\nDespite the high levels of diversity, mycobacteriophages can be grouped into distinct clusters based on their morphologic and genetic features. Some clusters are large and further divided into subclusters (cluster A, for example, with 11 subclusters and 246 members), while other are small and undivided (cluster S with two members and no subclusters). Some phages have no nearest neighbor to establish a cluster and are classified as singletons. Clusters are defined using four methods: dot-plot comparisons, pairwise average nucleotide identities, pairwise genome map comparisons and gene content analysis (Hatfull et al., 2010). However, it should be noted that the clustering scheme proposed for mycobacteriophages mainly serves to identify similarities in genome architecture. This clustering scheme, and our proposed methods of grouping based on tetranucleotide usage described below, are not true taxonomic representations of the mycobacteriophage population. Extensive horizontal gene transfer prevents accurate reconstruction of evolutionary history from purely phylogenetic information (Lawrence et al., 2002).\n\nMethods traditionally used to analyze mycobacteriophage genomes require sequence alignment or genome annotation. These analytical tasks can be effective, but they are not without drawbacks. Alignment-based methods can be biased by the choice of score parameters (Frith et al., 2010), and genome annotation may require significant manual input, including by-hand verification of automated gene calls before a mycobacteriophage genome is submitted to GenBank. It is especially difficult to build multiple-sequence alignment based phylogenetic trees from mycobacteriophage genomes because phages lack a common genetic element, such as 16S rRNA in bacteria (Doolittle, 1999). Alignment-free methods avoid many of the disadvantages associated with alignment-based inference. These methods typically use statistics based on the oligonucleotide composition of a sequence and are completely independent of alignment or annotation. Several methods have been developed for different applications; most are covered in the excellent review by Vinga (2007). Alignment-free methods are also less computationally intensive than multiple sequence alignment. While the complexity of sequence alignment algorithms scales at least as fast as the square of the number of sequences (at least O(n2) complexity), alignment free methods typically fall below O(n2) (Chan & Ragan, 2013).\n\nEven so, there are drawbacks to alignment-free methods for analyzing genomes, mostly related to the interpretation of statistics in an evolutionary context. It can be difficult to understand how oligonucleotide frequencies are modified in a population over time when selection usually takes place at the level of genes. Oligonucleotide frequencies can also be subject to convergent evolution: if two distantly related phages slowly converge to similar usage frequencies, these methods can give a false indication of common ancestry (Pride et al., 2003).\n\nAlignment-free methods have been used to study phage and bacterial genomes in a variety of contexts. For example, Pride et al. (2006) found tetranucleotide usage to carry a strong phylogenetic signal in bacteriophages and showed that tetranucleotide composition was similar among phages with common hosts. More recently, Ogilvie et al. (2013) surveyed metagenomic sequencing datasets using a tetranucleotide usage-based method and discovered several novel Bacteroidales-like phages which could not be identified with alignment-based methods. Oligonucleotide composition vectors have also been proposed as a method to root viral phylogenies (Simmons, 2008).\n\nStatistics based on nucleotide composition in a sliding window can theoretically be used to uncover horizontal gene transfer (HGT), based on the assumption that genomes have self-similar nucleotide composition and outlier regions could represent recent horizontal transfer events (Lawrence & Ochman, 1997). Guanine + Cytosine (GC) content in a sliding window was first used to look for pathogenicity islands within a genome (Hacker & Kaper, 2000). More recent methods have used nucleotide composition and Naïve Bayesian classifiers (Sandberg et al., 2001) or hidden Markov models (Waack et al., 2006). However, if horizontally transferred segments change in oligonucleotide composition to be more similar to the resident genome, a process known as amelioration, it can obscure truly horizontally transferred segments (Koski et al., 2001).\n\nThe number of sequenced mycobacteriophages has grown immensely in the past few years thanks to the Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course (Jordan et al., 2014). This program allows first year undergraduate students to isolate and characterize novel mycobacteriophages from the environment. It has also provided excellent opportunities for collaborative projects between undergraduates, resulting in the work presented here.\n\nAs the number of sequenced mycobacteriophages continues to increase, researchers need new methods to quickly make comparisons at many scales. Alignment-free methods are one possibility: they are independent of sequence alignment or genome annotation, less computationally complex than alignment-based methods and applicable to genomes without a common subsequence. We investigated tetranucleotide usage in mycobacteriophage genomes as an alignment-free alternative to traditional methods for genome comparison. Our findings support what is known about mycobacteriophage biology: phages form identifiable groups and subgroups, known as clusters, but have extensive differences between clusters. Tetranucleotide usage also highlights outliers in the population and can describe unique genomic features. All of the analyses here can be done in minutes on a personal laptop. Tetranucleotide usage is a powerful tool to quickly investigate features of the growing mycobacteriophage population.\n\n\nMethods\n\nWe obtained the genomic sequences of all 663 sequenced mycobacteriophages publicly available on the website PhagesDB.org as of April 2014. This dataset contains both unpublished genomes and genomes available on GenBank. There is not an easy way to download the mycobacteriophage database in its entirety, so we automated the process with a Python script available in the code accompanying this manuscript.\n\nTo compare mycobacteriophage genomes independently of sequence alignment, we first investigated the usage of k-mers, substrings of DNA of length k, in each genome. Observed values for each k-mer are counted in the genome using a sliding window of size k and step size of one. We normalized the k-mer frequencies using a zero-order Markov model, which removes biases from the background nucleotide composition and can be effective for analysis of prokaryotic genomes (Pride et al., 2003, Pride et al., 2006). The expected number of a k-mer W given the background nucleotide distribution is calculated by:\n\nE(W) = [(Aa * Tt * Cc * Gg) * N]\n\nwhere A,T,C,G are the frequency of each nucleotide in the genome, a,t,c,g are the number of each nucleotide in the k-mer W, and N is the length of the genome. Observed counts of each k-mer are then divided by the expected number to give the normalized k-mer usage. Before calculating k-mer usage, phage genomes are extended by the reverse complement.\n\nWe computed normalized k-mer usage for k in the range of two to seven. We chose to conduct further analyses with k=4, which we call tetranucleotide usage deviation (TUD). TUD is calculated for all possible 4-mers, leading to a vector of 44=256 values. This is equivalent to the “tetranucleotide usage departures from expectation” measure proposed by Pride et al. (2003). For a given 4-mer, a TUD value of one corresponds to the expected usage, while a value of two corresponds to usage twice as frequently as expected.\n\nPhage genomic sequences are extended by the reverse complement before calculation, leading to redundant values for a given tetranucleotide and its reverse complement. One of the redundant tetranucleotides was removed before distance calculations and Principal Components Analysis (PCA). We also removed tetranucleotides that were not present at least once in all phage genomes. Only ATAT and AATT were removed by this filter.\n\nTo compare phage genomes in terms of TUD, we calculated the pairwise Euclidean distance between all TUD vectors. This produces a distance matrix to use for tree building. For analysis of the subset of 60 phage in Hatful et al. (2010), we used the SplitsTree program (Huson & Bryant, 2006) to construct neighbor joining phylogenetic trees. This was done to facilitate easy comparisons between previously published figures and our alignment-free trees. Hierarchical clustering using the “average” method within R (version 3.1.0) was used to construct dendrograms for analyzing the entire phage database.\n\nPCA was used to visualize relationships between phage genomes in lower- dimensional space. PCA was done on log-transformed data in R using the ‘prcomp’ function and results were plotted using the ‘ggbiplot’ package.\n\nTo compare tetranucleotide usage within a phage genome, we used a sliding window of 2000bp (500bp step size). This window size was selected to balance two factors: a short window can detect differences in small regions, while a longer window is necessary to encounter the majority of tetranucleotides. 4-mers were counted and normalized to the nucleotide composition of a given window. A distance matrix was constructed from pairwise Euclidean distances of all windows and used to build heatmaps. Parts of the heatmap where windows overlapped were removed before plotting, leading to the white section along the diagonal in Figure 4.\n\n\nResults\n\nFirst, we investigated if TUD reflected relationships described from alignment-based analysis of phage genomes. In particular, does a grouping scheme based on tetranucleotide usage agree with previously assigned phage clusters? To test this hypothesis, we examined a subset of 60 mycobacteriophages first analyzed by Hatfull et al. (2010), where the authors propose a clustering scheme based on dot-plot comparisons, pairwise average nucleotide identities, pairwise genome maps and gene content analysis. We calculated the pairwise Euclidean distances between TUD vectors for the subset of 60 phages and used the SplitsTree program (Huson & Bryant, 2006) to construct a neighbor joining tree (Figure 1a). Our alignment-free tree has a striking resemblance to the tree from Hatfull et al. (2010), which is constructed from similarities in genomic architecture (Figure 1b). In every case, phages are placed in a monophyletic clade with members of their subcluster.\n\na) Neighbor joining phylogenetic tree constructed from pairwise Euclidean distances between TUD vectors for 60 mycobacteriophage genomes. Phage names are colored based on previously assigned cluster information. b) Neighbor joining phylogenetic tree constructed from gene presence data in mycobacteriophage genomes. Reproduced with permission from Figure 3 in Hatful et al. (2010). The TUD tree is similar to the alignment-based tree. Phages from the same subcluster form monophyletic clades. In clusters C, F and H, subclusters from the same parent cluster form monophyletic clades.\n\nHierarchically grouping phages into clusters and subclusters represents heterogeneity within the mycobacteriophage population. In the alignment-free tree, subclusters from parent clusters C, H and F are placed in a monophyletic clade. However, in some cases, tetranucleotide usage was vastly different between subclusters of a parent cluster. For example, subcluster B3 phages are most similar to cluster A phages in terms of tetranucleotide usage, but they are similar to other cluster B genomes when compared on genetic elements (Figure 1). We investigate this relationship further in a following section. Importantly, the relationships between the subset of 60 phages are consistent for varying values of k (Figure 2).\n\nNeighbor joining phylogenetic trees constructed from pairwise Euclidean distances between oligonucleotide usage deviation vectors for 60 mycobacteriophage genomes. Trees from k equal to two, five and seven are shown here. Trees show a high degree of similarity regardless of the k used. Trends observed in the tetranucleotide usage based tree (Figure 1), such as grouping of subcluster members into monophyletic clades, are conserved in these trees.\n\nHundreds of mycobacteriophages have been sequenced in the past few years, bringing the total to 663 genomes (PhagesDB.org as of April 2014), 21 clusters and 48 subclusters. We next examined TUD patterns in the entire database to see if the relationships observed for the subset of 60 phages were conserved. We used hierarchical clustering within R to analyze this larger dataset (see Methods). As observed for the subset of 60, almost all phages are grouped closely with members of their subcluster. Subclusters of cluster F, C, D, M and L form a monophyletic clade (Supplementary Figure 1). The relationships for cluster B genomes are also conserved – genomes within a given B subcluster are similar, but the subclusters themselves are different and placed in separate sections of the dendrogram.\n\nWe further investigated the ability of TUD to differentiate between predetermined phage clusters using PCA. PCA is useful for visualizing TUD, a 256-dimensional vector, in intuitive 2D space. PCA was applied to log-transformed TUD vectors for all 663 genomes. The first three principal components captured 29.3%, 15.6% and 12.9% of the variance, respectively. Comparing PC1 and PC2 highlighted groups of phage that corresponded well with assigned clusters (Figure 3a). Clusters that were similar in PC1/PC2 space could be separated further by including additional PCs.\n\na) Principal components analysis of all 663 mycobacteriophage genomes. Individual clusters of phages are well separated by PC1 and PC2 in most cases. Further separation can be achieved by incorporating additional principal components. b) Principal components analysis of cluster B phages. Individual subclusters are well separated. The outlier in B4 is KayaCho, a phage with different tetranucleotide usage but similar genome architecture when compared with other B4 phages.\n\nPCA was also useful to compare phages within a single cluster. When comparing cluster B phages, the first three components captured 44.6%, 31.4% and 10.3% of the variance present, respectively. Phage subclusters typically group tightly with each other in PC-space, which makes it easy to detect outliers in terms of TUD. A single member of B4, KayaCho, is placed far from the other genomes of that subcluster (Figure 3b). This indicates that KayaCho is dissimilar from other B4 phages, a finding that is supported through other methods of comparison. For example, KayaCho has a similar global genome architecture to other members of B4, but pairwise nucleotide identity is low in relation to other comparisons within the subcluster. TUD provides a quick and alignment-free way to detect genomes that are outliers within a subcluster.\n\nMycobacteriophage genomes are mosaic and heavily influenced by horizontal gene transfer (HGT) (Pedulla et al., 2003). We looked for sections within a phage genome that stood out in TUD as potential candidates for HGT events. Tetranucleotide usage was calculated in a 2000bp window with a 500bp step size. Heatmaps of pairwise Euclidean distances between all windows were plotted.\n\nObservation of these heatmaps revealed several interesting features. The last 5kb of cluster E phage “244” is self-similar, but different than the rest of the genome in terms of TUD (Figure 4a). This self-similar segment is present with >97% nucleotide identity in all cluster E phage and could represent a HGT event from a different phage cluster or organism. To search for potential transfer sources of this segment, we compared TUD in the region with other mycobacteriophages and searched for nucleotide similarity with BLAST (nr/nt database, blastn algorithm) (Altschul et al., 1997). However, we were unable to find regions of considerable homology with either method.\n\nComparing tetranucleotide usage in a sliding window (2000bp window, 500bp step size) across phage genomes. Each entry in the heatmap is the Euclidean distance between windows. a) 244, a cluster E phage, is relatively self-similar with low distance values (red) between most windows. The last 5kb of the genome is an exception: it is self-similar but different than the rest of the genome. This signature is not driven by repetitive sequences, and represents a putative HGT event. b) UPIE, a cluster L1 phage, also has a self-similar signature at the end of the genome. However, the difference in TUD in this window is driven by two cluster of repetitive k-mers (Figure 5).\n\nCluster L1 phages contain two small self-similar yet genome-different regions at the end of the genome (Figure 4b). We examined the genome of “UPIE” with the Repfind program (Betley et al., 2002) to search for repetitive sequences that could be driving the change in TUD. There are two blocks of repetitive GC-rich k-mers, from 68650-69050bp and 71100-71900bp, which match the regions in the heatmap (Figure 5). As the sliding window moves through each of these blocks, the TUD signal becomes dominated by the repetitive sequence and makes the regions appear self-similar yet genome different. The repetitive features don’t preclude the possibility of HGT in the region, but they do likely obscure a HGT signal carried by TUD. We found other self-similar yet genome-different repetitive regions in phages from clusters F1, H and O. Although the regions highlighted here have variations in GC content, TUD removes biases from the nucleotide composition using a zero-order Markov model (see Methods). Differences in TUD are not a result of variations in the underlying GC content.\n\nTwo clusters of GC-rich repetitive sequences at the end of the genome of UPIE (cluster L1). The repetitive sequences drive the differences in TUD and correspond with the self-similar yet genome-different sections in the within-genome heatmap (Figure 4). This image was reconstructed from the output of Repfind (Betley et al., 2002).\n\nFinally, we examined why B3 phages are not placed with other members of cluster B in the hierarchical clustering dendrogram, while most of the other clusters show this relationship. B3 genomes share greater than 60% average nucleotide identity with other members of cluster B. This is comparable with the relationship between B2 and B4 phages, which are placed close to each other in the dendrogram. The difference in TUD is not likely to be driven solely by differences in pairwise nucleotide identity. We investigated the individual k-mers making up the TUD vector to examine this relationship further.\n\nB3 phages used the 4-mer GATC four times more than expected by chance, greater than all other B subclusters (Figure 6a). The high abundance of GATC could be driven by a global increase in frequency or by discrete regions with very high usage of the 4-mer. To address this point, we compared normalized GATC usage in a sliding window across all cluster B genomes. GATC usage was increased genome-wide in B3 phages, refuting the hypothesis that the deviation was caused by a single genomic region (Figure 6c). This points to a genome-wide amelioration of GATC usage in cluster B3 genomes. Interestingly, some local peaks and valleys in GATC usage are persistent across all cluster B genomes, even though these genomes are unaligned.\n\na) Density plot of TUD values for the 4-mer GATC. Individual subclusters form well-defined groups. B3 phages have GATC usage four times what is expected, much higher than other B subclusters. b) Repeat of (a) with the 6-mer GGATCC. B3 phages use this 6-mer greater than four times what is expected. c) GATC usage deviation in a sliding window (5kb, 1kb step size). Each line represents the mean value in the specified subcluster. The increase in GATC usage is genome-wide, indicative of a global change in usage frequency. d) Repeat of (c) with the 6-mer GGATCC. Increased usage is also genome-wide.\n\nGiven the genome-wide increase in B3 GATC usage, it is possible that a higher-order signal could be driving the trend. We searched for highly used 6-mers in B3 phages and found GGATCC had a usage deviation value greater than four, while all other B genomes had a value less than one (Figure 6b). This increase was also genome-wide (Figure 6d). GATC and GGATCC are both palindromes, DNA sequences with identical reverse complements. Palindromes are typically underrepresented in bacteriophage and other prokaryotic genomes because they can be parts of recognition sites for restriction enzymes (Gelfand & Koonin, 1997, Karlin et al., 1992, Sharp, 1986).\n\nGATC is recognized by Dam methylase in E. coli (Marinus & Morris, 1973), but Mycobacterium species do not encode Dam methylase (Hemavathy & Nagaraja, 1995). If B3 phages recently accessed a host with an active Dam methylase, it could lead to a change in GATC frequency. Several restriction enzymes recognize GATC, like MgoI in Mycobacterium gordonae (Shankar & Tyagi, 1993), while others recognize GGATCC, such as BamHI in Bacillus amyloliquefaciens. However, the presence of a restriction/modification system in a host would theoretically lead to a decrease in usage of the recognized site. The finding that GATC and GGATCC occur in B3 genomes four times more than expected and significantly more frequently than in all other sequenced mycobacteriophages bears further investigation.\n\n\nDiscussion\n\nIn 2010, there were 60 sequenced mycobacteriophages. There are more than 660 as of April 2014. Alignment-based methods have been used to investigate the mycobacteriophage population, leading to interesting characterizations, such as hierarchical grouping into clusters and subclusters. However, as the number of published genomes continues to grow, there is a need for methods to quickly analyze the entire database of mycobacteriophage sequences.\n\nThroughout this paper, we apply oligonucleotide usage approaches to uncover relationships within the population of sequenced mycobacteriophages. Our findings support what is known about mycobacteriophage biology. Neighbor joining and hierarchical clustering from TUD place closely related phage in well-defined groups that correspond with assigned phage subclusters. In most cases, TUD supports grouping into larger clusters, such as cluster A, where all 246 members form a monophyletic clade in the hierarchical clustering dendrogram. The fact that members of cluster B do not form a clade in TUD-based comparisons does not invalidate grouping of phage into clusters, but rather serves as a way to highlight phages where TUD and gene or sequence comparisons capture different relationships.\n\nComparing TUD in a sliding window can highlight regions with dissimilar tetranucleotide composition and identify genomic segments that could have been horizontally transferred. We found self-similar yet genome-different regions at the end of cluster E and L genomes. The new TUD ‘space’ occupied by these segments could be from HGT – a recently transferred genomic section that had not yet ameliorated to the average genome TUD profile. At least for cluster L, we can say that HGT is likely not the cause. Two groups of repetitive sequences at the end of the genome are driving the difference in TUD.\n\nHowever, we found neither repetitive sequences nor a putative transfer candidate for the segment in cluster E. An improvement on our method could potentially detect legitimate HGT events, but we note that the concept of phams (Hatful et al., 2010) and the computer program Phamerator (Cresawn et al., 2011) are already efficient for detecting and visualizing these features.\n\nTUD vectors are similar between subcluster B3 phages but different from other members of cluster B. We found that the 4-mer GATC and 6-mer GGATCC were present over four times more than expected in B3 genomes. These sequences are palindromes and part of the BamHI restriction site, two characteristics of sequences that are typically underrepresented in prokaryotic genomes. GATC and GGATCC are highly used in all sections of B3 genomes, pointing to genome-wide amelioration of usage frequencies.\n\nOligonucleotide composition methods do not require knowledge of sequence alignment or gene content. They are ideal to compare mycobacteriophage genomes, which lack a common subsequence on which to make alignment-based inference. Alignment-free methods are also valuable when a reference sequence is not available. Recently, methods based on tetranucleotide usage were used to investigate sequences from a gut microbiome and uncovered a population of Bacteroidales-like phage that was previously unrepresented in metagenomic sequencing datasets (Ogilive et al., 2013). Statistics based on oligonucleotide usage are part of a broader class of alignment-free methods. These methods are easy to compute across large datasets: constructing the dendrogram in Supplementary Figure 1 from raw phage sequences takes less than two minutes on a personal laptop. Comparably, creating phylogenetic trees from pairwise global sequence alignment with the Needleman-Wunsch algorithm (Needleman & Wunsch, 1970) takes over 24 hours on a computing cluster. We envision oligonucleotide usage methods to be used alongside alignment-based techniques. Highlighting large trends and outliers is easy with these methods, but sequence alignment and gene annotation need to be applied to extract biological insights from the data.\n\nThe genomic sequences of all 663 sequenced mycobacteriophages are publicly available on the website PhagesDB.org as of April 2014. The authors obtained permission to use the data.\n\nThe code to download mycobacteriophage genome sequences and reproduce our analysis is freely available at https://github.com/bsiranosian/tango_final. Mycobacteriophage genome sequences are available at http://phagesdb.org.\n\nhttps://github.com/bsiranosian/tango_final\n\nhttps://github.com/F1000Research/tango_final\n\nhttp://dx.doi.org/10.5281/zenodo.14609 (Siranosian et al., 2015b).",
"appendix": "Author contributions\n\n\n\nBS designed the study. BS, SP, EW and CY performed the analysis. BS and CY prepared the figures. BS, SP, EW, CDG and PS wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by Brown University Biology Undergraduate Education and the HHMI SEA-PHAGES program.\n\n\nAcknowledgments\n\nWe would like to thank Sarah Taylor for instructing the Brown University Phage Hunters course and for her assistance during the development and presentation of this work. The present manuscript benefited from helpful comments by Dr. Graham Hatfull. We would also like to thank the hundreds of students from schools participating in the SEA-PHAGES program who have isolated, characterized and purified the mycobacteriophages we analyzed. Finally, we are deeply grateful to the SEA-PHAGES program and Howard Hughes Medical Institute for providing the resources to sequence hundreds of mycobacteriophage genomes, and PhagesDB.org for providing access to the unpublished material that formed the base of this work.\n\n\nSupplementary material\n\nHierarchical clustering dendrogram constructed on pairwise Euclidean distances between all 663 phages in the mycobacteriophage database. In almost every case, phages are placed in a monophyletic clade with members of their subcluster, highlighting the concordance between alignment-based and alignment-free methods for comparison for these genomes. Some clusters (F, C, D, M and L) form monophyletic clades, while others (B, for example) are grouped in different parts of the dendrogram. A larger version of this figure can be downloaded from here.\n\n\nReferences\n\nAltschul SF, Madden TL, Schäffer 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\nBetley JN, Frith MC, Graber JH, et al.: A ubiquitous and conserved signal for RNA localization in chordates. Curr Biol. 2002; 12(20): 1756–1761. PubMed Abstract | Publisher Full Text\n\nBohannan BJM, Lenski RE: Linking genetic change to community evolution: insights from studies of bacteria and bacteriophage. Ecology Letters. 2000; 3(4): 362–377. Publisher Full Text\n\nChan CX, Ragan MA: Next-generation phylogenomics. Biol Direct. 2013; 8: 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChibani-Chennoufi S, Bruttin A, Dillmann ML, et al.: Phage-host interaction: an ecological perspective. J Bacteriol. 2004; 186(12): 3677–3686. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCresawn SG, Bogel M, Day N, et al.: Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics. 2011; 12: 395. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDanelishvili L, Young LS, Bermudez LE: In vivo efficacy of phage therapy for Mycobacterium avium infection as delivered by a nonvirulent mycobacterium. Microb Drug Resist. 2006; 12(1): 1–6. PubMed Abstract | Publisher Full Text\n\nDoolittle WF: Phylogenetic classification and the universal tree. Science. 1999; 284(5423): 2124–2129. PubMed Abstract | Publisher Full Text\n\nFrith MC, Hamada M, Horton P: Parameters for accurate genome alignment. BMC Bioinformatics. 2010; 11: 80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGelfand MS, Koonin EV: Avoidance of palindromic words in bacterial and archaeal genomes: a close connection with restriction enzymes. Nucleic Acids Res. 1997; 25(12): 2430–2439. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHacker J, Kaper JB: Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol. 2000; 54: 641–679. PubMed Abstract | Publisher Full Text\n\nHatfull GF, Jacobs-Sera D, Lawrence JG, et al.: Comparative genomic analysis of 60 Mycobacteriophage genomes: genome clustering, gene acquisition, and gene size. J Mol Biol. 2010; 397(1): 119–143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHatfull GF: Mycobacteriophages: windows into tuberculosis. PLoS Pathog. 2014; 10(3): e1003953. PubMed Abstract | Publisher Full Text\n\nHemavathy KC, Nagaraja V: DNA methylation in mycobacteria: absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences. FEMS Immunol Med Microbiol. 1995; 11(4): 291–296. PubMed Abstract | Publisher Full Text\n\nHendrix RW: Bacteriophages: evolution of the majority. Theor Popul Biol. 2002; 61(4): 471–480. PubMed Abstract | Publisher Full Text\n\nHuson DH, Bryant D: Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006; 23(2): 254–267. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nPedulla ML, Ford ME, Houtz JM, et al.: Origins of highly mosaic mycobacteriophage genomes. Cell. 2003; 113(2): 171–182. PubMed Abstract | Publisher Full Text\n\nPride DT, Meinersmann RJ, Wassenaar TM, et al.: Evolutionary implications of microbial genome tetranucleotide frequency biases. Genome Res. 2003; 13(2): 145–158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPride DT, Wassenaar TM, Ghose C, et al.: Evidence of host-virus co-evolution in tetranucleotide usage patterns of bacteriophages and eukaryotic viruses. BMC Genomics. 2006; 7: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSandberg R, Winberg G, Bränden CI, et al.: Capturing whole-genome characteristics in short sequences using a naïve Bayesian classifier. Genome Res. 2001; 11(8): 1404–1409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShankar S, Tyagi AK: Purification and characterization of restriction endonuclease MgoI from Mycobacterium gordonae. Gene. 1993; 131(1): 153–154. PubMed Abstract | Publisher Full Text\n\nSharp PM: Molecular evolution of bacteriophages: evidence of selection against the recognition sites of host restriction enzymes. Mol Biol Evol. 1986; 3(1): 75–83. PubMed Abstract\n\nSimmons MP: Potential use of host-derived genome signatures to root virus phylogenies. Mol Phylogenet Evol. 2008; 49(3): 969–978. PubMed Abstract | Publisher Full Text\n\nSiranosian B, Herold E, Williams E, et al.: Tetranucleotide usage in mycobacteriophage genomes: alignment-free methods to cluster phage and infer evolutionary relationships. BMC Bioinformatics. 2015a; 16(Suppl 2): A7. Reference Source\n\nSiranosian B, Perera S, Williams E, et al.: Code to download mycobacteriophage genome sequences. Zenodo. 2015b. Data Source\n\nVinga S: Biological sequence analysis by vector-valued functions: revisiting alignment-free methodologies for DNA and protein classification in Advanced Computational Methods for Biocomputing and Bioimaging. (Nova Science Publishers). 2007; 71–107. Reference Source\n\nWaack S, Keller O, Asper R, et al.: Score-based prediction of genomic islands in prokaryotic genomes using hidden Markov models. BMC Bioinformatics. 2006; 7: 142. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "7811",
"date": "10 Mar 2015",
"name": "Oliver Bonham-Carter",
"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 article is nicely written but sadly, there are elements of discussion which are absent from the paper. If added, the paper's research on mycobacteriophages using alignment-free analysis would have much more support.The choice of TUD's as statistics for the alignment-free analysis is not fully explained /justified, nor is there much discussion about what algorithm or method is being employed by the analysis tools of the paper. Are TUD's frequencies? How do these software tools work? An simple example of how to calculate a TUD and apply it to a method is necessary to completely understand what they are and to see how they are different from any other motif frequency calculation applied to some other method. The assumptions of the methods are not discussed. Many methods from information theory, statistics and other kinds of mathematics require that the input data meets specific requirements (is normal, has a certain distribution, is a frequency, etc.). From the discussion in this paper, the function of analysis tool (the exact algorithm or method) is never clear and so we cannot be sure that the calculations from this work, as applied to these tools, is appropriate. For instance, many tools in information theory require that frequencies be used for their analysis. These frequencies must pass basic rules to be called as such (i.e., found on the scale of 0 to 1, all frequencies must sum to 1, 0 = false, 1 = true). This discussion is not mentioned and if it were, then the choice to used TUDs could be easily integrated into this discussion. The manuscript mentioned that k-mers in the range of two to seven were calculated (Methods Section). Where are the results for all these other values of k={2, 3, 5 and 7} which were not the k={4 and 6} results of the article? Although other sizes of motifs where apparently used in the analysis, the manuscript focuses on the length-4 motifs. The choice of k=4 for the size of motifs to study is not a very interesting statistic since the probability of a particular length-4 motif showing up randomly in a sequence not very high (1/(4^4) = 1/256). Given that the frequency of mutations, and all the evolutionary time during which to make changes to a sequence, these length=4 similar motifs are likely to randomly turn-up. The authors should consider using the occurrence of motifs which are at least seven since these frequencies begin to become less randomly placed. Length-4 words are already common in many many bacteria as restriction sites for restriction enzymes. The authors will also find that there are restriction sites of length-6 for the same purpose and so they will have to remove all restriction enzyme palindromes from their sets of k=4 or 6 sized motifs if they cannot continue with a longer motif length. However, if they are determining the level of conservation between organisms, then having longer motifs should not hurt their results.Once these issues are addressed, the manuscript will be much stronger.",
"responses": [
{
"c_id": "1668",
"date": "30 Oct 2015",
"name": "Benjamin Siranosian",
"role": "Author Response",
"response": "Thank you for reviewing the manuscript. I have considered the points you raised, and responded in order below. Changes to the manuscript are noted.The usage deviation-based statistics chosen for this paper are similar to those based on the composition vector of a sequence (Bonham-Carter et al., 2013). Usage deviation (tetranucleotide usage deviation, TUD, in the case of k=4) is a vector of the counts of the possible k-mers, normalized to the expected counts in a randomized genome with the same nucleotide composition. I have made additions to the methods section and included a new figure that makes the calculation of usage deviation more clear. The software tools used to perform these calculations have a description at the github page linked in the paper. I have added an example in the methods section that shows how to calculate TUD for a small sequence. Although this example outlines the method, the results are not very informative. The expected number of any 4-mer is very small in a short sequence, resulting in high TUD values for any 4-mers that do occur. We do not make any assumptions about the input data when calculating usage deviation or performing statistics in the paper. I showed trees constructed from other values of k in Figure 2. The relationships between phage genomes were consistent regardless of the value chosen for k. Other analyses mirrored this result, so we proceed exclusively with k={4, 6}. I agree that length-4 motifs are not interesting to study in isolation. Usage deviation, where values represent deviations from expected frequencies, overcome this point. Single occurrences or counts of any 4-mer are uninteresting. Only when counts are normalized and compared in aggregate do the trends that observed in the paper become meaningful. 7-mers would be less randomly placed in the phage genomes analyzed. Similar to the point above, however, the occurrences of singular k-mers are not considered. As k increases, the resulting usage deviation vectors become sparse. Up to 43% of the (4^7=16384) 7-mers are absent from individual genome sequences, and no 7-mer occurs at least once in every genome analyzed. The sparse nature of the data for 7-mers would not be well-suited to some of the analyses presented in this paper (PCA, searching for horizontally transferred segments). I acknowledge that many 4-mers and 6-mers are restriction sites. In fact, this makes the substrings more interesting. B3 mycobacteriophages have 4 times the expected usage of GATC, a restriction site in some bacteria. Biological sense dictates restriction sites would occur infrequently, but the results say the opposite. I do not feel it is necessary to remove restriction sites before the analysis, and doing so would be somewhat arbitrary. The set of restriction sites in mycobacteria species is not entirely characterized, and the host range for each mycobacteriophage has not been studied.We hope you find the answers to the points you raised and the revisions to the paper acceptable.References:Bonham-Carter, O., Steele, J. & Bastola, D. Alignment-free genetic sequence comparisons: a review of recent approaches by word analysis. Brief Bioinform 15, 890–905 (2014)."
}
]
}
] | 1
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https://f1000research.com/articles/4-36
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https://f1000research.com/articles/4-73/v1
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19 Mar 15
|
{
"type": "Research Article",
"title": "Weak and contradictory effects of self-medication with nectar nicotine by parasitized bumblebees",
"authors": [
"David Baracchi",
"Mark J. F. Brown",
"Lars Chittka"
],
"abstract": "The presence of antimicrobial secondary metabolites in nectar suggests that pollinators, which are threatened globally by emergent disease, may benefit from the consumption of nectars rich in these metabolites. We tested whether nicotine, a nectar secondary metabolite common in Solenaceae and Tilia species, is used by parasitized bumblebees as a source of self-medication, using a series of toxicological, microbiological and behavioural experiments. Caged bees infected with Crithidia bombi [TI1] had a slight preference for sucrose solution laced with the alkaloid and behavioural tests showed that the parasite infection induced an increased consumption of nicotine during foraging activity. When ingested, nicotine delayed the progression of a gut infection in bumblebees by a few days, but dietary nicotine did not clear the infection, and after 10 days the parasite load approached that of control bees. Moreover, when pathogens were exposed to the alkaloid prior to host ingestion the protozoan’s viability was not directly affected, suggesting that anti-parasite effects were relatively weak. Nicotine consumption in a single dose did not impose any cost even in food-stressed bees (starved) but the alkaloid had detrimental effects on healthy bees if consistently consumed for weeks. These toxic effects disappeared in infected bees suggesting that detoxification costs might have been counterbalanced by the advantages in slowing the progression of the infection. Nonetheless we did not find a benefit of nicotine consumption in terms of life expectancy of infected bees, making these findings difficult to interpret. Our results indicate that caution is warranted in interpreting impacts of plant metabolites on insect parasites and suggest that the conditions under which nicotine consumption provides benefits to either bees or plants remain to be identified. The contention that secondary metabolites in nectar may be under selection from pollinators, or used by plants to enhance their own reproductive success, remains to be confirmed.",
"keywords": [
"Bombus terrestris",
"Crithidia bombi",
"foraging",
"nicotine",
"pathogens",
"pollinators",
"pollinator-plant interactions",
"secondary metabolites"
],
"content": "Introduction\n\nParasites can have a dramatic impact on their hosts, and consequently provide a powerful selective force for host defence mechanisms. Molecular mechanisms (e.g. the innate and adaptive immune system) are traditionally considered the major anti-parasite defences in the animal kingdom. However, hosts can rely on a range of alternative defence mechanisms, such as morphological barriers (St Leger, 1991), changes in life-history traits (Michalakis, 2009), symbiont-mediated defences (Oliver et al., 2010) and altered behaviours (de Roode & Lefèvre, 2012; Moore, 2002).\n\nBehavioural immunity is an important modality of defence against diseases (de Roode & Lefèvre, 2012), and medication behaviour is a key immune mechanism in some animals (Clayton & Wolfe, 1993; de Roode et al., 2013). Medication behaviour has been defined as the use of anti-pathogenic substances found in the environment or produced by other species or individuals (Lozano, 1998). In therapeutic medication, sick individuals alter their behaviour to medicate themselves in response to parasites (Singer et al., 2009), while prophylaxis is displayed by healthy individuals in response to parasite risk rather than infection (Castella et al., 2008). For example, wood ants, bees and wasps behave prophylactically to incorporate conifer resin, propolis, or venom containing antimicrobial compounds into their nest, which inhibits the growth of bacteria and fungi (Baracchi & Turillazzi, 2010; Baracchi et al., 2011; Chapuisat et al., 2007; Castella et al., 2008; Simone et al., 2009), while, from a therapeutic perspective, ants apply antimicrobial venomous secretion to the cuticle of contaminated larvae to medicate their brood (Tragust et al., 2013). So far, most evidence for animal self-medication comes from the consumption of curative plants by vertebrates (Rodriguez & Wrangham, 1993). For example, chimpanzees, Pan troglodytes, alter their foraging to include medicinal substances (particular plant species) in their diets to cure helminth infections (Wrangham, 1995). Plants are good candidates for prophylactic or therapeutic foods as they often contain metabolites that display a wide range of biological activities (Cowan, 1999) which were originally evolved to combat herbivores or plant-parasites (Hadacek, 2002). This preferential ingestion of “non-nutritive” food and chemicals to self-medicate is known as pharmacophagy or zoopharmacognosy. Despite numerous studies investigating feeding plasticity with respect to plant nutrients and medicinal metabolites (reviewed in Mooney & Agrawal, 2008), investigations of potential pharmacophagy are rare, especially in insects. Exceptions concern self-medication behaviour described in two species of woolly bear caterpillars, which increase their preference for pyrrolizidine alkaloids or iridoid glycosides when parasitized, improving their chances of surviving parasitoid infection (Bernays & Singer, 2005; Singer et al., 2009; Smilanich et al., 2011). Similarly, wasp-infected fruit fly larvae preferentially consumed high-ethanol fly food as a medicine against their parasitoid wasp larvae, again increasing their survival (Milan et al., 2012), while no evidence for self-medication to nematode parasitism has been found in the fly Drosophila putrida (Debban & Dyer, 2013). Trans-generational medication, but not self-medication, has been described in the monarch butterfly (Lefevre et al., 2010) and self-medication has been hypothesized for honeybees that increase plant resin collection in response to a fungal infection (Simone-Finstrom & Spivak, 2012).\n\nAnimal societies arguably face the most intense pressure from pathogens. This pressure is enhanced in insect societies due to a suite of traits, including the high number of individuals living in high densities, relatively low genetic variability, and the relatively stable, high levels of humidity and temperatures of their nests (Schmid-Hempel, 1998). In addition, social pollinators, such as bumblebees and honeybees, are often exposed to an increased risk of infection via flowers (reviewed in McArt et al., 2014), which represent a shared “public place” where homo- and hetero-colonial conspecifics and other heterospecific pollinators feed repeatedly every day. Given the potential importance of parasites and disease in driving declines of managed honeybees (de Miranda & Genersch, 2010; Rosenkranz et al., 2010) and wild bumblebees (Cameron et al., 2011; Fürst et al., 2014; Schmid-Hempel et al., 2014), understanding the potential relevance of pharmacophagy to social pollinators may be a key to understanding and managing these declines.\n\nHere we use an important natural and managed pollinator, the bumblebee Bombus terrestris, and its parasite Crithidia bombi to investigate the potential for pharmacophagy in social pollinators. C. bombi, a trypanosome gut parasite, is the most prevalent parasite of bumblebees (Shykoff & Schmid-Hempel, 1991). The parasite, transmitted either vertically or horizontally (Durrer & Schmid-Hempel, 1994; Otterstatter & Thomson, 2007), infects adults per os, and two-three days post infection, infective cells are released through the faeces of bees (Schmid-Hempel & Schmid-Hempel, 1993). Queens infected by C. bombi have a reduced success in colony founding (Brown et al., 2003), and produce fewer reproductive offspring (Brown et al., 2003), while infected workers experience a higher mortality rate under stressful conditions (Brown et al., 2000). Moreover, infection impairs foraging success and learning abilities, inducing additional costs to the colony (Alghamdi et al., 2008; Gegear et al., 2006). Recent research (Manson et al., 2010; Richardson et al., 2015) has shown that several secondary metabolites such as alkaloids (including nicotine) and glycosides, reduce the C. bombi load after consumption by the bumblebee species Bombus impatiens, suggesting that these pollinators might exploit nectar toxins or other metabolites to self-medicate.\n\nTo test whether bumblebees are able to self-medicate using naturally occurring nectar secondary metabolites we conducted a series of toxicological, microbiological and behavioural experiments using a different species of Bombus (B. terrestris) and C. bombi as models and nicotine as a natural nectar alkaloid. Nicotine is encountered by pollinators at variable concentrations between 0.1 ng/μl and 3 ng/μl in floral nectar of Nicotiana species (native of South America and naturalised worldwide by humans) and Tilia species (native throughout most of the temperate Northern Hemisphere) (Detzel & Wink, 1993; Naef et al., 2004; Tadmor-Melamed et al., 2004).\n\n\nMethods\n\nAll experiments were performed with worker bumblebees (B. terrestris) obtained from a continuous rearing program (provided by Koppert B.V., The Netherlands) and conducted under standardized laboratory conditions. The insects were provided ad libitum with commercial pollen (provided by Koppert B.V., The Netherlands) and 30% sucrose solution as protein source and energy respectively. The parasites (the protozoan flagellates C. bombi) used for the experimental infections were taken from several naturally infected colonies that we started in the laboratory from infected queens.\n\nTo determine whether the nectar alkaloid nicotine influences the severity of C. bombi infections in bumblebees, we designed two experiments following (Manson et al., 2010). In the “Continuous Exposure” test, bees were first inoculated with C. bombi and then fed on a daily diet of a nicotine solution or sucrose solution (Control), simulating the continual ingestion of nectar constituents by an infected foraging bee. In the “Delayed Exposure” test, we first exposed directly C. bombi cells to nicotine or control solutions for two hours before inoculating bees, and then we fed them on a sucrose-only solution. We subsequently compared the parasite load in inoculated bumblebees.\n\nA mixture of different parasite strains was prepared by collecting faeces from 30 workers from three infected colonies. The faeces were mixed for one minute with a vortex mixer and the C. bombi cocktail was allowed to settle at room temperature for two hours, after which the supernatant was removed and mixed thoroughly. Cell counts were made using a haemocytometer. The faeces were then mixed with sugar water to produce an inoculum concentration of 2,000 parasite cells/μl. Prior to inoculation, bees were deprived of all food for two hours to facilitate infection. Bees derived from two different healthy colonies were screened to make sure that the bees were parasite-free. Each bee was then presented with a 10 μl drop of inoculum and observed until the inoculum was drunk. Thus, each bee ingested a total of 20,000 parasite cells. This dose falls within the range of C. bombi cells present in faeces from infected workers (Logan et al., 2005), and therefore simulates cells available for transmission to healthy bees.\n\nPost inoculation, in the “Continuous Exposure” test, bees from three colonies were kept individually in Petri dishes and received either a 0.5 ml solution of 2.5 ng/μl nicotine (nectar concentration in the natural range of this alkaloid) in 30% sucrose (Experimental bees, n = 20) or 0.5 ml of 30% sucrose only (Control bees, n = 20) along with a 1g pollen lump daily for 10 days. In the “Delayed Exposure” test the C. bombi inoculum was exposed to nicotine in the dark for two hours prior to host ingestion, simulating direct exposure of the pathogen to nectar in a flower. C. bombi cells were placed in a solution of 2.5 ng/μl nicotine in 30% sucrose (Experimental treatment), and in a solution of 30% sucrose only (Control treatment). Two hours later 20 Experimental bees and 20 Control bees were inoculated (for inoculum preparation see above). The treatment simulates the period between the deposition of Crithidia cells by infected bees and the next flower visit by a naïve bee. Post inoculation, bees of both groups were kept in individual Petri dishes and given 0.5 ml of 30% sucrose solution and a fresh pollen lump daily.\n\nIn both experiments, the infection levels were checked at day 7 and day 10 post inoculation (the period of time in which pathogen load is saturated (Schmid-Hempel & Schmid-Hempel, 1993)). Each bee was removed from its Petri dish and put into a small glass tube until it defecated. In cases when no or too little rectal fluid was obtained, the procedure was repeated for that bee a few hours later. Faeces were transferred to a haemocytometer to count the number of parasite cells.\n\nIn order to determine the impact of nicotine consumption on bumblebee survival and any possible interactive effects of dietary toxin consumption and physiological stress (for which we used starvation, as Crithidia has its biggest detrimental impacts on starved bees (Brown et al., 2000)), we conducted a series of experiments in which we exposed bumblebees to artificial nectars enriched with nicotine maintained either starved or provided with ad libitum food. “Starved bees” were moved individually from their nest into Petri dishes, starved for two hours and fed either with ad libitum 30% sucrose solution food for 30 minutes (Starved, Control) or 2.5 ng/μl nicotine in 30% sucrose (Starved, Nicotine). Survival censuses were conducted every hour until all bees were dead. “Ad libitum food bees” were kept individually in Petri dishes, and given 0.5 ml of 30% sucrose solution and a fresh pollen lump daily (Control ad libitum food), 2.5 ng/μl nicotine in 30% sucrose solution and a fresh pollen lump daily (Nicotine ad libitum food), 2.5 ng/μl nicotine in 30% sucrose solution on day 0 and 0.5 ml of 30% sucrose solution and a fresh pollen lump daily (Nicotine-once ad libitum food). Survival censuses were conducted daily until all bees had died. For each of the five treatments we chose bees from three different young healthy colonies and we randomised bees across treatment groups. Each treatment group was composed of 60 bees (20 bees per colony). Comparisons of the survival parameters of bumblebees in all treatments allowed us to evaluate the effect of nicotine, starvation, and colony membership on survival. Dead bees were immediately weighed using a microscale (Navigator N30330, Ohaus, Pine Brook, USA).\n\nIn order to evaluate whether infected bees benefit from the consumption of nicotine in terms of survival and/or parasite load we conducted two additional experiments in which infected bumblebees received artificial nectars enriched with nicotine or not and were maintained either starved (three groups of 30 bees, 10 bees from three different colonies, 90 bees in total) or provided with ad libitum food (three groups of 45 bees, 15 bees from three different colonies, 135 bees in total). In both experiments the three groups of bees were inoculated with C. bombi as described above and individually kept in Petri dishes under three types of diet (each diet consisted of two solutions dispensed by two different Eppendorf tubes): Control Group: 30% sucrose only in both dispensers (Suc-Suc group); Exp. Group 1: 2.5 ng/μl nicotine in 30% sucrose in both dispensers (Nic-Nic group); Exp. Group 2: 30% sucrose only in one dispenser and 2.5 ng/μl nicotine in 30% sucrose in the other one (Suc-Nic group). “Starved bees” were fed for 12 days and then starved until all bees were dead. The infection levels were checked at day 7 and day 10 post inoculation. Survival censuses were conducted every hour (starved bees) and every day (ad libitum food bees) until all bees were dead. At the end of the experiment we quantified total consumption of artificial nectars in each dispenser for each bee. Comparison of the survival parameters of bumblebees in all treatments allowed us to evaluate the effect of nicotine and starvation on survival.\n\nFor testing, each bee colony was housed in a wooden nest box (28 × 20 × 11 cm) connected to a wooden flight arena with a transparent, UV-transmitting Plexiglas lid (120 × 100 × 35 cm), by means of a transparent Plexiglas tube. Shutters along the length of this tube enabled control of the traffic of bees between nest boxes and flight arena (Chittka, 1998). Each bumblebee was individually marked with a coloured numbered disk.\n\nBees were pre-trained to forage on 12 square transparent plastic flowers of 24 × 24 mm (Perspex® Neutral) organized in two patches equidistant from the entrance of the nest. Plastic chips were placed on vertical transparent glass cylinders to raise them above the green floor of the flight arena. During the pre-training all flowers were rewarding with a 15 μl droplet of 30% sucrose solution, placed in a well in the centre of the flower (Raine & Chittka, 2008). This provided bees with an equal chance to associate both these patches (left and right) with reward during the pre-training period. Bees were allowed to forage freely on these flowers which were refilled as soon as the bees moved on a different artificial flower. In this way bees never experienced an empty flower with the exception of the last visited one. The number of foraging trips (bouts) made in the flight arena by each bee was observed to ensure only strongly motivated foragers visiting both patches (bees that did at least five consecutive foraging bouts) were selected for the experiment (Raine et al., 2006).\n\nAfter pre-training, the preference of both healthy and infected pre-trained bees was tested for blue plastic flowers (Perspex® 727) containing nicotine (one patch reward: 2.5 ng/μl nicotine in 30% sucrose solution; one patch reward: only 30% sucrose solution). Each bee (n = 31 infected bees; n = 28 healthy bees) was tested individually and one hundred consecutive choices were recorded after the first bout was initiated. Bees were regarded as choosing a flower when they landed and fed from it. Bees approaching or just briefly landing on a flower were not considered as choosing that flower. As in the pre-training, flowers were refilled after the bee moved to a different one so that bees never experienced an empty flower with the exception of the last visited one. Flowers were washed between subsequent bees in order to remove possible scent marks (Saleh & Chittka, 2006). The patch formed by nicotine-containing flowers was swapped from left to right for half the bees of each group (healthy and infected bees). Controlled illumination was provided by high frequency fluorescent lighting [(TMS 24F) lamp with HF-B 236 TLD (4.3 Khz) ballasts, Phillips, Netherlands fitted with Activa daylight fluorescent tubes, Osram] which simulated natural daylight (Dyer & Chittka, 2004). At the end of the experiment all the bees were sacrificed and the concentration of C. bombi in their hind gut was determined (see above).\n\nIn the infection experiments 10 out of 80 of bees died before day 10 for unknown causes. In total, we analysed the infection intensities of 40 (day 7) and 36 (day 10) bees in the “Continuous Exposure” experiment, and 37 (day 7) and 34 (day 10) bees in the “Delayed Exposure” experiment. To compare differences in parasite load between control and experimental bees at day 7 and day 10 post inoculation in both experiments we used a generalized linear mixed model (GLMM), with pathogen counts as the within-subject variable and C. bombi exposure to nicotine, time (day 7 and day 10), colony of origin, and bee body weight as explanatory factors. As the data were not normally distributed and homogeneity of variances and sphericity could not be assumed in several cases, we performed corrections according to Huynh-Feldt epsilon (Field, 2009). For the statistical evaluations in the survival experiments, we used the classical survival parameters including the survival distribution, percent survival at the end of the census period, median survival time (LT50), and the hazard ratio of death, using the Cox Proportional Regression analysis to generate the Wald Statistic. The hazard function characterized the instantaneous rate of death at a particular time while controlling for the effect of the other variables on survival. The following variables were entered in the regression model: colony of origin, body weight, nicotine treatment. The survival distributions for all treatments were computed and analysed with the Breslow Statistic (Mantel–Cox Test). For the behavioural experiment, a T test was used to examine differences between preferences for nicotine-rich nectar and control nectar in healthy and infected bees. Spearman rank correlation tests were used to correlate parasite load and nicotine preference. All statistical analyses were done on SPSS 13® for Windows.\n\n\nResults\n\nIn the “Continuous Exposure” test, a diet enriched with nicotine reduced the intensity of C. bombi infections in bumble bees (Dataset 1). GLMM analysis revealed significant main effects of nicotine and time since inoculation on infection intensity, but not colony of origin or bee body weight (Table 1). At both 7 days and 10 days post-inoculation, bees exposed to nicotine had infections that were, on average, 1.11 and 0.56 times respectively less intense than control bees (t test, day 7: n = 20-20, t = 5.2, df = 38, P < 0.001; day 10 n = 18-18, t = 3.47, df = 34, P = 0.001; Figure 1). Infection intensities increased significantly from day 7 to day 10, independently of nicotine treatment (no-significant Nicotine and Colony x Time effect; Table 1).\n\nFaeces were checked after 7 days and 10 days post inoculation. Box plots show medians, 25th and 75th percentiles (** P < 0.001; *P = 0.001).\n\nIn the “Delayed Exposure” test, exposing C. bombi to nicotine for two hours before inoculation had no effect on parasite load (Table 2) (Dataset 1). At 7 days and 10 days post-inoculation, bees exposed to nicotine had infections that on average were as intense as those of control bees (t test, day 7: n = 19-18, t = 0.16, df = 35, P = 0.87; day 10: n = 17-17, t = -0.69, df = 32, P = 0.5; Figure 2). Infection intensities increased significantly from day 7 to day 10, independently of nicotine treatment (there was no-significant Nicotine x Time and Colony x Time effects; Table 2). Taken together, these findings prove the antimicrobial activity of nicotine against the pathogen when ingested by bumblebees, but also indicate that when pathogens are exposed to the alkaloid prior to host ingestion the protozoan’s viability is not directly affected.\n\nFaeces were checked after 7 days and 10 days post inoculation. Box plots show medians, 25th and 75th percentiles (P = N.S.).\n\nIn the “Starved” test, statistical evaluation of the survivorship of control and experimental bumblebees revealed that a nicotine diet was not a significant predictor of mortality (Log-rank Mantel Cox test χ2 = 0.21, df = 1, P = 0.88; Figure 3A) (Dataset 2). Furthermore no effect of colony of origin and bee body weight on mortality was found (GLM, treatments: F = 1.1, df = 1, P = 0.29; Colony F = 0.46, df = 2, P = 0.63; body weight: F = 0.19, df = 1, P = 0.66). The median lethal time (LT50) for the two groups did not differ (control LT50: 39 hours, exp. bees LT50 = 37 hours).\n\nA: Cumulative survival of bees fed with a sucrose solution with (blue line) or without (green line) nicotine and starved. B: Cumulative survival of bees that received a daily diet of sucrose solution with (beige line), or without nicotine (blue line), or a single dose of nicotine on day one (green line).\n\nIn the “ad libitum food” test a Log-rank Mantel Cox test showed that a daily diet including nicotine was a significant predictor of mortality (χ2 = 11.56, df = 2, n = 180, P = 0.003; Figure 3B) (Dataset 2). Pairwise statistical comparisons revealed that bees fed consistently with nicotine had significantly lower survivorship than ‘Nicotine-once’ and ‘Control bumblebees’ (P = 0.001), while the latter two experimental groups did not differ (P = 0.86). LT50 of bees fed daily with nicotine was 39 days while ‘Nicotine-once’ bumblebees and control bees had a LT50 of 44 and 43 days respectively. Colony of origin and body weight did not affect bee mortality (GLM, Colony: F = 0.35, df = 2, P = 0.71; body weight: F = 1.90, df = 1, P = 0.16), but we found a significant interaction between body weight and treatment (larger bees were less susceptible to nicotine, GLM, F = 5.12, df = 1, P = 0.025). Taken together, these findings indicate that nicotine has some detrimental effects on healthy bumblebees if consistently consumed for weeks but also that these effects are possibly quite weak.\n\nIn both “ad libitum food bees” and “starved bees” tests, a nicotine diet was not a significant predictor of survival (Log-rank Mantel Cox test: “ad libitum food bees”: n = 135, Nic-Nic vs Nic-Suc χ2 = 0.3, P = 0.6; Nic-Nic vs Suc-Suc χ2 = 0.01, P = 0.9; Nic-Suc vs Suc-Suc χ2 = 0.7, P = 0.4; “Starved bees”, n = 76; Nic-Nic vs Nic-Suc χ2 = 0.4, P = 0.5; Nic-Nic vs Suc-Suc χ2 = 0.1, P = 0.7; Nic-Suc vs Suc-Suc χ2 = 0.01, P = 0.9) (Dataset 3). Furthermore no effect of colony of origin on mortality was found (GLM, “ad libitum food bees”: F = 1.4, df = 2, P = 0.24; “Starved bees”: GLM, F = 2.02, df = 2, P = 0.14). The median lethal time LT50 for the three groups did not differ (“ad libitum food bees”: Suc-Suc LT50: 22 days, Nic-Suc LT50 = 23, days, Nic-Nic LT50 = 22; “Starved bees”: Suc-Suc LT50: 25 hours, Nic-Suc LT50 = 28 hours, Nic-Nic LT50 = 31 hours).\n\nGLMM analysis revealed significant main effects of treatment (df = 2, F = 3.46, P = 0.03) and time since inoculation (df = 1, F = 57.3, P < 0.001) on infection intensity, but not colony of origin (df = 2, F = 1.64, P = 1.96). No interaction between diet, time and colony was significant. Overall bees caged in Petri dishes consumed less food over the entire duration of the experiment if exposed to nicotine (Anova test: F = 9.68, n = 90, df = 2, 87, P = 0.001; Dunnett T3 post hoc test: Suc-Suc vs Nic-Nic and Suc-Suc vs Nic-Suc P < 0.001) (Dataset 4). Infected bees showed a slight preference (54 ± 17 %) for sucrose solution laced with nicotine (Paired samples t test, t = 2.14, df = 29, n = 30, P = 0.04).\n\nOverall these findings indicate that, even though nicotine reduces the parasite load in infected bees, and such bees have a slight preference for sucrose solution laced with the alkaloid, there is no net benefit in term of survival for infected bees.\n\nInfected bumblebees allowed to forage on plastic flowers showed a significantly increased propensity to visit nicotine rewarding flowers when compared to healthy bees (t test, n = 31, 28, t = -2.4, df = 57, P = 0.016; Figure 4) (Dataset 5). Indeed on 100 consecutive choices infected bees visited the nicotine flowers on average 64.5 ± 13.8 (s.d.) times while healthy bees visited them 54.8 ± 19.4 (s.d.) times. Since test bees were introducing nicotine into the colony throughout testing, we controlled for prior exposure to nicotine effect on nicotine preference. Bees tested later in the experiment did not show a higher or lower nicotine preference (Spearman test, Infected bees: ρ = -0.21, n = 31, P = 0.3; Control bees n = 28, ρ = 0.041, P = 0.8). There was no correlation between pathogen load and the propensity of infected bees to visit flowers with nicotine-rich artificial nectar (Spearman test: n = 31, ρ = 0.19, df = 29, P = 0.28).\n\nInfected bees visited nicotine-containing flowers 64.5 ± 13.8 (s.d.) times while healthy bees visited them 54.8 ± 19.4 (s.d.) times.\n\n\nDiscussion\n\nHere we demonstrate that parasitized bumblebees modify their diet preference and foraging behaviour to delay the development of an infection. In our experimental setup the parasite infection induced an increased consumption of nicotine both in individually caged as well as in foraging bumblebees. Despite this preferential ingestion of a “non-nutritive” antimicrobial alkaloid by infected bees, the self-medication behaviour is not efficient since dietary nicotine does not fully cure C. bombi infection. Nonetheless bumblebees exhibited a reduced C. bombi load after daily consumption of the alkaloid. In nature, infection entails an array of costs (Alghamdi et al., 2008; Brown et al., 2000; Brown et al., 2003; Gegear et al., 2006). As a consequence, any reduction in the severity or progression of infection in bees, induced by mechanisms such as the consumption of nectar containing curative alkaloids (e.g. gelsemine (Manson et al., 2010), anabasine and nicotine (Richardson et al., 2015)), might be beneficial in terms of fitness for both bees and colonies.\n\nIn the same way as bumblebees have adapted their foraging behaviour to reduce the uptake of parasites (Fouks & Lattorff, 2011), bumblebees may be adapted to modify their diet with curative nectars once infected. The recent demonstration that honeybee nurse bees, infected with the microsporidian gut parasite Nosema ceranae, show different preferences for various types of honeys in a simultaneous choice test, preferring honeys with a higher antibiotic activity (Gherman et al., 2014), suggests that such behaviours may be widespread in social pollinators. However, our results suggest that a description of this behaviour as pharmacophagy may require further evidence. Indeed, although dietary nicotine slows the progression of infection by a few days, this effect does not induce any benefit in terms of life expectancy of infected bees. Even if we cannot completely exclude that the weak effect of nicotine is due to the initial challenge being too strong for the nicotine to have a measurable influence on life expectancy, both nicotine concentration and Crithidia inocula used in our study simulated natural doses. Additional field and mesocosm tests are thus needed to clarify the actual benefits of ingestion.\n\nNicotine also has a costly effect on uninfected individuals, as shown by our toxicological assays. A daily diet containing nicotine, lasting more than two months, reduced the life expectancy of bumblebees, and this effect was stronger in smaller bees. This might possibly be aggravated in the wild, where bees are exposed to other stressors and do not have access to ad libitum food. However, we note that differences in mortality rate between controls and nicotine-treated bees started to be evident only after 20 days from the first exposure suggesting that in nature this detrimental effect may be mitigated due to the relatively short lifespan of foragers in the wild (da Silva-Matos & Garófalo, 2000). Moreover, in nature, bees may not forage on a single nectar source continuously for weeks as we simulated in our experiments, further reducing the negative effect of nicotine intake. In infected bumblebees the detrimental effect of nicotine is no longer evident suggesting that detoxification costs might be counterbalanced by the advantages in slowing the progression of the infection. However, contrary to our prediction, we found no trade-off between costs and benefits in terms of survival, and infected bumblebee lifespan was not affected by the consumption on nicotine. Similar results have recently been found for the antimicrobial alkaloid anabasine that did not induce a significant fitness benefit in the bumblebee species B. impatiens despite its effectiveness in reducing the parasite load by up to 80 percent (Richardson et al., 2015).\n\nThe cost imposed by the consumption of nicotine in our experiments may explain why healthy bees did not constantly consume high doses of nicotine (Tiedeken et al., 2014). Similarly, infected bees kept in Petri dishes reduced the overall uptake of food if exposed to nicotine. This is surprising given that those bumblebees also had a slight preference for sucrose solution laced with the alkaloid, and free-flying healthy bumblebees were not repelled by artificial nectar laced with nicotine. While these behavioural preferences may be explained by the impact that some nectar alkaloids, including nicotine, have on learning and memory in bees (Chittka & Peng, 2013; Thany & Gauthier, 2005; Wright et al., 2013), the mechanism behind the overall reduced consumption caused by nicotine remains unexplained. In humans at least, it is well established that nicotine has appetite-reducing effects (Jessen et al., 2005).\n\nCurrently it is unclear how nicotine acts on C. bombi. Nicotine is a highly toxic molecule (Benowitz, 1998) that acts against a wide spectrum of bacterial and fungal pathogens (Pavia et al., 2000). House sparrows and several finch species, for example, add smoked cigarette butts retaining substantial amounts of nicotine to their nests to reduce mite infestations (Suárez-Rodríguez et al., 2013). While our in vivo microbiological experiments prove the antimicrobial activity of nicotine against the pathogen when ingested, they also suggest that nicotine does not directly interfere with the protozoan’s viability, at least when measured as infectivity. As suggested by Manson et al. (2010), who found similar effects of the natural alkaloid gelsemine, an alkaloid-rich diet might increase a bee’s excretion rate, as occurs for nectarivorous bird (Tadmor-Melamed et al., 2004), effectively “flushing” C. bombi cells from the gut. Another possibility might be that nicotine, or perhaps its metabolites, directly modify the mid-gut epithelium or the environment of its lumen, making it less suitable for the parasite.\n\nIn conclusion, we believe that our results suggest that a more careful approach to interpreting impacts of plant metabolites on insect parasites is warranted. Recent findings have suggested that the preferential ingestion of natural nectar secondary metabolites in pollinators might play a key role in mediating pathogen transmission within and between colonies (Richardson et al., 2015) or interactions among pollinators and their parasites (Manson et al., 2010). Similarly, our results and other recent studies (Gherman et al., 2014; Richardson et al., 2015) have suggested that bees may self-medicate by consuming plant secondary metabolites when they are infected with parasites. However, our study suggests that the conditions under which nicotine consumption provides benefits to either bees or plants remain to be identified. The contention that secondary metabolites in nectar may be under selection from pollinators, or used by plants to enhance their own reproductive success (Chittka & Peng, 2013; Thomson et al., 2014; Wright et al., 2013), should ideally be confirmed with further studies, which examine the impacts of these metabolites on both bee and plant fitness under field-realistic conditions.\n\n\nData availability\n\nF1000Research: Dataset 1. Infection experiments, 10.5256/f1000research.6262.d44610 (Baracchi et al., 2015a).\n\nF1000Research: Dataset 2. Laboratory toxicity bioassays, 10.5256/f1000research.6262.d44612 (Baracchi et al., 2015b).\n\nF1000Research: Dataset 3. Trade-off between detrimental and beneficial effects of nicotine, 10.5256/f1000research.6262.d44613 (Baracchi et al., 2015c).\n\nF1000Research: Dataset 4. Diet preference of caged bees, 10.5256/f1000research.6262.d44614 (Baracchi et al., 2015d).\n\nF1000Research: Dataset 5. Behavioural test, 10.5256/f1000research.6262.d44615 (Baracchi et al., 2015e).",
"appendix": "Author contributions\n\n\n\nD.B. conceived the study and carried out the experiments. D.B. and M.J.F.B. designed the experiments. LC made significant contributions to the interpretation of the data. All authors equally contributed in writing and revising the draft of the manuscript and have agreed the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nD.B. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme.\n\nThe authors confirm that the 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 Anna Woodhouse for helping in behavioural experiments, Dr. Gemma Baron for providing Crithidia samples, Dr. Caroline Brennan for nicotine solution, and Thomas Ingraham (an employee of F1000Research) for comments on the manuscript.\n\n\nReferences\n\nAlghamdi A, Dalton L, Phillis A, et al.: Immune response impairs learning in free-flying bumble-bees. Biol Lett. 2008; 4(5): 479–481. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaracchi D, Brown MJF, Chittka L: Dataset 1 in: Weak and contradictory effects of self-medication with nectar nicotine by parasitized bumblebees. F1000Research. 2015a. Data Source\n\nBaracchi D, Brown MJF, Chittka L: Dataset 2 in: Weak and contradictory effects of self-medication with nectar nicotine by parasitized bumblebees. F1000Research. 2015b. Data Source\n\nBaracchi D, Brown MJF, Chittka L: Dataset 3 in: Weak and contradictory effects of self-medication with nectar nicotine by parasitized bumblebees. F1000Research. 2015c. Data Source\n\nBaracchi D, Brown MJF, Chittka L: Dataset 4 in: Weak and contradictory effects of self-medication with nectar nicotine by parasitized bumblebees. F1000Research. 2015d. Data Source\n\nBaracchi D, Brown MJF, Chittka L: Dataset 5 in: Weak and contradictory effects of self-medication with nectar nicotine by parasitized bumblebees. F1000Research. 2015e. 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In Binnington K Retnakara A (eds) Physiology of the insect epidermis CSIRO. 1991.\n\nSuárez-Rodríguez M, López-Rull I, Garcia CM: Incorporation of cigarette butts into nests reduces nest ectoparasite load in urban birds: new ingredients for an old recipe? Biol Lett. 2013; 9(1): 20120931. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTadmor-Melamed H, Markman S, Arieli A, et al.: Limited ability of Palestine sunbirds Nectarinia osea to cope with pyridine alkaloids in nectar of tree tobacco Nicotiana glauca. Funct Ecol. 2004; 18(6): 844–850. Publisher Full Text\n\nThany SH, Gauthier M: Nicotine injected into the antennal lobes induces a rapid modulation of sucrose threshold and improves short-term memory in the honeybee Apis mellifera. Brain Res. 2005; 1039(1–2): 216–219. PubMed Abstract | Publisher Full Text\n\nThomson JD, Draguleasa MA, Tan MG: Flowers with caffeinated nectar receive more pollination. Arthropod-Plant Inte. 2015; 9(1): 1–7. Publisher Full Text\n\nTiedeken EJ, Stout JC, Stevenson PC, et al.: Bumblebees are not deterred by ecologically relevant concentrations of nectar toxins. J Exp Biol. 2014; 217(pt 9): 1620–1625. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTragust S, Mitteregger B, Barone V, et al.: Ants disinfect fungus-exposed brood by oral uptake and spread of their poison. Curr Biol. 2013; 23(1): 76–82. PubMed Abstract | Publisher Full Text\n\nWrangham RW: Relationship of chimpanzee leaf-swallowing to a tapeworm infection. Am J Primatol. 1995; 37(4): 297–303. Publisher Full Text\n\nWright GA, Baker DD, Palmer MJ, et al.: Caffeine in floral nectar enhances a pollinator’s memory of reward. Science. 2013; 339(6124): 1202–1204. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "8036",
"date": "27 Mar 2015",
"name": "James D. Thomson",
"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\nContent of paper. This paper reports a set of nicely designed experiments aimed at determining whether Bombus terrestris self-medicates against trypanosome (Crithidia bombi) infection by seeking and consuming nicotine-laced floral nectar. Using captive bee colonies and artificial flowers in laboratory conditions permits well-controlled manipulative experiments that would be effectively impossible in the field. The results are ambivalent, in that dietary nicotine does reduce the intensity of gut infection, and infected bees do seek out nicotine, but attempts to demonstrate improved health in terms of worker bee lifespan yield negative results. For the most part, I endorse both the interpretation of these results and the recognition by Baracchi et al. that further research is needed to settle the question of self-medication. I do have three reservations about the interpretations offered here, however, and I think the paper would be improved by some additional discussion of these issues. First, any anti-parasitic medicinal effects of toxic compounds will depend on dose rates. Too much medicine may harm the host; too little may exert no therapeutic effect. Baracchi et al. state that they have used “natural” dosages of nicotine, but what that means is that they have prepared solutions whose nicotine levels match those reported from some floral nectars. That is an appropriate starting point, but there may be little correspondence between the concentrations that flowers offer and those that bees are exposed to in a natural colony. Foraging workers typically collect floral nectar from various plant species that differ in nectar chemistry. Much of what they collect is not digested by the collectors themselves, but is transported back to the nest and regurgitated into communal honeypots that serve as energy stores for the larvae, the queen, and the many workers that do not forage. As this complicated cocktail is assembled, soluble compounds are concentrated by evaporation, diluted by mixing with other nectars, and probably further modified by enzymatic and microbial action. There is no reason to expect that the concentration of any particular compound in this brew bears any relationship to its concentration in one of the many floral nectars that have been pooled. Indeed, honeypots within a nest may hold different mixtures because particular foragers tend to discharge their collections into particular honeypots. I believe that explicit attention must be paid to honeypot composition if the question of bee medication is to advance. Second, by choosing to look at toxic effects on workers, Baracchi et al. are not able to detect possible benefits of medication on other members of the colony, specifically larvae or the queen. One can imagine that certain inputs of nicotine to the colony might have no net effects of worker survival but might allow the queen to lay more eggs or the larvae to prosper. Indeed, effects could conceivably be harmful for the foragers but still beneficial to the colony. There might also be different effects on workers that forage and those that serve as nurses. The social nature of these bees must be considered. Third, by considering nectar only, Baracchi et al. don’t consider the probability that secondary metabolites found in nectar are also likely to occur in the pollen of the same flower species. In nature, therefore, bees that choose to forage on nicotine-rich nectars are also likely to be collecting nicotine rich pollen from those flowers. To the extent that such correlations hold in nature, the larvae (who are the primary consumers of that pollen) may be receiving very different doses than adult bees. In summary, it would be very illuminating, although tedious, to consider experiments that measure whole-colony health as a response variable rather than worker longevity. Errors. In this review process, the lack of line numbers, or an editable form of the MS, makes it hard to flag such things as unclear phrases. Here are a few mistakes that should be addressed, however: In the abstract, the plant family Solanaceae is misspelled. In the caption for Figure 1, “bees received” should be rephrased. I believe that there must be a serious error in Dataset 2. Unless I am missing something, the data reported for “starved” and “ad libitum” treatments are identical. This looks like a cut-and-paste error. Column headings in the tables and data sets should be more explanatory. For example, in Table 2, experimental treatments are denoted simply as “time” and “nicotine.” Those labels are too cryptic. The \"Statistical Analysis\" section refers to several \"classical...parameters,\" but these are not picked up in the Results. Reconcile?",
"responses": [
{
"c_id": "1390",
"date": "29 Oct 2015",
"name": "Lars Chittka",
"role": "Author Response",
"response": "Referee comment: …Any anti-parasitic medicinal effects of toxic compounds will depend on dose rates. Too much medicine may harm the host; too little may exert no therapeutic effect. Baracchi et al. state that they have used “natural” dosages of nicotine, but what that means is that they have prepared solutions whose nicotine levels match those reported from some floral nectars. That is an appropriate starting point, but there may be little correspondence between the concentrations that flowers offer and those that bees are exposed to in a natural colony. Foraging workers typically collect floral nectar from various plant species that differ in nectar chemistry. Much of what they collect is not digested by the collectors themselves, but is transported back to the nest and regurgitated into communal honeypots that serve as energy stores for the larvae, the queen, and the many workers that do not forage. As this complicated cocktail is assembled, soluble compounds are concentrated by evaporation, diluted by mixing with other nectars, and probably further modified by enzymatic and microbial action. There is no reason to expect that the concentration of any particular compound in this brew bears any relationship to its concentration in one of the many floral nectars that have been pooled. Indeed, honeypots within a nest may hold different mixtures because particular foragers tend to discharge their collections into particular honeypots. I believe that explicit attention must be paid to honeypot composition if the question of bee medication is to advance. Author reply: We added a paragraph in the discussion section to address this point. Referee comment: By choosing to look at toxic effects on workers, Baracchi et al. are not able to detect possible benefits of medication on other members of the colony, specifically larvae or the queen. One can imagine that certain inputs of nicotine to the colony might have no net effects of worker survival but might allow the queen to lay more eggs or the larvae to prosper. Indeed, effects could conceivably be harmful for the foragers but still beneficial to the colony. There might also be different effects on workers that forage and those that serve as nurses. The social nature of these bees must be considered. Author reply: We agree and we stressed more clearly in the revised version that benefits from the consumption of nicotine may influence colony health and dynamics despite the lack of evident benefits for infected foragers. Referee comment: By considering nectar only, Baracchi et al. don’t consider the probability that secondary metabolites found in nectar are also likely to occur in the pollen of the same flower species. In nature, therefore, bees that choose to forage on nicotine-rich nectars are also likely to be collecting nicotine rich pollen from those flowers. To the extent that such correlations hold in nature, the larvae may be receiving very different doses than adult bees. Author reply: The argument raised by the referee is valid, and we briefly mentioned this possibility in the discussion Referee comment: In summary, it would be very illuminating, although tedious, to consider experiments that measure whole-colony health as a response variable rather than worker longevity. Author reply: We feel that considering a new set of experiments to measure whole-colony health would represent not simply an additional big experiment, but a new study itself. Instead, we now carefully discuss all these issues. Referee comment: In the abstract, the plant family Solanaceae is misspelled. In the caption for Figure 1, “bees received” should be rephrased. I believe that there must be a serious error in Dataset 2. Unless I am missing something, the data reported for “starved” and “ad libitum” treatments are identical. This looks like a cut-and-paste error. Author reply: Thank you for spotting these errors. In the revised version we have fixed them. Referee comment: Column headings in the tables and data sets should be more explanatory. For example, in Table 2, experimental treatments are denoted simply as “time” and “nicotine.” Those labels are too cryptic. Author reply: As suggested by the referee we labelled more effectively the figures and the date setsReferee comment: The \"Statistical Analysis\" section refers to several \"classical...parameters,\" but these are not picked up in the Results. Reconcile? Author reply: In the revised version we have made sure that the Results section addressed all the parameters mentioned in the Methods section."
}
]
},
{
"id": "8215",
"date": "10 Apr 2015",
"name": "Michael Simone-Finstrom",
"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 presented a fairly comprehensive set of experiments in order to elucidate the role that nicotine may have in self-medication of bumblebees against Chrithidia infection. The experiments progress well, starting with controlled lab infection studies to lab-based diet and lifespan analysis to a semi-field foraging test. I think the authors sufficiently described the methods and subsequent results and mostly had an appropriate discussion of the relevant findings. I just have a few small issues that the authors could address to improve and clarify aspects of the manuscript. But overall it is a strong paper. First, I do think “contradictory” could be removed from the title as I don’t think the results are necessarily contradictory, but do just show a weak effect. Just because the ingestion of nicotine reduces parasite infection and does not have a subsequent effect of increasing lifespan of infected bees, this does not mean that there are no other fitness-related benefits or that at different doses effects may not be more pronounced. I think “weak effect” aptly describes the findings. Generally, tables and figures could be labeled more effectively. For example, I think the fact that Figure 1 and 2 have the same x-axis is problematic given what the experimental treatments were. I think in Figure 2 it would be better to indicate that the Nicotine there is the Nicotine pre-treatment. Same for Table 2. Whereas in Figure 1 it would be nicotine diet. The legend for Figure 3 should explicitly state that these bees were all uninfected, since this is an important point, and since the lifespan data of infected bees is not represented in a figure. For the discussion and results, overall I think there just are a few other points that can be made. One is simply drawing stronger connections across the multitude of tests that were done. Infection experiments: would it be valuable to show statistically that there is no difference between the pre-treated Chrithidia and the standard (basically comparing figure 1 and figure 2)? Also is the 2-hour exposure time relevant? How was this decided upon? Same for dose of exposure. I think this needs to be discussed more thoroughly or at least a citation provided to justify this amount. Trade-off: Important to note that Chrithidia infection was found to reduce lifespan of bees (as compared to the bees in the toxicity part of the study). I think a discussion of the relevance of the doses used is important. Were any dose-response trials conducted? Presumably at a higher dose, it would be even more toxic to the bees and lower doses may not have much of an effect. Could ingestion of nectar alkaloids be a generalized response to sickness? So maybe it’s not as effective against Chrithidia, but this isn’t the only parasite bumblebees get. A larger context into how this might influence colony dynamics and health is important. This is hinted at in a couple sentences, but since all of these studies were really done with individual bees or individual behavior, this is a significant point. Perhaps lifespan analysis of forager bumblebees would be different in a social setting. Similarly perhaps fitness benefits aren’t seen since it’s just measured in terms of individual lifespan, but maybe a reduction in parasite load affects foraging efficiency or nursing ability and thus colony productivity.",
"responses": [
{
"c_id": "1391",
"date": "29 Oct 2015",
"name": "Lars Chittka",
"role": "Author Response",
"response": "Referee comment: First, I do think “contradictory” could be removed from the title as I don’t think the results are necessarily contradictory, but do just show a weak effect. Just because the ingestion of nicotine reduces parasite infection and does not have a subsequent effect of increasing lifespan of infected bees, this does not mean that there are no other fitness-related benefits or that at different doses effects may not be more pronounced. I think “weak effect” aptly describes the findings. Author reply: As suggested by the referee we removed the word “Contradictory” from the title and phrased it as a question instead. Referee comment: Generally, tables and figures could be labelled more effectively. For example, I think the fact that Figure 1 and 2 have the same x-axis is problematic given what the experimental treatments were. I think in Figure 2 it would be better to indicate that the Nicotine there is the Nicotine pre-treatment. Same for Table 2. Whereas in Figure 1 it would be nicotine diet. Author reply: As suggested by the referee we labelled more effectively the figures and the tables as outlined above. Referee comment: For the discussion and results, overall I think there just are a few other points that can be made. One is simply drawing stronger connections across the multitude of tests that were done. Author reply: We changed part of the discussion to address this concern.Referee comment: Infection experiments: would it be valuable to show statistically that there is no difference between the pre-treated Crithidia and the standard (basically comparing figure 1 and figure 2)? Author reply: As suggested by the referee we added the statistical test. Referee comment: Also is the 2-hour exposure time relevant? How was this decided upon? Same for dose of exposure. I think this needs to be discussed more thoroughly or at least a citation provided to justify this amount. Author reply: In the Materials and Methods section we provided a citation to justify the protocol used. Referee comment: Trade-off: Important to note that Crithidia infection was found to reduce lifespan of bees (as compared to the bees in the toxicity part of the study).Author reply: We stressed this point more clearly in the revised version. Referee comment: I think a discussion of the relevance of the doses used is important. Were any dose-response trials conducted? Presumably at a higher dose, it would be even more toxic to the bees and lower doses may not have much of an effect.Author reply: We agree and since the same point has been also raised by a second referee (James Thomson) we discuss carefully and more clearly this important point in the revised version. Referee comment: Could ingestion of nectar alkaloids be a generalized response to sickness? So maybe it’s not as effective against Crithidia, but this isn’t the only parasite bumblebees get. Author reply: The referee suggestion is valid and we now mention this in the discussion. Referee comment: A larger context into how this might influence colony dynamics and health is important. This is hinted at in a couple sentences, but since all of these studies were really done with individual bees or individual behavior, this is a significant point. Perhaps lifespan analysis of forager bumblebees would be different in a social setting. Similarly perhaps fitness benefits aren’t seen since it’s just measured in terms of individual lifespan, but maybe a reduction in parasite load affects foraging efficiency or nursing ability and thus colony productivity. Author reply: In line with the important comment, also raised by a second referee (James Thomson), we added a paragraph to further discuss these issues."
}
]
},
{
"id": "8216",
"date": "20 Apr 2015",
"name": "Marla Spivak",
"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\nDetermining the extent that bumble bees may self-medicate by consuming floral nectar containing antimicrobial secondary plant metabolites when infected with a pathogen is a fascinating line of study. The experiments presented are well conducted and analyzed, and I found the results not entirely surprising or contradictory. There was a temporary effect of nicotine (when provided in sugar syrup) against Crithidia bombi in Bombus terrestris workers. This effect might have been stronger and more long lasting, and even might have cleared the infection, if the dose of nicotine consumed was higher. This could be tested in future experiments using the high- and low-end concentrations found in floral nectar. Even though the infected bees preferred sucrose laced with nicotine in the clever foraging tests and in petri dishes, the authors say the results should be interpreted with caution because the life expectancy of the infected, nicotine-fed bees was not increased relative to controls. But as they also point out, bees don’t live very long, and many factors affect worker bee life expectancy. Possibly, at the colony level, reducing the infection level even for a few days in a certain number of workers might slow the rate of horizontal transmission among nest mates; this remains to be tested. I think it would be revealing to test the effects of these alkaloids, or other plant metabolites, on infected queen bumble bees. As Crithidia is also vertically transmitted, it would be interesting to know if the vertical transmission of this parasite could be reduced if the queen ingests nectar that contains antimicrobial metabolites. It also would be interesting to know if the infected workers that collect nectar containing these compounds feed the queen with them, potentially lowering her pathogen load, which would then might allow her to produce more reproductive male and gynes. These types of experiments are more difficult, but could yield more field-relevant results. Yet, the experiments presented here are great first steps in understanding a new area of research on the interaction of pollinators, pathogens and plant compounds. I greatly appreciate that the authors offer a cautionary stance in interpreting their results, but I do think, given the physiological tradeoffs involved in consuming potentially toxic compounds, that small temporary effects on individual bees might translate to larger effects at the colony-level.",
"responses": [
{
"c_id": "1392",
"date": "29 Oct 2015",
"name": "Lars Chittka",
"role": "Author Response",
"response": "Referee comment: Determining the extent that bumble bees may self-medicate by consuming floral nectar containing antimicrobial secondary plant metabolites when infected with a pathogen is a fascinating line of study. The experiments presented are well conducted and analyzed, and I found the results not entirely surprising or contradictory. Author reply: We agree both and we changed the title and the discussion accordingly. Referee comment: There was a temporary effect of nicotine (when provided in sugar syrup) against Crithidia bombi in Bombus terrestris workers. This effect might have been stronger and more long lasting, and even might have cleared the infection, if the dose of nicotine consumed was higher. This could be tested in future experiments using the high- and low-end concentrations found in floral nectar. Even though the infected bees preferred sucrose laced with nicotine in the clever foraging tests and in petri dishes, the authors say the results should be interpreted with caution because the life expectancy of the infected, nicotine-fed bees was not increased relative to controls. But as they also point out, bees don’t live very long, and many factors affect worker bee life expectancy. Possibly, at the colony level, reducing the infection level even for a few days in a certain number of workers might slow the rate of horizontal transmission among nest mates; this remains to be tested. I think it would be revealing to test the effects of these alkaloids, or other plant metabolites, on infected queen bumble bees. As Crithidia is also vertically transmitted, it would be interesting to know if the vertical transmission of this parasite could be reduced if the queen ingests nectar that contains antimicrobial metabolites. It also would be interesting to know if the infected workers that collect nectar containing these compounds feed the queen with them, potentially lowering her pathogen load, which would then might allow her to produce more reproductive male and gynes. These types of experiments are more difficult, but could yield more field-relevant results. Yet, the experiments presented here are great first steps in understanding a new area of research on the interaction of pollinators, pathogens and plant compounds. I greatly appreciate that the authors offer a cautionary stance in interpreting their results, but I do think, given the physiological tradeoffs involved in consuming potentially toxic compounds, that small temporary effects on individual bees might translate to larger effects at the colony-level. Author reply: We agree and we now explain in the discussion that our results provide potential evidence for self-medication. Yet, until measurable benefits for the bees or the colonies are found we think that caution is warranted."
}
]
}
] | 1
|
https://f1000research.com/articles/4-73
|
https://f1000research.com/articles/4-1177/v1
|
29 Oct 15
|
{
"type": "Review",
"title": "What do we know about the participation of hematopoietic stem cells in hematopoiesis?",
"authors": [
"Nina Drize",
"Nataliya Petinati",
"Nataliya Petinati"
],
"abstract": "The demonstrated presence in adult tissues of cells with sustained tissue regenerative potential has given rise to the concept of tissue stem cells. Assays to detect and measure such cells indicate that they have enormous proliferative potential and usually an ability to produce all or many of the mature cell types that define the specialized functionality of the tissue. In the hematopoietic system, one or only a few cells can restore lifelong hematopoiesis of the whole organism. To what extent is the maintenance of hematopoietic stem cells required during normal hematopoiesis? How does the constant maintenance of hematopoiesis occur and what is the behavior of the hematopoietic stem cells in the normal organism? How many of the hematopoietic stem cells are created during the development of the organism? How many hematopoietic stem cells are generating more mature progeny at any given moment? What happens to the population of hematopoietic stem cells in aging? This review will attempt to describe the results of recent research which contradict some of the ideas established over the past 30 years about how hematopoiesis is regulated.",
"keywords": [
"Hematopoiesis",
"Stem cells",
"colony-forming units"
],
"content": "A piece of history\n\nNature and Nature’s laws lay hid in night:\n\nGod said, “Let Newton be!” and all was light.\n\nAlexander Pope (1688–1744)\n\n\n\nIt did not last; the devil howling\n\n“Ho! Let Einstein be!” restored the status quo.\n\nSir John Collings Squire (1884–1958)\n\nHematopoietic cells arise from mesodermal precursors in the developing vertebrate embryo in multiple waves in different anatomic sites: allantois, yolk sac, and the aorta-gonad-mesonephros1–9. Cells able to maintain lifelong hematopoiesis emerge first from ventral aortic hemogenic endothelial cells and immediately enter the circulation10–13. These hematopoietic stem cells (HSCs) home to and expand within the fetal liver, spleen, thymus and eventually seed the bone marrow—the major blood-forming organ in the adult3,7. The precise number of HSCs at all stages of development remains poorly defined.\n\nThe modern era of HSC characterization started with the work of Till and McCulloch14. They described spleen colony-forming units (CFU-S). For many years thereafter, these cells were believed to have the properties of HSCs. Then it was discovered that most CFU-S can repopulate the hematopoietic system for only a short time and that they are descendants of phenotypically distinct cells that are able to repopulate the hematopoietic system for a long time15–18. The latter are usually detected by their ability to perpetually regenerate all blood cell types in a myeloablated recipient19 and, in the adult, these HSCs are quiescent most of the time20. Two general models have been put forth for how HSCs are recruited into proliferation and subsequent differentiation under normal physiological conditions. The “clonal succession model” proposes that small numbers of HSCs are sequentially recruited to leave the compartment and enter the cell cycle, and thereafter initiate an irreversible lineage commitment process16,21–23. The “clonal stability model” suggests that randomly selected self-maintaining HSCs continuously replenish the supply of mature blood cells throughout an organism’s lifetime24–26.\n\n\nMethods of marking individual HSCs\n\nIn order to understand how normal hematopoiesis is maintained, we require methods that can trace the separate long-term outputs of individual cells. One approach has been to permanently and uniquely mark individual HSCs. Radiation-induced chromosomal markers were used by Till and co-authors to show that all cells in each spleen colony derive from a single cell27,28. However, the small number of chromosomally marked cells obtained using this approach made it inadequate to resolve the HSC clones operative in long-term transplant experiments.\n\nThe genetic marking of mouse HSCs via the retroviral-mediated transfer of a foreign gene provided evidence that one cell is capable of differentiating into all major types of hematopoietic cells29,30 and allowed the composition of lifelong clones derived from adult mouse bone marrow and fetal liver HSCs to be examined22,31. It was also shown that the hematopoiesis in the lethally irradiated mice transplanted with the marked cells was polyclonal and was supported initially by a large number of short-lived, successively active clones22,32. Moreover, it was found that the formation of blood is polyclonal, not only at the level of early pluripotent progenitor, CFUs, but also at the level of terminally differentiated mature cells33.\n\nRetroviral marking is, however, limited to cells that are actively cycling. This method thus fails to label any HSCs that are not dividing at the time of retroviral exposure. Early studies that used this method thus had low resolution, leading to an underestimation of HSC numbers34. Moreover, the site where the retrovirus inserts into the host genome can have a major effect on the clonal output of the initially transduced HSC, ranging from a slight change to overt dominance and even leukemia35–38. These effects are reduced substantially if vectors are derived from lentiviruses, such as the human AIDS virus HIV39, and the use of lentiviruses to track the progeny regenerated from initially quiescent mouse HSCs has revealed many more clones than found earlier using retroviral marking40.\n\nA breakthrough in the study of the repopulating potential of mouse HSCs occurred when the use of large libraries of lentiviruses encoding short, randomly varied DNA marker sequences (called barcodes) was introduced41,42. Upon integration, each such vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each initially transduced cell to be tracked over time. By coupling the barcoding method to a high-throughput sequencing-based detection system, it became possible to identify even smaller clones42,43. The use of this sensitive methodology showed that many clones (about 70 per mouse) produced in mice transplanted with such marked cells can contribute to hematopoiesis over long periods of time, although their content of granulocytes, T cells, and B cells could be substantially different. Moreover, the contributions of individual clones to mature blood cells could change dynamically, with most clones either expanding or declining over time44, and not necessarily in a fashion concordant with their activity in the bone marrow. Nevertheless, many clones were observed for more than 12 weeks. Thus, the regeneration of the hematopoietic system from transplanted cells can involve a contribution from a large number of input HSCs and progenitor cells, including self-renewing HSCs, as well as more differentiated and lineage committed progenitor cells.\n\nThe stromal microenvironment niches for HSCs also play an important role in hematopoiesis. It is known that the niches for the HSC differ in different places of hematopoiesis. It has been reported that there are fewer niches in the long bones than in trabecular bones, and the properties of the HSCs in the niches in these different sites are different45–49.\n\n\nClonal composition of HSCs\n\nStudies of the clonal composition of HSCs in primates have shown results that are generally similar to those obtained in mice, albeit with some substantial differences. Tracking of thousands of HSCs and progenitor cells in rhesus macaques for up to 12 years revealed that approximately half of the analyzed clones contributed to long-term repopulation (over 3–10 years), arising in sequential groups and likely representing self-renewing HSCs50. Most of the remaining clones were observed only during the first year. A large number (43–71%) of clones were detected at an extremely low average frequency of <0.0002, contributing to <7% of total blood repopulation over the entire course of observation. The 5% of the most frequently detected clones contributed to an average of 49–72% of all regenerated blood cells, depending on the animal and cell type. About 330 clones per animal were detected. The sequential expansion of different groups of clones (over several months for the earliest clones and over several years for those clones appearing later after repopulation) was revealed. Approximately equal numbers of long-term and short-term clones were detected. Although the assay endpoint given to each animal to distinguish long-term and short-term clones ranged from 3–10 years, this variation in endpoints did not significantly affect the findings regarding the relative frequencies of these two populations in so far as the clonal kinetics in all animals became more stable after 1–2 years50.\n\nIn humans, HSC kinetic data derived from the clonal tracking of their activity in vivo have been obtained from gene therapy clinical trials for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich syndrome (WAS). These trials involved the infusion of genetically engineered HSCs whose progeny could then be followed over time by tracking the unique barcode integration sites (ISs) of the therapeutic vectors incorporated into the transplanted cells. Authors analyzed the timing of short, intermediate, and long-term HSC output showing that CD34+ clones active at 3–6 months after transplantation were not detectable at later times51. In the later steady-state, about 200 clones per person was the figure estimated by mark-recapture of transduced HSC clones that were stably contributing to the progenitor’s repertoire for up to 3 years after infusion of gene-corrected CD34+ cells. To evaluate the long-term preservation of activity by the transplanted HSCs, the authors exploited data derived from the IS-based tracking of 4,845 clones in ADA-SCID patients performed for up to 6 years after gene therapy. This analysis showed that identical ISs were consistently detected in multiple lineages at stable levels even several years after transplantation. Strikingly, semi-quantitative PCR used to measure specific vector-genome junctions revealed a fluctuating, but consistent, output of marked clones over a period of 5 years without evidence of transient inactivity. Additionally, since the gamma-retroviral vector used in ADA-SCID HSC-GT trial is able to transduce only actively replicating cells, these results provided the first evidence that in vitro activated human HSCs can still display long-term activity in vivo51,52. Thus, in humans and primates, hundreds of individual clones could be shown to contribute to different cell lineages with clonal stability established after an initial phase of instability during the first year after transplantation.\n\nIn all previously described transplantation studies, the progeny of individual HSCs were tracked using an ex vivo viral transduction of the cells prior to transplantation into myelosuppressed hosts. This approach is not effective for labeling HSCs in situ. To reduce the drawback of studying the behavior of cells stimulated to repopulate myeloablated recipients, mice were sublethally irradiated without hematopoietic cell transplantation. Day 10 CFU-S assays of bone marrow cells subsequently sampled throughout a 12– to 20–month period revealed chromosomally marked clones that remained active for at least 1.5 years of the life of the mice53,54. These long-lived clones produced 10% of all identified CFU-S. Interestingly, studies of immunophenotypically defined HSCs showed that only 10% of those that divided were able to return to a resting state55. In summary, some HSCs are able to maintain hematopoiesis for long periods and can return to a quiescent state after transient proliferative activity.\n\nRecent approaches used to track cells using a transposon system cloned into mice56 have now provided an opportunity to study the control of endogenous hematopoiesis without the use of transplantation57. The transposon is activated by a hyperactive “sleeping beauty” transposase whose expression is controlled by doxycycline. During the short time period when doxycycline is applied to a mouse, the transposon can randomly mobilize to a different genomic location. This transposition creates an inheritable genomic DNA insertion that is unique to individual cells and their progeny. Cells originating from a common ancestor can thus be identified by their shared unique transposon IS. The transposon system has produced strikingly different results than those from previous studies. At each of their measured time points, dramatically different clones appear to supply the blood. The authors estimated that thousands of clones contribute to blood formation at any time point. Thus, their data suggest that long-term hematopoiesis is sustained by the successive recruitment of a large number of clones. While their observed clonal dynamics are consistent with the “clonal succession model”, their estimated clonal complexity is much greater than could possibly be supported by the relatively small number of HSCs traditionally identified using transplantation methods. The clonal composition of hematopoietic cells derived from clonally marked donor mice when compared with that of transplanted recipients revealed that donors and recipients possessed different clonal repertoires. The authors also compared the clonal composition of the HSC compartment with that of the intermediate progenitors, and the mature blood cells present in individual mice. They found that fewer than 5% of HSC clones are subsequently represented in mature cell populations, whereas the clonal composition of the multipotent progenitor and myeloid progenitor populations did mirror the mature cell populations. Based on these two experiments, the authors conclude that the cells that supply blood under homeostatic conditions are not transplantable and are not found in the conventionally defined HSC pool. Moreover, they suggest that the large number of progenitor cells, including previously defined multipotent progenitors and myeloid progenitors, may be the major source of ongoing hematopoiesis. These data imply a need to change our idea of the regulation of steady-state hematopoiesis. However, it should be noted that these data do not exclude the possibility that HSCs participate in steady-state blood production but, if they do, they must be quickly depleted from the HSCs pool once they committed to differentiate.\n\n\nWhat happens during aging\n\nThe dynamics of hematopoiesis depend on the phase of ontogenesis from the cradle to the grave. What happens to the pool of HSCs during aging? The number of transplantable HSCs increases in the bone marrow of old mice58–60, although clonal analysis of their progeny has revealed many functional defects61. For example, the clonal outputs of HSCs from young mice are, on average, larger than those produced by HSCs from old mice44. These data appear to contradict the recent results of whole-exome sequencing of DNA in peripheral blood cells from aging humans62,63, where the development of oligoclonal hematopoiesis was found to be a relatively common condition and one associated with an increased risk of hematologic cancer. Based on deep whole-genome sequencing, it was estimated that approximately 450 somatic mutations had accumulated in the nonrepetitive portions of the genome present in healthy blood cells in a 115-year-old woman. The distribution of variant allele frequencies of these mutations suggests that the majority of the peripheral white blood cells were the offspring of two related HSC clones. Moreover, the telomere lengths of the white blood cells were significantly shorter than the telomere lengths from other tissues. Together, this suggests that the finite lifespan of HSCs, rather than somatic mutation effects, may lead to hematopoietic clonal evolution at extreme ages64. Exhaustion of the HSC pool leading to a reduced number of functioning clones might explain how such an oligoclonal picture of hematopoiesis develops in humans. It is thus becoming increasingly clear that what we know about the structure and functioning of the hematopoietic system is still just the tip of the iceberg, with much more to understand that is still under water.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have 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\nMoore MA, Owen JJ: Chromosome marker studies on the development of the haemopoietic system in the chick embryo. Nature. 1965; 208(5014): 956 passim. PubMed Abstract | Publisher Full Text\n\nMoore MA, Metcalf D: Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haematol. 1970; 18(3): 279–96. PubMed Abstract | Publisher Full Text\n\nDieterlen-Lièvre F: Emergence of haematopoietic stem cells during development. C R Biol. 2007; 330(6–7): 504–9. PubMed Abstract | Publisher Full Text\n\nMedvinsky AL, Samoylina NL, Müller AM, et al.: An early pre-liver intraembryonic source of CFU-S in the developing mouse. Nature. 1993; 364(6432): 64–7. 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PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTrumpp A, Essers M, Wilson A: Awakening dormant haematopoietic stem cells. Nat Rev Immunol. 2010; 10(3): 201–9. PubMed Abstract | Publisher Full Text\n\nKim S, Kim N, Presson AP, et al.: Dynamics of HSPC repopulation in nonhuman primates revealed by a decade-long clonal-tracking study. Cell Stem Cell. 2014; 14(4): 473–85. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBiasco L, Scala S, Dionisio F, et al.: Paper: Comprehensive Clonal Mapping of Hematopoiesis in Vivo in Humans By Retroviral Vector Insertional Barcoding. 2014. Reference Source\n\nBiasco L, Scala S, Basso Ricci L, et al.: In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells. Sci Transl Med. 2015; 7(273): 273ra13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nOlshanskaya Y, Drize N, Gerasimova L, et al.: Life-long hematopoiesis in sublethally irradiated as well as in reconstituted mice is maintained by multiple clones. Exp Hematol. 1998; 26: 733.\n\nDrize NJ, Olshanskaya YV, Gerasimova LP, et al.: Lifelong hematopoiesis in both reconstituted and sublethally irradiated mice is provided by multiple sequentially recruited stem cells. Exp Hematol. 2001; 29(6): 786–94. PubMed Abstract | Publisher Full Text\n\nWilson A, Laurenti E, Oser G, et al.: Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 2008; 135(6): 1118–29. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSun J, Ramos A, Chapman B, et al.: Clonal dynamics of native haematopoiesis. Nature. 2014; 514(7522): 322–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLu R: Sleeping beauty wakes up the clonal succession model for homeostatic hematopoiesis. Cell Stem Cell. 2014; 15(6): 677–8. PubMed Abstract | Publisher Full Text\n\nRossi DJ, Jamieson CH, Weissman IL: Stems cells and the pathways to aging and cancer. Cell. 2008; 132(4): 681–96. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMayack SR, Shadrach JL, Kim FS, et al.: Systemic signals regulate ageing and rejuvenation of blood stem cell niches. Nature. 2010; 463(7280): 495–500. PubMed Abstract | Publisher Full Text\n\nRetraction. Systemic signals regulate ageing and rejuvenation of blood stem cell niches. Nature. 2010; 467(7317): 872. PubMed Abstract | Publisher Full Text\n\nDykstra B, Olthof S, Schreuder J, et al.: Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells. J Exp Med. 2011; 208(13): 2691–703. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGenovese G, Kähler AK, Handsaker RE, et al.: Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med. 2014; 371(26): 2477–87. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nJaiswal S, Fontanillas P, Flannick J, et al.: Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med. 2014; 371(26): 2488–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolstege H, Pfeiffer W, Sie D, et al.: Somatic mutations found in the healthy blood compartment of a 115-yr-old woman demonstrate oligoclonal hematopoiesis. Genome Res. 2014; 24(5): 733–42. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "10982",
"date": "29 Oct 2015",
"name": "Connie Eaves",
"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",
"responses": []
},
{
"id": "10983",
"date": "29 Oct 2015",
"name": "Hector Mayani",
"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",
"responses": []
},
{
"id": "10984",
"date": "29 Oct 2015",
"name": "Leonid Bystrykh",
"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 review is an overview of the hematopoietic stem cells in hematopoiesis, and it is my opinion that there are still some issues with the review. For instance, I think that the abstract is not fully aligned with the body of the review and instead I feel that it should summarise the major statements made in the text. Moreover, in the main body of the review, there are a number of factual inaccuracies and the authors present the evolution of our knowledge as a series of mistakes which, in my view, is incorrect and they have not provided sufficient information on the methods and tools that they describe. I miss a more critical expert review of the evolution of our tools to study hematopoietic stem cells. As a result of these issues, I have given this review an “Approved with Reservations” rating.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1177
|
https://f1000research.com/articles/4-1167/v1
|
28 Oct 15
|
{
"type": "Review",
"title": "Should adults with type 2 diabetes be screened for atherosclerotic cardiovascular disease?",
"authors": [
"Yanglu Zhao",
"Nathan Wong",
"Yanglu Zhao"
],
"abstract": "Diabetes mellitus is associated with greater risks for cardiovascular diseases (CVD). Multiple noninvasive screening tools for CVD including cardiac CT, carotid intima-media thickness test, myocardial perfusion imaging have been examined in those with diabetes, but the prognostic value of these tests vary and issues remain regarding their cost-benefit ratios, potential harms of radiation, and how they fit into screening algorithms for CVD. We discuss in this report the needs and criteria for screening tests and summarize the evidence from observational studies and clinical trials. We also explore whether there should be more sensitive screening modalities to better detect both short and long-term cardiovascular risk among asymptomatic patients with diabetes.",
"keywords": [
"cardiovascular disease",
"non-invasive imaging",
"asymptomatic diabetes"
],
"content": "Introduction\n\nGlobally in 2014, 8.3% of the world population or nearly 400 million people had diabetes mellitus (DM)1. Although the prevalence of DM remains high in developed countries, developing countries now comprise the greatest increases in DM prevalence and burden of accompanying comorbidities. Patients with type 2 DM have up to four times the risk of atherosclerotic cardiovascular disease (ASCVD) events compared with those without DM2. Not only do patients with DM have an increased risk of developing ASCVD, but it is often silent3,4. However, once ASCVD becomes clinically identified, both shorter- and longer-term outcomes in persons with DM are worse than those in persons without DM, and this may be the result of other features associated with DM (e.g., inflammation and prothrombotic tendency). These observations establish the theoretical foundations for the early screening of ASCVD among those with DM. Although the concept for systematic ASCVD screening in DM is appealing, the benefits of such an approach have not been fully demonstrated. As there is ambiguity in outcomes regarding the benefits versus harm of such screening, major organizations, including the American Diabetes Association, currently do not recommend routine screening in those with DM5. Therefore, in this report, we summarize current evidence of pros and cons of screening modalities and propose a framework for screening for subclinical coronary atherosclerosis among asymptomatic patients with DM.\n\n\nNeed of individualized risk assessment in diabetes\n\nDespite the higher ASCVD risk among persons with diabetes, they are a highly heterogeneous population and diabetes often is not a coronary artery disease (CAD) equivalent. We have previously noted from data in the United States National Health and Nutrition Examination Survey that 32% of men and 48% of women with DM were deemed by the Framingham Risk Score (FRS) to be at low to intermediate risk6. Refining risk estimates in patients with DM may aid in implementing prevention strategies in an efficient and cost-saving manner. Although the Framingham and European risk scores and more recently the American Heart Association/American College of Cardiology (AHA/ACC) Pooled Cohort risk scores emphasize the classic ASCVD risk factors, they are only moderately accurate for the prediction of short- and long-term risk of CVD events7. In addition, the United Kingdom Prospective Diabetes Study (UKPDS) risk engine shows no better performance than FRS and tended to overestimate the coronary heart disease (CHD) risk8,9. Diabetes in fact can often attenuate the protective effect of optimal levels from other risk factors (dyslipidemia, hypertension, obesity, and so on), and thus the number of traditional risk factors may not be useful in identifying risk in those with DM10. On the other hand, directly examining for subclinical ASCVD, such as by coronary artery calcium (CAC), carotid intima-media thickness (CIMT), endothelial dysfunction, and myocardial ischemia, holds the potential to more accurately discriminate risk in those with DM11,12.\n\n\nKey screening methods for detecting subclinical cardiovascular disease for persons with diabetes mellitus\n\nA good screening test should have the following features: (1) accurately discriminate low- and high-risk persons, (2) produce reliable and reproducible results, (3) provide incremental value to risk predicted by office-based risk assessment, and (4) detect individuals for whom early intervention is likely to have a beneficial impact7. Additional criteria that have been proposed include: (1) ensuring the test identifies a high enough prevalence of disease so that a reasonable number of persons can be identified for intervention and (2) exhibiting high cost-effectiveness13. Currently used modalities may not satisfy all of the criteria completely and instead may vary in providing support for each criterion, thus warranting more studies to provide further validation.\n\nCAC assesses the extent of calcified atherosclerotic plaques in the coronary arteries and is exquisitely sensitive for detection of atherosclerosis14. CAC scanning has emerged as the most powerful tool for refining risk assessment on top of global risk assessment in asymptomatic individuals15. In those subjects with DM, a CAC score of 0 is associated with ASCVD event rates as low as or lower than those of many persons without DM and increasing CAC scores are associated with progressively higher ASCVD event rates; those with DM who have CAC scores of at least 400 have 10-fold greater event rates (CHD incidence of 4% per year) than those with CAC of 0 (38% of our DM subjects)11. Subjects who undergo scanning for CAC also appear to have improved outcomes in terms of improved risk factor control, including blood pressure, low-density lipoprotein cholesterol, and waist circumference, compared with those not scanned16. This observation may be explained by greater adherence to lifestyle modifications and medical therapy on the basis of visualizing their disease17–20. Also, CAC screening has been noted to be more cost-effective than myocardial perfusion imaging (MPI); it was estimated that CAC scanning can prevent one event at a cost of $71,249, about a third of the cost of MPI and half that of no screening (treating everyone)21.\n\nMost guidelines have suggested that CAC be considered for screening and risk stratification of patients with DM. Both the 2010 AHA risk assessment guidelines (level IIa) and the 2014 position statement of the Brazilian Diabetes Society (level A) recommend CAC scanning for those who are at intermediate risk or who have diabetes22,23. The 2012 American Association of Clinical Endocrinology (AACE) Lipid Management Guideline also stated that CAC can be used in those with DM to refine risk stratification and the need for more aggressive preventive strategies24. Most recently, the ACC/AHA guideline on the assessment of cardiovascular risk25 identifies CAC screening (as well as family history of premature ASCVD, ankle brachial index, and high-sensitivity C-reactive protein) as a tool that can be used when, after quantitative risk assessment, a risk-based treatment decision is uncertain. Although current guidelines recommend that all DM patients who are 40 or over be on statin therapy, the intensity of therapy (or possible consideration of therapy in those younger than 40 years of age) may be guided by the use of such testing.\n\nObservational investigations of MPI have shown high sensitivity (86%) in those with DM and even higher sensitivity among those at higher risk (94%)26,27. However, the Detection of Ischemia in Asymptomatic Diabetics (DIAD) randomized clinical trial demonstrated that screening patients with DM does not improve clinical outcomes28, even when ischemia is present upon repeat testing (at 3 years)29. The negative result may be due to the low prevalence of CAD and thus a low incidence of coronary events. Although some guidelines have advocated screening for silent myocardial ischemia in high-risk asymptomatic patients with DM22, it is no longer routinely recommended in current guidelines5,23.\n\nCoronary computed tomography angiography (CCTA) allows the evaluation of the full spectrum of CAD from totally normal arteries to non-obstructive disease to significant coronary stenosis and total occlusion. It also allows plaque characterization, including calcified and non-calcified plaque, spotty calcification, positive remodeling, and the napkin ring sign30. Some of these features are associated with a higher likelihood of near-term major adverse cardiovascular events. Several studies have shown the prognostic value of CCTA findings in subjects with asymptomatic DM31,32. Whether clinical outcomes can be impacted by CCTA screening, however, was only recently reported by the FACTOR-64 randomized clinical trial, which enrolled 900 asymptomatic patients with diabetes; while showing a 20% reduced risk of subsequent adverse cardiovascular events in those screened with CCTA, the findings were not statistically significant and this was due in part to the fairly low event rates33. Due to a lack of sufficient supportive evidence, CCTA is not conventionally recommended for screening asymptomatic individuals with DM in current guidelines34–36.\n\nCIMT is an indicator of atherosclerosis in the carotid artery, measuring the combined thickness of the intima and media with B-mode ultrasound. Although CIMT is related to higher CVD event risk37, the meaning of measuring CIMT alone has recently been questioned, as meta-analysis and pooled cohort studies showed that the addition of common CIMT to traditional risk models was associated with only a modest improvement and is unlikely to be of clinical importance38,39. Similar findings in a cohort of 4,220 patients with DM demonstrated that common CIMT did not add predictive value to the FRS during a median follow-up of 8.7 years40. However, if combined with CAC, ankle brachial index, high-sensitivity C-reactive protein, and family history, the predictive ability for future CHD events may supersede the traditional FRS and UKPDS risk engine41. Importantly, the presence of carotid plaques alone and in combination with CIMT does add to risk prediction beyond FRS, as demonstrated by Nambi and colleagues42, although this question was not specifically evaluated in those with DM in that study.\n\n\nWhat is the future of screening for atherosclerotic cardiovascular disease in diabetes mellitus?\n\nThe appropriate use of multimodality screening in those with DM depends on: (1) clinical history of other risk factors, (2) whether the use of a second or third method can address residual risk not addressed by the first method, and (3) determining the correct order to conduct such tests to maximize clinical utility and cost-effectiveness. For instance, CAC scores can accurately reflect the possibility of abnormal stress MPI findings (and with much less radiation), suggesting a role for CAC scoring as a gatekeeper for patients who may benefit from further risk stratification with stress MPI. Expert consensus opinion recommends stress testing imaging in individuals whose CAC score exceeds 400, given that 25% of such subjects will have significant asymptomatic ischemia on MPI. In addition, CAC screening and MPI are complementary for risk assessment since CAC is usually an indicator of anatomic CAD whereas MPI is a physiological test for CAD. Although different algorithms of screening have been proposed, more complete and detailed protocols should be developed and tested for effectiveness to be used in guidelines. Persons with DM are at an increased mortality risk because of CVD, but many receive inadequate treatment for CVD risk factors43. Patients with DM require individualized risk assessment before appropriate intensity of treatment can be implemented. Current screening methods have proven to be effective in predicting future coronary events, but limitations remain in that: (1) few studies concerning the cost-effectiveness of various scanning modalities have been carried out; (2) large randomized clinical trials should be designed to directly look into the impact of screening tests on CVD outcomes as well as the impact on downstream clinical decisions, risk factor changes, and the total medical costs; and (3) few screening methods have directly compared predictive efficacy. The results of such trials will allow us to better identify which screening methods should be employed in patients with DM and help inform therapeutic decision making.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have 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\nIDF Diabetes Atlas. Sixth Edition. Update International Diabetes Federation, 2014. Reference Source\n\nHaffner SM, Lehto S, Rönnemaa T, et al.: Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med. 1998; 339(4): 229–34. PubMed Abstract | Publisher Full Text\n\nZellweger MJ, Pfisterer ME: Silent coronary artery disease in patients with diabetes mellitus. Swiss Med Wkly. 2001; 131: 427–32. Reference Source\n\nBarthelemy O, Le Feuvre C, Timsit J: Silent myocardial ischemia screening in patients with diabetes mellitus. Arq Bras Endocrinol Metabol. 2007; 51(2): 285–93. PubMed Abstract | Publisher Full Text\n\nAmerican Diabetes Association. Standards of medical care in diabetes--2014. Diabetes care. 2014; 37(Suppl 1): S14–80. PubMed Abstract | Publisher Full Text\n\nWong ND, Glovaci D, Wong K, et al.: Global cardiovascular disease risk assessment in United States adults with diabetes. Diab Vasc Dis Res. 2012; 9(2): 146–52. PubMed Abstract | Publisher Full Text\n\nPasternak RC, Abrams J, Greenland P, et al.: 34th Bethesda Conference: Task force #1--Identification of coronary heart disease risk: is there a detection gap? J Am Coll Cardiol. 2003; 41(11): 1863–74. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGuzder RN, Gatling W, Mullee MA, et al.: Prognostic value of the Framingham cardiovascular risk equation and the UKPDS risk engine for coronary heart disease in newly diagnosed Type 2 diabetes: results from a United Kingdom study. Diabet Med. 2005; 22(5): 554–62. PubMed Abstract | Publisher Full Text\n\nBannister CA, Poole CD, Jenkins-Jones S, et al.: External validation of the UKPDS risk engine in incident type 2 diabetes: a need for new type 2 diabetes-specific risk equations. Diabetes care. 2014; 37(2): 537–45. PubMed Abstract | Publisher Full Text\n\nScognamiglio R, Negut C, Ramondo A, et al.: Detection of coronary artery disease in asymptomatic patients with type 2 diabetes mellitus. J Am Coll Cardiol. 2006; 47(1): 65–71. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMalik S, Budoff MJ, Katz R, et al.: Impact of subclinical atherosclerosis on cardiovascular disease events in individuals with metabolic syndrome and diabetes: the multi-ethnic study of atherosclerosis. Diabetes care. 2011; 34(10): 2285–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCortigiani L, Bigi R, Sicari R, et al.: Comparison of prognostic value of pharmacologic stress echocardiography in chest pain patients with versus without diabetes mellitus and positive exercise electrocardiography. Am J Cardiol. 2007; 100(12): 1744–9. PubMed Abstract | Publisher Full Text\n\nMiller TD, Redberg RF, Wackers FJ: Screening asymptomatic diabetic patients for coronary artery disease: why not? J Am Coll Cardiol. 2006; 48(4): 761–4. PubMed Abstract | Publisher Full Text\n\nWon KB, Chang HJ, Niinuma H, et al.: Evaluation of the predictive value of coronary artery calcium score for obstructive coronary artery disease in asymptomatic Korean patients with type 2 diabetes mellitus. Coron Artery Dis. 2015; 26(2): 150–6. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nYeboah J, McClelland RL, Polonsky TS, et al.: Comparison of novel risk markers for improvement in cardiovascular risk assessment in intermediate-risk individuals. JAMA. 2012; 308(8): 788–95. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRozanski A, Gransar H, Shaw LJ, et al.: Impact of coronary artery calcium scanning on coronary risk factors and downstream testing the EISNER (Early Identification of Subclinical Atherosclerosis by Noninvasive Imaging Research) prospective randomized trial. J Am Coll Cardiol. 2011; 57(15): 1622–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrakzai RH, Nasir K, Orakzai SH, et al.: Effect of patient visualization of coronary calcium by electron beam computed tomography on changes in beneficial lifestyle behaviors. Am J Cardiol. 2008; 101(7): 999–1002. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKalia NK, Miller LG, Nasir K, et al.: Visualizing coronary calcium is associated with improvements in adherence to statin therapy. Atherosclerosis. 2006; 185(2): 394–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTaylor AJ, Bindeman J, Feuerstein I, et al.: Community-based provision of statin and aspirin after the detection of coronary artery calcium within a community-based screening cohort. J Am Coll Cardiol. 2008; 51(14): 1337–41. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKalia NK, Cespedes L, Youssef G, et al.: Motivational effects of coronary artery calcium scores on statin adherence and weight loss. Coron Artery Dis. 2015; 26(3): 225–30. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDiamond GA, Kaul S, Shah PK: Screen testing cardiovascular prevention in asymptomatic diabetic patients. J Am Coll Cardiol. 2007; 49(19): 1915–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGreenland P, Alpert JS, Beller GA, et al.: 2010 ACCF/AHA guideline for assessment of cardiovascular risk in asymptomatic adults: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2010; 122(25): 2748–64. PubMed Abstract | Publisher Full Text\n\nBertoluci MC, Pimazoni-Netto A, Pires AC, et al.: Diabetes and cardiovascular disease: from evidence to clinical practice - position statement 2014 of Brazilian Diabetes Society. Diabetol Metab Syndr. 2014; 6: 58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJellinger PS, Smith DA, Mehta AE, et al.: American Association of Clinical Endocrinologists' Guidelines for Management of Dyslipidemia and Prevention of Atherosclerosis. Endocr Pract. 2012; 18 Suppl 1: 1–78. PubMed Abstract | Publisher Full Text\n\nGoff DC Jr, Lloyd-Jones DM, Bennett G, et al.: 2013 ACC/AHA guideline on the assessment of cardiovascular risk: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014; 63(25 Pt B): 2935–59. PubMed Abstract | Publisher Full Text\n\nKang X, Berman DS, Lewin H, et al.: Comparative ability of myocardial perfusion single-photon emission computed tomography to detect coronary artery disease in patients with and without diabetes mellitus. Am Heart J. 1999; 137(5): 949–57. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRajagopalan N, Miller TD, Hodge DO, et al.: Identifying high-risk asymptomatic diabetic patients who are candidates for screening stress single-photon emission computed tomography imaging. J Am Coll Cardiol. 2005; 45(1): 43–9. PubMed Abstract | Publisher Full Text\n\nYoung LH, Wackers FJ, Chyun DA, et al.: Cardiac outcomes after screening for asymptomatic coronary artery disease in patients with type 2 diabetes: the DIAD study: a randomized controlled trial. JAMA. 2009; 301(15): 1547–55. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWackers FJ, Chyun DA, Young LH, et al.: Resolution of asymptomatic myocardial ischemia in patients with type 2 diabetes in the Detection of Ischemia in Asymptomatic Diabetics (DIAD) study. Diabetes care. 2007; 30(11): 2892–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSato A, Aonuma K: Role of cardiac multidetector computed tomography beyond coronary angiography. Circ J. 2015; 79(4): 712–20. PubMed Abstract | Publisher Full Text\n\nMin JK, Labounty TM, Gomez MJ, et al.: Incremental prognostic value of coronary computed tomographic angiography over coronary artery calcium score for risk prediction of major adverse cardiac events in asymptomatic diabetic individuals. Atherosclerosis. 2014; 232(2): 298–304. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nFaustino A, Providência R, Mota P, et al.: Can cardiac computed tomography predict cardiovascular events in asymptomatic type-2 diabetics?: results of a long term follow-up. BMC Cardiovasc Disord. 2014; 14: 2. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMuhlestein JB, Lappé DL, Lima JA, et al.: Effect of screening for coronary artery disease using CT angiography on mortality and cardiac events in high-risk patients with diabetes: the FACTOR-64 randomized clinical trial. JAMA. 2014; 312(21): 2234–43. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTaylor AJ, Cerqueira M, Hodgson JM, et al.: ACCF/SCCT/ACR/AHA/ASE/ASNC/NASCI/SCAI/SCMR 2010 appropriate use criteria for cardiac computed tomography. A report of the American College of Cardiology Foundation Appropriate Use Criteria Task Force, the Society of Cardiovascular Computed Tomography, the American College of Radiology, the American Heart Association, the American Society of Echocardiography, the American Society of Nuclear Cardiology, the North American Society for Cardiovascular Imaging, the Society for Cardiovascular Angiography and Interventions, and the Society for Cardiovascular Magnetic Resonance. J Am Coll Cardiol. 2010; 56(22): 1864–94. PubMed Abstract | Publisher Full Text\n\nSingapore Ministry of Health: Screening for cardiovascular disease and risk factors. MOH Clinical Practice Guidelines 1/2011. Reference Source\n\nChinese Society of Cardiology of Chinese Medical Association: [Chinese expert consensus for assessment of cardiovascular risk in asymptomatic adults]. Zhonghua Xin Xue Guan Bing Za Zhi. 2013; 41(10): 820–4. PubMed Abstract\n\nYokoyama H, Katakami N, Yamasaki Y: Recent advances of intervention to inhibit progression of carotid intima-media thickness in patients with type 2 diabetes mellitus. Stroke. 2006; 37(9): 2420–7. PubMed Abstract | Publisher Full Text\n\nvan den Oord SC, Sijbrands EJ, ten Kate GL, et al.: Carotid intima-media thickness for cardiovascular risk assessment: systematic review and meta-analysis. Atherosclerosis. 2013; 228(1): 1–11. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDen Ruijter HM, Peters SA, Anderson TJ, et al.: Common carotid intima-media thickness measurements in cardiovascular risk prediction: a meta-analysis. JAMA. 2012; 308(8): 796–803. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nden Ruijter HM, Peters SA, Groenewegen KA, et al.: Common carotid intima-media thickness does not add to Framingham risk score in individuals with diabetes mellitus: the USE-IMT initiative. Diabetologia. 2013; 56(7): 1494–502. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nYeboah J, Erbel R, Delaney JC, et al.: Development of a new diabetes risk prediction tool for incident coronary heart disease events: the Multi-Ethnic Study of Atherosclerosis and the Heinz Nixdorf Recall Study. Atherosclerosis. 2014; 236(2): 411–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNambi V, Chambless L, Folsom AR, et al.: Carotid intima-media thickness and presence or absence of plaque improves prediction of coronary heart disease risk: the ARIC (Atherosclerosis Risk In Communities) study. J Am Coll Cardiol. 2010; 55(15): 1600–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWong ND, Patao C, Wong K, et al.: Trends in control of cardiovascular risk factors among US adults with type 2 diabetes from 1999 to 2010: comparison by prevalent cardiovascular disease status. Diab Vasc Dis Res. 2013; 10(6): 505–13. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "10979",
"date": "28 Oct 2015",
"name": "Michael Shapiro",
"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",
"responses": []
},
{
"id": "10980",
"date": "28 Oct 2015",
"name": "Peter Wilson",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1167
|
https://f1000research.com/articles/4-1162/v1
|
28 Oct 15
|
{
"type": "Review",
"title": "Cancer Genomics",
"authors": [
"Elaine Mardis"
],
"abstract": "Modern cancer genomics has emerged from the combination of the Human Genome Reference, massively parallel sequencing, and the comparison of tumor to normal DNA sequences, revealing novel insights into the cancer genome and its amazing diversity. Recent developments in applying our knowledge of cancer genomics have focused on the utility of these data for clinical applications. The emergent results of this translation into the clinical setting already are changing the clinical care and monitoring of cancer patients.",
"keywords": [
"cancer",
"genomics"
],
"content": "Introduction\n\nEven before we knew of DNA’s role in determining cellular function and biology, even before we knew chromosomes were made of DNA, there was speculation that the source of cancer somehow was determined by profound changes in the chromosomes1. Early pioneers in cancer genomics, such as Janet Rowley (cited in this review), provided substantial evidence of a role for the genome in cancer’s development by observing microscopically that patients with specific subtypes of leukemia shared specifically altered chromosomes2–4. Initially, these chromosomal translocations were used to provide diagnostic evidence of the specific subtype, and as our characterization of these translocations became more precise, the fusion gene drivers of oncogenesis such as BCR-ABL and PML-RARα were identified and defined according to their mechanisms. Ultimately, several of the recurrent genomic events in hematologic malignancies have been targeted by highly specific and effective therapies, rendering them manageable from a clinical standpoint and permitting patients either to survive cancer as a chronic disease, especially with the development of specific second- and third-line therapies that address acquired resistance mutations in the targeted fusion proteins, or to be cured outright (for example, approximately 94% of patients with acute promyelocytic leukemia are cured by all-trans retinoic acid or arsenic consolidation therapies).\n\nAs the Human Genome Project drew to a close in the early 2000s, scientists had a template or keystone with which they could compare and characterize changes to the genome in disease states such as cancer5. Initially, however, sequencing technology did not permit the sequencing of the entire genome at reasonable cost and throughput, so several groups began to design pipelines for high-throughput polymerase chain reaction (PCR) amplification and sequencing of known cancer genes in an effort to catalogue cancer-specific (“somatic”) mutations. During this same time frame, pharmaceutical companies began to perform clinical trials of drugs for solid tissue malignancies in major cancer centers that targeted specific proteins or protein families thought to be drivers of oncogenesis. In some but not all cases, these tyrosine kinase inhibitors (TKIs) were highly successful at achieving dramatic reductions of tumor burden in some (but not all) advanced metastatic patients. Given these remarkable results and the differential patient responses, focused efforts began to identify whether specific mutations could be correlated with response. In 2004, three groups published independently that, in non-small cell lung adenocarcinomas (NSCLCs), approximately 80% of responders to TKI therapy could be correlated with patients having mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR)6–8. As remarkable as these responses were, patients frequently relapsed, often with more aggressive and widespread disease after several months of treatment. As initially defined by Engelman and colleagues, these examples of acquired resistance to targeted therapy were due to new mutations in EGFR that conferred a lack of response to the TKIs because of reduced binding affinity9.\n\nIn the midst of these efforts to catalogue the mutations in cancer genes, transformative sequencing technologies were emerging. So-called “massively parallel” sequencing (MPS) technologies, they coupled the molecular biology of polymerase-catalyzed sequencing with light-based detection to report the incorporated nucleotides for each of several hundred thousand sequencing reactions taking place simultaneously10,11. These technologies further streamlined the sequencing library preparation steps and permitted pooled PCR products to be sequenced in the same instrument run, thereby accelerating throughput, reducing sequencing costs, and introducing a “digital” type of data that sequenced individual DNA molecules (after in situ amplification). Although these platforms introduced new challenges into data analysis based on the initially short reads relative to capillary sequencers, early efforts12–15 defined methods for whole genome sequencing of tumor and normal genomes and their comparison in order to identify somatic mutations in an unbiased way. A “middle ground” between directed PCR of genes and whole genome sequencing was developed and reported by several groups to capture by hybridization the exonic portion of the genome (“exome”), providing a more conscripted yet easier to analyze and interpret subset of the genome16–18. What has followed during the time period from around 2009 to the present is large-scale discovery, by MPS-based methods, of somatic alterations in thousands of cancer genomes, including comparisons of the tissue site-specific range and diversity of mutational load genome-wide19, the identification of phenomena such as chromothripsis20 and kataegis21, and a broad-based recognition that cancer genomes find myriad and different ways to create themselves.\n\nSeveral early studies pioneered the notion of using high-depth digital MPS-based sequencing and clustering of mutation sites with shared variant fractions of reads to evaluate the changes in clonal heterogeneity that occur between primary and metastatic or recurrent disease22,23. Recent comparisons of this type have explored changes to the cancer genome in the transition from treatment-naïve to post-therapy recurrent disease24–29. One challenge that has limited these types of studies in solid tissue malignancies has been the difficulty in obtaining post-treatment biopsies, which often cannot be obtained as the standard of care and/or may have associated risk or morbidities.\n\nGiven the range and scope of discovery that have taken place over the past five years, a basic understanding of the tumor genome landscape has been defined for most of the prevalent tumor types and a few rare ones as well. There is ample evidence that, given this body of knowledge and pertinent clinical questions that may be further informed by genomics, the clinical translation of genomics is an obvious next step. This review will focus on three pertinent aspects of clinical translation for cancer genomics in an effort to highlight the trends and add evidence from the existing body of translational work that genomics already is impacting and will continue to impact on cancer medicine.\n\n\nTumor evolution and changes in genomic heterogeneity\n\nSeveral groups have built upon early studies and methods that evaluated deep coverage at mutation sites to build models of founder and subclonal cell population genotypes. As mentioned, recent studies have focused on the comparison of primary with metastatic or of treatment-naïve with recurrent post-treatment tumors. The comparisons of primary with metastatic disease in solid tissue malignancies have illustrated the persistence of the founder or trunk mutations into metastases, with new mutations being acquired in different metastatic sites. These studies30–32 build upon, but somewhat differ in their conclusions when compared with, the earlier work by Gerlinger and colleagues33, who reported comparisons of primary with metastatic renal cell carcinomas.\n\nSimilar studies have evaluated treatment-naïve to recurrent disease in the setting of DNA-damaging chemotherapy, establishing a mutational “signature” in the recurrent disease setting that defines the resulting DNA damage and results in an elevated mutation rate. Our early work describing this result in recurrent acute myeloid leukemias22 was recently followed by a study of post-temozolomide-treated pediatric gliomas, illustrating a profound increase in the number of mutations from exome sequencing-based comparisons34. In both cases, the emergent disease has a mutational landscape akin to carcinogen-associated mutational processes, such as those observed in lung cancer due to smoking or in melanomas due to ultraviolet (UV) exposure. Another study of platinum-resistant high-grade serous ovarian cancer has identified post-therapy resistance signatures akin to BRCA (breast cancer, early onset)-associated mismatch repair (MMR) defects35 or, in a minority of samples, the apolipoprotein B mRNA editing enzyme-related (APOBEC) defects36. However, the predominant impact in high-grade serous ovarian disease for platinum resistance appears to be due to gene breakage defects at tumor suppressor loci, discernable only by the integration of whole genome and transcriptome data performed in this study35. Interestingly, the sequencing results revealed a higher mutational burden measured as single-nucleotide variants and insertion-deletion variants when comparing the platinum-resistant recurrent tumor cells derived from ascites fluid with the primary tumor. A significant relationship between the number of non-coding mutations and the numbers of courses of platinum-based chemotherapy the patient received also was described.\n\nRecent genomic comparisons of matched treatment-naïve disease with post-therapy recurrent tumors have mainly studied patients emerging with acquired resistance to targeted therapy treatment24,37,38. The results have elucidated the nature and types of mutations that are conferring therapy resistance and give rise to the hope that pinpointing the genomic source(s) of acquired resistance to targeted therapies might be more straightforward and less complicated than to chemotherapies. In one report regarding the genomics of therapy-resistant EGFR-mutated NSCLCs, the mechanism for a rarely observed transition of NSCLCs into small cell lung carcinoma was elucidated as being due to loss of RB1, solving a long-standing puzzle39.\n\nTherefore, it is important to understand the genomic alterations that might lead to treatment resistance, where possible. When these alterations are identified as the means by which the tumor cells can evade the mechanism of therapeutic action, real-time blood-based monitoring for the rise and fall of the acquired resistance alteration(s) may be possible. This genomics application addresses the difficulty of obtaining recurrent tumor biopsy material for genomic testing. Often, the sensitivity of blood-based monitoring or “liquid biopsy” over imaging-based detection of recurrent tumor growth is quite desirable as well. In the next section, the concepts and practices of liquid biopsy will be addressed as a means of introducing this alternative approach to tumor progression and treatment response monitoring.\n\n\nLiquid biopsy\n\nAlthough some of the cancer genomics discovery work that was discussed above has contributed substantially to our understanding of the genomic relationships between primary and metastatic disease in the same patient, the reality is that obtaining a metastatic resection or biopsy sample is often not the standard of care and therefore is not reimbursable by private insurance payors. Beyond these practical considerations, metastases can be inaccessible and therefore difficult to sample. Small studies of multiple metastatic lesions have indicated that there are differences in the genomes of metastases in different sites that must be considered in tracking the progression or stability of the cancers present in the individual. There also can be associated morbidity and risk with biopsy procedures that diminish the enthusiasm of study participants to undergo the procedure. Suffice it to say that a proxy for detecting solid tumor progression-associated changes is badly needed in cancer medicine.\n\nIn this regard, an opportunity may be present in assays referred to collectively as “liquid” or blood-based biopsy, whereby a blood sample is obtained from a patient at diagnosis and compared with sequential temporal blood samples obtained during treatment for the purposes of monitoring tumor burden, often as a function of response to therapy40–45. From these blood samples, one can study the DNA shed from tumors as cells turn over, in the form of mutation-specific assays of circulating free DNA (cfDNA), or DNA from isolated circulating tumor cells (CTCs) or from tumor-derived exosomes.\n\nEach approach has its own nuances, including specialized isolation approaches and assay types, as follows. CTCs are rare cell types that can be isolated from the blood and indeed may fluctuate in their prevalence and representation of the mutational landscape according to disease stage, tissue site, and other factors required for isolation such as cell surface markers46–48. Typically CTCs require specialized instrumentation to isolate, of which several types are available commercially, any one of which may be more applicable to different tumor types. Also, the rarity of CTCs requires higher amounts of blood input, which can impose a practical/clinical limitation. After isolation and cell lysis, whole genome amplification of CTC DNA is followed by whole genome, exome, or targeted sequencing. cfDNA, by contrast, requires isolation from plasma within a few hours of blood draw to minimize degradation and varies in amount according to disease stage and tissue site. Mutational assay of cfDNA requires focused PCR of known or suspect mutations, due to the degraded state of tumor DNA in the circulation, followed by high-depth sequencing to overcome the background of cfDNA provided by normal cell apoptosis49–51. Exosomes, which are small (950–1000 μm) vesicles containing DNA, RNA, and protein components from apoptotic tumor cells, also are shed at lower amounts by normal cells. Owing to the contents of exosomes, evaluation may occur by multiple assay types to identify DNA, RNA, or protein related to tumor monitoring. There are several different isolation procedures for obtaining purified exosomes from blood, ranging from low-throughput differential ultracentrifugation to size- or affinity-based purification52.\n\nRegardless of the type of blood biopsy, there is increasing evidence that this approach will be broadly applicable to monitoring patient response to neo-adjuvant therapy, to surgery, or to surgery followed by chemo-, radiation, or targeted therapy. With the genomic characterization of acquired resistance mutations arising in the targeted therapy setting, precise mutational analyses can detect patients who are developing acquired resistance in a much more sensitive way than by conventional imaging, which can often be misleading regarding objective response to a therapeutic intervention51. Depending upon the approach, blood-based monitoring also is quite rapid and inexpensive relative to imaging, yet more studies are required to fully understand its applicability and limitations.\n\n\nImmunogenomics\n\nImmunogenomics is a somewhat broad term that refers to numerous genomics-based inquiries that (1) may assay specific immune components in their interaction with established cancers, (2) may indicate the likelihood of a tumor to respond to immunotherapy, or (3) may be used to design personalized vaccines for individual patients deemed likely to respond to an immune modulatory therapy. Much of the foundational work in immunogenomics stems from studies of melanoma, a tumor type long recognized as having extensive immune system interactions53,54. Sequencing of DNA isolated from melanomas has defined the signature of UV-associated DNA damage55 and has identified that melanomas have overall one of the highest mutation rates of any tumor type, as a result of UV damage19,56. In 2010, the first results of clinical trials in melanoma testing a new class of immunotherapeutic, called “checkpoint blockade immunotherapy”, were announced, showing dramatic responses in some advanced metastatic patients57. In 2011, the US Food and Drug Administration (FDA) approved the use of anti-CTLA4 immunotherapy (ipilimumab or Yervoy™; Bristol-Myers Squibb Company, New York, NY, USA) for the treatment of metastatic melanoma. Subsequent FDA approvals have been granted for immunotherapies targeting another checkpoint blockade protein, PD1, in melanoma (nivolumab and pembrolizumab). These therapies have expanded into single-agent clinical trials of other cancer sites, including non-small cell lung and bladder cancers, and also are showing significant response rates when used in combination58–60. Nivolumab was recently approved by the FDA for previously treated advanced or metastatic NSCLCs. Like melanomas, these tumors are associated with the carcinogens in cigarette smoke and have a correspondingly high mutation rate across the genome. Whether combination checkpoint blockade therapies in smoker-associated lung adenocarcinomas will have increased efficacy as seen in melanomas remains to be tested.\n\nStudies of mouse models of sarcomas induced by a chemical carcinogen, methylcholanthrine (MCA), have been used to study the interaction between the immune system and cancer61. A genomic study of these mouse model tumors revealed an MCA-specific mutational signature and a high mutational load. Combined exome sequencing with neoantigen prediction algorithms (based on major histocompatibility complex [MHC] binding avidity comparing mutated to wild-type peptides) identified those tumor-specific mutant antigens (TSMA) or “neoantigens” that were specifically targeted by the immune system to effect elimination of growing tumors62. More recently, this MCA model and the same genomics-based approach were used to demonstrate that TSMA were also the proteins targeted by anti-CTLA4 or anti-PD1 antibodies, and importantly that synthetic peptides corresponding to TMSA could be used as a prophylactic or therapeutic vaccine63.\n\nIn human cancers, exome sequencing and neoantigen prediction have now characterized that patients with melanoma who responded to anti-CTLA4 checkpoint blockade have a high number of non-synonymous mutations64. Similar results were described on the basis of only exome sequencing data for lung cancer patients with anti-PD1 responses65, for bladder cancer and other high mutational load cancers with anti-PD-L1 responses66,67, and recently for MMR-deficient colon and other MMR-deficient cancers treated with anti-PD1 therapy68. These results, though exciting, raise the issue of whether this high mutation rate is a biomarker of sorts for gauging which patients will respond to these therapies. Likely, it is more complicated since even in the small number of MMR-deficient patients who received anti-PD1 therapy, there were a small number of non-responders. To state the question in another way, will all tumors with a significant mutational load respond to checkpoint blockade? Or should the mutations be further evaluated algorithmically for their antigenic potential as neoantigens? How does the predictive quality of mutational load characterization or neoantigen load compare with immunohistochemistry-based evaluation of PD1 and PD-L1 protein expression? These open questions require further study and the requisite comparisons of predictive power. Regardless of the answers, the notion that the mutational load of non-synonymous mutations in the tumor exome can predict therapeutic response fundamentally changes our definition of an “actionable mutation”.\n\nUsing an analytical approach similar to that described above for the MCA mouse models to predict neoantigens, we combined exome sequencing with algorithmic prediction of MHC binding to compare tumor-unique peptides with their wild-type counterparts in a small clinical trial of patients with melanoma. This approach identified the neoantigens most likely to stimulate tumor-specific T cells, which were further evaluated for RNA expression of the mutant alleles and then evaluated with patient-derived immune components in vitro. The neoantigenic peptides were synthesized and used to condition patient-derived dendritic cells to create personalized vaccines for three patients69. In all three patients receiving vaccines and post-vaccine monitoring to date, three of the seven tumor-specific peptides elicited a T-cell response that was measurable after vaccination. In determining the neoantigens to include in each patient’s vaccine, we evaluated inter-metastatic heterogeneity by producing exome sequencing from multiple biopsies in two of the three patients. We also used T-cell receptor-specific PCR and MPS to characterize the resulting T-cell repertoire from blood. Here, we determined that for the three peptides eliciting an enhanced T-cell expansion in each patient, the T-cell receptor repertoire was very diverse, representing multiple clonotypes. These studies demonstrate how cancer genomics-based approaches are being used to characterize the mutational load of tumors that do or do not respond to checkpoint blockade immunotherapies or to design personalized immunotherapies and monitor the resulting T-cell repertoire in vaccinated patients.\n\n\nFuture forward\n\nCancer genomics has progressed dramatically in its application to clinical questions of cancer care in just a few short years. This translational trajectory has been demonstrated in several ways. Firstly, the use of deep sequencing and analysis to evaluate the evolution of cancers via clonal heterogeneity changes has revealed important information about the nature of acquired resistance to targeted therapies and chemotherapies. Secondly, the concept of tracking emerging resistance to therapy has led to the notion of blood-based monitoring via “liquid biopsy” as a sensitive and inexpensive proxy for tumor response. Thirdly, a surprising application for cancer genomics has emerged from studies of the immune system’s interaction with cancer, supporting the notion that mutational load via genomics may be a predictor of response to checkpoint blockade therapy. Importantly, if mutational or neoantigen load is a predictor of checkpoint blockade response, we may, in our clinical use of DNA-damaging chemotherapy as the standard of care for many patients, be creating an opportunity to use immunotherapies as a second-line therapeutic approach. This is predicted by genomic studies of post-therapy recurrent tumors or metastases that indicate a signature of DNA damage and a correspondingly higher mutation rate resulting from DNA-damaging chemotherapies22,34.\n\nGenomics also is contributing to personalized vaccine development efforts by identifying tumor-specific neoantigens that potentially can stimulate T-cell memory against cancer cells. Though still in development, the vaccine “angle” provided by genomics may provide an important possibility to cancer patients who have exhausted other treatment approaches, including other types of immunotherapy. Although cancer remains a significant and as-yet-unsolved disease, modern cancer genomics is contributing to clinical diagnosis and to therapeutic decision-making. Taken together, impactful clinical translational efforts involving cancer genomics should continue for some time to come. It will be exciting to see the results!",
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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLong GV, Stroyakovskiy D, Gogas H, et al.: Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: a multicentre, double-blind, phase 3 randomised controlled trial. Lancet. 2015; 386(9992): 444–51. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHu-Lieskovan S, Mok S, Homet Moreno B, et al.: Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma. Sci Transl Med. 2015; 7(279): 279ra41. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAtefi M, Titz B, Avramis E, et al.: Combination of pan-RAF and MEK inhibitors in NRAS mutant melanoma. Mol Cancer. 2015; 14: 27. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSchreiber RD, Old LJ, Smyth MJ: Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science. 2011; 331(6024): 1565–70. PubMed Abstract | Publisher Full Text\n\nMatsushita H, Vesely MD, Koboldt DC, et al.: Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting. Nature. 2012; 482(7385): 400–4. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGubin MM, Zhang X, Schuster H, et al.: Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. 2014; 515(7528): 577–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSnyder A, Wolchok JD, Chan TA: Genetic basis for clinical response to CTLA-4 blockade. N Engl J Med. 2015; 372(8): 783. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRizvi NA, Hellmann MD, Snyder A, et al.: Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015; 348(6230): 124–8. 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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "10965",
"date": "28 Oct 2015",
"name": "Timothy J. Triche",
"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",
"responses": []
},
{
"id": "10966",
"date": "28 Oct 2015",
"name": "Caroline Dive",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1162
|
https://f1000research.com/articles/4-1158/v1
|
28 Oct 15
|
{
"type": "Review",
"title": "Conservation in the face of climate change: recent developments",
"authors": [
"Joshua Lawler",
"James Watson",
"Edward Game",
"James Watson",
"Edward Game"
],
"abstract": "An increased understanding of the current and potential future impacts of climate change has significantly influenced conservation in practice in recent years. Climate change has necessitated a shift toward longer planning time horizons, moving baselines, and evolving conservation goals and targets. This shift has resulted in new perspectives on, and changes in, the basic approaches practitioners use to conserve biodiversity. Restoration, spatial planning and reserve selection, connectivity modelling, extinction risk assessment, and species translocations have all been reimagined in the face of climate change. Restoration is being conducted with a new acceptance of uncertainty and an understanding that goals will need to shift through time. New conservation targets, such as geophysical settings and climatic refugia, are being incorporated into conservation plans. Risk assessments have begun to consider the potentially synergistic impacts of climate change and other threats. Assisted colonization has gained acceptance in recent years as a viable and necessary conservation tool. This evolution has paralleled a larger trend in conservation—a shift toward conservation actions that benefit both people and nature. As we look forward, it is clear that more change is on the horizon. To protect biodiversity and essential ecosystem services, conservation will need to anticipate the human response to climate change and to focus not only on resistance and resilience but on transitions to new states and new ecosystems.",
"keywords": [
"climate change",
"conservation",
"restoration",
"assisted colonization",
"biodiversity",
"extinction"
],
"content": "Introduction\n\nClimate change is one of the largest threats to biodiversity and to natural systems in general1,2. Recent changes in climate are driving shifts in the timing of ecological events3, the distribution of species4,5, and the functioning of ecosystems6. Models project even greater changes for the future7–10.\n\nThese climate-driven changes challenge the way the planning and practice of conservation have traditionally been done. Most conservation has been predicated on the fact that the environment is relatively stable over the time frames of management planning (generally less than 50 years). However, projected changes in species distributions and ecosystem functions present obvious challenges to this assumption. Over the last decade, there has been a growing literature on what climate change means for biodiversity11 and the implications this has for conservation planning and action12. As a consequence, there have been some notable shifts in the way conservation is being conducted, from the goals and structure of conservation organizations to the planning and execution of conservation projects on the ground12,13.\n\nClimate change is increasingly integrated into the daily operations of conservation organizations. The anticipated impacts of climate change have driven planners and managers to consider longer time horizons and to anticipate the potentially synergistic effects of climate change and other threats and the need to address them quickly. Likewise, conservation biologists have begun to acknowledge the importance of planning for extreme weather events in addition to slow, long-term climatic change. These shifts have led to new perspectives on, and alterations to, approaches used to conserve biodiversity. Here, we describe some of the more prominent changes in the approaches conservation planners and practitioners have taken to help address the threat of climate change. Some of these approaches, such as restoration and the prioritization of species for conservation actions, have been around for quite some time but needed reframing as a consequence of the likely impacts of climate change. Others, such as assisted colonization, are new twists on old practices and have arisen directly out of the challenges posed by climate change.\n\n\nChanging conservation approaches to address climate change\n\nRestoration is one of the basic tools of a conservation practitioner. It has traditionally involved returning a system to its state prior to some disturbance14. However, climate change challenges the very nature of restoration based on such a definition15. It brings into question the utility of historical benchmarks as restoration targets, the species or seed sources to be used, and the time frame for planning.\n\nRethinking how restoration efforts are applied in light of climate change has led to shifts in thinking as well as in practice. Most fundamentally, practicing restoration in a changing climate requires embracing uncertainty and accepting that the goals of a project may need to change over time16. Instead of relying on historical benchmarks, restoration efforts will likely need to look into the future and anticipate change—perhaps relying on a dynamic reference process that accounts for variability in reference ecosystems16,17. Looking forward will also include rethinking the mix of species to be planted and potentially focusing on ecosystem function rather than particular assemblages of species. Restoration efforts have begun to make use of the same niche modelling methods that have been used to assess potential species responses to climate change18, but there is clear recognition that better models are needed19.\n\nMany recent climate-adaptation efforts have involved restoration. Of the projects funded by the Wildlife Conservation Society’s Climate Adaptation Fund, at least 70% have involved restoration20. These projects have included riparian restoration to enhance connectivity, coastal restoration to prevent storm damage, forest restoration to reduce fire risk, and prairie restorations to enhance watershed function. In addition to the projects that clearly engage in restoration, several projects are aimed at converting one type of ecosystem to another—not necessarily restoring a historical condition but preparing an ecosystem to function differently in a future climate. An example of this type of project involved converting forested areas to grasslands to facilitate marsh establishment in the face of sea-level rise. Whether such actions are classified as restoration could be debated, but clearly the lessons learned from decades of restoration will be essential to these new types of adaptation projects.\n\nSystematic conservation planning21 has been used widely around the world to help prioritize conservation efforts, particularly the location of protected areas22. Most conservation planning has been based on “static” representations of biodiversity across a region, an approach that is clearly challenged by climate-driven changes in the distribution of species and communities23. Consequently, substantial thought has been put into how best to incorporate climate change into the conservation-planning process.\n\nThe cornerstone of initial approaches to integrate climate change into conservation planning was the use of correlative niche models to predict future distributions of species and ensure that these were adequately represented by present-day conservation efforts. The more sophisticated niche-based planning efforts included the ability of species to track changing habitat conditions through space and time24,25 and consideration of uncertainties in predicted distributions26,27. Climate, however, is only one of many factors determining the distributions of species, and the relationship is complex, uncertain, and in many cases evolving28. The magnitude of these ecological uncertainties compounded by the uncertainties associated with climate predictions29 led to calls to integrate climate change into planning using approaches that were more robust to uncertainty in predicted climate impacts30–32.\n\nAn approach to planning that has gained some traction for conservation in a dynamic climate involves conserving the underlying geophysical variation in a region, also referred to as “conserving nature’s stage”33. The rationale for this approach is that, theoretically, there should be a strong relationship between species distributions and geophysical settings (for example, elevation and geology)34 such that conserving representative examples of geophysical settings will protect representative ecological communities under both current and future climates35. A similar approach that focuses on current and known patterns in a region emphasizes conserving connectivity between climatically diverse areas31,36,37.\n\nIncreasing the connectivity of landscapes to allow species to move in response to climate change is the most-often cited climate change adaption strategy38–40. Traditionally, connectivity planning has focused on connecting patches of habitat with what amount to linear strips or stepping-stones of more habitat. Although this approach may allow species to move through the landscape, it may do little to facilitate movement into what might become newly suitable habitat. Early attempts to model connectivity expressly for addressing climate change involved projecting shifts in species distributions through time and either identifying overlap of current and future distributions or mapping pathways that tracked those shifting distributions41,42. A more mechanistic application of this approach involved mapping potential corridors through climate-driven shifts in suitable levels of snowpack for wolverines in the northwestern United States43. Other studies have taken different approaches to identifying important areas for species movement in the face of climate change—approaches that are less reliant on projected future changes in climate and species distributions. Brost and Beier44 and Beier45 focused on the geophysical settings mentioned in the previous section, and charted routes across the landscape that either connected similar geophysical setting or linked a diversity of settings. Nuñez and colleagues46 mapped routes through landscapes that connected slightly warmer patches of intact land to slightly cooler ones with routes that followed gentle temperature gradients and avoided human-impacted landscapes.\n\nAlthough efforts continue to develop more relevant climate-connectivity methods, the critical question of whether corridors are really needed—or whether there might be other strategies and approaches that would achieve the same result—has been repeatedly raised37. Some argue that the focus on corridors is misguided and that, alternatively, protecting large intact ecosystems should be prioritized47,48. Others have argued that the benefits of increasing the size and number of corridors are fewer than those resulting from simply increasing the amount of protected land, which, if regularly distributed, would increase connectivity49.\n\nAlthough a growing wealth of studies predict increased extinction risk for species because of climate change50,51, many of these vary enormously in their estimations. It is also increasingly recognized that the predictions of extinction risk do not reflect the number of species that have become vulnerable (or extinct) to date, nor do they match the number identified as threatened because of climate change on the International Union for the Conservation of Nature (IUCN) Red List (only 10.5% of the 22,176 species)52. A fundamental challenge has involved integrating the projections of species niche models (the most-often used tool for assessing the impacts of climate change on species) into the processes of real-world extinction assessments.\n\nSimple measures of population size, geographic range size, and other indicators of current status already used as IUCN Red List criteria are likely to be good predictors of climate change-associated extinction risk53. Such IUCN Red List criteria have been used to predict the risk of extinction in the absence of conservation action and the time lag between assessment and extinction54,55. This time lag amounts to a warning period in which adaptation efforts can be taken to prevent extinction. Although there is a warning period, it is finite and thus delays in developing and implementing conservation plans after a species is identified as being threatened could be costly. Roughly half of listed species are likely to go extinct within 20 years of being listed as critically endangered54.\n\nAn additional challenge is that most species risk assessments treat climate change as a problem driven by relatively slow, predictable, and continuous change in environmental conditions and fail to account for other important components of climate change, such as increasing extreme weather and climate events (for example, cyclones, floods, and drought)56,57. It is increasingly recognized that the increases in frequencies and intensities of extreme events are critical determinants of patterns of biological diversity and will affect it differently from impacts resulting from steady climate change58. A good example of this is how climate change will impact bat species: extreme maximum temperature is now considered a critical factor in the vulnerability of bats to climate change59, but many studies (for example, 60) fail to consider it in projections of species distributions under climate change.\n\nEven though our understanding of which extremes are most important and how they are shifting is limited61, there are good examples of assessments that do account for extremes. Recently, Ameca y Juárez and colleagues62 produced a comprehensive analysis of the impacts of cyclones and droughts on terrestrial mammals, one of the few large-scale studies to consider exposure to extreme events. They followed this exposure analysis with an assessment of terrestrial mammal sensitivity to extreme weather and climate events, identifying biological traits that make large terrestrial mammals more susceptible to climate-induced population declines.\n\nAlongside species risk assessments, the assessment of the vulnerability of species to climate change—as well as the climate-related vulnerability of places and natural resources in general—has emerged as an important step in the adaptation process63. As with extinction risk assessments, many different approaches to assessing vulnerability have been proposed (for example, 64), each evaluating some subset or combination of sensitivity, exposure, and adaptive capacity65. These vulnerability assessments serve not only to determine which species are likely to be most vulnerable but also to identify the factors that make a species vulnerable and thus potential conservation actions—adaptation measures—that can be taken to reduce vulnerability.\n\nOne of the relatively new tools in the conservation toolbox is assisted colonization—broadly defined by the IUCN as the movement of an organism outside of its native range to avoid extinction of populations due to current or future threats66. There are some species, particularly endemics with relatively specific habitat requirements and poor dispersal abilities, that will be unable to move to suitable climates. When these species are threatened with extinction—because of either climate change or some other factor—it may become necessary to move them to prevent their loss.\n\nThe question of whether assisted colonization should even be considered as an option spawned a lively debate67–72. Those opposed to assisted colonization argue that the history of invasive species has taught us that the potential impacts on the ecosystems into which organisms would be moved could be too great68. Those in favour of keeping the option of assisted colonization on the table argue that it would likely be necessary for preventing the extinction of certain species69, that the potential impact of translocated species is likely overstated for several reasons69,71 (including that the traits of species that will need to be moved are not those traits generally associated with invasive species73), and that the amount of change that systems will experience over the coming decades will likely overshadow the impacts of translocated individuals of a rare and declining species74.\n\nAs the debate worked its ways through the scientific literature, many researchers started to ask more productive questions—facing the reality that assisted colonization was already being used. There were, for example, several early efforts to develop frameworks for determining under what circumstances assisted colonization would be a viable conservation option75,76. Other studies have highlighted the importance of the timing of assisted colonization efforts77, developed advanced modelling approaches for identifying potential sites for translocations78, and explored the situations in which invasions will be less likely and hence assisted colonization a less risky venture79. Furthermore, calls for the development of policies to address assisted colonization80 have begun to be met81,82. Overall, it appears that, at least in the scientific literature, assisted colonization is gaining acceptance as a tool in the conservation toolbox and one that may not differ so much from other movements of species for conservation reasons83,84.\n\n\nFuture trends\n\nThe practice, and to some degree the study, of conservation is currently undergoing a major shift—a shift from a focus on nature to a focus on nature and people. The idea that people are a part of ecosystems and that conservation needs to include the social sciences is not new and this is not the shift to which we are referring. This new shift is one from conserving nature for nature’s sake to conserving nature both for nature’s sake and for the use and enjoyment by people85. This shift has resulted in an apparent change in the missions and the actions of several major non-governmental conservation organizations (for example, The Nature Conservancy, the World Wildlife Fund, and Conservation International). Like the subject of assisted colonization, however, this shift has not been well received by all in the conservation community and there remains a heated debate in conservation circles as well as in the literature about the degree to which conservation should focus on the needs of people85–88.\n\nThe impact of this shift can be clearly seen in the way that conservation organizations are addressing climate change and is reflected in the application of all of the approaches mentioned above. With respect to restoration, conservation planning, and connectivity, conservation practitioners have begun to target efforts that consider the roles that natural systems play in protecting people against the potential impacts of climate change89,90. These ecosystem-based adaptation strategies may be more cost-effective than hard infrastructure-based solutions. One striking example of this approach is The Nature Conservancy’s “flood plains by design” strategy in which stretches of river are restored in places that will simultaneously reduce flooding of nearby communities and restore fish habitat. Another example is the active protection of coastal habitats, which has the potential to reduce the risks and the costs of sea-level rise, providing a critical service in the face of climate change91.\n\nBecause climate-driven changes are likely to be so large in some places, climate change is in part causing conservation practitioners to question their goals as well as the approaches they use7. These new goals are beginning to take people’s needs into account. For example, restoration efforts are now being refocused toward ecosystem function and ecosystem services instead of the specific set of species in a given ecosystem19. In addition, assisted colonization may be called on not just to preserve threatened species, but also to provide certain functions—and perhaps to allow ecosystems to provide certain services73,92.\n\nGiven that our understanding of climate change impacts is still evolving, the theory and practice of conservation will likely continue to change at a relatively fast pace. One of the greatest future challenges to the conservation of biodiversity will likely come from how people respond to climate change93. Sea-level rise is forcing human populations to consider radical adaption action, including the construction of massive sea walls and the migration of coastal and island communities94,95. Water shortages and crop failures will similarly result in human migrations, shifts in agriculture, and increased water withdrawals. There is increasing recognition that, in many places, human responses to climate change may further constrain options for biodiversity conservation, and therefore planning needs to simultaneously consider both human and biodiversity responses93. The tools that conservation practitioners have to address climate change (for example, conservation planning, restoration, species risk assessments, and assisted colonization) will likely be most effective if their application takes human responses to climate change into account.\n\nContinued rapid climate change will also necessitate a shift from discussions of resistance and resilience to more strategies that embrace change and foster transitions96. Particularly if society hopes to continue to be the recipient of essential ecosystem services and to enjoy a diversity of plants and animals, conservation efforts will need to focus on smoothly transitioning ecosystems from one state to another. The enormity of that challenge necessitates policies and actions that reduce greenhouse-gas emissions and increase carbon sequestration. Unless adaptation is accompanied by meaningful mitigation efforts, it will be hard for conservation practitioners to accomplish even their shifting and evolving goals.\n\n\nAbbreviation\n\nIUCN, International Union for the Conservation of Nature.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nJoshua J. Lawler thanks the Wilburforce Foundation and the Denman endowment at the University of Washington for funding.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nSala OE, Chapin FS, Armesto JJ, et al.: Global biodiversity scenarios for the year 2100. Science. 2000; 287(5459): 1770–4. PubMed Abstract | Publisher Full Text\n\nMillennium Ecosystem Assessment: Ecosystems and Human Well-being Biodiversity Synthesis. Washington DC, World Resources Institute. 2005. Reference Source\n\nGregory RD, Willis SG, Jiguet F, et al.: An indicator of the impact of climatic change on European bird populations. PLoS One. 2009; 4(3): e4678. 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Publisher Full Text\n\nKlenk NL: The development of assisted migration policy in Canada: An analysis of the politics of composing future forests. Land Use Policy. 2015; 44: 101–9. Publisher Full Text\n\nKlenk NL, Larson BMH: The assisted migration of western larch in British Columbia: A signal of institutional change in forestry in Canada? Glob Environ Change. 2015; 31(0): 20–7. Publisher Full Text\n\nThomas CD: Translocation of species, climate change, and the end of trying to recreate past ecological communities. Trends Ecol Evo. 2011; 26(5): 216–21. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSeddon PJ, Griffiths CJ, Soorae PS, et al.: Reversing defaunation: restoring species in a changing world. Science. 2014; 345(6195): 406–12. PubMed Abstract | Publisher Full Text\n\nKareiva P, Marvier M: What Is Conservation Science? BioScience. 2012; 62(11): 962–9. Publisher Full Text\n\nSoule M: Also seeking common ground in conservation. Conserv Biol. 2014; 28(3): 637–8. PubMed Abstract | Publisher Full Text\n\nSoulé M: The \"new conservation\". Conserv Biol. 2013; 27(5): 895–7. Publisher Full Text\n\nTallis H, Lubchenco J: Working together: A call for inclusive conservation. Nature. 2014; 515(7525): 27–8. PubMed Abstract | Publisher Full Text\n\nJones HP, Hole DG, Zavaleta ES: Harnessing nature to help people adapt to climate change. Nat Clim Change. 2012; 2: 504–9. Publisher Full Text\n\nHobbs RJ, Higgs E, Hall CM, et al.: Managing the whole landscape: historical, hybrid, and novel ecosystems. Front Ecol Environ. 2014; 12(10): 557–64. Publisher Full Text\n\nArkema KK, Guannel G, Verutes G, et al.: Coastal habitats shield people and property from sea-level rise and storms. Nat Clim Change. 2013; 3(10): 913–8. Publisher Full Text\n\nLunt ID, Byrne M, Hellmann JJ, et al.: Using assisted colonisation to conserve biodiversity and restore ecosystem function under climate change. Biol Conserv. 2013; 157: 172–7. Publisher Full Text\n\nWatson JEM: Human Responses to Climate Change will Seriously Impact Biodiversity Conservation: It’s Time We Start Planning for Them. Conserv Lett. 2014; 7(1): 1–2. Publisher Full Text\n\nWyett K: Escaping a Rising Tide: Sea Level Rise and Migration in Kiribati. Asia Pac Policy Stud. 2014; 1(1): 171–85. Publisher Full Text\n\nBirk T, Rasmussen K: Migration from atolls as climate change adaptation: Current practices, barriers and options in Solomon Islands. Nat Resour Forum. 2014; 38(1): 1–13. Publisher Full Text\n\nChornesky EA, Ackerly DD, Beier P, et al.: Adapting California’s Ecosystems to a Changing Climate. BioScience. 2015; 65(3): 247–62. Publisher Full Text"
}
|
[
{
"id": "10948",
"date": "28 Oct 2015",
"name": "Paul Beier",
"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",
"responses": []
},
{
"id": "10949",
"date": "28 Oct 2015",
"name": "A Townsend 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",
"responses": []
},
{
"id": "10950",
"date": "28 Oct 2015",
"name": "Bruce Stein",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1158
|
https://f1000research.com/articles/4-1157/v1
|
28 Oct 15
|
{
"type": "Review",
"title": "Early lessons from schistosomiasis mass drug administration programs",
"authors": [
"W. Evan Secor"
],
"abstract": "Mass drug administration using praziquantel is the backbone of the current strategy for the control of schistosomiasis. As the theoretical plans have moved into practical application, certain challenges with this approach have surfaced, and it is likely that annual mass drug administration alone may not be sufficient to achieve program goals. However, mass drug administration is still the only available intervention that can be readily used in the wide variety of settings where schistosomiasis is endemic. The task then becomes how to improve this approach and identify what adjuncts to mass drug administration are effective, as programs move from morbidity control to elimination goals. Other aspects worthy of consideration include how best to employ new diagnostic tools to more easily identify where treatment is needed, and new formulations of praziquantel to extend the availability of treatment to all age groups. The aim of this review is to highlight both areas of challenge and of opportunity to improve the public health impact of schistosomiasis control programs.",
"keywords": [
"schistosomiasis",
"mass drug administration",
"praziquantel",
"schistosome"
],
"content": "A new focus on schistosomiasis control and elimination\n\nOver the last decade, there has been an increased emphasis on schistosomiasis control, especially with respect to using mass drug administration (MDA) to reduce its prevalence and the intensity of infections. This shift has largely been driven by the introduction of the preventative chemotherapy approach for neglected tropical diseases and the passage of World Health Assembly (WHA) resolutions 54.19 (2001; http://www.who.int/neglected_diseases/mediacentre/WHA_54.19_Eng.pdf?ua=1) and 65.21 (2012; http://www.who.int/neglected_diseases/mediacentre/WHA_65.21_Eng.pdf) that set control and elimination goals for schistosomiasis, respectively. In addition, access to treatment for those persons in need has dramatically increased as a result of aid agency purchase and manufacturer donation of praziquantel, the only drug currently available for the treatment of schistosome infections, along with efforts of groups such as the Schistosomiasis Control Initiative (SCI, http://www3.imperial.ac.uk/schisto) to work with ministries of health to distribute praziquantel1. Finally, increased support for operational research on how best to distribute treatment has facilitated the posing of questions that have direct public health impact in a more widespread approach than was previously possible. While there have been clear public health benefits associated with schistosomiasis MDA in certain settings2,3, it has not been an unqualified success4–6. This review will focus on some of the practical questions that have surfaced with the introduction of MDA for schistosomiasis and issues that have implications for the successful implementation of schistosomiasis control and elimination programs.\n\n\nChallenges with providing MDA\n\nDespite the dramatic expansion of praziquantel availability, data from 2013 indicate that of the more than 260 million people in need of treatment for schistosomiasis, less than 40 million received it7. The shortfall can be attributed to a number of factors, including a remaining deficit (about 120 million treatments) in the amount of available praziquantel1. Furthermore, even with significant price reduction or donation of the drug, the costs associated with identifying where MDA is needed and the delivery of the drugs create barriers for many national control programs in the absence of external funding to support these activities. Other obstacles include the lack of compliance with treatment programs by persons needing to take the drug. While health education increases community cooperation with praziquantel delivery programs, the side effects (fever, nausea, abdominal pain, diarrhea, fatigue) associated with dying worms, especially the first time someone is treated when worm burdens tend to be the highest, can lead to wariness about receiving follow-up treatments8–11. Further research on how best to promote participation in treatment campaigns, some of which will need to be tailored to specific countries, languages, or ethnic groups, is essential. MDA strategies are geared towards school age children but adults and pre-school aged children also contribute to ongoing transmission, suggesting that achieving elimination will require treating these other age groups as well. There is also a growing recognition that even very young children can become infected with schistosomes and suffer health consequences. However, current formulations of praziquantel are not appropriate for MDA in this age group because of the size and taste of the tablets. The therapeutic dose younger children need may also differ from that required by older individuals, as suggested by recent studies in Uganda12. Fortunately, efforts are currently underway to develop a pediatric formulation of praziquantel and to define the dosing regimen. As this formulation becomes available, operational research on how to carry out treatments targeted to young children will be needed, in parallel with the development of treatment delivery approaches to improve compliance among school children and adults.\n\n\nAdvances in determining where to treat\n\nThe initial requirement for any control program is to accurately determine the areas where treatment is needed. For schistosomiasis, this has traditionally been done by parasitologic assessment of stool or urine samples, depending on the schistosome species endemic in the area. Although parasitologic methods have recognized limitations in sensitivity, they were (until very recently) the only feasible option for estimating the prevalence of intestinal schistosomiasis and the only way to monitor the intensity of human infection for any of the species. As a result, the current World Health Organization (WHO) guidelines for schistosomiasis control are heavily dependent on detection of eggs in stool for Schistosoma mansoni and S. japonicum or urine for S. haematobium13. Recently, a point of contact (POC) test that detects a S. mansoni carbohydrate antigen in the urine of infected individuals has become commercially available. This circulating cathodic antigen (CCA) POC test can indicate a relative intensity of infection and distinguish active infection, or reinfection, from cure following treatment. A large number of studies have evaluated the POC-CCA in comparison to stool examination by the Kato-Katz method and found that it is at least as good as traditional stool examination for mapping areas in need of MDA14–19. In general, the POC-CCA appears to be more sensitive than traditional stool examination methods but questions remain about whether disparities in results obtained when comparing the two methods are attributable to the known insensitivity of the Kato-Katz method or imperfect specificity of the POC-CCA test20. However, when considering all the expenses associated with laboratory testing and treatment-associated expenditures, the costs of using either test are comparable19,21. Because the POC-CCA does not require equipment, it should be easier to deploy than the Kato-Katz method in areas that need mapping for S. mansoni prevalence. Nevertheless, training for POC-CCA use and interpretation will be needed and there is a distinct need to develop bench aids for this test. It is also not possible to simply apply the WHO guidelines that were written for morbidity control based on stool exam prevalence levels to the POC-CCA, which consistently detects higher prevalence levels. Thus, while the introduction of the POC-CCA is perhaps the most important technical advance for S. mansoni control since the release of praziquantel, more operational research is needed before it can achieve its full potential and be incorporated into WHO recommendations. The POC-CCA may also be useful for detecting S. japonicum infections22.\n\nThe paradox of the POC-CCA test is that, even though it uses a urine sample for the assay, it is not a reliable test for S. haematobium infections. Fortunately, another carbohydrate, the circulating anodic antigen (CAA), is effective for detecting both urogenital and intestinal schistosomiasis23,24. It is also considered more sensitive and specific for S. mansoni than the POC-CCA. The drawback of the CAA test is that it involves equipment-requiring processing of samples prior to testing and the output is a non-visual signal that requires an automated reader. However, research is ongoing to develop the CAA into a more field applicable test that would have the advantage of detecting both intestinal and urinary schistosomiasis.\n\nThe detection of specific antibodies may also become important for control and elimination programs for schistosomiasis, although they are likely to be employed in different settings, or at different phases of the program than egg or antigen detection tools25. An advantage of antibody assays is the ability to directly observe the collection of finger stick blood from the population being surveyed. Although urine collection is easier than stool collection, it is not feasible or culturally acceptable to directly observe the collection of either and therefore both present an increased risk of sample sharing. In addition, small quantities of blood can be used in multiplex assays that may simultaneously test for a variety of neglected tropical diseases, other infectious agents, and monitor vaccine coverage26. Thus, a single sample can be used for several public health programs, thereby providing cost savings, compared to performing an independent survey for each infection or vaccine response of interest. Many low-cost rapid diagnostic tests are based on antibody detection and could be adapted for schistosomiasis, provided the proper antigen was selected. It is also theoretically possible to develop pan-schistosome or species-specific antigens depending on the intended use of the assay. A major drawback with using antibody-based assays is that the current antigens that are used for immunodiagnosis are recognized by host sera even after successful cure. Thus, it is not possible to distinguish former infections from active infections with great certainty, and therefore not possible to monitor decreases in prevalence levels as a control program progresses. Antibodies are also a less reliable indicator of intensity of infection than egg or antigen detection methods. However, antibody detection will likely be very useful for schistosomiasis elimination programs; children born after cessation of transmission would not be exposed to infection and thereby become very sensitive sentinels to confirm that elimination has been achieved. Further, as immunodiagnostic tests are developed that use individual antigens rather than antigen mixtures, immunoassays that are positive during active infection but become negative shortly after treatment may become available. One antigen that shows promise in this regard is recombinant SP-13 from S. japonicum27. Detection of parasite DNA in stool or urine could also be a sensitive method for specifically identifying active infections28.\n\n\nMDA plus what?\n\nCurrent schistosomiasis control guidelines are based on different frequencies and target populations of MDA, with the most intensive intervention being annual MDA of all community members29. This approach is predicated in large part on the benefits of annual MDA for reducing prevalence of lymphatic filariasis, onchocerciasis, and blinding trachoma30,31. However, growing evidence from schistosomiasis control efforts suggest that yearly MDA alone may not be sufficient to achieve program goals, especially when the objective is the elimination of transmission32–34. Research into what other interventions are both cost effective and environmentally acceptable is needed. A vaccine for schistosomiasis is quite desirable and the identification of novel vaccine targets that are responsible for vital biologic functions are providing intriguing new strategies to attack the parasite35–37. However, as has been the case for the last 3 decades, it seems that a vaccine for schistosomiasis is still 5–10 years and millions of dollars away from being a reality. Therefore, in the near term it is likely that other interventions involving water are the most likely adjuncts to MDA for reducing infection38,39.\n\nAn unfortunate side effect of the introduction and initial success of praziquantel was the assumption that treatment alone would be adequate to reach program goals. This belief contributed to reduced support for research into other control approaches. Because of the expense of water and sanitation systems, efforts to reduce urine or fecal contamination of water, as well as limiting exposure of people to contaminated water, will rely heavily on health education and behavioral modification. These approaches may need to be specifically tailored to individual communities and are therefore difficult to readily apply across endemic areas. The control of intermediate host snails can be highly effective but most interventions that have demonstrated success involve the introduction of molluscicides that also kill other aquatic species or entail the introduction of non-native snail predators, both of which have limited acceptability for local populations or groups with environmental concerns. One exciting idea that does not suffer from these limitations is represented by The Upstream Alliance project, which will reintroduce native Macrobrachium vollenhoveni prawns in the Senegal River upstream of the Daima Dam. Following completion of this dam in 1986, there was an outbreak of new schistosome infections that has in part been attributed to the interruption of the prawn’s ability to move up the river from its breeding grounds in brackish water. M. vollenhoveni are voracious predators of the schistosome intermediate host snails. The restriction of their migration led to an expansion of snail populations, which in turn led to increased transmission of schistosomiasis40. The Upstream Alliance hopes to couple prawn aquaculture with the reintroduction of prawns above the dam to create an economically self-sustaining intervention to reduce schistosome infection prevalence41. If successful, it will be a robust model for snail control programs in other areas with native Macrobrachium spp. populations.\n\n\nAdapting programs as they progress\n\nIn the “staged control” strategy for schistosomiasis, program objectives change from morbidity control to reduction of infection to elimination of transmission to post transmission surveillance, depending on the infection levels of the population42. One of the biggest unknowns for schistosomiasis control is what prevalence cutoffs merit changes in treatment strategies and, in fact, what those different strategies should be as the goals of the program change. As mentioned above, the current WHO guidelines for schistosomiasis were developed at a time when praziquantel was less abundant and more expensive, and therefore the primary goal was the reduction of severe hepatosplenic disease for intestinal schistosomiasis and the prevention of bladder and kidney complications for urogenital schistosomiasis. Now that praziquantel is more readily available, it is recognized that treatment should be extended more generally, as it is not only those with the most severe manifestations of schistosomiasis that suffer morbidity. In fact, a strong case has been made that there is no such thing as an asymptomatic schistosome infection43. Thus, there may really be no practical differences between reducing morbidity and reducing infection.\n\nMonitoring of control program progress, like mapping, has traditionally been performed by measuring egg prevalence and the intensity of schistosome infections in school age children because they provide a useful barometer of the level of infection in other age groups in the community44. This age group also tends to have the highest intensities of infection, so decreases in their egg output would result in fewer eggs that could contaminate fresh water and infect snails. Fewer infected snails would lead to the release of fewer infectious cercariae and a theoretical reduction in the “force of transmission”. A test to measure force of transmission in areas where people come into contact with water would be a more timely way to assess the impact of control efforts, rather than having to rely solely on measuring changes in infection levels in people. Unfortunately, previous attempts to measure the number of cercariae in water have not been successful. Sentinel mice and snail sampling, followed by cercarial shedding or PCR, have been somewhat informative but have not yet been incorporated as ways to monitor the impact of control programs. A method that has been somewhat successful in assessing water bodies for cercariae of avian schistosomes, ultrafiltration followed by real-time PCR, can detect as few as 5 cercariae in 100 liters of water45. If this method will also work for detecting cercariae of schistosomes infectious for humans, control programs may have a technique to more directly assess the impact of different control efforts.\n\n\nIs praziquantel sufficient?\n\nAnother potential concern for treatment programs is the incomplete efficacy of praziquantel. The introduction of MDA for schistosomiasis has, fortunately, not resulted in evidence of widespread clinical resistance to drugs. However, it has long been recognized that a single treatment, especially for persons with high intensities of infection, is not adequate to kill all the worms. This finding has been further highlighted in recent studies comparing parasitologic and antigen detection assays following treatment46,47. Many individuals become egg negative but retain antigen positivity, suggesting that viable adult worms remain even if egg excretion has stopped. From a transmission perspective, and perhaps even from a morbidity standpoint, if egg laying stops and does not resume, the treatment has accomplished its goal. However, worms that are not killed may only be temporarily affected and subsequently resume egg laying. Interpretation of post treatment data is also complicated by the decreased efficacy of praziquantel against juvenile worms that can produce eggs once they mature. Provision of a second dose of praziquantel to target worms that may not have been killed by the first treatment produces increased cure rates and greater egg reduction than a single treatment48. These studies highlight the need to continue research into improved formulations of praziquantel, if there are more effective dosing schedules than annual MDA depending on a location’s force of transmission or schistosomiasis species, as well as control versus elimination program goals. There is also concern that reliance on a single drug is risky, which argues for continued investigation into new or repurposed drugs for treatment49–52.\n\n\nConclusions\n\nThe prospects for a global reduction in schistosomiasis prevalence and intensity are better now than ever before. Sub-Saharan Africa remains the biggest challenge, although even more developed countries like Brazil and China that have had control programs in place for many years still have much work remaining to achieve elimination. In addition to the WHA resolutions, the formation of groups such as SCI, the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE), the Coalition for Operational Research on Neglected Tropical Diseases (COR-NTD) and, most recently, the Global Schistosomiasis Alliance (GSA) has provided better opportunities for researchers to interact with WHO, ministry of health officials, and schistosomiasis control program managers to identify and test practical solutions for challenges encountered where MDA has been used, and to begin to define and test the strategies for effecting and verifying elimination where appropriate. The pending completion of multi-year operational research studies should provide strong data for the development of updated evidenced-based guidelines for schistosomiasis in the near future. However, progress and answers will take time, requiring patience from donors and governmental aid agencies. Similarly, when prevalence levels decrease and schistosomiasis becomes a lower public health priority, ministries of health in endemic countries will need to maintain control activities amidst competing agendas to achieve elimination. Diagnostic tools with improved sensitivity and specificity, as well as operational research on how to employ them, are critical needs for elimination strategies in areas with decreasing prevalence and intensity of infection. Continued coordination of efforts, along with innovative thinking to identify effective interventions to complement MDA, will be necessary to reduce the public health burden and ultimately eliminate schistosomiasis.\n\n\nAbbreviations\n\nCAA, circulating anodic antigen; CCA, circulating cathodic antigen; MDA, mass drug administration; POC, point of contact; WHA, World Health Assembly; WHO, World Health Organization.\n\n\nDisclaimer\n\nThe findings and conclusions in this report are those of the author and do not necessarily represent the official position of the Centers for Disease Control and Prevention.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has 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\nThe author would like to thank Susan P. Montgomery, Kimberly Y. Won and Laurence Slutsker for critical reading of the manuscript and helpful comments.\n\n\nReferences\n\nFenwick A: Praziquantel: do we need another antischistosoma treatment? Future Med Chem. 2015; 7(6): 677–80. PubMed Abstract | Publisher Full Text\n\nFrench MD, Churcher TS, Gambhir M, et al.: Observed reductions in Schistosoma mansoni transmission from large-scale administration of praziquantel in Uganda: a mathematical modelling study. PLoS Negl Trop Dis. 2010; 4(11): e897. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSesay S, Paye J, Bah MS, et al.: Schistosoma mansoni infection after three years of mass drug administration in Sierra Leone. Parasit Vectors. 2014; 7: 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLelo AE, Mburu DN, Magoma GN, et al.: No apparent reduction in schistosome burden or genetic diversity following four years of school-based mass drug administration in Mwea, central Kenya, a heavy transmission area. PLoS Negl Trop Dis. 2014; 8(10): e3221. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTuhebwe D, Bagonza J, Kiracho EE, et al.: Uptake of mass drug administration programme for schistosomiasis control in Koome Islands, Central Uganda. PLoS One. 2015; 10(4): e0123673. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoss AG, Olveda RM, Li Y: An audacious goal: the elimination of schistosomiasis in our lifetime through mass drug administration. Lancet. 2015; 385(9983): 2220–1. PubMed Abstract | Publisher Full Text\n\nSchistosomiasis: number of people treated worldwide in 2013. Wkly Epidemiol Rec. 2015; 90(5): 25–32. PubMed Abstract\n\nOmedo M, Ogutu M, Awiti A, et al.: The effect of a health communication campaign on compliance with mass drug administration for schistosomiasis control in western Kenya--the SCORE project. Am J Trop Med Hyg. 2014; 91(5): 982–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWon KY, Abudho B, Blackstock AJ, et al.: Assessment of quality of life as a tool for measuring morbidity due to Schistosoma mansoni infection and the impact of treatment. Am J Trop Med Hyg. 2014; 90(2): 322–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZwang J, Olliaro PL: Clinical efficacy and tolerability of praziquantel for intestinal and urinary schistosomiasis-a meta-analysis of comparative and non-comparative clinical trials. PLoS Negl Trop Dis. 2014; 8(11): e3286. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMuhumuza S, Olsen A, Katahoire A, et al.: Uptake of preventive treatment for intestinal schistosomiasis among school children in Jinja district, Uganda: a cross sectional study. PLoS One. 2013; 8(5): e63438. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nStothard JR, Sousa-Figueiredo JC, Betson M, et al.: Schistosomiasis in African infants and preschool children: let them now be treated! Trends Parasitol. 2013; 29(4): 197–205. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWorld Health Organization: Helminth Control in School-Age Children: A Guide for Managers of Control Programmes. Geneva: World Health Organization; 2011. Reference Source\n\nCoulibaly JT, Knopp S, N'Guessan NA, et al.: Accuracy of urine circulating cathodic antigen (CCA) test for Schistosoma mansoni diagnosis in different settings of Côte d'Ivoire. PLoS Negl Trop Dis. 2011; 5(11): e1384. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTchuem Tchuenté LA, Kueté Fouodo CJ, Kamwa Ngassam RI, et al.: Evaluation of circulating cathodic antigen (CCA) urine-tests for diagnosis of Schistosoma mansoni infection in Cameroon. PLoS Negl Trop Dis. 2012; 6(7): e1758. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nNavaratnam AM, Mutumba-Nakalembe MJ, Stothard JR, et al.: Notes on the use of urine-CCA dipsticks for detection of intestinal schistosomiasis in preschool children. Trans R Soc Trop Med Hyg. 2012; 106(10): 619–22. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nColley DG, Binder S, Campbell C, et al.: A five-country evaluation of a point-of-care circulating cathodic antigen urine assay for the prevalence of Schistosoma mansoni. Am J Trop Med Hyg. 2013; 88(3): 426–32. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nErko B, Medhin G, Teklehaymanot T, et al.: Evaluation of urine-circulating cathodic antigen (Urine-CCA) cassette test for the detection of Schistosoma mansoni infection in areas of moderate prevalence in Ethiopia. Trop Med Int Health. 2013; 18(8): 1029–35. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAdriko M, Standley CJ, Tinkitina B, et al.: Evaluation of circulating cathodic antigen (CCA) urine-cassette assay as a survey tool for Schistosoma mansoni in different transmission settings within Bugiri District, Uganda. Acta Trop. 2014; 136: 50–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFoo KT, Blackstock AJ, Ochola EA, et al.: Evaluation of point-of-contact circulating cathodic antigen assays for the detection of Schistosoma mansoni infection in low-, moderate-, and high-prevalence schools in western Kenya. Am J Trop Med Hyg. 2015; 92(6): 1227–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorrell CM, Bartoces M, Karanja DM, et al.: Cost analysis of tests for the detection of Schistosoma mansoni infection in children in western Kenya. Am J Trop Med Hyg. 2015; 92(6): 1233–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Dam GJ, Odermatt P, Acosta L, et al.: Evaluation of banked urine samples for the detection of circulating anodic and cathodic antigens in Schistosoma mekongi and S. japonicum infections: a proof-of-concept study. Acta Trop. 2015; 141(Pt B): 198–203. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCorstjens PL, De Dood CJ, Kornelis D, et al.: Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels. Parasitology. 2014; 141(14): 1841–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnopp S, Corstjens PL, Koukounari A, et al.: Sensitivity and Specificity of a Urine Circulating Anodic Antigen Test for the Diagnosis of Schistosoma haematobium in Low Endemic Settings. PLoS Negl Trop Dis. 2015; 9(5): e0003752. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSolomon AW, Engels D, Bailey RL, et al.: A diagnostics platform for the integrated mapping, monitoring, and surveillance of neglected tropical diseases: rationale and target product profiles. PLoS Negl Trop Dis. 2012; 6(7): e1746. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLammie PJ, Moss DM, Brook Goodhew E, et al.: Development of a new platform for neglected tropical disease surveillance. Int J Parasitol. 2012; 42(9): 797–800. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nXu X, Zhang Y, Lin D, et al.: Serodiagnosis of Schistosoma japonicum infection: genome-wide identification of a protein marker, and assessment of its diagnostic validity in a field study in China. Lancet Infect Dis. 2014; 14(6): 489–97. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMeurs L, Brienen E, Mbow M, et al.: Is PCR the Next Reference Standard for the Diagnosis of Schistosoma in stool? A Comparison with Microscopy in Senegal and Kenya. PLoS Negl Trop Dis. 2015; 9(7): e0003959. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Schistosomiasis: progress report 2001–2011 and strategic plan 2012–2020. Geneva: World Health Organization; 2013. Reference Source\n\nRamaiah KD, Ottesen EA: Progress and impact of 13 years of the global programme to eliminate lymphatic filariasis on reducing the burden of filarial disease. PLoS Negl Trop Dis. 2014; 8(11): e3319. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEvans DS, Alphonsus K, Umaru J, et al.: Status of Onchocerciasis transmission after more than a decade of mass drug administration for onchocerciasis and lymphatic filariasis elimination in central Nigeria: challenges in coordinating the stop MDA decision. PLoS Negl Trop Dis. 2014; 8(9): e3113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnopp S, Stothard JR, Rollinson D, et al.: From morbidity control to transmission control: time to change tactics against helminths on Unguja Island, Zanzibar. Acta Trop. 2013; 128(2): 412–22. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNjenga SM, Mutungi FM, Wamae CN, et al.: Once a year school-based deworming with praziquantel and albendazole combination may not be adequate for control of urogenital schistosomiasis and hookworm infection in Matuga District, Kwale County, Kenya. Parasit Vectors. 2014; 7: 74. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRoss AG, Olveda RM, Chy D, et al.: Can mass drug administration lead to the sustainable control of schistosomiasis? J Infect Dis. 2015; 211(2): 283–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCarvalho-Queiroz C, Nyakundi R, Ogongo P, et al.: Protective Potential of Antioxidant Enzymes as Vaccines for Schistosomiasis in a Non-Human Primate Model. Front Immunol. 2015; 6: 273. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nYou H, Gobert GN, Cai P, et al.: Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential. PLoS Negl Trop Dis. 2015; 9(5): e0003730. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLi XH, de Castro-Borges W, Parker-Manuel S, et al.: The schistosome oesophageal gland: initiator of blood processing. PLoS Negl Trop Dis. 2013; 7(7): e2337. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nEvan Secor W: Water-based interventions for schistosomiasis control. Pathog Glob Health. 2014; 108(5): 246–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrimes JE, Croll D, Harrison WE, et al.: The roles of water, sanitation and hygiene in reducing schistosomiasis: a review. Parasit Vectors. 2015; 8: 156. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSavaya Alkalay A, Rosen O, Sokolow SH, et al.: The prawn Macrobrachium vollenhovenii in the Senegal River basin: towards sustainable restocking of all-male populations for biological control of schistosomiasis. PLoS Negl Trop Dis. 2014; 8(8): e3060. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSokolow SH, Huttinger E, Jouanard N, et al.: Reduced transmission of human schistosomiasis after restoration of a native river prawn that preys on the snail intermediate host. Proc Natl Acad Sci U S A. 2015; 112(31): 9650–5. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nEngels D, Chitsulo L, Montresor A, et al.: The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Trop. 2002; 82(2): 139–46. PubMed Abstract | Publisher Full Text\n\nKing CH: It's time to dispel the myth of \"asymptomatic\" schistosomiasis. PLoS Negl Trop Dis. 2015; 9(2): e0003504. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMwinzi PN, Muchiri G, Wiegand RE, et al.: Predictive Value of School-Aged Children's Schistosomiasis Prevalence and Egg Intensity for Other Age Groups in Western Kenya. Am J Trop Med Hyg. 2015; pii: 15–0467. PubMed Abstract | Publisher Full Text\n\nJothikumar N, Mull BJ, Brant SV, et al.: Real-time PCR and sequencing assays for rapid detection and identification of avian schistosomes in environmental samples. Appl Environ Microbiol. 2015; 81(12): 4207–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMwinzi PN, Kittur N, Ochola E, et al.: Additional Evaluation of the Point-of-Contact Circulating Cathodic Antigen Assay for Schistosoma mansoni Infection. Front Public Health. 2015; 3: 48. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLamberton PH, Kabatereine NB, Oguttu DW, et al.: Sensitivity and specificity of multiple Kato-Katz thick smears and a circulating cathodic antigen test for Schistosoma mansoni diagnosis pre- and post-repeated-praziquantel treatment. PLoS Negl Trop Dis. 2014; 8(9): e3139. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKing CH, Olbrych SK, Soon M, et al.: Utility of repeated praziquantel dosing in the treatment of schistosomiasis in high-risk communities in Africa: a systematic review. PLoS Negl Trop Dis. 2011; 5(9): e1321. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nCioli D, Pica-Mattoccia L, Basso A, et al.: Schistosomiasis control: praziquantel forever? Mol Biochem Parasitol. 2014; 195(1): 23–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLee EF, Fairlie WD: Repurposing apoptosis-inducing cancer drugs to treat schistosomiasis. Future Med Chem. 2015; 7(6): 707–11. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRamamoorthi R, Graef KM, Dent J: Repurposing pharma assets: an accelerated mechanism for strengthening the schistosomiasis drug development pipeline. Future Med Chem. 2015; 7(6): 727–35. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGelmedin V, Dissous C, Grevelding CG: Re-positioning protein-kinase inhibitors against schistosomiasis. Future Med Chem. 2015; 7(6): 737–52. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "10945",
"date": "28 Oct 2015",
"name": "Don McManus",
"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",
"responses": []
},
{
"id": "10946",
"date": "28 Oct 2015",
"name": "Alan Fenwick",
"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",
"responses": []
},
{
"id": "10947",
"date": "28 Oct 2015",
"name": "David Rollinson",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1157
|
https://f1000research.com/articles/4-1146/v1
|
27 Oct 15
|
{
"type": "Review",
"title": "Bugs, genes, fatty acids, and serotonin: Unraveling inflammatory bowel disease?",
"authors": [
"Jonathan D. Kaunitz",
"Piyush Nayyar",
"Piyush Nayyar"
],
"abstract": "The annual incidence of the inflammatory bowel diseases (IBDs) ulcerative colitis and Crohn’s disease has increased at an alarming rate. Although the specific pathophysiology underlying IBD continues to be elusive, it is hypothesized that IBD results from an aberrant and persistent immune response directed against microbes or their products in the gut, facilitated by the genetic susceptibility of the host and intrinsic alterations in mucosal barrier function. In this review, we will describe advances in the understanding of how the interaction of host genetics and the intestinal microbiome contribute to the pathogenesis of IBD, with a focus on bacterial metabolites such as short chain fatty acids (SCFAs) as possible key signaling molecules. In particular, we will describe alterations of the intestinal microbiota in IBD, focusing on how genetic loci affect the gut microbial phylogenetic distribution and the production of their major microbial metabolic product, SCFAs. We then describe how enteroendocrine cells and myenteric nerves express SCFA receptors that integrate networks such as the cholinergic and serotonergic neural systems and the glucagon-like peptide hormonal pathway, to modulate gut inflammation, permeability, and growth as part of an integrated model of IBD pathogenesis. Through this integrative approach, we hope that novel hypotheses will emerge that will be tested in reductionist, hypothesis-driven studies in order to examine the interrelationship of these systems in the hope of better understanding IBD pathogenesis and to inform novel therapies.",
"keywords": [
"Inflammatory bowel disease",
"ulcertative colitis",
"crohn's diseas",
"IBD"
],
"content": "Introduction\n\nInflammatory bowel disease (IBD) is a term encompassing two major types of disorders—ulcerative colitis and Crohn’s disease—that are characterized by chronic relapsing intestinal inflammation1. The incidence and prevalence of IBD has increased globally over the past few decades: in a systematic review of population-based IBD data, the average annual incidence rate was reported as 1.2–23.3% for Crohn’s disease and 2.4–18.1% for ulcerative colitis from 1920–20102. Recent estimates of the total annual financial burden (including direct and indirect costs) of IBD in the US are $14.6–$31.6 billion3–6. Although newer therapies that have improved quality-of-life for a subset of patients have emerged in recent years, the underlying causes of and preventative measures against IBD remain unknown.\n\nMajor scientific advances over the last decade in the fields of genetics, immunology, and microbiology have increased our understanding of the underlying pathways involved in IBD. Although the specific pathophysiology continues to be elusive, it is hypothesized that IBD results from an aberrant and persistent immune response directed against microbes or their products in the gut, facilitated by the genetic susceptibility of the host and intrinsic alterations in mucosal barrier function. Figure 1 depicts the historical trends of increasing IBD-related research articles that focus on genetics and gut microbiome since the year 20007.\n\nThe graph depicts increasing IBD-related research articles that focus on genetics and gut microbiome. Adapted from 7.\n\nWe will describe advances in the understanding of how the interaction of host genetics and the intestinal microbiome contribute to the pathogenesis of IBD, with a focus on bacterial metabolites such as short chain fatty acids (SCFAs) as possible key signaling molecules.\n\n\nGenetics\n\nThe argument for a genetic predisposition to IBD begins with the observation that family members of affected persons have a greatly increased risk for developing IBD, with a relative risk 8–10 times higher amongst first-degree relatives of IBD patients compared with the general population8,9. Subsequent epidemiological data, which include differences in prevalence amongst different ethnic groups, familial aggregation of IBD, concordance in twins, and association with genetic syndromes, further confirmed the influence of genetics in IBD8–13. These instrumental early studies preceded the era of modern IBD genetic research with the discovery of the nucleotide-binding oligomerization domain containing 2 (NOD2) gene in 2001, the first susceptibility gene discovered for Crohn’s disease14–16. In an analysis of 75,000 IBD cases and controls, including data from 15 different genome-wide association scans (GWAS) for ulcerative colitis and Crohn’s disease, the International IBD Genetics Consortium (IIBDGC) identified 71 new causative regions, increasing the total number of independent IBD risk loci to 163: 110 associated with both diseases, 30 classified as Crohn’s disease-specific, and 23 as ulcerative colitis-specific. The notable overlap of genetic loci suggests that Crohn’s disease and ulcerative colitis share many biological mechanisms: 43 of the 53 disease specific loci have the same direction of effect in both diseases, suggesting concordance for many of the biological mechanisms implicated in both diseases. Insight into biological differences is supported by the observation that two risk loci for Crohn’s disease, NOD2 and PTPN22, are protective for ulcerative colitis17–19. These strategies have identified several important signaling pathways that have consistently been associated with susceptibility to IBD. Figure 2 depicts genetic loci associated with IBD, grouped by disease specificity and involved pathways19.\n\nIBD loci are represented by lead gene name and grouped by disease specificity and involved pathways. Loci associated with inflammatory bowel disease are shown in black, Crohn’s disease (CD) in blue, ulcerative colitis (UC) in green and both UC and CD in black. Adapted from 19.\n\nSome of these pathways highlight the interaction between the host, the microbiome, and their products. Genetic analysis has highlighted the importance of autophagy in immune responses in IBD. Autophagy, involved in intracellular homeostasis, facilitates the degradation and recycling of cytosolic contents and organelles, and also helps resist microbial infection by removing intracellular microbes20. In Crohn’s disease, several genes (ATG16L1, IRGM, and LRRK) regulate the autophagy pathway, including NOD2, further supporting the theory of defective microbial degradation in some patients with Crohn’s disease21–26.\n\nGenetic loci also affect innate and adaptive immunity and epithelial function. Specifically, NOD2 modulates innate and adaptive immune responses14,15. Further, adaptive immune genes that regulate the interleukin (IL)-17 and IL-23 receptor pathways are implicated in IBD risk, including genes associated with risk for ulcerative colitis and Crohn’s disease (e.g., IL23R, IL12B, STAT3, JAK2, and TYK2) and those implicated in Crohn’s disease only (e.g., IL-27, TNFSF15). A number of genes associated with epithelial barrier function are also specifically associated with only ulcerative colitis and not with Crohn’s disease (e.g., OCTN2, ECM1, CDH1, HNF4A, LAMB1, and GNA12)13,27,28,30,31. Genes that control Paneth cell biology and the endoplasmic reticulum (ER) stress/unfolded protein response are also associated with Crohn’s disease (e.g., Xbp-1; Nod2)32.\n\nDifferent compositions of gut microbiota affect epigenetic regulation of genes through microbial products such as SCFAs33. Butyrate, one type of SCFA, influences epigenetic methylation of SCFA receptors, especially the promoter region of the free fatty acid receptor 3 (FFAR3) with consequent effects on gene expression and function34. We will further discuss SCFAs and their specific pathways later in this review.\n\nDespite the above-mentioned advances, no genetic associations can be confirmed in 77% of Crohn’s disease patients and in up to 84% of ulcerative colitis patients30. Alterations in 163 distinct single genes confer only a modest effect in and of themselves, suggesting that an aggregate effect at several loci may be responsible for the IBD phenotype35. For instance, as many as 20 to 30% of patients with Crohn’s disease may have a variant NOD2, though the penetrance is not more than 5% of homozygous and roughly 0.5% in heterozygous persons31. This indicates that disease-related allelic variants of the gene may be present in a large number of persons who do not have Crohn’s disease.\n\nWhile the increasing number of susceptibility gene loci described in IBD reflects their importance, the loci discovered so far account for only 20–25% of IBD heritability30. Further, the remarkable rise of the incidence of IBD over the past few decades cannot be sufficiently explained by only genetic risk or increased diagnosis and accessibility of care2, which has opened the doors for immunological, environmental and particularly microbial-based research in this field.\n\n\nMicrobiome\n\nA microbial etiology for IBD has long been hypothesized, starting with descriptions of potential infectious agents associated with ulcerative colitis in the 19th century and Crohn’s disease in the early 20th century36,37. In the 1920s, Rettger et al. studied the effects of Bacillus acidophilus on IBD, while in the 1940s Kirsner evaluated the possible correlation between streptococci and ulcerative colitis38–40. In the late 1990s, the association between fecal microbiota and Crohn’s disease was apparent when recurrent inflammation was observed after the fecal stream was reestablished in post-operative Crohn’s disease patients41,42. Despite these associations, no specific microbe(s) were identified to be the cause of IBD.\n\nWith recent advances in bioinformatics and culture-independent methods used for bacterial identification, there has been a resurgence of interest in the 21st century in studying the phylogeny and function of the gut microbiome in IBD. One popular proposed mechanism is the development of dysbiosis, which is defined as an imbalance between protective and harmful intestinal bacteria causing disease. Figure 3 depicts proposed microbial composition changes underlying dysbiosis and associated pathways modulating gut inflammation, including regulation by T cells, SCFAs, sphingolipids and antimicrobial factors as reviewed recently by Huttenhower et al.43.\n\nThe schematic depicts consistent observations of changes in microbial composition underlying dysbiosis and associated pathways modulating gut inflammation. The lumen (yellow), mucus layer (brown), epithelium (purple brush-border-containing cells), and lamina propria (bottom purple section) are indicated. Multiple mechanisms depicted include regulation by T cells, short chain fatty acids (SCFAs), sphingolipids and antimicrobial factors. Adapted from 85.\n\nThe diversity of the intestinal microbiome is 30–50% lower in IBD subjects than in controls. In the past year, a study investigating twin pairs discordant for IBD revealed a reduction in microbial diversity in the healthy sibling, mirroring the changes in the ulcerative colitis-affected twin44. Furthermore, individuals who are steroid-responsive have a more diverse microbiota when compared to non-responders (Shannon index 338 ± 62 versus 142 ± 49; P = 0.013)45.\n\nThere is also evidence that upregulation and downregulation of the abundance of certain bacterial species correlates with disease activity. Recent studies have demonstrated a significant reduction of Faecalibacterium prausnitzii and Roseburia hominis in active ulcerative colitis patients versus control subjects. Moreover, a significant inverse correlation between disease activity and the abundance of R. hominis and F. prausnitzii is present even in quiescent ulcerative colitis46,47. F. prausnitzii is widely regarded as one of the main fecal bacterial groups involved in colonic saccharolytic fermentation which produces SCFAs, in particular, butyrate48.\n\nFurther validation of the protective function of some microbial genera of the microbiome in acute and chronic colitis was confirmed by the improvement of inflammatory markers after intragastric administration of F. prausnitzii. In their mouse studies, Sokol et al. reported the protective effect of F. prausnitzii in a trinitrobenzene sulfonic acid (TNBS)-induced acute colitis model and, more recently, in a model of dinitrobenzene sulfonic acid (DNBS)-induced chronic colitis, in which a reduction of inflammatory markers, such as myeloperoxidase (MPO) and pro-inflammatory colonic cytokines (IL-6, IL-9, TNF-α, IFN-α), was reported, indicating a decreased severity of inflammation associated with an alteration of the microbiome49,50. The findings were particularly notable in that further analysis indicated that butyrate was not implicated in this protective effect, presumably due to the limitations of the TNBS colitis model employed, but nonetheless suggesting other protective mechanisms are present. Figure 4 briefly summarizes the numerous proposed anti-inflammatory mechanisms mediated by F. prausnitzii, either by its metabolites or by direct contact with the mucosa47. These pathways, ranging from production of anti-inflammatory matrix components to SCFAs to regulation of the immune system, to activation of the inflammatory cascade and the enteric nervous system, represent the complexities inherent in elucidating the biological mechanisms relating the microbiome to IBD pathogenesis47,51–53.\n\n1. The supernatant of F. prausnitzii blocks NF-κB activation induced by inflammation. 2. Butyrate produced by F. prausnitzii inhibits NF-κB activation in the mucosa. 3. F. prausnitzii may interact with CD103+ dendritic cells (DCs) in the lamina propria and stimulate their migration to mesenteric lymph nodes (MLN) and the induction of Tregs. 4. M cell transcytosis of F. prausnitzii in organized lymphoid structures may induce Tregs. 5. F. prausnitzii may induce IL-10 in antigen-presenting cells to enhance the suppressive activity of Foxp3+ Tregs and block Th17 cells. Adapted from 93.\n\nA multicenter cohort study that enrolled treatment-naïve and newly diagnosed patients with Crohn’s disease reported increased abundance of Enterobacteriaceae, Pasteurellacaea, Veillonellaceae, and Fusobacteriaceae and decreased abundance of Erysipelotrichales, Bacteroidales, and Clostridiales in ileal and rectal biopsies54. The complexities underlying the interpretation of such simple microbial associations through their production of SCFA are evident in conflicting observations of increased Enterobacteriaceae and Fusobacteriacea in Crohn’s disease, both of which are implicated as the main SCFA-producing bacterial groups48.\n\nThe importance of the luminal contents and the microbiome in the foregut is also illustrated in a recent study by Said et al. reporting that dysbiosis in the oral cavity is associated with inflammatory responses in IBD patients. The salivary microbiome of patients with IBD had higher proportions of Prevotella, Bacteroidetes, and Veillonella and lower proportions of Streptococcus, Neisseria, Haemophilus, Proteobacteria, and Gemella55. Although the study reported changes in several bacterial groups that may have obscured the effect of a single group, Bacteroidetes is one of the major groups producing SCFAs48, which are thought to protect against inflammation.\n\nDysbiosis is also suggested by the observations of increased prevalence of bacteria that may be implicated in the pathogenesis of IBD. For instance, in ulcerative colitis patients, the population of sulfite-reducing bacteria such as Desulfovibrio is increased, whereas low amounts of thiosulfate sulfur transferase (TST), an enzyme responsible for hydrogen sulfide (H2S) detoxification, are present56–58. The consequent increased intestinal H2S content can impair DNA repair and inhibit SCFA oxidation and its protective properties59,60, further implicating microbial products such as SCFAs as key mediators.\n\nSome aspects of the specific changes of microbial composition triggering IBD, however, continue to be elusive: for instance, it is yet to be established whether the gut microbiome is stable or continuously changing during the course of the disease. Furthermore, the impact of diet, standard medical therapy, and other environmental factors on the gut microbiome is not well understood. Most importantly, it is as yet undetermined if microbial imbalance is a cause or a consequence of IBD development.\n\nAlthough contemporary research has focused mostly on descriptive study of the compositional changes in gut microbiota in IBD, studies of the functional impact of microbial communities in IBD will be necessary to gain further insight into disease pathogenesis. On the basis of metagenomic and metaproteomic studies, only 2% of genera changes in stool and intestinal biopsy specimens may have a much larger functional impact, affecting up to 12% of total metabolic pathways in active IBD patients compared to controls61.\n\n\nIntegrating the microbiome and the genome\n\nIsolated research on the genetic and microbial factors affecting IBD manifestations and pathogenesis over the past decades has provided valuable insights and strong associative relationships between IBD, genetics, and dysbiosis, but has been unable to provide mechanistic explanations for these associations. There have been limited studies of the co-association of complex host genetic factors with microbial composition and metabolism in IBD patients or other populations. IBD-associated genetic variants associated with alterations in the intestinal microbiome, particularly in individuals carrying polymorphisms in NOD2 and FUT2, have been reported62,63. The mechanisms of host genome-microbiome disease pathways are largely unknown.\n\nThere is mounting evidence that genetic loci across the human genome are instrumental in shaping the gut microbiome64,65. Knights et al., in a systematic analysis of the effect of 154 IBD-associated polymorphisms on microbial composition in three cohorts of patients with IBD (152 to 162 patients in each cohort) using multivariate linear models, reported that 49/154 IBD-associated genes significantly and concordantly affected microbial taxa in at least two of the cohorts, implicating the innate immune response, the inflammatory response, and the JAK-STAT cascade64. In a separate analysis, the NOD2 risk allele count also influenced the overall microbial composition and abundance of Enterobacteriaceae. These data not only support an intricate link between host genetics and microbial dysbiosis in IBD, but also illustrate the ability to uncover novel associations from paired genome-microbiome data, opening the possibility that an unexpected number of genetic factors act directly on microbial composition, modulating immune pathways and metabolic phenotypes in host physiology and disease. Further studies are necessary to understand if these variants contribute to disease phenotype through their direct influence on microbiome selection, which in turn can affect disease pathophysiology either through elaboration of metabolic products or through direct mucosal interaction. These studies may involve investigating whether genetic polymorphisms concordantly affect microbial composition in healthy individuals in addition to those with IBD.\n\n\nBiological reductionism\n\nThe systems-based studies of genome and microbiome in the pathogenesis of IBD have only yielded associations without a causal mechanism. Traditional reductionist experimentation is necessary to validate associations in system-based approaches. With the advent of new statistical methods, computing and technological advances termed “systems biology”, this approach has become dominant. Since complex models with emergent properties are arguably difficult to explain with a reductionist approach, the systems approach looks broadly for correlations in comprehensive data sets, building models based on these correlations. The statistical approaches used to “mine” systems-based data sets are tools from which hypotheses can be developed. These hypotheses should then be tested in specific (and often reductionist) experiments. Thus, experimental verification of the systems-based approaches will be important to establish if the statistical approaches employed in data analysis are robust. It is therefore the marriage of systems-based approaches with traditional reductionist experimentation that will be needed to advance the field.\n\n\nSCFAs: pathway to IBD\n\nFew studies have addressed the gap between intestinal microbes and inflammatory biological pathways in the understanding of IBD pathogenesis in a human host. Dysbioses in IBD are not simply structural changes in the gut microbiota, but are instead associated with major impairments of many fundamental microbial metabolic functions with potential impact on the host. Profound disturbances have been reported in the metabolic pathways associated with gut microbiota in IBD, including major shifts in oxidative stress pathways, decreased amino acid biosynthesis, increased mucin degradation, and decreased SCFA production61.\n\nA promising route to further understanding the pathogenesis of IBD involves the investigation of the interactions of gut microbiota with the host, particularly through the bacterial fermentation products N-butyrate and other SCFAs. Not only do SCFAs provide essential nutrition for colonocytes, but they are also sensed by enteroendocrine and enterochromaffin cells in addition to possessing anti-inflammatory activity in vitro and in vivo48,66.\n\nSCFAs have been studied for decades for their effects on IBD. Although early clinical trials reported beneficial effects of SCFA enemas in ulcerative colitis patient subpopulations (e.g., distal ulcerative colitis, mild-to-moderate distal ulcerative colitis67,68), several large randomized studies reported no significant effects of exogenous SCFA treatment of ulcerative colitis patients69,70. These early trials were confounded by the now known epigenetic regulation of multiple host factors by SCFAs. Other limitations include the unknown utility of a transient rise of SCFA concentration in the distal gut achievable through enemas compared to sustained foregut elevations made possible by specific microbiome compositions of specific gut segments.\n\nSCFAs, fermented from dietary fiber resistant to mammalian digestion, are actively produced by anaerobic microbiota in the intestine and colon. The concentration of SCFA in hindgut lumen can reach 100 mM, which provides sufficient driving force for absorption by or transport into colonocytes71. SCFAs activate specific G-protein-coupled receptors (GPCRs), in particular GPR43 (FFA2), expressed by leukocytes, adipocytes and enterochromaffin (EC) cells, myenteric nerves, and GPR41 (FFA3), expressed by adipose tissue, spleen, bone marrow, lymph nodes, enteroendocrine cells, and peripheral blood mononuclear cells. GPR signaling can regulate cell activation, proliferation, and differentiation through the release of hormones or other bioactive molecules, or possibly through direct effects on enteric nerves48.\n\nAnother mechanism of SCFA action is inhibition of histone deacetylase (HDAC) activity, with subsequent modification of gene expression in human cells48,50,72. Because HDAC inhibition increases the acetylation of histone and other proteins, it can impact multiple genes and proteins. SCFAs also regulate cell metabolism through the Krebs cycle intermediates and mechanistic target of rapamycin (mTOR) activation regulating T cells72.\n\nOur laboratory has pursued experimental studies aimed at decoding how chemosensing of luminal microbial products, including SCFAs, can generate host responses. We have shown that the duodenum possesses specialized chemosensing functions that alert the distal gut to proximal conditions. The presence of SCFA in the proximal gut lumen activates mucosal defense mechanisms, including increased mucosal blood flow and mucus, bicarbonate secretion, and release of gut hormones73–77. One notable mechanism implicates the expression of SCFA receptors in luminal-facing projections of rat duodenal EC cells and enteroendocrine L-cells. FFA3 colocalises with glucagon-like peptide (GLP)-1 in enteroendocrine cells, whereas FFA2 colocalises with 5-hydroxytryptamine (serotonin; 5-HT) in EC cells. Activation of FFA2 receptor expressed on EC cells releases 5-HT and acetylcholine (ACh). These activate 5-HT4 and muscarinic receptors respectively, which are expressed on enteric nerves, afferent nerves, and epithelial cells. Activation of duodenal epithelial cells by these signals increases the rate of HCO3− secretion.\n\nThe contribution of 5-HT and ACh was confirmed subsequently when a synthetic selective FFA2 agonist dose-dependently increased HCO3− secretion, but was inhibited by atropine and a 5-HT4 antagonist. Similarly, SCFAs are thought to activate FFA2 receptors expressed on L cells, releasing GLP-2, which activates GLP-2 receptors expressed in myenteric neurons, enhancing HCO3- secretion, inhibited by GLP-2 receptor antagonists but enhanced by dipeptidyl peptidase (DPP) IV inhibition. These novel pathways have an inherent ability to locally regulate hormone release, implying that they are important in mucosal homeostasis74. Dysregulation of these pathways may contribute to intestinal inflammation through possible mechanisms detailed below, highlighting 5-HT and GLP-2 mediated effector pathways.\n\nSerotonin (5-HT) mediates many GI functions, including secretion and peristalsis, presumably through its activation of the five known gut-expressed 5-HT receptors out of the seven 5-HT receptors so far described78. Agonists and antagonists to 5-HT3 and 5-HT4 receptors are particularly well studied, with many drugs in clinical use, with utility in the management of diarrhea, constipation, and gut associated pain syndromes78. The contribution of 5-HT and its most recently discovered 5-HT7 receptors to intestinal homeostasis and inflammation is less well understood. Initial studies reported alterations in 5-HT signaling in IBD; differences in EC cell and 5-HT content have been reported with ulcerative colitis and Crohn’s disease79–85. 5-HT released from EC cells can act on proximal gut 5-HT7 receptors expressed by smooth muscle cells, enteric neurons, enterocytes, and immune cells. Activation of 5-HT7 receptors can influence muscle tone and enteric neuron excitation, inhibit serotonin transporter (SERT) activity, and modulate inflammation through dendritic cells (DCs) in the lamina propria, which are highly involved in host immune pathways.\n\nGuseva et al. investigated the enhanced expression and distribution of 5-HT7R in the intestinal tissue of IBD patients, based on an experimental model of dextran sulfate sodium (DSS)-induced colitis in which 5-HT7R expression was upregulated on CD11c/CD86 double-positive dendritic cells obtained from cecal and rectal tissue samples. The authors reported a similarly high expression of 5-HT7R in analogous dendritic cells obtained from large intestinal tissue samples of patients diagnosed with Crohn’s disease. Pharmacological blockade or genetic ablation of 5-HT7R increased the severity of acute and chronic DSS-induced colitis, whereas receptor stimulation was anti-inflammatory. These experiments supported the hypothesis that 5-HT7R expressed on CD11c/CD86-positive myeloid cells is an important component of intestinal inflammatory pathways86. Contrary to these results, Kim et al. in 2013 reported that pharmacologic blockade or genetic ablation of 5-HT7R actually alleviated intestinal inflammation in two separate chemical models of colitis (DSS and DNBS), which was confirmed histopathologically and also associated with decreased concentrations of pro-inflammatory markers, including myeloperoxidase (MPO) and cytokines IL-1β, IL-6, and TNF-α. Mice that received hematopoietic stem cells from 5-HT7 receptor-deficient donors exhibited decreased histopathological damage and disease activity87. These apparently discordant effects may be due to differences in dosing of the 5-HT7R antagonist despite substantial differences in experimental design and housing condition of the animals. In separate experiments by Kim et al. in which a lower dose (20mg/kg) of the antagonist was used, no significant differences were detected compared to control, though a higher dose (80mg/kg) significantly decreased colitis severity and inflammatory markers. Kim further acknowledged that the antagonist dosages were higher and dosing periods longer in his studies than those used by Guseva et al.85. Thus, 5-HT7R expressed on CD11c/CD86-positive myeloid cells, which modulate the severity of intestinal inflammation in experimental models of acute and chronic colitis, may serve as a potential therapeutic target for the treatment of inflammatory disorders such as Crohn’s disease.\n\nThe other hormone released through the action of SCFA, GLP-2, promotes mucosal growth, decreases barrier permeability and reduces inflammation in the intestine88. After recently being approved for treatment of short bowel syndrome, there has been a surge in interest in the non-metabolizable GLP-2R agonist teduglutide due to its multiple beneficial effects, including effects on glucose homeostasis88, and on intestinal inflammation in Crohn’s disease. Pediatric patients with acute ileal Crohn’s disease have lower postprandial GLP-2 release and higher intestinal permeability, which both reverse as the disease improves, suggesting a crucial link between the inflammatory state and GLP-2 meal-stimulated release89. In another study, GLP-2 treatment was associated with a significantly reduced neutrophil infiltration and microscopic colitis scores in the TNBS model of colitis in mice. They also reported that GLP-2 contributed to protecting the enteric nervous system under basal conditions or inflamed states90. GLP-2 is not only trophic for the intestine but also has other salutatory effects. The GLP-2R, expressed by pericryptal myofibroblasts, releases growth factors in response to GLP-2R activation91. GLP-2 decreases gut inflammation by downregulation of Th1 cytokines cells via an IL10-independent pathway, altering the mucosal response of inflamed intestinal epithelial cells and macrophages92. GLP-2 also may activate enteric nerves, since the GLP-2R is localized to myenteric neurons in addition to the myofibroblasts, but not to the intestinal epithelium93.\n\nThe contribution of specific gut microbiota is evident in studies that demonstrate their influence on 5-HT biosynthesis and on the increase in endogenous GLP-2 production—both of which are implicated in modulation of gut inflammation—although the signaling pathways are yet to be fully understood94–96. SCFAs may represent the biological mediators of these findings as well. SCFAs that promoted Tph1 (tryptophan hydroxylase 1) transcription in BON cells (human EC cell model) were the key link between gut microbiota regulating enteric 5-HT production and homeostasis in a recent study94. Figure 5 outlines the proposed mechanisms through which SCFAs regulate gut inflammation.\n\nSCFAs are present in the diet and actively produced by gut microbiota as fermentation products of dietary materials. SCFAs exert their effects directly on epithelial cells, antigen-presenting cells, and T cells. Multiple mechanisms depicted above include metabolic regulation, HDAC inhibition, and GPR activation by SCFAs releasing mediators including 5-HT and GLP-2 hypothesized to modulate gut inflammation.\n\n\nOther pathways\n\nThere are many other pathways being investigated using the reductionist approach to help integrate genetic, microbial, and biochemical pathways. In a breakthrough study of IBD- protective single nucleotide polymorphisms (SNPs) in the MAP3K8 gene, Roulis et al. reported that MAP3K8 encodes tumor progression locus-2 (Tpl2) kinase in intestinal myofibroblasts, in addition to promoting arachidonic acid metabolism and COX-2/PGE-2 activation, which are important in the compensatory proliferative response of the intestinal epithelium to injury97.\n\nMice with complete knockout of Tpl2, and conditional knockout of Tpl2 targeted to intestinal myofibroblasts, were highly susceptible to DSS-induced colitis, with greater tissue damage when compared to wild-type mice despite similar DSS-induced levels of inflammation. Tpl2 expression was downregulated in intestinal myofibroblasts isolated from the inflamed ileum of nine patients with IBD.\n\n\nConclusions\n\nCurrently, the data acquisition rate in traditional IBD research has been far outpaced by the massive data generated by bioinformatics. Despite the considerable ongoing efforts of investigators across the globe, reductionist studies are still needed to help explain the basic causal mechanisms that underlie the exponentially increasing number of correlations detected through systems-based approaches in IBD. Further integration of the study of host genetics and the gut microbiome in the setting of clinical metadata has already reduced the number of confounding variables and has elicited new associations. Efforts to integrate complicated genetics and the gut microbiome in further reductionist experiments to determine causal associations may be best served by an emphasis on bacterial metabolic products, such as SCFAs, as the logical mediators of the interactions between genetic variables and the microbiota as well as key molecules of the biological etiopathogenic pathway to gut inflammation. Further reductionist studies exploring the interactions between diet, microbiome, SCFAs and serotonin can help guide our understanding of the pathogenesis of colitis.",
"appendix": "Author contributions\n\n\n\nDr. Nayyar performed the initial literature search, composed Figure 4 and wrote the draft. Dr. Kaunitz originated the underlying concept and drafted the overall organization, revised and edited the draft, prepared the final version, and submitted the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no disclosures.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbraham C, Cho JH: Inflammatory bowel disease. N Engl J Med. 2009; 361(21): 2066–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMolodecky NA, Soon IS, Rabi DM, et al.: Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012; 142(1): 46–54. e42; quiz e30. PubMed Abstract | Publisher Full Text\n\nThe Facts about Inflammatory Bowel Diseases. Crohn’s & Colitis Foundation of America, 2014. 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Genome Biol. 2012; 13(9): R79. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLi E, Hamm CM, Gulati AS, et al.: Inflammatory bowel diseases phenotype, C. difficile and NOD2 genotype are associated with shifts in human ileum associated microbial composition. PLoS One. 2012; 7(6): e26284. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTong M, McHardy I, Ruegger P, et al.: Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism. ISME J. 2014; 8(11): 2193–206. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKnights D, Silverberg MS, Weersma RK, et al.: Complex host genetics influence the microbiome in inflammatory bowel disease. Genome Med. 2014; 6(12): 107. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGoodrich JK, Waters JL, Poole AC, et al.: Human genetics shape the gut microbiome. Cell. 2014; 159(4): 789–99. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSoldavini J, Kaunitz JD: Pathobiology and potential therapeutic value of intestinal short-chain fatty acids in gut inflammation and obesity. Dig Dis Sci. 2013; 58(10): 2756–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScheppach W, Sommer H, Kirchner T, et al.: Effect of butyrate enemas on the colonic mucosa in distal ulcerative colitis. Gastroenterology. 1992; 103(1): 51–6. PubMed Abstract\n\nVernia P, Marcheggiano A, Caprilli R, et al.: Short-chain fatty acid topical treatment in distal ulcerative colitis. Aliment Pharmacol Ther. 1995; 9(3): 309–13. PubMed Abstract | Publisher Full Text\n\nSteinhart AH, Hiruki T, Brzezinski A, et al.: Treatment of left-sided ulcerative colitis with butyrate enemas: a controlled trial. Aliment Pharmacol Ther. 1996; 10(5): 729–36. PubMed Abstract | Publisher Full Text\n\nBreuer RI, Soergel KH, Lashner BA, et al.: Short chain fatty acid rectal irrigation for left-sided ulcerative colitis: a randomised, placebo controlled trial. Gut. 1997; 40(4): 485–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRechkemmer G, Rönnau K, von Engelhardt W: Fermentation of polysaccharides and absorption of short chain fatty acids in the mammalian hindgut. Comp Biochem Physiol A Comp Physiol. 1988; 90(4): 563–8. PubMed Abstract | Publisher Full Text\n\nKim CH, Park J, Kim M: Gut microbiota-derived short-chain Fatty acids, T cells, and inflammation. Immune Netw. 2014; 14(6): 277–88. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAkiba Y, Kaunitz JD: Duodenal luminal chemosensing; acid, ATP, and nutrients. Curr Pharm Des. 2014; 20(16): 2760–5. PubMed Abstract | Publisher Full Text\n\nAkiba Y, Inoue T, Kaji I, et al.: Short-chain fatty acid sensing in rat duodenum. J Physiol. 2015; 593(3): 585–99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAkiba Y, Watanabe C, Mizumori M, et al.: Luminal L-glutamate enhances duodenal mucosal defense mechanisms via multiple glutamate receptors in rats. Am J Physiol Gastrointest Liver Physiol. 2009; 297(4): G781–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInoue T, Wang JH, Higashiyama M, et al.: Dipeptidyl peptidase IV inhibition potentiates amino acid- and bile acid-induced bicarbonate secretion in rat duodenum. Am J Physiol Gastrointest Liver Physiol. 2012; 303(7): G810–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang JH, Inoue T, Higashiyama M, et al.: Umami receptor activation increases duodenal bicarbonate secretion via glucagon-like peptide-2 release in rats. J Pharmacol Exp Ther. 2011; 339(2): 464–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMawe GM, Hoffman JM: Serotonin signalling in the gut--functions, dysfunctions and therapeutic targets. Nat Rev Gastroenterol Hepatol. 2013; 10(8): 473–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhonen A, Kyösola K, Penttilä O: Enterochromaffin cells in macrophages in ulcerative colitis and irritable colon. Ann Clin Res. 1976; 8(1): 1–7. PubMed Abstract\n\nBelai A, Boulos PB, Robson T, et al.: Neurochemical coding in the small intestine of patients with Crohn's disease. Gut. 1997; 40(6): 767–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEl-Salhy M, Danielsson A, Stenling R, et al.: Colonic endocrine cells in inflammatory bowel disease. J Intern Med. 1997; 242(5): 413–9. PubMed Abstract | Publisher Full Text\n\nMagro F, Vieira-Coelho MA, Fraga S, et al.: Impaired synthesis or cellular storage of norepinephrine, dopamine, and 5-hydroxytryptamine in human inflammatory bowel disease. Dig Dis Sci. 2002; 47(1): 216–24. PubMed Abstract | Publisher Full Text\n\nCoates MD, Mahoney CR, Linden DR, et al.: Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology. 2004; 126(7): 1657–64. PubMed Abstract | Publisher Full Text\n\nBishop AE, Pietroletti R, Taat CW, et al.: Increased populations of endocrine cells in Crohn's ileitis. Virchows Arch A Pathol Anat Histopathol. 1987; 410(5): 391–6. PubMed Abstract | Publisher Full Text\n\nKim JJ, Khan WI: 5-HT7 receptor signaling: improved therapeutic strategy in gut disorders. Front Behav Neurosci. 2014; 8: 396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuseva D, Holst K, Kaune B, et al.: Serotonin 5-HT7 receptor is critically involved in acute and chronic inflammation of the gastrointestinal tract. Inflamm Bowel Dis. 2014; 20(9): 1516–29. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKim JJ, Bridle BW, Ghia JE, et al.: Targeted inhibition of serotonin type 7 (5-HT7) receptor function modulates immune responses and reduces the severity of intestinal inflammation. J Immunol. 2013; 190(9): 4795–804. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBaldassano S, Amato A: GLP-2: what do we know? What are we going to discover? Regul Pept. 2014; 194–195: 6–10. PubMed Abstract | Publisher Full Text\n\nSigalet DL, Kravarusic D, Butzner D, et al.: A pilot study examining the relationship among Crohn disease activity, glucagon-like peptide-2 signalling and intestinal function in pediatric patients. Can J Gastroenterol. 2013; 27(10): 587–92. PubMed Abstract | Free Full Text | Faculty Opinions Recommendation\n\nSigalet DL, Wallace L, De Heuval E, et al.: The effects of glucagon-like peptide 2 on enteric neurons in intestinal inflammation. Neurogastroenterol Motil. 2010; 22(12): 1318–e350. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nEl-Jamal N, Erdual E, Neunlist M, et al.: Glugacon-like peptide-2: broad receptor expression, limited therapeutic effect on intestinal inflammation and novel role in liver regeneration. Am J Physiol Gastrointest Liver Physiol. 2014; 307(3): G274–85. PubMed Abstract | Publisher Full Text\n\nIvory CP, Wallace LE, McCafferty DM, et al.: Interleukin-10-independent anti-inflammatory actions of glucagon-like peptide 2. Am J Physiol Gastrointest Liver Physiol. 2008; 295(6): G1202–10. PubMed Abstract | Publisher Full Text\n\nPedersen J, Pedersen NB, Brix SW, et al.: The glucagon-like peptide 2 receptor is expressed in enteric neurons and not in the epithelium of the intestine. Peptides. 2015; 67: 20–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nReigstad CS, Salmonson CE, Rainey JF 3rd, et al.: Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells. FASEB J. 2015; 29(4): 1395–403. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYano JM, Yu K, Donaldson GP, et al.: Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015; 161(2): 264–76. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCani PD, Possemiers S, Van de Wiele T, et al.: Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability. Gut. 2009; 58(8): 1091–103. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRoulis M, Nikolaou C, Kotsaki E, et al.: Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis. Proc Natl Acad Sci U S A. 2014; 111(43): E4658–67. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "10942",
"date": "27 Oct 2015",
"name": "Enzo Ierardi",
"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",
"responses": []
},
{
"id": "10943",
"date": "27 Oct 2015",
"name": "Rebeca Martin",
"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",
"responses": []
},
{
"id": "10944",
"date": "27 Oct 2015",
"name": "Waliul I Khan",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1146
|
https://f1000research.com/articles/4-153/v1
|
16 Jun 15
|
{
"type": "Review",
"title": "Non-invasive imaging to monitor lupus nephritis and neuropsychiatric systemic lupus erythematosus",
"authors": [
"Joshua M. Thurman",
"Natalie J. Serkova",
"Natalie J. Serkova"
],
"abstract": "Systemic lupus erythematosus (SLE) is an autoimmune disease that can affect multiple different organs, including the kidneys and central nervous system (CNS). Conventional radiological examinations in SLE patients include volumetric/ anatomical computed tomography (CT), magnetic resonance imaging (MRI) and ultrasound (US). The utility of these modalities is limited, however, due to the complexity of the disease. Furthermore, CT and MRI contrast agents are contraindicated in patients with renal impairment. Various radiologic methods are currently being developed to improve disease characterization in patients with SLE beyond simple anatomical endpoints. Physiological non-contrast MRI protocols have been developed to assess tissue oxygenation, glomerular filtration, renal perfusion, interstitial diffusion, and inflammation-driven fibrosis in lupus nephritis (LN) patients. For neurological symptoms, vessel size imaging (VSI, an MRI approach utilizing T2-relaxing iron oxide nanoparticles) has shown promise as a diagnostic tool. Molecular imaging probes (mostly for MRI and nuclear medicine imaging) have also been developed for diagnosing SLE with high sensitivity, and for monitoring disease activity. This paper reviews the challenges in evaluating disease activity in patients with LN and neuropsychiatric systemic lupus erythematosus (NPSLE). We describe novel MRI and positron-emission tomography (PET) molecular imaging protocols using targeted iron oxide nanoparticles and radioactive ligands, respectively, for detection of SLE-associated inflammation.",
"keywords": [
"Systemic lupus erythematosus",
"imaging",
"kidney",
"brain"
],
"content": "Introduction\n\nSystemic lupus erythematosus (SLE) is an autoimmune disease that can affect any organ throughout the body1. SLE is associated with a loss of immunologic tolerance to multiple nuclear antigens and the production of autoantibodies specific for these self-antigens. The treatment of SLE almost always employs immunomodulatory therapies that suppress this autoimmune response. Immunosuppressive drugs, such as cyclophosphamide and mycophenolate mofetil (MMF), reduce tissue inflammation and injury, and the mortality for patients with SLE has improved in recent decades2,3.\n\nSLE is a lifelong disease marked by flares and remissions. Aggressive and prolonged immunosuppression reduces - but does not eliminate - the risk of future flares. Consequently, even patients who have remained in remission for prolonged periods should continue to be monitored periodically for evidence of a disease flare. Active SLE may be clinically apparent, and several serologic tests are also helpful for monitoring disease. However, the definitive diagnosis of activity within a specific tissue requires a tissue biopsy. Unfortunately, biopsies sample only a small portion of a given tissue. In general, the implementation of repetitive biopsies in a clinical setting of immunosuppressive treatment trials remains low. Furthermore, it may not be feasible to biopsy lesions in some organs, such as the brain and spinal cord.\n\nRadiologic assessment and imaging end-points currently have only a limited role in monitoring disease in patients with SLE. Computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US) are frequently used to assess end organ damage in patients with specific manifestations4–6. A major limitation to the use of these studies, however, is that MRI and CT contrast-agents (iodine and gadolinium based, respectively) are contraindicated in patients with renal impairment. It has become evident that for chronic inflammatory and autoimmune diseases, radiologists need tools that go beyond the standard anatomical imaging protocols. Generally, 2-deoxy-2-[18F]fluoro-d-glucose (FDG) is considered an excellent PET tracer for most inflammatory pathologies (including osteomyelitis, inflammatory bowel disease, atherosclerotic plaques) since activated granulocytes and monocytes have elevated glucose metabolism. However, antibodies can deposit in tissues prior to infiltration with granulocytes, causing inflammatory tissue injury without high 18FDG uptake7. Hence, physiological and molecular imaging methods are being developed to detect organ dysfunctional and locate specific molecular markers in affected tissues in autoimmune diseases8–11. These methods could potentially allow clinicians to non-invasively monitor lupus disease activity.\n\n\nThe unpredictable course of SLE\n\nOne of the hallmarks of lupus is that the manifestations vary between patients, and an individual patient’s disease will often vary over time. Because immunosuppressive drugs carry the risk of infection and other toxicities, the choice of treatment depends upon a patient’s specific manifestations. LN and NPSLE are two of the most severe manifestations of lupus3,12–14. Consequently, patients who present with NPSLE or LN are frequently treated with potent immunosuppressive agents, and immunosuppression is usually continued for prolonged periods15,16.\n\nLupus nephritis (LN). More than 50% of patients with SLE develop renal involvement during their lives17. SLE patients with LN have a higher mortality than those without renal involvement18,19, but the prognosis among patients with LN varies widely20. Several histologic findings predict those patients whose disease is most likely to progress to renal failure17, and this has led to the development of histologic scoring systems. The World Health Organization (WHO) classification system was published in 1982, and was revised by the International Society of Nephrology and the Renal Pathology Society (ISN/RPS) in 200421. The prognostic value of these systems has been validated, and the ISN/RPS system is now widely employed21,22. Several clinical and laboratory findings are also of prognostic importance (such as hypertension, an elevated serum creatinine, and a low serum C3 level)20, but a renal biopsy is still considered essential for deciding whether a patient requires treatment23.\n\nThe standard treatment for proliferative LN involves three to six months of induction therapy with either MMF or cyclophosphamide24–27. Maintenance therapy can last for years, and the optimal duration of maintenance therapy is unknown. The response to immunosuppression is quite variable, with only ~50% of patients responding to treatment in some large trials24,25,27. Unfortunately, most patients are treated with a “one size fits all” approach. Patients are treated according to the protocols used in the large trials, and clinicians can only determine whether a given patient will respond to treatment after months of therapy. Thus, new methods to select patients for treatment and to monitor the response to treatment are greatly needed.\n\nNeuropsychiatric systemic lupus erythematosus (NPLSE). Up to two-thirds of patients with SLE may have some form of neurologic or psychiatric manifestations of their disease15,28–30. Involvement of the central nervous system (CNS) can be caused by direct immune-mediated injury of tissues, systemic inflammatory mediators, vascular disease, and/or thromboembolic insults. Clinically, NPSLE has a broad range of presentations, including headaches, mood disorders, cognitive dysfunction, cerebrovascular accidents, transverse myelitis, and neuropathy15. As with LN, these symptoms and findings can change over time31.\n\nGiven the non-specific nature of these neurologic manifestations, it can be difficult to determine whether findings suggestive of NPSLE are caused by autoimmune mechanisms, side effects from medications, infectious complications of the disease, medications used to treat the disease, or are incidental. Obviously the CNS is less accessible for biopsy than the kidney. Consequently, the underlying pathology is less well characterized than LN. Much of the data points to vascular processes, and micro-infarcts are a common finding in autopsy series of patients with SLE32. However, cerebral vascular injury may be caused by thrombotic and/or inflammatory lesions. Patients with antiphospholipid syndrome are at increased risk of stroke33. Even in patients without detectable CNS lesions, antiphospholipid antibodies may be associated with cognitive abnormalities34. Immunoglobulin G (IgG) and C3 deposits are detected in the brains of mice with lupus-like disease35. Anti-neuronal autoantibodies have been detected in cerebrospinal fluid, and have also been detected in in brain tissue at autopsy36. Overall, however, the clinical-pathologic correlation of specific CNS lesions with different clinical syndromes is unknown.\n\nGiven the wide variety of etiologies and manifestations of NPSLE, it is likely that the optimal treatment of different patients requires different approaches. The treatment of anti-phospholipid antibody syndrome, for example, primarily involves anti-coagulation. In some patients, NPSLE is likely caused by autoimmune or inflammatory factors and might effectively be treated by immunomodulatory drugs, and several case reports support this approach37–40. For patients with mild cognitive impairment, a small trial reported that prednisone may be beneficial41. A randomized trial of 32 patients with severe NPSLE manifestations (e.g. optic neuritis, transverse myelitis, or coma) reported that cyclophosphamide was superior to methylprednisolone42, a result similar to what is found in severe LN43. Rituximab has also been effective in patients with severe NPSLE44,45. Nevertheless, little is known about which patients should be treated, what the most effective treatment is, and the optimal duration of therapy. Specific biomarkers of NPSLE would therefore make it much easier to conduct clinical trials and to identify specific patients who are likely to benefit from immunomodulatory treatment.\n\n\nThe need for better biomarkers of disease activity in LN and NPSLE\n\nBecause the intensity and duration of treatment is different in patients with LN and/or NPSLE than in SLE patients who do not have renal or neurologic involvement, it is important to accurately detect involvement of these organs. The primary utility of biomarkers in this setting is to distinguish: i) patients with mild disease who do not need treatment, ii) patients with active disease who do need treatment, and iii) patients who have developed irreversible organ injury and who will not benefit from immunomodulatory treatment.\n\nClinically, renal involvement is detected by elevations in the urine protein excretion, erythrocytes or white blood cells in the urine, or an elevation in serum creatinine levels. Objective activity indices have also been created that incorporate these findings in order to objectively monitor patients’ renal disease and response to therapy, but these tools are more useful for clinical studies and do not replace the clinician’s assessment46. As outlined above, the diagnosis of neurologic involvement is usually made on clinical grounds. The American College of Rheumatology has created classification criteria for neuropsychiatric syndromes47. These definitions do not distinguish whether SLE is the underlying cause of the findings, however, and are not designed to measure disease severity.\n\nThe reactivity of particular autoantibodies has been associated with the involvement of particular organs. Unfortunately, the serologic biomarkers that are currently in use do not specifically report on lupus activity within the kidneys or CNS. Anti-double-stranded DNA antibodies48, anti-ribosomal P antibodies49, anti-chromatin antibodies50,51, anti-nucleosome antibodies52, and decreases in C3 and C4 levels have all been associated with LN53. Overall, however, the absolute levels of these markers and changes in their levels do not accurately predict a disease flare or a response to therapy. Anti-C1q antibodies also associate with LN54. They have a high negative predictive value for active disease but they have a poor positive predictive value54. Anti-ribosomal P antibodies55, anti-glial fibrillary acidic protein antibodies56, anti-N-methyl-D:-aspartate antibodies, and anti-microtubule-associated protein 2 antibodies57,58 may be associated with NPSLE. C3 and C4 levels are increased in the CSF of patients with NPSLE59. Similar to the case with LN, however, the accuracy of these biomarkers for diagnosing and monitoring NPSLE has been variable in clinical studies and their role in clinical care is currently very limited15,60.\n\n\nThe role of conventional radiologic imaging in LN and NPSLE\n\nRadiologic imaging (US, CT, MRI and nuclear medicine, Figure 1) has presently only a minor role in the assessment of disease activity in patients with LN or NPSLE, but possesses promising potential for future molecular assessment. CT is based on scattering and absorbing of X-ray beams while passing through the tissues; CT has a great anatomical discrimination (spatial resolution 5 mm, Figure 1) but rather a limited soft tissues contrast. It has limited application in LN patients with renal impairment since iodine-based CT contrast dyes (which are necessary due to the poor intrinsic soft tissue contrast by CT) are often contra-indicated. US sends out pulses of high-frequency sound waves and detects returning echoes scattered from the tissues. It has a very good anatomical resolution and an excellent potential for dynamic scans (Doppler). US was recently reported as a valuable platform to identify sub-clinical joint manifestations (to predict the risk of chronic deformities such as Jaccoud’s Arthropathy) in SLE patients6. US is also frequently performed to examine the kidneys of patients with renal abnormalities, and it is also routinely used to guide kidney biopsies. Conventional US can detect gross changes in the kidney size and contour. Radiologically small kidneys have likely sustained chronic, irreversible changes. Decreased blood flow by Doppler might indicate irreversible disease, and it has been postulated that inflammation can initially manifest with increased blood flow. Beyond this, however, US is not useful for detecting or staging LN.\n\nThe anatomic spatial resolution and sensitivity for different molecular imaging methods are shown. CT and MRI based methods have excellent anatomic resolution, whereas PET/CT and SPECT/CT have very high sensitivity.\n\nMRI is based on visualization of the physical properties of proton nuclei in tissue water in response to excitation by radio-frequency waves in a strong magnetic field. MRI has evolved as the method of choice for both LN and as well as NPSLE patient sub-populations. It has high spatial resolution (comparable to CT, Figure 1) combined with high intrinsic soft tissue contrast, allowing for non-contrast image protocols since gadolinium-based MRI contrast is also prohibited in patients with renal impairment. Even though standard MRI also has a limited role in the assessment of LN, it can detect kidney edema in patients with glomerulonephritis61,62. Most importantly, MRI allows for non-contrast physiology-based imaging which increasingly plays an important role for assessing renal function. Tissue oxygenation in the cortex and medulla has been assessed by blood-oxygenation-level-dependent (BOLD) MRI10. Glomerular function can be assessed by arterial spin labeling (ASL) perfusion MRI protocols. Diffusion-weighted MRI (DWI) is based on random microscopic motion of tissue water (the Brownian motion = diffusion) which provides quantitative imaging end-points (so-called apparent diffusion coefficients, ADCs). DWI helps to characterize interstitial diffusion and to some extent renal fibrosis9,10,63.\n\nMRI is also the main modality used in neuroradiology. Because NPSLE is frequently caused by vascular disease (thromboembolic, hemorrhagic, or inflammatory), radiologic imaging is frequently performed in these patients. Conventional MRI and especially DWI are sensitive for the detection of strokes and transverse myelitis64. A number of other CNS abnormalities detected by MRI have been reported in lupus patients, including subcortical focal lesions, cortical atrophy, ventricular dilation and cerebral edema64. Some MRI findings may indicate acute, reversible processes, including diffuse, high intensity lesions, as well as hyperintensity in gray matter adjacent to the lesion and brain atrophy11,15,64. Similar to the kidney, brain perfusion can be assessed by non-contrast ASL. More recently, a novel sophisticated MRI platform, called vessel size imaging (VSI, using commercially available T2-contrast agents, usually iron nanoparticles) has been used to precisely characterize brain vascularization, cerebral blood volume and vessel permeability65. Since iron oxide contrast is not associated with toxicities in patients with renal impairment, VSI holds promise for the future. VSI has been used in pre-clinical models of stroke and oncology (particularly for gliomas) and is being recently translated into clinical oncology trials; it has not yet been applied in NPSLE or LN research.\n\nNuclear medicine methods include the gamma camera, positron emission tomography (PET) and single photon emission tomography (SPECT). These modalities permit in vivo detection of free isotopes or more complex compounds labeled with radioisotopes. Because of their low spatial resolutions (in the range of 15 mm), PET and SPECT are usually performed in combination with CT for anatomical alignment. PET is the most promising technique for molecular imaging: its sensitivity to the target lies in a picomolar range for PET-based tracers as compared to the millimolar range for MRI (Figure 1). PET detects a decay of positron-emitting radionucleotides (such as 18F-, 11C-, 124I-, 64Cu-) by capturing a pair of gamma rays. The most commonly used PET tracer is 18FDG which accumulates in inflammatory cells and can be used to detect tissue inflammation. It has been used to monitor renal inflammation in a pre-clinical model66, but has not been formally tested in patients with LN. 18FDG-PET abnormalities are very common in patients with NPSLE67. These abnormalities may represent prior injury to the CNS, however, and do not distinguish active from chronic injury15, thus a more specific inflammatory probe is highly desirable. The main advantages of PET is that radiolabeled proteins and peptides can be synthetized for conferring imaging visibility of targets and their activities. It can detect these markers with high sensitivity and localize the signal to specific anatomic sites, particularly when the images are co-registered with MRI or CT images.\n\n\nThe promise of molecular imaging for monitoring LN and NPSLE\n\nThere is strong evidence that most of the MRI abnormalities described above are related to tissue inflammation, which is frequently present in SLE. Therefore, an idea of imaging the molecular features of SLE-driven inflammation represents an attractive and direct approach for visualizing “active” SLE. Molecular imaging is a fast developing radiological area and, without doubt, PET and SPECT are the two modalities with the highest potential in the area of molecular imaging. When using radioactive probes, both nuclear medicine techniques have higher sensitivity and specificity for targets than does MRI, and the scans are obtained relatively quickly. Importantly, routine radiolabeling protocols are available and/or can be designed rapidly. However, compared to MRI (which frequently uses targeted iron oxide nanoparticles), nuclear medicine based molecular imaging requires high dose radioactivity (especially for a slow kinetic probes such as 124I) and has low spatial resolution.\n\nSeveral molecular imaging probes have been developed to detect markers of tissue inflammation [reviewed in 68,69]. Pre-clinical studies have used radiolabelled antibodies against granulocytes, lymphocytes, as well as anti-TNF-alpha, anti-CD20, anti-CD2, anti-CD3, and anti-CD4 monoclonal antibodies, for both PET (124I-based) as well as SPECT imaging (123I, 99mTc, 111In)70,71. Considerable success has been reported with peptide imaging, such as radiolabelled cytokines and interleukins, as well as peptide ligands for somatostatin receptors. For the most part, these probes have not yet been tested in pre-clinical models of lupus or in lupus patients. However, many of these molecular imaging probes have the potential to detect immune proteins that deposit in affected tissues. For LN, these imaging agents and methods could enable non-invasive staging of kidney disease using these validated markers. Given the wealth of existing data regarding the deposition of immunoglobulin and complement proteins, one can infer that these molecules will likely be of diagnostic and prognostic importance. Because percutaneous renal biopsies are regularly performed, new molecular imaging probes can be compared to the biopsies in order to test the correlation of the molecular imaging method with the “gold standard” of disease staging.\n\nCurrently, the approach to patients with signs and symptoms of NPSLE is to search for underlying thromboembolic, infectious, metabolic causes, and to treat those factors15,60. Findings suggestive of antiphospholipid syndrome and/or active SLE can also inform the treatment of these patients. For NPSLE, tissue biopsies are not routine, and the decision to treat patients is based upon clinical findings. Because there is less biopsy data for comparison it is harder to foresee what molecular imaging probes that detect inflammatory markers would reveal. It is difficult to predict the extent and abundance of particular inflammatory molecules, or prognostic significance of inflammatory markers. The dearth of knowledge regarding the underlying pathology of NPSLE, however, increases the importance of developing new tools for classifying patients. It is the authors’ belief that molecular imaging methods will provide new methods for detecting and quantifying inflammation within the CNS, and could provide a means of selecting which patients to treat.\n\nComplement C3 as an imaging target. Our own molecular imaging efforts have focused on the development of probes to detect tissue-bound C3 fragments. There are several aspects of C3 that make it particularly useful as a biomarker of SLE. First, during complement activation by immune complexes, millions of C3 molecules are cleaved and covalently fixed to nearby tissues72,73. These fragments provide a durable tissue biomarker, and biopsies from patients with SLE are usually stained for C3. Interestingly, the detection of glomerular C3 within a renal biopsy is predictive of progression of renal disease74. The abundance of deposited C3 is likely to be, therefore, a sensitive and dynamic marker of inflammation. C3 is deposited in a wide range of renal diseases, however, so it is not specific for LN75.\n\nA major difficulty in developing probes to detect tissue bound C3 is that the probe must distinguish C3 fragments that are fixed to tissues from intact C3 protein in blood. During complement activation C3 undergoes conformational shifts and fragments of the protein are cleaved by proteases76. Thus, there are epitopes on the cleavage fragments that are not present on intact C3. We have developed two classes of imaging probes to detect C3. We have used a recombinant form of complement receptor-2 (CR2) to bind C3 fragments. CR2 is a complement receptor expressed on B cells and follicular dendritic cells. CR2 binds the C3d cleavage fragment of C3 with a KD of approximately 0.5 μM77. Because CR2 does not bind intact plasma C3 it can be used to target therapeutic and diagnostic agents to sites of complement activation8,78–80. We have also developed several monoclonal antibodies to C3d that do not bind to intact C3 or to C3b81. These antibodies bind C3d with a high affinity (<1 nM) and target sites of complement activation when injected systemically81.\n\nMRI-based detection of C3 deposits. To test whether tissue C3 deposits can be detected by MRI, we conjugated recombinant CR2 to the surface of superparamagnetic iron-oxide nanoparticles (SPIONs)8. Superparamagnetic iron-oxide causes rapid dephasing of nearby protons and, as a result, accelerates the spin-spin relaxation rate (R2)82. Thus, T2 relaxation times decrease producing a drop in T2-weighted signal intensity (negatively enhanced) in areas of SPION accumulation.\n\nWe injected wild-type and lupus-prone (MRL/lpr) mice with CR2-targeted SPIONs and with untargeted SPIONs. We performed T2-weighted MRI before and after injection with the nanoparticles and analyzed the signal in the kidneys. In MRL/lpr mice, the injection of CR2-targeted SPIONs caused a significant decrease in the T2 signal within the kidneys8. The T2 signal did not decrease in age-matched MRL/lpr mice injected with untargeted SPIONs, however, nor in healthy control mice injected with CR2-targeted SPIONs. These results indicate that the CR2-targeted SPIONs can be used to non-invasively detect active glomerulonephritis by T2-MRI based on tissue-bound C3-complement activation.\n\nTo determine whether this method can be used to assess disease severity, we repeated the protocol at four week intervals in MRL/lpr mice80. Kidney disease worsens as MRL/lpr mice age, and the abundance of C3 fragments in the glomeruli increases until the terminal stages of the disease80. The degree of negative enhancement in the kidneys of the mice increased between 12 and 20 weeks of age. These results suggest that MRI-based detection of glomerular C3 can be used to monitor the severity of the underlying disease, although this method still has limited sensitivity for detecting small differences in glomerular C380. A study to determine whether this method can detect the response of MRL/lpr mice to immunosuppressive treatment is currently underway.\n\nPET-based detection of C3 deposits. As outlined above, PET probes can be detected with higher sensitivity than SPIONs (Figure 1), and we have developed high-affinity anti-C3d monoclonal antibodies that accumulate at sites of complement activation after systemic injection81. Factor H deficient (fH-/-) mice develop spontaneous glomerulonephritis characterized by abundant glomerular C3 fragments83. When injected systemically into fH-/- mice, the anti-C3d antibodies bound to C3 fragments located within the glomeruli. We have also performed pilot PET experiments in which one of the anti-C3d mAbs was radiolabeled with 124I and injected into fH-/- mice, MRL/lpr mice, and control mice, and PET/CT scans were performed 4 to 144 hrs after injection with the antibody. A high degree of signal was seen in the kidneys of fH-/- mice and MRL/lpr mice after injection with the antibody (unpublished data).\n\nThese pilot experiments confirm that radiolabeled C3 probes can detect glomerular C3 fragments in mice with lupus-like glomerulonephritis. Future experiments will test the sensitivity of the method to distinguish mice with disease of varying severity.\n\n\nFuture directions\n\nThe treatment of patients with SLE requires a continual reevaluation of the risks of the disease versus the risks of immunomodulatory treatment. Ideally, clinicians employ aggressive immunosuppression (e.g. cytotoxic drugs) for treating patients with severe disease, but do not use these agents in patients with mild disease or with renal damage that cannot be salvaged (Figure 2). Currently, the assessment of lupus disease activity and prognosis is based upon a number of clinical, serologic, radiographic, and histologic findings.\n\nIdeally, aggressive immunosuppression is reserved for those with severe renal disease. Lupus nephritis is associated with the presence of serologic changes, proteinuria, hematuria, and elevated serum creatinine levels. Unfortunately, these changes are not accurate for identifying patients with severe disease that is amenable to treatment. The abundance of glomerular C3 deposits increases with disease severity but falls off in end stage disease80, raising the possibility that non-invasive detection of glomerular C3 will be useful for guiding treatment of patients with lupus nephritis.\n\nLN and NPSLE are two of the most serious manifestations of SLE, and accurate assessment is critical in patients with renal or neurologic involvement. In the case of LN, tissue biopsies provide crucial information for treatment decisions, and the patterns of disease are well characterized. Unfortunately, biopsies can be subject to sampling error, and their invasive nature limits their repeated use. Molecular imaging methods may, therefore, provide a more comprehensive picture of inflammation within the kidney and will enable serial assessments as patients are treated. In the case of NPSLE, the difficulty of obtaining tissue biopsies (let alone serial tissue biopsies) is a major barrier to the full characterization of the disease process and segmentation of patients. Molecular imaging methods will enable a clearer sense of the role of inflammation in this disease, and the establishment of clinical-pathologic correlations of CNS inflammation with the broad range of neurologic symptoms that patients develop. The first clinical applications and the FDA-approval of radiotracers for detecting neurodegeneration clearly show that PET molecular imaging is feasible. The recent development of multimodality PET/MRI scanners provides the opportunity for high-resolution functional and molecular brain imaging research.\n\nSLE is a disease that is notorious for its variable presentation and its unpredictable course. Molecular imaging biomarkers will improve our ability to care for individual patients, and our ability to evaluate the efficacy of new treatments. For individual patients, better methods of monitoring the response to therapy will allow clinicians to adjust the doses of drugs and the duration of treatment. In some cases higher treatment doses may be necessary to eliminate tissue inflammation, whereas in other patients the lower doses may be required to control tissue inflammation, and medication toxicity can be avoided.\n\nFor clinical trials, the evaluation of new drugs can take several years. Furthermore, the complex nature of SLE and the need to treat high-risk patients with established drugs has made it particularly difficult to evaluate new drugs. Treatment response is usually based on urine protein and serum creatinine measurements, and the cutoffs used to define complete and partial responses differ among trials24,27,84. New diagnostic methods – particularly companion diagnostics for biologic therapies – will facilitate the evaluation of new drugs. Thus, new methods for detecting and monitoring inflammation within the kidneys and CNS are expected to improve the care of individual patients and to facilitate the development of new therapeutic agents.",
"appendix": "Author contributions\n\n\n\nJMT and NJS both contributed to the design, conduct and interpretation of the experiments included in this manuscript. Both authors contributed to the preparation of this manuscript. All authors have approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nJMT receives royalties from Alexion Pharmaceuticals, Inc. and has received consulting fees from Baxter Pharmaceuticals, Inc.\n\n\nGrant information\n\nThe original studies reported in this review article were supported by the University of Colorado Cancer Center P30 grant CA046934, and the Colorado Clinical and Translational Sciences Institute UL1 award RR025780. This work was also supported in part by the KIDNEEDS Foundation (JMT) and the Lupus Research Institute (JMT).\n\n\nReferences\n\nTsokos GC: Systemic lupus erythematosus. N Engl J Med. 2011; 365(22): 2110–21. 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}
|
[
{
"id": "9062",
"date": "24 Jun 2015",
"name": "Dorin Bogdan Borza",
"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 well-written review article by Thurman and Serkova focuses on the utility and the potential of non-invasive imaging techniques to monitor two common yet severe manifestations of systemic lupus erythematosus (SLE), lupus nephritis (LN) and neuro-psychiatric SLE (NPSLE). The authors summarize the clinical challenges posed by the unpredictable course of SLE, explain the need for better biomarkers of disease activity in LN and NPSLE, critically review both the utility and limitations of conventional radiologic imaging, and discuss the promise of molecular imaging for monitoring LN and NPSLE.Minor points to consider:When introducing lupus nephritis (page 3), the WHO classification is mentioned only in passing. More detail will help orient the reader and provide context when discussing \"proliferative LN\" in the next paragraph. Page 4, when discussing particular autoAbs associated with LN, the authors may consider referencing very recent studies identifying IgG2 autoAbs to alpha-enolase or annexin AI are a major component of immune deposits in kidney biopsies from LN patients (Bruschi et al, 2014). Page 5, C3b and C3d are introduced without providing much background information how this fragments arise. On page 3, paragraph 7: NPLSE should be NPSLE.",
"responses": [
{
"c_id": "1660",
"date": "19 Oct 2015",
"name": "Joshua Thurman",
"role": "Author Response",
"response": "Thank you for the thoughtful comments. We will revise the manuscript to address these points."
}
]
},
{
"id": "9535",
"date": "11 Aug 2015",
"name": "Patrick Cunningham",
"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 article by Thurman and Serkova is a well-written, thorough, interesting review of the potential of newer, noninvasive imaging techniques to follow disease activity in lupus.The preliminary experiments of the authors (references 8 and 80) showing the utility of this technology in mouse models is the most interesting part. The article would benefit from much more detail of these experiments, perhaps with images if that would be illustrative. However, is there any preliminary data to suggest this approach would work in the brain, as well as kidneys? Similarly, more detail of the predictive power of blood tests in patients with SLE to predict outcomes would be illustrative – I suspect these biomarkers do relatively poorly. Would the techniques of labeling complement, Abs, cells, etc. have applicability to other autoimmune or inflammatory diseases? It is not entirely clear at first that the paramagnetic nanoparticles described on page 6 are the same as listed under MRI for figure 1. The paramagnetic nature of the technology should be described better in the section that correlates with Figure 1.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-153
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https://f1000research.com/articles/3-283/v1
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18 Nov 14
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{
"type": "Case Report",
"title": "Case Report: \"ADHD Trainer\": the mobile application that enhances cognitive skills in ADHD patients",
"authors": [
"Gonzalo Ruiz-Manrique",
"Kazuhiro Tajima-Pozo",
"Francisco Montañes-Rada",
"Gonzalo Ruiz-Manrique",
"Francisco Montañes-Rada"
],
"abstract": "We report the case of a 10 year old patient diagnosed with attention deficit hyperactivity disorder (ADHD) and comorbid video game addiction, who was treated with medication combined to a novel cognitive training method based on video games called TCT method. A great risk of developing video game or internet addiction has been reported in children, especially in children with ADHD. Despite this risk, we hypothesize that the good use of these new technologies might be useful to develop new methods of cognitive training. The cognitive areas in which a greater improvement was observed through the use of video games were the visuospatial working memory and fine motor skills. TCT method is a cognitive training method that enhances cognitive skills such as attention, working memory, processing speed, calculation ability, reasoning, and visuomotor coordination. The purpose of reviewing this case is to highlight that regular cognitive computerized training in ADHD patients can improve some of their cognitive symptoms and can help treating video game addition.",
"keywords": [
"ADHD",
"mobile app",
"TCT method",
"working memory"
],
"content": "Introduction\n\nAttention deficit hyperactivity disorder (ADHD) is the most commonly diagnosed neurodevelopmental disorder in childhood, which affects 3% to 7% of the population worldwide1. ADHD is characterized by distractibility, hyperactivity and impulsivity. The standard treatment for ADHD includes mainly medication, psychosocial and behavioral treatment, and cognitive training exercises.\n\nCognitive training exercises are especially useful when cognitive impairment is observed and when a regular and personalized cognitive training is performed2. Studies in participants with cognitive impairment have shown that regular and daily cognitive training can improve some of their cognitive symptoms3,4. In addition, recent studies have demonstrated that computerized working memory and executive function training programs lead to better results than ordinary cognitive training methods in children with ADHD5–7.\n\nChildren’s use of electronic devices, Internet and video games, has noticeably increased in the last 10 years. Since the first case of Internet addiction was described in 1996 by Young6, several other pathologies have been proposed including pathological gambling and dependence7. Despite extensive research literature available, the prevalence and proper diagnostic criteria for pathological gaming are still debated among the scientific community8. Gaming addiction represents part of the postulated construct of Internet addiction, and is the most widely studied specific form of Internet addiction to date9. Prevalence estimates range from 2%10 to 15%11, depending on the respective socio-cultural context, sample, and assessment criteria utilized. A great risk of developing video game or Internet addiction has been reported in children, and especially in those with ADHD8. Despite this risk, we hypothesize that good use of these new technologies can be useful to develop new methods of cognitive training.\n\n\nCase report\n\nThis case study involves a 10 year old child born in Madrid (Spain) who received treatment in a childhood psychiatry unit for 2 years due to behavioral disorders and ADHD. No other previous medical history was reported. His mother, aged 35, received psychological treatment for anxiety 3 years ago. His father, aged 36, works as an engineer and presented no relevant medical history. The patient was their only son. The parents described a great addiction to video games in the last year, a, referring 4 hours per day of video game playing, affecting his social interaction, and causing a lack of imaginative play and poor academic scores. Teachers at the school reported deterioration in his academic performance over the past year. At that time, the child was treated with methylphenidate 40 mg per day. The patient’s parents reported to the psychiatrist that the only significant change from the previous year was a major addiction to a war videogame.\n\nTo reduce the exposure to video games, we used a novel technique, cognitive stimulation with a mobile/tablet application designed specifically to treat ADHD, based on the method of Tajima Cognitive Method (TCT) cognitive training called “ADHD Trainer”.\n\nBehavioral and academic improvements were rated on the Conners Parent and Teacher Rating Scales (brief version) and Barkley School Situations Questionnaire.\n\nADHD diagnosis was made according to DSM V criteria9,10. Attention was rated with CPT Conners Continuous Performance Test.\n\nA differential diagnosis between oppositional defiant disorder and ADHD disorder was considered, because most of the symptoms were observed at home, however not angry or irritable mood was observed.\n\nThe patient was treated with a combination of methylphenidate and a cognitive training method based in the TCT method. The patient received daily treatment with 40 mg of methylphenidate, and at least 10 minutes of daily cognitive training with the “ADHD Trainer” app.\n\nThe TCT is a type of computer adaptive test (CAT), as it adapts to the individual’s cognitive strengths and weaknesses, based on his own scores over time, as well as those of his peers. Users receive separate scores in different cognitive areas, including simple calculation, attention, perceptual reasoning, and visuomotor coordination (Figure 1). The goal of the daily training is to reach a pre-set individualized score in different cognitive domains, in order to complete a week of successful training\n\nDuring the first month of cognitive training therapy, the patient was only allowed to play with specific games based on the TCT Method, using the “ADHD Trainer” (Figure 2). The patient had to use the app every day at the same time, provided the other targets that were assigned in therapy, such as the progressive reduction in the number of hours to play other games and just being able to play with them once a week, were met. During the first month, he was allowed to play this game to a maximum range of 4 hours per day. No addiction to this videogame was observed during the first month. The average number of hours that the child played the video game was 1 hour a day. In the following months the objective was to play the game at least 10 minutes per day.\n\nIn less than two months the video games abuse was substantially reduced, limiting their use to weekends, and always for periods not exceeding 4 hours in total.\n\nBehavioral and academic improvement was rated on the Conners Parent and Teacher Rating Scales and Barkley School Situations Questionnaire. The initial score of the Conners was 19 for the teachers and 20 for the parents, and after the cognitive training the scores were 15 for the teachers and 16 for the parents.\n\nBoth the school and the family reported a significant improvement in the patient after 6 months of TCT cognitive training, which included important improvements of both academic and behavioral outcomes.\n\n\nDiscussion\n\nMost of the studies reported so far emphasize the potential addictive risk of new technologies and the influence they have on children's interpersonal development, by reducing the time children spend outside home and increasing the time they spend alone playing in front of a television or a computer screen12–14. It is also known that the new technologies may affect children's academic performance by reducing the number of hours that they dedicate to studying.\n\nThere are few studies which focus on the positive aspects of new technologies and the opportunities that they offer new ways of interaction between professionals and users as well as the development of new therapeutic methods, capable of reaching the young.\n\nNew technologies, in particular video games, can be used as therapeutic tools to train executive functions6,7. As they generate greater motivation in children and adolescents they will increase the frequency of performing cognitive tasks oriented to enhance executive functions, especially the working memory.\n\nThere are key advantages for children practicing the TCT Method relative to traditional cognitive training therapies which include:\n\n1) Increased motivation in children for completing cognitive training therapy. This increase in motivation comes from: entertainment value (these games are designed to be similar to regular video games that children enjoy) and feedback on performances relative to own and peer scores (which improves children’s sense of agency and self-efficacy, as demonstrated by documented research on motivation and learning)11,12.\n\n2) Ease of accessing the application. Children can play the games at any place or time, day and night.\n\n\nConclusion\n\nADHD patients are especially vulnerable to develop video gaming addition. ADHD patients often suffer from working memory and executive function dysfunctions, but we have observed that very few cognitive training techniques have been developed for ADHD patients in the last years. Poor completion rates of cognitive training in children with ADHD have been observed. We conclude that a daily cognitive computerized training in ADHD patients can improve some of their cognitive symptoms, and can help treating the video gaming addition.\n\n\nConsent\n\nWritten informed consent to publish this report was obtained by the patient’s parents.\n\nDr. Tajima takes responsibility for the integrity of the data and informed consent.",
"appendix": "Author contributions\n\n\n\nDr. Gonzalo Ruiz wrote the manuscript, supervised by Dr. Kazuhiro Tajima-Pozo and Dr. Francisco Montañes-Rada. All authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nDr. Kazuhiro Tajima-Pozo, participated in the development of “ADHD Trainer”, and other mental health applications at TKT Brain Solutions, which is a Spanish startup, integrated by medical doctors and engineers, whose aim is to develop mental health applications.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWeinstein A, Weizman A: Emerging association between addictive gaming and attention-deficit/hyperactivity disorder. Curr Psychiatry Rep. 2012; 14(5): 590–7. PubMed Abstract | Publisher Full Text\n\nMartinussen R, Hayden J, Hogg-Johnson S, et al.: A meta-analysis of working memory impairments in children with attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry. 2005; 44(4): 377–84. PubMed Abstract | Publisher Full Text\n\nVoelbel G, Ceceli A, Georgieva S, et al.: C-40Computerized Neuroplasticity Training Increases Processing Speed of Verbal Information: A Pilot Study of Adults with Traumatic Brain Injury. Arch Clin Neuropsychol. 2014; 29(6): 589. PubMed Abstract | Publisher Full Text\n\nJungblut M, Huber W, Mais C, et al.: Paving the way for speech: voice-training-induced plasticity in chronic aphasia and apraxia of speech--three single cases. Neural Plast. 2014; 2014: 841982. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShaw R, Grayson A, Lewis V: Inhibition, ADHD, and computer games: the inhibitory performance of children with ADHD on computerized tasks and games. J Atten Disord. 2005; 8(4): 160–8. PubMed Abstract | Publisher Full Text\n\nBioulac S, Arfi L, Bouvard MP: Attention deficit/hyperactivity disorder and video games: a comparative study of hyperactive and control children. Eur Psychiatry. 2008; 23(2): 134–41. PubMed Abstract | Publisher Full Text\n\nLim CG, Lee TS, Guan C, et al.: Effectiveness of a brain-computer interface based programme for the treatment of ADHD: a pilot study. Psychopharmacol Bull. 2010; 43(1): 73–82. PubMed Abstract\n\nWalther B, Morgenstern M, Hanewinkel R: Co-occurrence of addictive behaviours: personality factors related to substance use, gambling and computer gaming. Eur Addict Res. 2012; 18(4): 167–74. PubMed Abstract | Publisher Full Text\n\nKeith Conners C: PhD inventor; Conners 3rd Edition™ (Conners 3™). 2013. Reference Source\n\nDupaul GJ, Barkley RA: Situational Variability of Attention Problems: Psychometric Properties of the Revised Home and School Situations Questionnaire. J Clin Child Adolesc Psychol. 1992; 21(2): 178–188. Publisher Full Text\n\nDovis S, Van der Oord S, Wiers RW, et al.: Can motivation normalize working memory and task persistence in children with attention-deficit/hyperactivity disorder? The effects of money and computer-gaming. J Abnorm Child Psychol. 2012; 40(5): 669–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilkinson N, Ang RP, Goh DH: Online video game therapy for mental health concerns: a review. Int J Soc Psychiatry. 2008; 54(4): 370–82. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "6739",
"date": "25 Nov 2014",
"name": "Aviv Weinstein",
"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 on the usefulness of cognitive computer training for a child who is diagnosed with ADHD and concurrent videogame addiction. The rationale for the study, methods and findings are fine but I would like to make some additional comments.First, the usefulness of treatment of ADHD and internet addiction by using methylphenidate was reported by Han D et al., 2009, please add it to the introduction.Second, what evidence have you got that the child is not addicted to the educational game?Third, 4 hours of play of a videogame post-treatment is still a lot, this should be mentioned as a limitation.Fourth, why were the Conners ratings after treatment for parents and teachers lower compared with pre-treatment?Fifth, the authors should be commended for the use of advanced computer games for treatment for ADHD. There are other tools for this purpose that are worthwhile mentioning such as ONTRAC (Mishra et al., 2013) and the game reported by Prins PJ et al., 2011.",
"responses": [
{
"c_id": "1117",
"date": "07 May 2015",
"name": "Kazuhiro Tajima-Pozo",
"role": "Author Response",
"response": "We have included the following new references and made the changes according to the reviewer comments. Mishra J, Merzenich MM, Sagar R. Accessible online neuroplasticity-targeted training for children with ADHD. Child Adolesc Psychiatry Ment Health. 2013 Nov 14;7(1):38 Prins PJ, Dovis S, Ponsioen A, ten Brink E, van der Oord S. Does computerized working memory training with game elements enhance motivation and training efficacy in children with ADHD? Cyberpsychol Behav Soc Netw. 2011 Mar;14(3):115-22. Han DH1, Lee YS, Na C, Ahn JY, Chung US, Daniels MA, Haws CA, Renshaw PF.The effect of methylphenidate on Internet video game play in children with attention-deficit/hyperactivity disorder."
},
{
"c_id": "1292",
"date": "07 May 2015",
"name": "Kazuhiro Tajima-Pozo",
"role": "Author Response",
"response": "We have included the following new references and made the changes according to the reviewer comments. Mishra J, Merzenich MM, Sagar R. Accessible online neuroplasticity-targeted training for children with ADHD. Child Adolesc Psychiatry Ment Health. 2013 Nov 14;7(1):38 Prins PJ, Dovis S, Ponsioen A, ten Brink E, van der Oord S. Does computerized working memory training with game elements enhance motivation and training efficacy in children with ADHD? Cyberpsychol Behav Soc Netw. 2011 Mar;14(3):115-22. Han DH1, Lee YS, Na C, Ahn JY, Chung US, Daniels MA, Haws CA, Renshaw PF.The effect of methylphenidate on Internet video game play in children with attention-deficit/hyperactivity disorder."
}
]
}
] | 1
|
https://f1000research.com/articles/3-283
|
https://f1000research.com/articles/4-1104/v1
|
23 Oct 15
|
{
"type": "Research Article",
"title": "Embryo-lethal phenotypes in early abp1 mutants are due to disruption of the neighboring BSM gene",
"authors": [
"Jaroslav Michalko",
"Marta Dravecká",
"Tobias Bollenbach",
"Jiří Friml",
"Jaroslav Michalko",
"Marta Dravecká",
"Tobias Bollenbach"
],
"abstract": "The Auxin Binding Protein1 (ABP1) has been identified based on its ability to bind auxin with high affinity and studied for a long time as a prime candidate for the extracellular auxin receptor responsible for mediating in particular the fast non-transcriptional auxin responses. However, the contradiction between the embryo-lethal phenotypes of the originally described Arabidopsis T-DNA insertional knock-out alleles (abp1-1 and abp1-1s) and the wild type-like phenotypes of other recently described loss-of-function alleles (abp1-c1 and abp1-TD1) questions the biological importance of ABP1 and relevance of the previous genetic studies. Here we show that there is no hidden copy of the ABP1 gene in the Arabidopsis genome but the embryo-lethal phenotypes of abp1-1 and abp1-1s alleles are very similar to the knock-out phenotypes of the neighboring gene, BELAYA SMERT (BSM). Furthermore, the allelic complementation test between bsm and abp1 alleles shows that the embryo-lethality in the abp1-1 and abp1-1s alleles is caused by the off-target disruption of the BSM locus by the T-DNA insertions. This clarifies the controversy of different phenotypes among published abp1 knock-out alleles and asks for reflections on the developmental role of ABP1.",
"keywords": [
"Auxin-binding protein 1",
"ABP1",
"BELAYA SMERT",
"BSM",
"embryo-lethality",
"allelism",
"short read DNA mapping"
],
"content": "Introduction\n\nThe plant hormone auxin plays a central role in plant growth and development. Sensing and interpreting the fluctuating cellular auxin levels is crucial for mediating the corresponding physiological and developmental responses (Enders & Strader, 2015; Grunewald & Friml, 2010). Currently, two main auxin receptor/co-receptor systems are known and have been proposed to activate a range of cellular responses; among them the Auxin Binding Protein 1 (ABP1) has been considered as a prime candidate for the extracellular auxin receptor (Grones & Friml, 2015).\n\nThe first notion of ABP1 was based on the auxin-binding activity at the plant cell surface and in the endoplasmic reticulum in crude membrane preparations of etiolated coleoptiles (Hertel et al., 1972). This binding activity was characterized over the next decade by detailed biochemical studies (Batt et al., 1976; Ray et al., 1977). ABP1 was firstly purified from maize coleoptile cells (Lobler & Klambt, 1985) as a soluble 22-kDa large glycoprotein and later its crystal structure was elucidated (Woo et al., 2002).\n\nThe biological function and importance of ABP1 has been investigated extensively. Early studies have demonstrated that APB1 is involved in the rapid regulation of the membrane potential and ion fluxes at the plasma membrane and that it mediates the auxin-induced cell swelling, cell elongation, and cell division (Braun et al., 2008; Steffens et al., 2001; Tromas et al., 2009; Yamagami et al., 2004). In the presence of auxin, ABP1 activates the H+ pump ATPase that acidifies the extracellular space, presumably triggering cell wall loosening (Cosgrove, 2000).\n\nWith the advent of the genomic era and Arabidopsis thaliana as a model system, genetic tools have been adopted to facilitate the studies of the ABP1 developmental roles. Using a reverse genetic approach, two independent Arabidopsis thaliana knock-out alleles of ABP1 gene were identified, abp1-1 and abp1-1s (Chen et al., 2001; Tzafrir et al., 2004) and both were reported to be allelic and embryo-lethal, arresting the embryo development at the globular stage. This defined ABP1 as an essential protein required from early embryonic development with functions in cell division and elongation (Chen et al., 2001). However, embryo-lethal phenotype of abp1 knock-out mutants has hampered its further functional characterization. This was rectified by the generation of transgenic lines conditionally downregulating ABP1 levels by ethanol-inducible immuno-modulation or antisense approaches that, despite entirely different technologies used, show the same phenotypes confirming the role of ABP1 in cell division and cell expansion (Braun et al., 2008; David et al., 2007).\n\nBoth downregulation lines along with the weak abp1-5 point mutation allele showed defects in clathrin-mediated endocytosis of PIN auxin export proteins (Dhonukshe et al., 2007; Petrášek et al., 2006) and their inhibition by auxin (Paciorek et al., 2005). In contrast, the ABP1 gain-of-function mutation has an opposite effect, promoting PIN internalization in tobacco cultured cells and stable Arabidopsis lines (Robert et al., 2010). Opposite effects of ABP1 loss- and gain-of-function lines were observed also for auxin effects on arrangements of microtubules (Xu et al., 2014) and for controlling morphogenesis and shape of leaf epidermal pavement cells (Nagawa et al., 2012; Xu et al., 2010). Furthermore, the importance of auxin binding to ABP1 for gain-of-function phenotypes has been demonstrated by analysis of ABP1 variants with introduced mutations in the auxin binding pocket (Grones et al., 2015). Thus various types of loss- and gain-of-function strategies show an internally consistent picture of ABP1 signaling being involved in a range of physiological and cellular processes.\n\nAn important breakthrough came with the finding that the auxin-bound ABP1 docks on the extracellular domain of the Transmembrane Kinase 1 (TMK1) (Dai et al., 2013; Xu et al., 2014) which added the missing piece to the puzzle of how the auxin signal is transmitted from the cell surface to the cytosol and further confirmed involvement of the ABP1/TMK1 pathway in auxin-mediated development, particularly in pavement cell morphology (Grones & Friml, 2015).\n\nSurprisingly enough, shortly after these studies had been published, Gao et al. (2015) described two independent, full loss-of-function abp1 mutants of A. thaliana (abp1-c1 and abp1-TD1) that show no apparent developmental defects under normal growth conditions. This directly contradicts the embryo-lethal phenotypes of the originally described abp1-1 and abp1-1s lines (Chen et al., 2001; www.seedgenes.org/SeedGeneProfile?geneSymbol=ABP+1) and also questioned the relevance of the aforementioned studies.\n\nThe explanation of these contradictory results is therefore of crucial importance to understand the biological role of ABP1. In this study we aim to clarify the discrepancy between the dramatic embryolethal phenotype of the originally described abp1 knock-out mutants and the newly identified loss-of-function alleles.\n\n\nMaterial and methods\n\nArabidopsis thaliana mutants used in this study were: abp1-1 (Chen et al., 2001), abp1-1s (NASC accession N16148), abp1-c1, abp1-TD1 (Gao et al., 2015), bsm1-1 (Babiychuk et al., 1997). A. thaliana Col-0 wild type seeds were obtained from The Nottingham Arabidopsis Stock Centre (NASC, http://arabidopsis.info). The seeds were vernalized for 3 days in the dark at 4°C. Plants were grown under long-day conditions (16-h light/8-h dark cycles) at 22°C in soil (Potgrond P) : perlit 4 : 1 substrate and watered regularly with tap water.\n\nFor selection of abp1-1, abp1-1s and bsm1-1 heterozygous plants, seeds were surface-sterilized with chlorine vapor and plated on 1/2 MS agar medium (pH 5.9) containing 1% (w/v) sucrose and 25 µg/mL kanamycin according to Harrison et al. (2006).\n\nData from two publicly available short read libraries (NCBI Short Reads Archive accessions SRX759525 and SRX703650) from A. thaliana re-sequencing experiments were downloaded and mapped to the deposited reference genome TAIR10 (accessions NC_003070, NC_003071, NC_003074, NC_003075, NC_003076) using the Bowtie 2 plugin within the Geneious software, version 7.1.7 (http://www.geneious.com). For the first analyzed short read library (accession SRX759525), mapping parameters were set to a local alignment (“--very-fast-local”) with multi-mapping allowed (value k = 10). For the second analyzed short read library (accession SRX703650), mapping parameters were set to end-to-end alignment and the best-match mapping option (k = 1) was used. In both cases the seed length (L) was set to 22 and number of mismatches (N) to 1. To maximize the mapping coverage short reads were mapped single-end. For all the remaining parameters the default Bowtie 2 values were used. For both alignments, the median coverage at the ABP1 locus (bases 1 319 656 - 1 321 477 of NC_003075) was compared to the surrounding 20 Kbp region (Figure 1A and 1B) and to the whole chromosome 4 (Figure 1C and Dataset 2 for SRX759525; Figure 1D and Dataset 3 for SRX703650). Mapping coverage is defined as the number of reads mapped to a given base. Coverage data from the Geneious software were exported and visualized using Matlab (v. R2011b).\n\nTo test the hypothesis of hidden ABP1 duplicates in the genome of Arabidopsis thaliana, two short reads libraries from A. thaliana re-sequencing projects (NCBI accession numbers SRX759525 and SRX703650) were separately aligned to the reference Arabidopsis genome (TAIR10) using the BOWTIE 2 suit using different mapping parameter sets (see the Methods section). (A, B) The coverage of the 20 kbp consensus sequence within the chromosome 4 that surrounds ABP1 is shown for the two libraries: SRX759525 (A) and SRX703650 (B). (C, D) The overall distribution of the base coverage within the chromosome 4 is shown below. The median coverage at the ABP1 locus is highlighted in light blue and is well within the expected coverage values for both SRX759525 (C) and SRX703650 (D).\n\nThe T-DNA insertional mutants were genotyped by using a PCR-based method (Alonso et al., 2003). Amplification of PCR products was made using Phire Plant Direct PCR Kit (obtained from Thermo Scientific by Finnzymes, Espoo, Finland) following manufacturer’s instructions for the dilution protocol and using Bio-Rad T100 Thermal Cycler. The PCR conditions were as follows: initial denaturation for 5 min. at 98°C and subsequent 40 cycles: denaturation for 5 s at 98°C, annealing for 10 s at the respective annealing temperature for each primer set (calculated with the Thermo Scientific Tm calculator; available at www.thermofisher.com), elongation for 30 s at 72°C and final elongation for 1 min. at 72°C.\n\nGenotyping primers were as follows: cdsBSM_F and nptII_R for the bsm mutation, ABP1_3UTR_FOR and WiscDsLoc_REV for the abp1-1 mutation, WiscDsLoc_REV and qBSM_R2 for the abp1-1s mutation. Genotyping of abp1-c1 and abp1-TD1 mutants was described previously (Gao et al., 2015). Sequences of all primers used in this study are listed in Table 1. Purified PCR products from plants positive for the presence of the T-DNA insertion were sequenced using a commercial service (LGC genomics, www.lgcgenomics.com). Primers used for sequencing were the same as for PCR. Sequence reads were aligned against A. thaliana genome (TAIR 10) using the BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The position of the mutation was identified at the border of the sequence part aligned to ABP1 locus and the sequence part aligned to the transformation vector. Position of individual mutations was mapped to the sequence of ABP1/BSM locus acquired from the TAIR database (https://www.arabidopsis.org), visualized with the SnapGene Viewer software version 2.8.2 (http://www.snapgene.com/products/snapgene_viewer) and edited in MS Power Point 2010.\n\nFor the RNA extraction approximately twenty 8 day-old seedlings were frozen in liquid nitrogen. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. For digestion of genomic DNA the following modification was incorporated into the protocol: in step 7, first 350 µL of RW1 buffer was added to the RNeasy spin column and centrifuged (8000 rpm, room temperature, 15 s). Subsequently, 40 µL of the RNase-free recombinant DNase I incubation mix (mixture of 5 µL DNase I and 35 µL buffer RDD) (Roche) was added to the column and incubated for 15 min at room temperature. Next, another 350 µL of RW1 buffer was added to the column and centrifuged again (8000 rpm, room temperature, 15 s). Two µg of purified DNase I pre-treated total RNA was used for a reverse transcription reaction using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA). Primers used for the quantitative RT-PCR were designed using QuantPrime (http://www.quantprime.de). For amplification of the BSM cDNA fragment (88 bp in length) primers qBSM_F2 and qBSM_R2 were used. The ABP1 cDNA fragment (84 bp in length) was amplified with A2E and ABP1-586R primers. Arabidopsis tubulin beta chain 2 (TUB2, At5g62690) was used as a reference gene and amplified with TUB-2_F3 and TUB-2_R3 primer set (Table 1, Dataset 1). All primers were synthesized by commercial service (https://www.eurofinsgenomics.eu/). qRT-PCR was performed using the LightCycler 480 SYBR Green I Master chemistry (Roche) in a LightCycler 480 II thermal cycler (Ser. no. 5659, Roche) according to manufacturer’s instructions. A 1:10 cDNA dilution was used as a template to prepare 5 µL reaction mixture (final volume). The cycling conditions were as follows: pre-incubation for 10 min. at 95°C and subsequent 45 cycles: denaturation for 10 s at 95°C, annealing for 15 s at 60°C, elongation for 15 s at 72°C followed by high resolution melting analysis. Gene expression was calculated with the 2-ΔΔCT method (Livak & Schmittgen, 2001). Individual experiments were made in a technical triplicate.\n\nTo compare the stage of embryo arrest, seeds from siliques of the heterozygous abp1-1 and bsm plants in the 7th to 8th developmental stage were used. For evaluation of embryo development, seeds from the siliques in the 3rd to 5th developmental stage were used. Siliques were dissected using a sharp needle and seeds were extracted and transferred onto microscopic slides into the drop of the Hoyer’s solution (http://www.seedgenes.org/Tutorial.html) and cleared for 1 h similarly as described (Friml et al., 2003). The embryos were analyzed under 20 × magnification using a digital camera system of the Olympus BX53 microscope by Normanski optics (Differential Interference Contrast (DIC) light microscopy). Images were processed in the ImageJ software, v. 1.48 (http://imagej.nih.gov/ij/) and mounted in Adobe Illustrator CS5.1.\n\nApproximately 4 week-old plants were used for crossing. Flowers of recipient plants were emasculated 2 days before pollination. Crosses were generated by manual pollination. Green siliques were dissected 8 days after pollination and number of white and green seeds was counted and their ratio calculated.\n\n\nResults\n\nOne of the possibilities explaining at least some of the recent ABP1 controversies, in particular the strong phenotypes of the conditional lines versus no apparent phenotypes of the abp1-c1 and abp1-TD1 alleles, is the presence of a second, non-annotated ABP1 gene copy in the genome of A. thaliana that is functionally redundant and not disrupted in the abp1-c1 and abp1-TD1 alleles. Highly similar copies of the ABP1 gene are present in the annotated genomes of maize (Zea mays), rice (Oryza sativa) or poplar (Populus trichocarpa) (http://phytozome.jgi.doe.gov). During genome assembly, highly similar sequences such as transposons or recently duplicated genes could collapse into one copy resulting in no annotation of the duplicated copy (Stephane Rombauts, personal communication, 30th January 2015). A non-annotated second copy of the ABP1 gene is also present in the sequenced genome of the moss Physcomitrella patens (http://phytozome.jgi.doe.gov) raising the possibility that a similar situation could take a place in the A. thaliana genome.\n\nWe hypothesized that in case of a second non-annotated copy of ABP1 in the A. thaliana genome, the short reads coverage at the ABP1 locus would be doubled compared to the neighboring DNA regions and to the coverage of most other loci on the chromosome. To address this question we mapped short reads from two publicly available short read libraries from A. thaliana re-sequencing projects to the annotated Arabidopsis reference genome (TAIR 10) and analyzed the coverage at the ABP1 locus.\n\nAs depicted (Figure 1), the number of aligned short reads at the ABP1 locus is comparable with the short read coverage of the neighboring sequences and is well within the range of most common coverage values for loci on chromosome 4 indicating that no hidden copy of ABP1 is present in the A. thaliana genome.\n\nAnother possibility to explain phenotypic discrepancies between different abp1 mutant alleles is a disruption of another gene in addition to ABP1 that would be responsible for the strong phenotypes in the abp1-1 and abp1-1s alleles. As it can be seen on the ABP1 locus map (Figure 2A), the ABP1 gene is closely located to its neighboring gene BSM in a head-to head orientation with translational start codons and 5’-UTR regions of the two genes being just 708 bp and 381 bp respectively apart from each other. BSM encodes the mitochondrial transcription termination factor (mTERF)-like protein responsible for mitochondrial transcription termination in humans. In plants, mutants disrupting the BSM gene have been shown to be embryo-lethal (Babiychuk et al., 2011), therefore we suspected that the BSM gene is the most likely off-target of mutations in the abp1-1 and abp1-1s alleles.\n\n(A) Map of the ABP1/BSM locus showing the mapped position of existing loss-of-function mutant alleles (red arrowheads) relative to the ABP1 translation start. Note that the T-DNA insertion in abp1-1s allele is located in the 5’-UTR region of the BSM gene. Positions of the forward primers ABP1-586R (F1) and qBSM_F2 (F2) and reverse primers A2E (R1) and qBSM_R2 (R2) used for the qRT-PCR are indicated by red arrows. (B) Quantitative real-time PCR of the ABP1 (left graph) and BSM (right graph) shows relative transcript levels in wild-type Col-0, abp1-c1 and abp1-TD1 seedlings. The reference gene was TUB2 (At5g62690). The transcript levels were calculated using 2-ΔΔCT method. The data represent average relative quantity values of three technical replicates, and the bars denote standard errors. For wild-type Col-0 plants data from 2 biological replicates are shown.\n\nTo look into this possibility closely, we mapped the relative position of individual mutations within the ABP1/BSM locus (Figure 2A). Surprisingly, we found that the T-DNA insertion in abp1-1s mutant is in fact located in the 5’-UTR of the BSM gene. Because ABP1 and BSM share a common promoter region, we hypothesized that the T-DNA insertion in abp1-1 could also disrupt the expression of the BSM gene thus causing the described embryonic lethality. Notably, the T-DNA insertion in the wild type-like allele abp1-TD1 is located just 24 bp apart from the abp1-1 T-DNA insertion and is even closer to the BSM gene. We speculated that the dramatic phenotypic difference between both closely positioned T-DNA-based insertions might be caused by the presence of multiplied 35S enhancers within the T-DNA construct in the abp1-TD1, because this allele was recovered from the activation tagging Arabidopsis mutant collection (SASKATOON) and harbors multiplied enhancers near the right T-DNA border on the inserted T-DNA element (Figure 2B).\n\nTo see whether the expression of the BSM gene in homozygous abp1-TD1 plants was affected by the T-DNA insertion, we performed a qRT-PCR analysis. Surprisingly, the BSM transcript levels in abp1-TD1 were not substantially affected by the T-DNA insertion and were comparable to wild type Col-0 plants (Figure 2B). On the other hand, the expression of the ABP1 gene was abolished in both abp1-c1 and abp1-TD1 homozygous plants as reported previously (Gao et al., 2015). For abp1-1, abp1-1s or bsm mutants it was only possible to evaluate the BSM expression in the heterozygous plants (due to the lethality of homozygous mutants) and this showed no substantial differences compared to wild type plants (Dataset 1).\n\nTherefore, it is possible that while in the abp1-TD1 allele the expression of the BSM gene is rescued by its ectopic expression driven by 35S enhancers within the T-DNA, in the abp1-1 allele, where no such a mechanism exists, the expression of both genes may be disrupted. In addition, the abp1-c1 null allele only contains a small 5 bp deletion at the end of the first exon of ABP1 which is less likely to have any effect on the BSM gene compared to a few kB long T-DNA insertions in other alleles. Thus it is plausible that in abp1-1 and abp1-1s, both ABP1 and BSM genes are disrupted.\n\nTo compare the embryo-lethal phenotypes of abp1-1 and abp1-1s alleles with the embryo-lethal phenotype of the bsm1-1 T-DNA insertional knock-out allele, we analyzed the embryo development in siliques of abp1-1, abp1-1s and bsm1-1 heterozygous plants along with homozygous new abp1 knock-out alleles.\n\nThe inactivation of BSM gene by T-DNA insertion has been shown to cause an embryo arrest at the late globular stage of development (Babiychuk et al., 2011). In the abp1-1 mutants the embryo arrest has been reported at the early globular 32-cell stage in 25% of immature seeds (Chen et al., 2001). In both cases the homozygous mutant embryos never reach the heart-shape stage, indicating prevention in vertical elongation and subsequent anticlinal placement of division planes in the lower tier of cells. Other characteristic features reported in abp1-1 embryos have been connected with failure in degeneration of the suspensor structure and ectopic anticlinal divisions of basal cells resulting in longer suspensors at the later stages (Chen et al., 2001).\n\nInspection of embryos at different stages from the homozygous abp1-c1 and abp1-TD1 plants did not reveal any aberrations in the embryo development as compared to the wild type embryos (Figure 3) consistent with the lack of obvious postembryonic phenotypes (Gao et al., 2015). Also, no white aborted seeds were found in the older siliques of these plants (Supplementary Figure 1).\n\nSeeds from the siliques of abp1-1+/- (A-H), bsm+/- (I–L), abp1-TD1 -/- (M–P), and abp1-c1 -/- (R–U) plants at different developmental stages were isolated and cleared in Hoyer’s solution and screened for defects in embryo development using Normanski optics. Wild type-looking embryos in the seeds of abp1-1 mutant (A–D) showed normal developmental progression: the late globular stage (A), early heart (B), torpedo (C) as well as the mature embryo stage (D). At the globular stage we started to observe differences in embryo development between different mutants earliest manifested by periclinal divisions in protodermal cells (arrows) (A, E, I, M). When most of the wild-type embryos entered the early heart stage (B) some abp1-1 embryos, from the same silique as (B) showed abnormal cell divisions (F), also visible in bsm (J) (arrows) but not in the abp1-TD1 (N) or abp1-c1 mutant (S). At the later stages of embryo development, when wild-type embryos in abp1-1 (C, D) as well as embryos in abp1-TD1 (O, P) and abp1-c1 (T, U) reached the torpedo (C, O, T) and mature stage (D, P, U), the mutant abp1-1 (G, H) and bsm (K, L) embryos were still arrested at the late globular stage. While the bsm mutant embryos show signs of apical basal polarity, abp1-1 embryos formed ball-shaped symmetric structure. Also, abnormal cell divisions in the suspensor cells were visible (arrows). Scale bars, 25 µm.\n\nOpening and inspection of the older siliques in abp1-1, abp1-1s and bsm1-1 heterozygous plants did not reveal any visible differences between those alleles. In all three cases, the siliques carried approximately 25% of white aborted seeds (see Figure 4) containing developmentally arrested embryos (Figure 3). Analysis of the younger embryonic stages did not reveal any significant differences between the analyzed alleles. The obvious embryo defects at the early globular stage were infrequent, and in both abp1 and bsm mutants started to be more pronounced at the late globular stage manifested by the failure of directional cell elongation leading to ball-shaped embryos at later stages. Another characteristic feature for both mutants was a disrupted cell division pattern with frequent periclinal divisions of outer layer cells (Figure 3B). At the later stages of embryo development, bsm mutant embryos showed some degree of apical-basal polarity by forming sometimes oval-shaped structures while abp1-1 mutant embryos continue to divide non-directionally forming more symmetrically ball-shaped embryos. These differences could be explained either by different backgrounds of the two mutant alleles (C24 in the case of bsm and Wassilewskija in the case of abp1-1, respectively) or by simultaneous disruption of both ABP1 and BSM genes in the abp1-1 mutant that may produce stronger effect as compared to a single BSM loss-of-function mutation in bsm1-1. Despite these minor differences at the very late embryo stages, both mutations generated undistinguishable cell elongation and cell division pattern defects starting in both cases at the globular stage resulting in similar embryo-lethal phenotypes.\n\nThe embryolethal phenotype was detected in siliques 8 days after pollination by the presence of white seeds (arrested embryo development). It was not possible to complement the embryo-lethal phenotype of bsm mutant (A, B) with either abp1-1 or abp1-1s mutant (C, D). Similarly, abp1-1 and abp1-1s mutants did not mutually complement (E). As a control Col-0 plants were crossed into the abp1-1 (F), abp1-1s (G) or bsm (H) mutants showing complementation of the embryo-lethal phenotype. Genetic crosses of the recently identified abp1 null mutants (abp1-c1, abp1-TD1) with bsm (I), abp1-1s (J, K) or abp1-1 (L, M) lines resulted in complementation of the embryo-lethal phenotypes showing that in these lines the disruption of the BSM and not the ABP1 gene is responsible for the embryo-lethal phenotype. Arrows indicate the position of white seeds in (C). Number of scored seeds for each cross and the percentage of white seeds are listed in the table below. Note that in not complementing crosses, the segregation of embryo-lethal seeds follows the Mendelian genetic laws as expected.\n\nAs the embryo-lethal phenotypes of abp1 and bsm mutants are indistinguishable from each other, we tested whether these phenotypes might be due to the mutations affecting the same gene or they are independently embryolethal. To address this hypothesis we performed allelic crosses between the bsm and abp1-1 as well as abp1-1s line and looked for the presence of white seeds carrying defective embryos within immature siliques 8 days after pollination of emasculated flowers. We assumed that if the mutations affect the same gene it will not be possible to complement the embryolethal phenotype of abp1-1 or abp1-1s mutants with a functional ABP1 allele from the bsm mutant which will result in the segregation of approximately 25% not developing (white) seeds in the F1 generation.\n\nAfter dissection of siliques of bsm1-1 × abp1-1, bsm1-1 × abp1-1s or control crosses bsm1-1 × bsm1-1 and abp1-1s × abp1-1, we observed the presence of clearly distinguishable white seeds (Figure 4A–E). The segregation ratio of 1:4 white:green seeds was observed (Table in Figure 4, Supplementary Table 1) which is in perfect agreement with the Mendelian genetic laws implying that abp1-1 and abp1-1s mutants do not carry a functional BSM gene. On the other hand, when crossed with wild type (Col-0), abp1-c1 or abp1-TD1 mutants, more than 99% of seeds in the siliques were green, indicating that a functional copy of the BSM gene present in these lines was able to complement the embryolethal phenotype of bsm1-1, abp1-1 and abp1-1s lines (Figure 4F–M).\n\nIn summary, these observations show that the described embryolethal phenotype of the abp1-1 and abp1-1s lines is caused by a disruption in the BSM gene.\n\n\nDiscussion\n\nSince its discovery, the biological importance of the ABP1 protein as a plasma membrane auxin receptor has been a matter of debate, in part because of its predominant ER localization in plant cells where the conditions for auxin binding are unfavorable (Habets & Offringa, 2015; Napier et al., 2002). Nonetheless, after the two independently isolated Arabidopsis abp1 loss-of-function alleles were reported to be embryolethal (Chen et al., 2001; www.seedgenes.org/SeedGeneProfile?geneSymbol=ABP+1), the crucial importance of this protein in cell division and elongation was accepted. This view was challenged by the isolation of two new knock-out alleles (Gao et al., 2015) that harbor mutations in a close vicinity to the previously published insertions and show no obvious phenotypes.\n\nThe phenotype of the originally reported abp1-1 mutant was reported to be complemented by the 35S::ABP1 overexpression construct (Chen et al., 2001). However, despite multiple attempts it was not possible to repeat this complementation either with constructs for overexpression of the wild type ABP1 copy or by the genomic fragment (Grones et al., 2015). Altogether, these findings implied that there exists another cause for the drastic phenotypes present in the abp1-1 and abp1-1s mutants.\n\nHere we show that the neighboring gene BSM, which has been shown previously to be crucial for embryogenesis in Arabidopsis (Babiychuk et al., 2011) is likely to be also disrupted by the T-DNA insertions in the original abp1-1 and abp1-1s mutants, but not in the newly isolated loss-of-function lines abp1-c1 and abp1-TD1. Furthermore, we demonstrate that the mutant embryos of abp1-1 and abp1-1s as well as the loss-of-function bsm1-1 allele showed similar embryo phenotypes and are arrested at the globular stage as it was shown in the original studies (Babiychuk et al., 2011; Chen et al., 2001).\n\nBy allelic complementation experiments we showed that the embryolethal phenotype of the original abp1-1 and abp1-1s alleles can be complemented by the functional copy of the BSM gene that is present in the new abp1-c1 and abp1-TD1 alleles, but not by the bsm loss-of-function or the abp1-1 mutants themselves. Therefore, the originally described abp1 mutants are, indeed, loss-of-function alleles of the BSM gene and at least abp1-1 is a double loss-of-function mutant of ABP1 and BSM. Thus, the embryo-lethal phenotype previously attributed to the abp1 loss-of-function alleles is a result of the mutation in the neighboring BSM gene. For this reason, we also propose to re-annotate the abp1-1 and abp1-1s alleles as abp1-1/bsm1-2 and bsm1-3 (for this line it remains unclear if the insertion disrupts both ABP1 and BSM expression), respectively.\n\nThe abp1 and bsm allelic test together with no embryonic defects in the true abp1 knock-out lines makes it clear that no role has been identified for ABP1 in early embryogenesis. However, this clarification of abp1 knock-out genotypes does not per se undermine all experimentation with ABP1 genetic tools. The other ABP1 genotypes comprise conditional and constitutive gain-of-function alleles, two types of conditional knock-downs, as well as a weak abp1-5 point mutation (Braun et al., 2008; Covanova et al., 2013; Grones et al., 2015; Robert et al., 2010; Tromas et al., 2009; Xu et al., 2010). In several analyzed cellular processes including auxin-dependent ROP-GTPase activation, auxin-regulated endocytosis, E3 ligase-mediated ubiquitination, or cortical microtubule reorientation (Robert et al., 2010; Sassi et al., 2014; Tromas et al., 2013; Xu et al., 2010; Xu et al., 2014) this genetic material produced fully internally consistent data sets. Furthermore, loss-of-function mutants in TMK receptor-like protein kinases, which interact with ABP1 in an auxin-inducible manner, show largely overlapping phenotypes with abp1 mutants (Xu et al., 2014). Altogether, these data support the importance of the ABP1/TMK auxin perception and signaling mechanism in plant development and physiology. One possible explanation for the absence of obvious phenotype defects in true abp1 knock-outs is a presence of another copy of the ABP1 gene in the genome of A. thaliana that could escape the gene annotation during genome assembly and that could stay undetected by Southern blot analysis if the two copies were located close together. However, by in silico analysis of the ABP1 locus coverage we excluded this possibility. Other possible ways to reconcile the absent phenotype defects in the knock-outs with observations from the other genetic material include a genetic compensation mechanism that can be triggered by independent deleterious loss-of-function mutations but not by the genetic knock-downs of the same genes as shown for example in zebrafish (Rossi et al., 2015). Such compensation machinery could involve previously largely overlooked ABP1-like proteins from the germin family that are not a close sequence homologs of ABP1 but share some common characteristic features and some of them have been identified by their binding to auxin (reviewed in Napier et al., 2002).\n\nImportantly, with the controversy of the embryonic phenotypes in different abp1 mutant alleles clarified, we can move on and with the updated genetic toolbox clarify the role of ABP1 in auxin signaling and plant development. The high affinity binding of ABP1 to auxin, universal occurrence of the ABP1 genes in the genomes from algae to higher plants and its highly conserved structure argue for its importance throughout the plant kingdom and promise further interesting discoveries.\n\n\nData availability\n\nF1000Research: Dataset 1. Dataset 1, 10.5256/f1000research.7143.d104552\n\nF1000Research: Dataset 2. Dataset 2, 10.5256/f1000research.7143.d104553\n\nF1000Research: Dataset 3. Dataset 3, 10.5256/f1000research.7143.d104554",
"appendix": "Author contributions\n\n\n\nJF and JM designed the experiments and wrote the manuscript, JM performed all experiments and analyzed the data except the short read coverage experiment, MD performed the genome coverage experiment, analyzed the data and helped with writing the manuscript, TB helped with planning and evaluating the genome coverage experiment. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by ERC Independent Research grant (ERC-2011-StG-20101109-PSDP to JF). JM internship was supported by the grant “Action Austria – Slovakia”.\n\n\nAcknowledgements\n\nWe would like to thank Elena Babyichuk and Sergei Kushnir for providing bsm1-1 mutant seeds. We also like to thank Stephane Rombauts for useful advices for genome coverage analysis. We are thankful to Daniel von Wangenheim for final processing of the images.\n\n\nSupplementary materials\n\nAllelic test between A. thaliana bsm and abp1 knock-out mutants. Complete table with number of scored seeds for all crosses made. The percentage of white seeds from the total number of scored seeds is shown in brackets for each tested combination. N/A = data not available,♀ = plant was used as a pollen recipient, ♂ = plant was used as a pollen donor.\n\nClick here to access the data.\n\nDissected immature siliques of abp1-TD1 and abp1-c1 mutants.\n\nClick here to access the data.\n\n\nReferences\n\nAlonso JM, Stepanova AN, Leisse TJ, et al.: Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003; 301(5633): 653–657. PubMed Abstract | Publisher Full Text\n\nBabiychuk E, Fuangthong M, Van Montagu M, et al.: Efficient gene tagging in Arabidopsis thaliana using a gene trap approach. Proc Natl Acad Sci U S A. 1997; 94(23): 12722–12727. 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}
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[
{
"id": "10911",
"date": "02 Nov 2015",
"name": "Lars Ostergaard",
"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 provides a clear introduction to the current controversy regarding the Auxin Binding Protein1 (ABP1). The authors have carried out a thorough genetic analysis which shows that the embryo lethality of the abp1-1 and abp1-1s mutants previously assigned to the loss of ABP1 function is in fact due to loss of the neighbouring gene, BELAYA SMERT (BSM).",
"responses": []
},
{
"id": "10914",
"date": "02 Nov 2015",
"name": "Richard M. Napier",
"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 examines the genotypic basis behind distinctly different phenotypes which have been reported for arabidopsis mutant lines each annotated as abp1 knock-out/loss-of-function alleles. The various mutant lines as homozygotes have been reported over time both as conferring embryo lethality and as displaying no obvious phenotype. Naturally, each outcome translates into very different interpretations of the function and relevance of ABP1. Consequently, clarification of the genotypes behind each phenotype is welcome. This paper maps the mutation sites in each allele and ties embryo lethality to disruption of the adjacent gene BSM. Thus, ABP1 is not necessary for embryo development in arabidopsis. I applaud the clarity of the conclusion and the suggestion to rename the misleading lines. The title is appropriate and factual. The abstract is appropriate. The experimental strategy is direct and sound, methods are described well and suitable statistical analyses have been employed. All the data are presented clearly and are accessible. The text is clear and well structured.Following the conclusion drawn from the data presented here, there is a discussion on how this and the report of Gao et al affect our understanding of the biological role of ABP1 in planta. It is clear that in the case of ABP1 molecular genetic techniques have provided misleading genotypes. Thank goodness this is now cleared up. The case made in the discussion is that these specific false leads do not undermine all ABP1 results where the results are based on different genotypes and complementary tools. This is in stark contrast to the case suggested by the somewhat provocative title of the Gao paper. The phenotypes considered differ in timing and detail, but this paper and that of Gao et al are linked by revelations clarifying ABP1 mutant genotypes. The contrasting perspective offered by the discussion contributes to a healthy, objective dialogue. Minor points:Throughout I believe ‘Normanski’ should read Nomarski optics. In the results section, text associated with the 35S inserts from SASKATOON accessions should refer to Fig 2A (not 2B).Technical replicates are used for the qPCR data, and where biological replicates are shown there is clearly more variation than between technical replicates, but interpretations of the result are not affected by this level of uncertainty. However, I am not sure the use of “Surprisingly” is necessary in the text (page 5, right column) considering the qPCR data for BSM transcripts in Fig 2B. “In contrast to the loss of ABP1 transcript…” might be more appropriate at this stage of the narrative. Perhaps reordering consideration of the two sets of data (especially given the ABP1 data are shown first in the figure) would help here.",
"responses": []
},
{
"id": "10913",
"date": "02 Nov 2015",
"name": "Christian Luschnig",
"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 publication of an article by Gao and colleagues earlier this year, several published results dealing with the role of ABP1 in auxin signaling and plant development became a matter of doubt. This also concerns the embryo-lethal phenotypes that have been attributed to a loss of ABP1 function in abp1-1 and abp1-1s alleles - quite the opposite of the wild type appearance of the likely abp1-1c and abp1-TD1 null alleles, described by Gao and colleagues. These contradicting results predict off-target effects that would cause phenotypes associated with abp1-1 and abp1-1s, and the authors of this study attempted to identify such targets. From the results presented in this manuscript it appears that functional disruption of the BSM gene, located proximal to ABP1, is responsible for abp1-1/abp1-1s embryo-lethal phenotypes. This is indicated by genetic complementation analyses, demonstrating that neither abp1-1 nor abp1-1s complement segregation of bsm1-1 embryo-lethal phenotypes, when analyzing progeny of crosses. On the other hand, when introducing bsm1-1 into abp1-c1 and abp1-TD1, the authors observed complementation of embryo-lethal phenotypes. This provides genetic evidence for a scenario in which phenotypes associated with abp1-1 and abp1-1s result from disruption of the BSM1 locus. This is an important and timely report, summarizing experiments that certainly will contribute to our understanding of ABP1 function in Arabidopsis. Specifically, it finally establishes an off-target candidate locus, responsible for abp1 mutant phenotypes that have been an integral part of a number of published studies.With respect to the title:\"early abp1 mutants\" sounds a bit ambiguous. The authors might consider \"original abp1 mutants\" instead",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1104
|
https://f1000research.com/articles/4-1102/v1
|
23 Oct 15
|
{
"type": "Research Article",
"title": "Oxygen changes drive non-uniform scaling in Drosophila melanogaster embryogenesis",
"authors": [
"Steven G. Kuntz",
"Michael B. Eisen",
"Michael B. Eisen"
],
"abstract": "We previously demonstrated that, while changes in temperature produce dramatic shifts in the time elapsed during Drosophila melanogaster embryogenesis, the relative timing of events within embryogenesis does not change. However, it was unclear if this uniform scaling is an intrinsic property of developing embryos, or if it is specific to thermal fluctuations. To investigate this, here we characterize the embryonic response to changes in oxygen concentration, which also impact developmental rate, using time-lapse imaging, and find it fundamentally different from the temperature response. Most notably, changes in oxygen levels drive developmental heterochrony, with the timing of several morphological processes showing distinct scaling behaviors. Gut formation is severely slowed by decreases in oxygen, while head involution and syncytial development are less impacted than the rest of development, and the order of several developmental landmarks is inverted at different oxygen levels. These data reveal that the uniform scaling seen with changes in temperature is not a trivial consequence of adjusting developmental rate. The developmental rate changes produced by changing oxygen concentrations dwarf those induced by temperature, and greatly impact survival. While extreme temperatures increase early embryo mortality, mild hypoxia increases arrest and death during mid-embryogenesis and mild hyperoxia increases survival over normoxia.",
"keywords": [
"oxygen",
"hypoxia",
"hyperoxia",
"embryo",
"development",
"time-lapse",
"temperature",
"Drosophila"
],
"content": "Introduction\n\nAfter discovering that the time elapsed during different morphological stages of Drosophila embryogenesis scale uniformly as temperature changes the overall time of embryogenesis1, several colleagues questioned whether the result was surprising, suggesting instead that it was a natural and trivial consequence of physical and chemical laws. To explore this possibility, and to provide orthogonal insight into the mechanisms of the control of developmental timing, we sought to manipulate developmental rate in a temperature-independent manner.\n\nIt has long been known that oxygen levels affect the rate of animal development2. In D. melanogaster mild hypoxia (10% oxygen) slows time to eclosion relative to normoxia (21% oxygen), while hyperoxia (41% oxygen) accelerates time to eclosion in a temperature-dependent manner. This suggested to us that studying the effects of varying oxygen levels on embryogenesis might provide an ideal complement to our earlier studies of the effects of temperature.\n\nAlthough the scaling behavior of embryos grown at different oxygen concentrations has not been previously characterized, there have been extensive studies of the effect of oxygen on the D. melanogaster embryo. In normal development, oxygen sensation plays a crucial role in cellular differentiation, organogenesis, and growth rate. It is known to influence Notch, Wnt, and OCT4 pathways3 and at low levels slows growth by driving components of the Tor pathway4,5. It is critical for hematopoiesis6, myogenesis7,8, and notochord and liver formation in vertebrates9,10.\n\nHypoxic Drosophila syncytial embryos arrest at a metaphase checkpoint11 and resume development under normoxia if the hypoxic period is not too long12. Cellularized embryos survive longer hypoxic periods, up to several days13. However, hypoxic arrest is not entirely benign, as even brief periods of hypoxia lead to smaller bodies and wings, driven in part by decreased cell size14–16. Active oxygen sensing and nitric oxide signaling drive this arrest, which is independent of the electron transport chain13,17,18. Hypoxia tolerance also varies between tissues19 and possibly between stages of embryonic development. Hyperoxia, on the other hand, is toxic20,21 and drives malformation of mitochondria22.\n\nThe response of the D. melanogaster embryo to oxygen is highly conserved, in both function and molecular mechanism21,23–25. Drosophila, like other animals, regulate metabolism and gene expression in response to changes in oxygen levels through the HIF-1α pathway, which communicates with the Tor and VEGF pathways. Under normal conditions, proline residues of simalar (sima/hif-1/HIF-1α) are hydroxylated by prolyl hydroxylase (Hph/egl-9/EGLN) to both inactivate sima/hif-1/HIF-1α and target it for Vhl-dependent degradation19,26. The prolyl hydroxylase Hph/egl-9 is itself negatively regulated under hypoxia by the cystathionine β-synthase Cbs/cysl-1/CBS, an ambient oxygen sensor via hydrogen sulfide signaling. sima has an oxygen-dependent degradation domain with a nuclear export sequence27. Thus, only during hypoxia does sima escape degradation and accumulate in the nucleus26. Rather than serving as a switch, the process is dynamic, with greater levels of oxygen accelerating both the degradation and nuclear export of sima27.\n\nOver the course of development, changes in insulin levels, the metabolic state of the embryo, and temperature may impact the oxygen response24,28–31, either directly or through its dependence on transcription, nuclear-import and export, prolyl hydroxylation and Vhl-dependent degradation32.\n\nHere we use time-lapse imaging of embryos under a range of oxygen concentrations with precise temperature control to monitor the effects on developmental timing and morphology. In covering hypoxic through hyperoxic and warm through cold conditions, we have collected dynamic data on how the developing embryo responds to oxygen, and how that response is affected by temperature.\n\n\nMethods\n\nDrosophila melanogaster, OreR, were reared and maintained on standard fly media at 25°C. Egg-lays were performed in medium cages on 10 cm molasses plates for 1.5 hours at the temperature at which the lines were maintained after pre-clearing. Embryos were collected and dechorionated with fresh 50% bleach solution (3% hypochlorite final) for 60 seconds in preparation for imaging.\n\nEmbryos were monitored by modifying a temperature control system1 in which an aluminum bar was embedded in an acrylic box (TAP Plastics). Both ends of the aluminum bar were external to the box and bound to Peltier heat pumps and heat sinks. A thermistor connected to the aluminum bar provided feedback to maintain the temperature using an H-bridge temperature controller (McShane Inc., 5R7-570). Embryos were glued33 to oxygen-permeable film (lumox, Greiner Bio-one), covered with Halocarbon 700 oil (Sigma), and placed over holes drilled in the aluminum for imaging. An oxygen sensor (Grove Gas sensor (O2)) was placed in the box and connected to an external computer (Arduino-style Seeeduino V3.0 (Atmega 328P)). Finally, the box was sealed with two gas inputs and an over-pressure release. The computer utilized the oxygen sensor input and controlled two valves via NPN transistors, one connected to an oxygen tank and regulator and one connected to a nitrogen tank and regulator, to maintain specific oxygen concentrations in the box (Figure 1A).\n\nTime-lapse imaging with bright field transmitted light was performed on a Leica M205 FA dissecting microscope with a Leica DFC310 FX camera using the Leica Advanced Imaging Software (LAS AF6000 version 2.3.5) platform. Greyscale images were saved from pre-cellularization to hatch. Z-stacked images were saved every two minutes (five minutes at 17.5°C). Analysis data available from http://dx.doi.org/10.6084/m9.figshare.1572474 and imaging data available from http://dx.doi.org/10.6084/m9.figshare.1582639.\n\nZ-stack and image analysis were conducted as previously described1. Events selected for measurement (pole-bud appearance, membrane reaching yolk, pole cell invagination, amnioproctodeal invagination, amnioserosa exposure, clypeolabrum retraction, clypeolabrum and ventral lobes being even, heart-shaped midgut, and the filling of the trachea) were identified by hand using a graphical user interface. Oxygen dependent trends were analyzed with least-squares regression. Significant differences between events in their response to oxygen changes were determined by comparing the pooled estimate of the variation about the regression line using a t-test with a Bonferonni multiple testing correction. For modeling total developmental response to oxygen and temperature changes, least-squares regression was used based on linear, exponential, logarithmic, polynomial (up to cubic), and inverse proportional models, with the models consistently yielding the best Pearson product-moment correlation coefficient being selected. For the combined effect of both oxygen and temperature, all possible combinations of exponential and inverse-proportional models identified for each component were attempted with least squares surface regression. The curve fit with the best adjust correlation coefficient (R¯2) across all available data was selected. All scripts are available at github.com/sgkuntz/OxygenCode.\n\n\nResults\n\nWe used automated time-lapse imaging in an airtight box with oxygen concentration control (±1%) and precise temperature control (±0.1°C) to track development using previously described methods1. We investigated embryos raised at constant oxygen concentrations (29%, 25%, 21%, 17%, 14%, and 10% O2) and kept at three different temperatures (17.5°C, 22.5°C, and 27.5°C), giving a total of eighteen specific conditions with over 800 embryos. A schematic of the setup is provided in Figure 1A. The actual setup is shown in Figure 1B.\n\nIn agreement with previous research, developmental rate correlates with oxygen concentrations (Figure 1C). Hyperoxia accelerates development, allowing embryos to hatch sooner than they would under normal atmospheric conditions. Hypoxia slows development in a dose-dependent fashion. As oxygen levels fall, an increasing fraction of embryos die or arrest their development. Therefore, there are fewer embryos shown in Figure 1C at lower oxygen concentrations due to low rates of successful development, despite similar numbers of animals being prepared for imaging (Table S1).\n\n(A) The oxygen control schematic. A thermistor embedded in the aluminum bar provides temperature data to the temperature controller, which in turn adjusts the voltage to the thermo-electric controllers (Peltier). An oxygen sensor in the airtight box provides feedback on oxygen concentrations to the gas controller, which opens and closes oxygen and nitrogen valves accordingly. Embryos are imaged in the center of the aluminum bar within the airtight box, indicated by the black dots in the schematic. (B) Image of the oxygen control setup mounted on the microscope at 20% oxygen and 17.5°C. (C) Developmental rate across all stages changes with the oxygen concentration, performed at 27.5°C. Each animal is represented with a dot, with averages represented with a large diamond. Developmental times here are zeroed on the end of cellularization.\n\nBy tracking and analyzing nine morphological stages as oxygen concentrations change, we identified significant differences in scaling between major morphological events. While all morphological events speed up with increasing oxygen concentrations (Figure 1C), their changes in speed are notably different. Syncytial development, as measured by the time between the appearance of the pole bud and the end of cellularization, takes proportionally less time as oxygen concentrations decrease, indicating that this stage is not slowed as much by decreasing oxygen (Figure 2A). The stages of gastrulation (end of cellularization, pole cell invagination, and amnioproctodeal invagination) are relatively uniformly affected. Germ band retraction, as measured by amnioserosa exposure, tracks subtly but inversely with syncytial development. More striking are the oxygen-dependent changes observed in head involution (clypeolabral retraction and advancing of the ventral lobe to match the clypeolabrum) and gut formation (heart-shaped midgut). While head involution takes proportionally more time as oxygen levels increase—meaning it does not slow as much as overall development in hypoxia—gut formation does the opposite. The midgut takes proportionally less time to form as oxygen levels increase, meaning it responds more strongly to increases in oxygen than overall development. This juxtaposition of behaviors leads to an inversion of when the cephalic lobes are even versus heart-shaped midgut formation. While hypoxia leads to head involution stages finishing first, hyperoxia results in the heart-shaped midgut forming first.\n\nSurprisingly, the point of inversion varies with temperature (Figure 2). At 27.5°C, the inversion takes place at 29% oxygen, while at 17.5°C the inversion falls around 19% oxygen. This may be due to an overall shift in the oxygen response curve of heart-shaped midgut formation to proportionally later in development as temperatures fall. Supplementary Figure S1 reveals how each stage of development at each oxygen concentration changes with temperature.\n\nOxygen levels affect the stage at which embryos arrest or die. Higher concentrations of oxygen (29%) lead to more animals dying during early development, including death in the syncytium and a failure to properly gastrulate. This point of failure is similar to that observed at high temperatures with normal oxygen levels1. Lethality at 25% oxygen is actually lower than that at 21%, which approximates atmospheric levels. Problems with development may be aggravated by the dechorionation and mounting procedure. At high temperatures (32.5°C) and high oxygen (29%), almost all embryos die very early in development (Table S1).\n\nAt lower oxygen levels there is a major shift from very early developmental arrest and death to mid-embryogenesis arrest (Figure 3). This holds true at all temperatures (especially at 10% O2), but is most pronounced at 27.5°C, where the effects are still seen at 14% O2. Frequently development halts during germ band retraction, preventing full exposure of the amnioserosa. The midgut primordia in these embryos routinely migrates haphazardly after arrest, coinciding with the embryo falling into morphological disarray. In embryos that pass these mid-embryogenesis stages, trachea formation often proves problematic. Commonly the trachea fails to form, which coincides with arrest late in midgut formation, following the heart-shaped midgut stage. These animals generally form functional muscle, with some twitching observed.\n\nGut formation and head involution are the most strikingly oxygen concentration dependent processes, but germ band retraction and syncytial development are also affected. Both syncytial development and head involution take proportionally more time as oxygen concentrations increase. Gut formation and germ band retraction, in contrast, take proportionally less time as oxygen concentrations are raised. These trends hold true across all temperatures, though the rates of change as a function of oxygen do vary. Development is normalized here between the end of cellularization and the filling of the trachea.\n\nHigher, but still hypoxic, oxygen levels (14% and 17%) have a significant fraction of embryos that fail to hatch. While embryonic development appears to be completed, including the filling of the trachea with air, larvae struggle to break out of their vitelline membrane yet fail to escape. While seen in all conditions, this behavior is most prevalent in these mildly hypoxic conditions.\n\nDecreasing oxygen concentrations from 29% to 10% at any temperature lead to an additional sixteen to eighteen hours of embryogenesis (Figure 4A). This results in a different proportional change at each temperature, with nearly a 100% increase at 27.5°C and only a 50% increase at 17.5°C. This contrasts with changes in temperature, where developmental time roughly doubles over a 10°C range, regardless of the oxygen concentration (Supplementary Figure S2).\n\nChanges in oxygen concentration have an inverse proportional effect on developmental rate. Least squares curve fitting was attempted with multiple models, including exponential models, for changes in oxygen concentration. The data most closely matched a model based on the Monod equation. The parameters of the response for embryogenesis as a whole change significantly with temperature, however the qualitative response is the same (Figure 4A):\n\nLethality at different concentrations are shown for three different temperatures (27.5°C, 22.5°C, and 17.5°C). Lower concentrations of oxygen are more likely to exhibit failure during pre-tracheal development, with a particularly large increase in mortality between gastrulation and completion of the heart-shaped midgut (shown in red). A substantial increase in late development before trachea fill is also seen (shown in orange). Developmental arrest is frequently at germ band retraction. This is in contrast with higher oxygen concentrations, where failure is almost exclusively very early in development (shown in brick red), prior to the completion of gastrulation, or during difficulties hatching following trachea filling (shown in yellow). Highest survival is interestingly at 25% oxygen.\n\nt17.5=280.92[O2]+30.13t22.5=167.39[O2]+17.58t27.5=204.03[O2]+8.55\n\nFitting at each oxygen concentration (Supplementary Figure S2) yields relatively good fits using an exponential Arrhenius model. These different methods of fitting can be combined and yield the best fit as a multivariable non-additive model. The overall effect of oxygen and temperature can be combined to yield (Figure 4B):\n\n(A) Fitting at different temperatures (27.5°C, 22.5°C, 17.5°C). The shift is very temperature-dependent. The solid red line represents a fit at each particular temperature, with 90% confidence of reproduction marked with the dashed orange line. The blue dashed line represents the fit across all temperatures, which departs from the individual temperature fits. (B) Total developmental time is affected by both temperature and oxygen levels. Each point represents an individual embryo at a given temperature and oxygen level. Color contours help visualize the transitions of increased heat (yellow to red contour) and increased oxygen (purple to blue contour).\n\nt=e0.47[O2]+31T(65.407[O2]+1.0)\n\nBased on the fit, oxygen appears to have an effect on both the linear and exponential coefficients. This model is empirical and does not predict effective oxygen concentration as a function of temperature-dependent changes in oxygen solubility and diffusion. Increased oxygen may allow some additional growth acceleration, but the acceleration of growth rate appears to be leveling off, asymptotically approaching a maximum. At lower oxygen levels, the prevalence of arrest is expected to overtake the observed response curve.\n\n\nDiscussion\n\nWe tracked embryogenesis at different oxygen concentrations to determine its effect on development, performing these experiments in conjunction with precise temperature control. We found that developmental rate is highly dependent on oxygen and exhibits a complex relationship with temperature. Embryos are not as robust to oxygen changes and have much less of a dynamic response than is seen with temperature. We observed significant differences in oxygen responsiveness across tissues and morphological events. These changes can be aggravated by temperature (long known to interact with oxygen consumption24,34) to reveal situations in which embryogenesis loses its uniform thermal scaling.\n\nThe prevalence and timing of developmental failure depend strongly on oxygen. Under hypoxia, failure is largely concentrated in mid-embryogenesis at germ band retraction. Commonly the germ band fails to fully retract to expose the amnioserosa. It is possible that this stage either requires more oxygen or its complexity makes it prone to failure. A checkpoint at this stage that hypoxic embryos fail to pass may explain this phenotype. Rapid hypoxic arrests are not frequently observed under our conditions. Oxygen concentrations of 10% and 14% may fail to trigger complete hypoxic arrest in a subset of embryos yet serve to slow development enough to cause problems. Increasing oxygen levels would likely restart development, but the manner in which it restarts would depend on the stage of arrest, duration of hypoxia, and revived oxygen levels. Thermal tolerance has been previously linked to oxygen concentration34–36. Likewise, we see increased hypoxic mortality with increased temperature.\n\nHypoxia’s mid to late embryogenesis failure contrasts with high heat and high oxygen, where failure occurs during early development, during either the syncytium or early gastrulation. Under conditions with high oxygen tension, death frequently resembles, at least qualitatively, high temperature normoxia death. Failure during syncytial development commonly involves mass migration of nuclei throughout the embryo, making it difficult to distinguish the point of failure between pre-gastrulation death resulting in nuclei migration and premature gastrulation that causes death.\n\nThe syncytium responds differently to oxygen levels than other embryonic stages. Perfect scaling collapses in the syncytium at high temperatures1, so it is not surprisingly that a difference is seen with the oxygen response as well. Interestingly, while syncytial development is less responsive to changes in oxygen than other stages across the range we tested, it is more responsive to excess heat than other stages. The difference may be aggravated by the lack of transcriptional responses available at that stage and the limited repertoire of maternally deposited genes and mRNAs. This may lead to the syncytium lacking high heat mitigations and prophylactic hypoxic responses. This implies that transcriptionally active embryos deliberately slow development either under high heat when kinetics are accelerating or under hypoxia to conserve energy.\n\nThe developmental rate response to oxygen is more subtle, yet causes more problems, across the range we tested than is seen with a moderate change in temperature. We measured ambient oxygen, meaning the exact oxygen concentration in the embryo microenvironment may differ. Across a 10°C differential, developmental time doubles with minimal change in viability. This is virtually invariant, regardless of oxygen concentrations. However, across a 19% change in oxygen concentration, development time experiences an absolute, rather than proportional, change of sixteen to eighteen hours. Changes in oxygen thus provide a proportionally smaller change in developmental time with enormous consequences for viability. While changes in temperature follow the Arrhenius equation, changes with oxygen appear to follow Monod’s equation. Rather than a logarithmic curve, developmental time is inversely proportional to oxygen concentration. This comparatively shallow oxygen response undermines the hypothesis, which had previously been refuted for thermal limits37,38, that oxygen availability explains temperature-dependent changes. Changes in temperature will affect oxygen diffusion in the embryo, with a 10°C change shifting the effective oxygen concentration by ∼4%. However, the difference in developmental time between 21% and 17% oxygen at 27.5°C is dwarfed by the dramatically larger difference between 27.5°C and 17.5°C at 21%. Therefore, basic energy metabolism is not solely responsible for the changes in developmental rates seen across temperature. Our results show that the embryo’s developmental program is robust to small changes in oxygen. This suggests some leeway in respiration efficiency; nevertheless, there is a notable biological response.\n\n\nData availability\n\nFigshare: Raw data for Kuntz and Eisen, 2015 ‘Oxygen changes drive non-uniform scaling in Drosophila melanogaster embryogenesis’. 10.6084/m9.figshare.158263939\n\nFigshare: Oxygen imaging. 10.6084/m9.figshare.157247440",
"appendix": "Author contributions\n\n\n\nConceived and designed the experiments: SGK MBE. Performed the experiments: SGK. Analyzed the data: SGK. Algorithms used in analysis: SGK. Contributed reagents/materials/analysis tools: SGK MBE. Wrote the paper: SGK MBE.\n\n\nCompeting interests\n\n\n\nMBE is on the International Advisory Board for F1000.\n\n\nGrant information\n\nThis work was supported by a Howard Hughes Medical Institute investigator award to MBE and by NIH grant HG002779 to MBE. SGK was supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award (F32-FGM101960A) from the National Institute of General Medical Sciences.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nStocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. We thank BCK and EDKK for their support.\n\n\nSupplementary material\n\nAs oxygen concentrations decrease, gut-development is proportionally delayed, as can be seen with the timing of the heart-shaped midgut shifting later in development. Proportional changes in development are more severe at higher temperatures. At very low oxygen concentrations (10% oxygen), development becomes highly irregular. In many animals, development ceases during germ band retraction at very low oxygen concentrations. Development is normalized here between the end of cellularization and the filling of the trachea.\n\nCurve fitting at each oxygen concentration is shown with the red line, with the 90% confidence interval delineated by the orange dashed line. The overall curve fitting is identified with the blue dashed line. Differences between different oxygen concentrations are much smaller than the range covered by temperature changes. The change in developmental time from high to low temperatures remains proportional across oxygen concentrations.\n\n\nReferences\n\nKuntz SG, Eisen MB: Drosophila embryogenesis scales uniformly across temperature in developmentally diverse species. PLoS Genet. 2014; 10(4): e1004293. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCruz SR, Romanoff AL: Effect of oxygen concentration on the development of the chick embryo. Physiol Zool. 1944; 17(2): 184–187. Reference Source\n\nSimon MC, Keith B: The role of oxygen availability in embryonic development and stem cell function. Nat Rev Mol Cell Biol. 2008; 9(4): 285–296. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReiling JH, Hafen E: The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila. Genes Dev. 2004; 18(23): 2879–2892. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nDiGregorio PJ, Ubersax JA, O’Farrell PH: Hypoxia and nitric oxide induce a rapid, reversible cell cycle arrest of the Drosophila syncytial divisions. J Biol Chem. 2001; 276(3): 1930–1937. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeinrich EC, Farzin M, Klok CJ, et al.: The effect of developmental stage on the sensitivity of cell and body size to hypoxia in Drosophila melanogaster. J Exp Biol. 2011; 214(Pt 9): 1419–1427. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarrison JF, Shingleton AW, Callier V: Stunted by developing in hypoxia: Linking comparative and model organism studies. Physiol Biochem Zool. 2015; 88(5): 455–470. Publisher Full Text\n\nPeck LS, Maddrell SH: Limitation of size by hypoxia in the fruit fly Drosophila melanogaster. J Exp Zool A Comp Exp Biol. 2005; 303(11): 968–975. PubMed Abstract | Publisher Full Text\n\nWingrove JA, O’Farrell PH: Nitric oxide contributes to behavioral, cellular, and developmental responses to low oxygen in Drosophila. Cell. 1999; 98(1): 105–114. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeodoro RO, O’Farrell PH: Nitric oxide-induced suspended animation promotes survival during hypoxia. EMBO J. 2003; 22(3): 580–587. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArquier N, Vigne P, Duplan E, et al.: Analysis of the hypoxia-sensing pathway in Drosophila melanogaster. Biochem J. 2006; 393(Pt 2): 471–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams CM, Beecher HK: Sensitivity of Drosophila to poisoning by oxygen. Am J Physiol. 1944; 140: 566–573. Reference Source\n\nClark AM, Herr EB Jr: The sensitivity of developing Habrobracon to oxygen. Biol Bull. 1954; 107(3): 329–334. Publisher Full Text\n\nWalker DW, Benzer S: Mitochondrial \"swirls\" induced by oxygen stress and in the Drosophila mutant hyperswirl. Proc Natl Acad Sci U S A. 2004; 101(28): 10290–10295. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Farrell PH: Conserved responses to oxygen deprivation. J Clin Invest. 2001; 107(6): 671–674. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRogers E: The effect of temperature on the oxygen consumption of an insect, Melano-plus differentialis. Physiol Zool. 1929; 2(2): 275–283. Reference Source\n\nGarside ET: Some effects of oxygen in relation to temperature on the development of lake trout embryos. Can J Zool. 1959; 37(5): 689–698. Publisher Full Text\n\nLavista-Llanos S, Centanin L, Irisarri M, et al.: Control of the hypoxic response in Drosophila melanogaster by the basic helix-loop-helix PAS protein similar. Mol Cell Biol. 2002; 22(19): 6842–6853. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIrisarri M, Lavista-Llanos S, Romero NM, et al.: Central role of the oxygen-dependent degradation domain of Drosophila HIFalpha/Sima in oxygen-dependent nuclear export. Mol Biol Cell. 2009; 20(17): 3878–3887. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDekanty A, Lavista-Llanos S, Irisarri M, et al.: The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima. J Cell Sci. 2005; 118(Pt 23): 5431–5441. PubMed Abstract | Publisher Full Text\n\nVan Voorhies WA: Metabolic function in Drosophila melanogaster in response to hypoxia and pure oxygen. J Exp Biol. 2009; 212(19): 3132–3141. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClare MR: A study of oxygen metabolism in Drosophila melanogaster. Biol Bull. 1925; 49(6): 440–460. Publisher Full Text\n\nTennessen JM, Thummel CS: Coordinating growth and maturation - insights from Drosophila. Curr Biol. 2011; 21(18): R750–R757. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe Renzis S, Elemento O, Tavazoie S, et al.: Unmasking activation of the zygotic genome using chromosomal deletions in the Drosophila embryo. PLoS Biol. 2007; 5(5): e117. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTechnau GM: Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster. I. The method. Roux’s Arch Dev Biol. 1986; 195(6): 389–398. Publisher Full Text\n\nFrazier MR, Woods HA, Harrison JF: Interactive effects of rearing temperature and oxygen on the development of Drosophila melanogaster. Physiol Biochem Zool. 2001; 74(5): 641–650. PubMed Abstract | Publisher Full Text\n\nBjelde BE, Miller NA, Stillman JH, et al.: The role of oxygen in determining upper thermal limits in Lottia digitalis under air exposure and submersion. Physiol Biochem Zool. 2015; 88(5): 483–493. Publisher Full Text\n\nLiang L, Sun BJ, Ma L, et al.: Oxygen-dependent heat tolerance and developmental plasticity in turtle embryos. J Comp Physiol B. 2015; 185(2): 257–263. PubMed Abstract | Publisher Full Text\n\nMcCue MD, De Los Santos R: Upper thermal limits of insects are not the result of insufficient oxygen delivery. Physiol Biochem Zool. 2013; 86(2): 257–265. PubMed Abstract | Publisher Full Text\n\nMölich AB, Förster TD, Lighton JR: Hyperthermic overdrive: oxygen delivery does not limit thermal tolerance in Drosophila melanogaster. J Insect Sci. 2012; 12: 109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuntz S, Eisen M: Raw data for Kuntz and Eisen, 2015 ‘Oxygen changes drive non-uniform scaling in Drosophila melanogaster embryogenesis’. Figshare. 2015. Data Source\n\nKuntz S: Oxygen imaging. Figshare. 2015. Data Source"
}
|
[
{
"id": "10904",
"date": "10 Nov 2015",
"name": "Sofia Lavista Llanos",
"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\nMotivation:Authors have previously shown for various Drosophila species, that the relative timing of events within embryogenesis is temperature indifferent (ref 1 in manuscript). To test if this uniform scaling with varying temperature is an intrinsic property of developing embryos, or rather a specific response to thermal fluctuations, authors soughed a temperature-independent manner to manipulate the embryogenesis development rate. Proposed study:Authors studied the development scale of embryos grown at different oxygen concentrations and its interaction with varying temperatures. Main result found:In contrast to the uniform scaling of development with temperature variation, authors found heterochrony of the developmental events with changes in oxygen levels. There was an interaction between temperature and oxygen. Conclusions:These data reveal that the uniform scaling seen with changes in temperature is not a trivial consequence of adjusting developmental rate. Authors inaccurately raise conclusions on the behaviour of developing embryos with fluctuating temperature (referred as to the previous study), based on experiments performed with varying oxygen in the present study. Instead, the data presented in this study supports the hypothesis that a uniform scaling of development is not an intrinsic property of developing embryos. Rather, embryos adjust their developmental program in different ways depending on variable ambient conditions. Relevance of the study:Considering the importance of studies on tissue and cellular responses to hypoxia, for human health (i.e.: tumour growth and cardiovascular disease, among others) this study could potentially become of great impact. Control aspects that should be addressed:Authors assume that O2 is delivered uniformly through the embryo, as most probably temperature does. Are the results here obtained simply a consequence of a differential diffusion of O2 through the embryonic tissues?Does temperature affect O2 diffusion, explaining the interaction between both variables here shown?Is there an effect of nitrogen changing concentration in the hypoxia treatments?Derived biological questions & suggestions:Is the oxygen dependency of developmental rate conserved in the different Drosophila species?The non-uniform scaling of development in hypoxia might indicate that oxygen plays a signalling role during the developmental process (see Jarecki et al., 1999).Might mild physiological hypoxia in embryonic deep tissues explain the differential reluctance to oxygen deprivation of different tissues?Is the embryonic developmental program a coordinated hierarchical chain of events, being the chronological appearance of each one dependent on the occurrence of the previous event? Or, rather, is the embryonic developmental program a sum of independent events each following an autonomous synchrony and/or independent scaling that adjusts to the ambient conditions? Or a combination of both? These aspects could actually be addressed in the manuscript based on the results obtained with varying temperature (previous study) and varying oxygen (results shown here).Authors could put their results into an ecological context, comparing species with different habitats and ovipositon sites.These studies were done on de-chorionated embryos. Authors could address the fact that chorion might serve as a physiological barrier to fluctuating oxygen concentrations, which would prevent desynchronising the embryonic development with varying ambient conditions.Proposed experiments:To reinforce the evidence of the role of oxygen/hypoxia on the development scale, authors could use mutants of the oxygen cellular sensing machinery (i.e.: Hph (CG31543), sima (CG7951); see Centanin et al., 2005) in time-lapse imaging experiments. Such experiments could serve to rule out a mere physic-chemical effect of hypoxia on the embryonic development scale and at the same time test if oxygen has an active signalling role during embryogenesis.Authors could follow with fluorescent markers the dividing cells (e.g.: bromouridine) and or apoptotic cells (e.g.: TUNEL) during development at varying oxygen levels to get an insight on the cellular/molecular mechanisms sustaining the adjustment of the development to varying oxygen or temperature fluctuations.Authors could try to address the embryonic internal oxygen concentrations by indirect labelling of populations of synchronized embryos (30 min egg-laying) fixed after the relevant oxygen/temperature treatments (e.g.: lac-Z/GFP staining using hypoxia reporter genes, NADPH diaphorase staining).To test if the presence of chorion affects the response to varying oxygen, authors could repeat the experiments with synchronized chorioned eggs (30 min egg-laying) & immediately dechorionated + fixed after treatment. This would test if indeed the ambient variation in oxygen affects development in nature.Having overcome the challenge of obtaining developmental staged-matched samples for different species in their previous study (ref 1 in manuscript), authors could include different species to complement this study. Minor comments:Introduction paragraph-5 line-6: simalar should read ‘similar’.Methods: Egg-lays were performed in medium cages on 10 cm molasses plates for 1.5 hours at the temperature at which the lines were maintained after pre-clearing. This phrase is not clear. Were adult flies maintained at the same temperature as eggs? Does this affect the stage at which eggs are laid? With 1.5 hours collection eggs have an error of ± 45 minutes, which at the beginning of embryogenesis might have huge effects on development.How was the proportion of development calculated? Data quantification methods should be clearly detailed in methods section.Figure 1: Y axis is not in scale. Authors should also indicate in the figure legend the number of eggs (n) for each experiment.Results paragraph-3 line-5: Syncytial development, as measured by the time between the appearance of the pole bud and the end of cellularization, takes proportionally less time as oxygen concentrations decrease, indicating that this stage is not slowed as much by decreasing oxygen.The event ‘end of cellularization’ is not shown on figure 1C legend.Authors might want to re-phrase the passive voice in ‘indicating that this stage is not slowed as much by decreasing oxygen’ for ‘ indicating that decreasing oxygen does not slow this stage’.I don’t see the dependence of one argument with the other. Authors might want to rephrase the entire argument.Results paragraph-3: The events mentioned in the text do not correspond to those shown in the figure legend. Authors might want to make it easier for the reader to follow their descriptions by choosing a uniform way of representing the data and referring to it in the text.Figure 2: Legend reference does not correspond to the events described in the figure legend, making difficult for the reader to follow the arguments.Table S1: legend missing; it might aid clarification to provide a legend to this table.Figure S1: Title is not clear: Changes in stages react differently as temperature changes. What do they react to?Figure S1: No reference legend is provided!Discussion pagraph-2 line-6: A checkpoint at this stage that hypoxic embryos fail to pass may explain this phenotype. Phrase not clear.Discussion: pagraph-5 line-3: Authors might suggest experiments to grasp this aspect.Discussion: final conclusion does not correspond to the aim stated at the beginning of the manuscript.Final comment: This study addresses an important topic relevant to biological and medical fields. Despite the fact that the conclusions raised are rather superficial, and regrettably inadequate to the objective aimed in the first place after major revision (!), this manuscript could make an important contribution to Drosophila embryogenesis and, more general, to homeostasis of animal development.",
"responses": []
},
{
"id": "11218",
"date": "16 Nov 2015",
"name": "Angela H. DePace",
"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 assesses the effect of oxygen level and temperature on developmental timing in Drosophila embryos. The methods are adequately described to interpret the experiments. Oxygen and temperature are controlled externally in a custom built apparatus. Developmental timing is assessed by time lapse imaging under bright field; developmental milestones were manually annotated from this data. The statistical analysis used to compare across conditions is adequately described. This experimental set up does not make any internal measurements of oxygen level or temperature, which may be of interest for future studies, but this is a substantial technical undertaking. The conclusions are primarily descriptive—the relative duration and timing of developmental stages varies in a non-uniform way with oxygen level and the effect depends on temperature. This result contrasts with the effect of varying temperature alone, where developmental stages maintain precise relative timing despite variation of the overall time of development, as described in a previous study by the same authors. The authors also pinpoint which stages and tissues are most sensitive to changes in oxygen level. These phenomenological conclusions will be useful for future studies. There are also some secondary interpretations based on their phenomenological observations. First, because the blastoderm stage is more responsive to changes in temperature than other stages, but less responsive to changes in oxygen, the authors speculate that this is because blastoderm embryos have a limited ability to mitigate heat stress via transcription of new genes or translation of existing mRNA. Reciprocally, they state that “transcriptionally active embryos” (I read this to mean non-blastoderm embryos) “deliberately slow development either under high heat when kinetics are accelerating or under hypoxia to conserve energy.” In my opinion, this interpretation doesn’t immediately follow from the previous observation about the heat and oxygen sensitivity of blastoderm embryos. It is also not clear what the authors mean by “deliberately”; perhaps they mean that the embryos have pathways dedicated to a regulated response to these conditions, rather than the effect arising passively from global biochemical parameters. Second, the authors state that the thermal response is not due to changes in oxygen delivery as the two responses have distinct dynamics and phenotypic outcomes. This generalizes previous results about oxygen availability under extreme temperatures, as pointed out by the authors. In my view, this interpretation is sound. In my opinion, one of the most interesting directions for this line of research is how variation in temperature and oxygen level are sensed and how responses are executed. In the previous paper, the authors stated that they expected non-uniform scaling in response to temperature, because chemical reactions are known to exhibit different temperature sensitivity. Under this line of thinking, pathways specific to distinct developmental stages would respond differently to temperature, resulting in heterochrony. Instead, they do find uniform scaling of developmental timing in response to temperature. In this work, specific pathways are known to have differential sensitivity to oxygen (as described in the introduction), and indeed they observe heterochrony. What underlies this difference? Are developmental pathways not differentially sensitive to temperature (or stated reciprocally, do they all respond to temperature the same way)? Or are different molecular or pathway level effects of temperature buffered by other systems? Both of these are interesting possibilities. Deciphering whether uniform temperature sensitivity and/or buffering are under selection will also be exciting.",
"responses": []
},
{
"id": "11301",
"date": "23 Nov 2015",
"name": "Pablo Wappner",
"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\nKuntz and Eisen have analysed the effect that variations of oxygen concentrations and temperature exert on Drosophila embryonic development. A previous study from the same group had shown that temperature variations affect embryogenesis preserving the order and relative duration of a list of morphological events during this developmental process. The question addressed in this work is as whether variations of oxygen levels also preserve the order and relative duration of events during embryogenesis. They have designed and built a special device to monitor embryogenesis while controlling oxygen levels and temperature simultaneously. The work is technically sound and the results are truly unexpected: The different morphological events of the embryogenesis are not affected by oxygen levels to a similar extent, but rather, each morphological event responds to such variations in an unequal manner. Strikingly, the order in which some of the morphological events occur is altered when oxygen levels are modified. These effects are modulated by the temperature, so that the order and relative duration of each of the events of the embryogenesis is independently modulated by oxygen and temperature simultaneously. The approach and the whole concept is highly original and adds a new dimension to our understanding of metazoan development that paves the road for further studies aimed to the definition of the molecular and genetic mechanisms that underlie this phenomenon.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1102
|
https://f1000research.com/articles/4-159/v2
|
23 Jul 15
|
{
"type": "Case Report",
"title": "Case Report: Severe acute respiratory distress by tracheal obstruction due to a congenital thyroid teratoma.",
"authors": [
"Jose Colleti Junior",
"Uenis Tannuri",
"Felipe Monti Lora",
"Eliana Carla Armelin Benites",
"Walter Koga",
"Janete Honda Imamura",
"Patricia Rute Moutinho",
"Werther Brunow de Carvalho",
"Uenis Tannuri",
"Felipe Monti Lora",
"Eliana Carla Armelin Benites",
"Walter Koga",
"Janete Honda Imamura",
"Patricia Rute Moutinho",
"Werther Brunow de Carvalho"
],
"abstract": "Congenital teratoma is a rare condition and is a germ cell tumor composed of elements from one or more of the embryonic germ layers and contain tissues usually foreign to the anatomic site of origin. We report a case of a neck tumor diagnosed during pregnancy, initially thought to be a goiter. After birth the neck mass kept growing until it compressed the trachea and produced respiratory failure. The infant had a difficult tracheal intubation because of the compressing mass. The staff decided to surgically remove the neck mass. After that, the infant became eupneic. The histological analysis showed a mature teratoma with no atypias.",
"keywords": [
"thyroid teratoma",
"pediatric neck mass",
"pediatric respiratory distress",
"hypothyroidism",
"children case report"
],
"content": "Introduction\n\nCongenital thyroid teratoma is a rare condition1,2. We report a case of an infant with a neck mass diagnosed by ultrasound during pregnancy which was initially supposed to be a congenital goiter. Two doses of levothyroxine were administered into the amniotic fluid. The goiter kept growing after birth until it caused severe respiratory distress by compressing the trachea, necessitating immediate tracheal intubation. The tumor was surgically resected and the patient went eupneic for the first time in his life. The histological analysis demonstrated a mature teratoma with no atypias. Thyroid hormone substitute therapy was started and the infant is thriving well.\n\n\nCase report\n\nA 2-months-and-20-days-old Brazilian white male infant weighing 4.2 kg was admitted to the pediatric intensive care unit of our hospital (Santa Catarina Hospital, São Paulo, Brazil) in acute respiratory distress and was immediately intubated and placed in mechanical ventilation.\n\nFrom a routine ultrasound during pregnancy, the fetus had been diagnosed with a cervical mass, considered initially to be a goiter (Figure 1) by doctors at another institution. Family history of the mother uncovered a cousin with hypothyroidism. The mother was previously healthy, but after diagnosis of the cervical mass of the fetus, she was tested for thyroid hormones and had hypothyroidism diagnosed during pregnancy (TSH: 5.0 mUI/mL – normal: 0.2 to 3.0 mUI/mL; free T4: 0.7 ng/dL – normal: 0. To 1.3 ng/dL; antithyroglobulin antibodies: 65 U/mL – normal: inferior to 60 U/mL and thyroid antiperoxidase antibodies: 166 UI/mL – normal: inferior to 9 UI/mL). Two single doses of 200µg of levothyroxine were administered into the amniotic fluid, one during the 28th and one during the 31st week of pregnancy, in order to treat the supposed fetal thyroid hormone deficiency. Chorioamnionitis appeared after the second levothyroxine administration which triggered a premature cesarean birth which was undertaken in the other hospital. The premature newborn had sepsis due to maternal infection (chorioamnionitis) and remained in mechanical ventilation for 10 days. After tracheal extubation, he remained in nasal continuous positive airway pressure (CPAPn) for 7 more days, and after that was kept on oxygen therapy for 10 days. He was discharged from the hospital 50 days after birth, still presenting with a laryngeal stridor that was attributed to tracheal malacia by the doctors that initially treated the patient.\n\nAfter hospital discharge, he was observed by a pediatric endocrinologist who started research on thyroid disorders. Meanwhile, the infant maintained a euthyroid state, receiving no treatment, waiting for more investigation on the cause of the neck mass. However, the cervical mass kept visibly growing, was palpable and the infant presented a laryngeal stridor that was still attributed, by the pediatrician who followed the infant, to laryngomalacia. In the few days preceding hospitalization at our institution, the infant became increasingly dispneic each day, as related by his mother. One day, after choking and vomiting during breastfeeding he became hypotonic and went into acute respiratory distress.\n\nHe was admitted to our pediatric intensive care unit 25 days after he had been discharged from the other hospital, and was immediately intubated. An X-ray showed a small amount of interstitial infiltrate, compatible with aspiration pneumonia. However, the respiratory distress was attributed mainly to an upper airway obstruction. It was difficult to tracheally intubate the infant; only an uncuffed 2.5 mm endotracheal tube (ETT) was able to be inserted into the trachea and it was difficult to place this in the right position. The X-ray after intubation showed the ETT in a high position and the trachea displaced to the right (Figure 2). Magnetic resonance imaging (MRI) revealed the extent of the cervical mass and its compression on the trachea, and the latter’s subsequent displacement (Figure 3a and 3b).\n\na. Sagittal MRI (T2) of the neck showing the teratoma. b. Axial MRI (T2) of the neck showing the teratoma and the tracheal displacement.\n\nMeanwhile, we started investigation into the cause of the neck mass and performed blood tests on the infant: thyroid hormones were in the normal range (free thyroxine (T4): 1.3 ng/dL and thyroid-stimulating hormone (TSH): 4.7 ng/dL). Calcitonin levels, for investigations into potential malignance, were normal (calcitonin: 21 pg/mL), as was the alpha-fetoprotein: 505 µg/L.\n\nWe decided to remove the cervical mass, since it was causing the tracheal obstruction. The surgery lasted 35 minutes and was uneventful. The mass was well circumscribed and could be easily dissected, weighed 20 grams and measured 33×61×45 mm Figure 4. The infant returned from surgery in good condition. A bronchoscopy was performed the next day after surgery, during tracheal extubation, which revealed no malacia or any other disorders on the trachea or the upper respiratory tract. The patient has been eupneic since then. The histological analysis revealed a mature teratoma with no atypias or signs of malignancy.\n\nLevothyroxine was started (25µg, once a day) as thyroid hormone substitute therapy and the infant is thriving well according to the pediatric endocrinologist that continues following the patient.\n\n\nDiscussion\n\nTeratomas originate from multipotent primitive germ cells and result in different tissues, diverging from the anatomical site of origin2,3. They are most common during early childhood and the most common location is the sacrococcygeal region in children and the gonadal region in adults2–4. The frequency of these embryonic tumours is about 1:20,000–40,000 live births. However, only 1.5% to 5.5% of all pediatric teratomas are placed in the neck region. These tumours are usually solitary, with no other associated congenital malformations or chromosomal abnormalities4. Although 95% of all teratomas are benign, the cervical teratomas if not properly treated, lead to death in 80% of the cases due to obstructive respiratory distress3–5.\n\nIn this case, the acute clinical presentation of the neck mass with severe respiratory distress, needing ready intervention and immediate tracheal intubation should alert all pediatricians to the risk of these neck masses, and consider it as a potentially fatal case. In the presented case, surgical removal of the neck mass was both diagnostic and therapeutic.\n\n\nConclusion\n\nThyroid teratoma is rare in infants, it is usually benign, and can cause airway compression depending on the site and size of the mass. The likelihood of a malignant thyroid teratoma is low in infants, however it could be fatal by causing upper airway obstruction. Therefore, surgical resection is required both for diagnosis and treatment. If the surgical removal is a success, the long-term outcome and quality of life should be good5.\n\n\nConsent\n\nWritten informed consent was obtained from parents of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the editor of this journal.",
"appendix": "Author contributions\n\n\n\nJCJ drafted the first manuscript. UT is the pediatric surgeon and helped with the decision of resecting the tumor. FML did the endocrinological research and helped with the draft. ECB is from the pediatric oncology team and helped with the diagnosis and the draft. WK, JHI and PRM contributed to treating the patient. WBC revised and edited the manuscript. All authors have read and approved the content of the final manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have 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\nOak CY, Kim HK, Yoon TM, et al.: Benign teratoma of the thyroid gland. Endocrinol Metab (Seoul). 2013; 28(2): 144–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHasiotou M, Vakaki M, Pitsoulakis G, et al.: Congenital cervical teratomas. Int J Pediatr Otorhinolaryngol. 2004; 68(9): 1133–9. PubMed Abstract | Publisher Full Text\n\nFichera S, Hackett H, Secola R: Perinatal germ cell tumors: a case report of a cervical teratoma. Adv Neonatal Care. 2010; 10(3): 133–9. PubMed Abstract | Publisher Full Text\n\nKocarslan S, Dorterler ME, Koçarslan A, et al.: Asymptomatic cervical mature teratoma in a child: an unusual presentation. J Clin Diagn Res. 2015; 9(2): EL01–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSheikh F, Akinkuotu A, Olutoye OO, et al.: Prenatally diagnosed neck masses: long-term outcomes and quality of life. J Pediatr Surg. 2015; 50(7): 1210–3. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "9516",
"date": "25 Aug 2015",
"name": "Francisco Eulógio Martinez",
"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\nColetti et al described in a case report of severe acute respiratory distress by tracheal obstruction due to congenital thyroid teratoma. This case report adds knowledge to the literature, not only by tumor description, but also the clinical management of the case.Tumor description is interesting, however I believe that the authors should also discuss in this article the clinical management of malacia in premature, since this is very common in neonatal clinic. I believe that this discussion would make the most interesting article and is also an opportunity to develop the discussion of the article, which I believe is short. For this reason there are some points which need to be considered and are necessary clarificationsAdd data:The patient was born with what gestational age?After birth was not performed control with imaging for the mass?What are the results of thyroid hormones tests after birth?The staff investigated other causes for laryngeal stridor before discharge?Suggestions:The discussion could discuss the differential diagnosis of stridor. The staff not investigated a patient with stridor, attributing the malacia. Many patients in neonatology have stridor and I believe that this article could show that there is need for investigation in stridor, because sometimes the differential diagnosis can be serious pathologies.Case reports are interesting, but I believe it should have a good discussion of the various aspects involved in the pathology, in which case I would like that you develop the discussion and mainly develop the discussion about the differential diagnosis.",
"responses": [
{
"c_id": "1593",
"date": "10 Sep 2015",
"name": "Jose Colleti Junior",
"role": "Author Response",
"response": "I would like to thank Dr Martinez and colleague for the remarks and suggestions above.Answering the questions regarding this case report:The patient was born with 31 weeks of gestational age, immediately after the second administration of levothyroxine in the amniotic fluid due to a chorioamnionitis. After birth new imaging control was done in the previous hospital and in our medical center (figure 3). After first hospital discharge, the patient was followed by a pediatric endocrinologist who asked for thyroid hormone tests which resulted normal for the age (TSH=1.8 mUI/mL – normal: 0.8 to 6.0 mUI/mL; free T4: 0.9 ng/dL – normal= 0.7 to 1.5 ng/dL). Since the patient was born in other hospital we could not know if the staff did any other investigation for other causes of laryngeal stridor. In our medical center, the patient was admitted in a critical clinical status and the surgery was promptly performed.I hope I have clarified your doubts and concerns about this case report. Thank you for the review."
}
]
},
{
"id": "10593",
"date": "30 Sep 2015",
"name": "Daniel Garros",
"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 an interesting case, with a positive outcome despite some delay in diagnosis.The title and the description of the case are all well written.However, the discussion and the conclusion require more work. This case brings up an important and not so rare clinical scenario in Paediatric Critical Care, i.e. the difficulties inherent to extrinsic airway compression by a tumour. The authors, on their discussion, should take advantage of this case and discuss the approach to airway extrinsic compression, the use of extra-corporeal life support (ECLS) as a back-up plan, who should be present in such situations (ENT, anaesthesia), etc… The clinician could barely secure the child’s airway, and the child survived and was able to have the teratoma resected. But it could have been a disastrous outcome. There are protocols for such scenarios, and they should be mentioned and described for the reader to be prepared when facing with similar situations",
"responses": []
}
] | 2
|
https://f1000research.com/articles/4-159
|
https://f1000research.com/articles/4-28/v1
|
28 Jan 15
|
{
"type": "Software Tool Article",
"title": "AGA: Interactive pipeline for reproducible genomics analyses",
"authors": [
"Michael Considine",
"Hilary Parker",
"Yingying Wei",
"Xaio Xia",
"Leslie Cope",
"Michael Ochs",
"Elana Fertig",
"Hilary Parker",
"Yingying Wei",
"Xaio Xia",
"Leslie Cope",
"Michael Ochs"
],
"abstract": "Automated Genomics Analysis (AGA) is an interactive program to analyze high-throughput genomic data sets on a variety of platforms. An easy to use, point and click, guided pipeline is implemented to combine, define, and compare datasets, and customize their outputs. In contrast to other automated programs, AGA enables flexible selection of sample groups for comparison from complex sample annotations. Batch correction techniques are also included to further enable the combination of datasets from diverse studies in this comparison. AGA also allows users to save plots, tables and data, and log files containing key portions of the R script run for reproducible analyses. The link between the interface and R supports collaborative research, enabling advanced R users to extend preliminary analyses generated from bioinformatics novices.",
"keywords": [
"automated",
"genomic",
"analysis",
"datasets",
"DNA",
"methylation",
"expression",
"arrays"
],
"content": "Introduction\n\nWhile high dimensional genetic data have increased in availability at reduced cost, robust analyses remain labor intensive and costly. Numerous automated software pipelines have been developed in an effort to increase the rate and decrease the costs at which analyses can be completed, including SVAw10, Partek3, InSilicoDB17, and cBioPortal4. Automated Genomics Analysis (AGA) provides a more dynamic experience, allowing the user to start with raw data and a text file containing corresponding sample annotations from either a single or multiple studies. AGA performs all necessary normalization and batch correction, and then enables the user to interactively determine the samples to contrast in the analysis based on the sample annotations. AGA is implemented in R to facilitate adaptation of state-of-the art genomics analysis techniques. Linking R to a web browser-based interface through RStudio’s shiny also facilitates collaborative analyses in research teams with diverse bioinformatics expertise.\n\nAGA bridges the gap between interactive and reproducible analyses for several platforms, including expression arrays, methylation arrays, and processed RNAseq data. Through the interface, the user determines the size and scope of the analyses. AGA first performs data normalization, including the ComBat6 and SVA8 batch correction algorithms to enable comparison across multiple datasets for non-methylation platforms. The software then performs differential analysis15, and gene set analyses1,15 based upon defined sample groups. Users obtain standard visualization of genomics data, including hierarchical clustering, boxplots and heatmaps as part of the default analysis. Plots and tables summarizing the results from each analysis are customizable through the interface. The figures and tables in AGA are interactive and customizable. In contrast to other point and click software, AGA logs the R code, and exports the workspace with each figure and table, ensuring that each analysis can be reproduced and further customized.\n\n\nMethods\n\nThe AGA application is run through R and interactive through web browsers. AGA is implemented with RStudio’s shiny12, integrating the R code used in the analysis with HTML and JavaScript, for the interactive user interface. Usage requires R version 3.0.1 or higher, and either Mozilla Firefox or Google Chrome, and R packages described in the AGA User’s Manual. The program is divided into seven tabs. Clicking the respective Update button generates the results to be displayed in each tab and clicking the Download buttons save the plots and data.\n\nAGA supports analyses of DNA methylation and gene expression data. Currently, AGA supports DNA methylation analysis on Illumina 450k arrays. It also supports gene expression analysis of any human Affymetrix expression platform, including exon arrays, and normalized gene counts from RNAseq data. Notably, the flexible format for normalized RNAseq data may be adapted to analyze normalized data from other platforms measuring continuous data, many of which we plan to incorporate in future versions of AGA.\n\nUsers of AGA select to load annotation files and high throughput genomic data from files in a specified directory. AGA accepts raw CEL files and iDat files for Affymetrix and DNA methylation arrays, respectively. It is assumed that normalized RNAseq data are formatted as individual text files for each sample, containing gene names and normalized counts for each sample. More details about the format for each data type are provided in the User’s manual. Sample annotations are specified in a CSV file, whose first column matches the names of the data files. By default, it is assumed the annotation file defines the sample batch; however, this can be updated by editing the annotation files to contain a ‘Batch’ column with unique identifiers for each respective batch within the dataset. Further details about the sample annotations are also provided in the User’s manual.\n\nAfter loading in the annotation files, AGA users select categories from the annotation for differential expression analysis. AGA automatically groups samples with common levels in each category as groups for differential analysis. Samples may be further subset from the complete dataset from the criteria selected for each group. When selected, AGA updates the display to output the sample size for each group. Samples are set for analysis by clicking the “Run the Analysis!” button. In cases for which samples span multiple batches, the analysis automatically performs ComBat and SVA batch correction protecting for the biological groups in the annotation selected by the user. Help boxes are available to clarify each input field with further details in the User’s manual.\n\nThe Dendrogram Plot tab in displays unsupervised hierarchical clustering based upon the complete correlation between values of genes (rows) and samples (columns). The Heatmap Plot tab provides an interactive Javascript heatmap of the genomic data, allowing users to customize genes plotted and color rows by sample annotations. For both Dendrograms and Heatmaps, an option is available to view the pre-batch corrected data to show the effects of batch on and efficacy of correction of the data. The Gene Box Plot tab creates boxplots to summarize values of a user-selected gene in the selected groups.\n\nThe Differential Results tab displays the results from the differential analysis using empirical Bayes moderated t-statistics with the Bio-conductor Package limma15. Statistics are computed on data that have been batch corrected by combining ComBat with SVA, protecting for the biological groups selected for comparison9. The p-values are adjusted utilizing the Benjamini-Hotchberg method for multiple hypothesis testing7. Optionally, gene set statistics can be performed for each gene set defined in Biocarta and Gene Ontology using a Wilcoxon rank-sum test comparing the t-statistics from the most differentially expressed probe for genes in the set to similarly selected t-statistics for genes outside of the set. If selected, results from gene set analysis are displayed in the GSA Results tab.\n\n\nExample\n\nAs an example, we perform analysis on sample datasets containing gene expression of primary head and neck squamous cell carcinoma (HNSCC) tumors. We downloaded measurements from a combination of frozen tumor samples from two distinct studies in GEO available under accession numbers GSE103002 and GSE679111, representing two distinct batches. Raw CEL files and annotation csv files were obtained as described in the User’s manual. We initialize AGA by selecting the directory containing these data. Once loaded, we check the HPV and Tumor.Source.Type columns to group the samples into primary HPV-positive and HPV-negative tumors for differential expression analysis. We then click “Run the Analysis” to normalize the CEL files with fRMA5, batch correct the data with ComBat and SVA, and perform differential expression analysis. The plot in the Dendrogram Plot tab confirms that the batch effects are apparent between these datasets but removed after batch. The heatmap generated in the Heatmap Plot tab (Figure 1) demonstrates that the batch correction nonetheless preserves gene expression difference between HPV-positive and HPV-negative tumors. Moreover, performing differential expression analysis comparing HPV-positive and HPV-negative HNSCC in the “Differential Analysis” tab confirms the well-established overexpression (p=8.74e-9) of CDKN2A (p16) in HPV-positive HNSCC13,14.\n\n\nDiscussion\n\nAGA provides an interface to enable users who may be unfamiliar with R to perform reproducible genomics class comparison analysis. Unlike other automated pipelines, experienced R users can reproduce, extend or modify preliminary analyses. Thus, AGA facilitates collaborations between novice and expert R users for genomics analysis. Future work will extend the AGA pipeline to encode normalization routines to DNA methylation, and analysis routines for other genomics platforms, including copy number data.\n\n\nSoftware availability\n\nhttps://gist.github.com/78f566e1a51d745fac3b\n\nhttps://gist.github.com/F1000Research/9d2acc6aca8ba2d1cc76\n\nhttp://dx.doi.org/10.5281/zenodo.1405618\n\nGNU GPL V2",
"appendix": "Author contributions\n\n\n\nMFO and EJF conceived the software and EJF and MC designed the web interface. MC designed and coded implemented the software application, and prepared the manuscript. HSP researched and composed cross-study normalization techniques. XXX standardized annotation files for the two example data sets. YW and LC assisted by providing the initial coding for alternative analyses. All authors helped prepare the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding was provided by the JHU Head and Neck SPORE, NCI (CA141053) to EJF, and NLM (LM011000) to MFO.\n\nI confirm that the 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 Joe Cheng and Winston Chang of RStudio for their support with shiny. Alla Guseynova, Michael Fox and Louis Franceschi are very much appreciated their technical support and implementations of various iterations of the project. We thank Thomas Considine for his assistance in proofreading this manuscript; and Bahman Afsari and Thomas Considine for testing the application and User Manual. Finally, we also thank Luigi Marchionni and Jean-Philippe Fortin for collaborative efforts.\n\n\nSupplementary material\n\nAGA User’s Manual: available here.\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\nCohen EE, Zhu H, Lingen MW, et al.: A feed-forward loop involving protein kinase Calpha and microRNAs regulates tumor cell cycle. Cancer Res. 2009; 69(1): 65–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDowney T: Analysis of a multifactor microarray study using Partek genomics solution. Methods Enzymol. 2006; 411: 256–270. PubMed Abstract | Publisher Full Text\n\nGao JB, Aksoy A, Dogrusoz U, et al.: Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013; 6(269): pl1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIrizarry RA, Hobbs B, Collin F, et al.: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003; 4(2): 249–264. PubMed Abstract | Publisher Full Text\n\nJohnson WE, Li C, Rabinovic A: Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics. 2007; 8(1): 118–127. PubMed Abstract | Publisher Full Text\n\nKlipper-Aurbach Y, Wasserman M, Braunspiegel-Weintrob N, et al.: Mathematical formulae for the prediction of the residual beta cell function during the first two years of disease in children and adolescents with insulin-dependent diabetes mellitus. Med Hypotheses. 1995; 45(5): 486–490. PubMed Abstract | Publisher Full Text\n\nLeek JT, Johnson WE, Parker HS, et al.: The sva package for removing batch effects and other unwanted variation in high-throughput experiments. Bioinformatics. 2012; 28(6): 882–883. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParker H: Practical Statispractical Statistical Issues in Translational Genomical Issues in Translational Genomics (doctoral dissertation). Johns Hopkins University, Baltimore. 2013.\n\nPirooznia M, Seifuddin F, Goes FS, et al.: SVAw - a web-based application tool for automated surrogate variable analysis of gene expression studies. Source Code Biol Med. 2013; 8(1): 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPyeon D, Newton MA, Lambert PF, et al.: Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers. Cancer Res. 2007; 67(10): 4605–4619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRStudio and Inc: shiny: Web Application for R. R package version 0.7.0. 2012. Reference Source\n\nRobinson M, Sloan P, Shaw R: Refining the diagnosis of oropharyngeal squamous cell carcinoma using human papillomavirus testing. Oral Oncol. 2010; 46(7): 492–6. PubMed Abstract | Publisher Full Text\n\nSmeets SJ, et al.: A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer. 2007; 121(11): 2465–72. PubMed Abstract | Publisher Full Text\n\nSmyth GK: Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2012; 3: 1. Article3. PubMed Abstract | Publisher Full Text\n\nSubramanian A, Tamayo P, Mootha VK, et al.: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102(43): 15545–15550. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTaminau J, Steenhoff D, Coletta A, et al.: inSilicoDb: an R/Bioconductor package for accessing human Affymetrix expert-curated datasets from GEO. Bioinformatics. 2011; 27(22): 3204–3205. PubMed Abstract | Publisher Full Text\n\nConsidine M, Parker HS, Wei Y, et al.: Automated Genomics Analysis. Zenodo. 2015. Data Source"
}
|
[
{
"id": "7514",
"date": "02 Feb 2015",
"name": "Subha Madhavan",
"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 described Automated Genomics Analysis (AGA), an interactive program to analyze high-throughput genomic data sets on a variety of platforms.The software is implemented in R with web app using Shiny.Specific comments are noted below:cBIOPortal is listed as an example for reducing cost of genomic analysis using AGA. cBioPortal's purpose is to help researchers mine analyzed results and it is available for free for non-commercial use. cBIOPortal is not in the same class of software as AGA and is an inappropriate comparison. Title needs to be changed - current AGA software supports expression and methylation analysis only. The title is very broad, especially given that there is not support for genomic variant analysis in the software. Describe any quality checks performed on CEL and idat files briefly beyond batch correction. How does the software deal with missing values? Address scalability. How does the software scale for large studies?",
"responses": [
{
"c_id": "1606",
"date": "21 Oct 2015",
"name": "Michael Considine",
"role": "Author Response",
"response": "1) cBIOPortal is listed as an example for reducing cost of genomic analysis using AGA. cBioPortal's purpose is to help researchers mine analyzed results and it is available for free for non-commercial use. cBIOPortal is not in the same class of software as AGA and is an inappropriate comparison.We have removed reference to cBIOPortal in the revised manuscript.2) Title needs to be changed - current AGA software supports expression and methylation analysis only. The title is very broad, especially given that there is not support for genomic variant analysis in the software.The title has been revised to \"AGA: Interactive pipeline for reproducible gene expression and DNA methylation data analyses\"3) Describe any quality checks performed on CEL and idat files briefly beyond batch correction. How does the software deal with missing values?We have revised the Initiation subsection of the Methods section to note that: “For gene expression microarrays, AGA performs RMA normalization implemented in the Bioconductor package affy 5. Probe-level estimates of DNA methylation are computed from iDat files using Illumina standards with the minfi package 1. RNAseq data are formatted as individual text files for each sample, assumed to contain gene names and normalized counts for each sample.4) Address scalability. How does the software scale for large studies?We have revised the Introduction section to include more information on the length of analyses, “The runtime of analyses will depend largely on the desktop hardware, but also on the data platform and optional analyses selected. On a Mac Pro workstation, containing a 3.2 GHz Quad-Core Intel Xeon processor and 10Gb 1066 MHz DDR3 RAM, analyses containing under 100 samples were completed in under 30 minutes.”"
}
]
},
{
"id": "8835",
"date": "22 Jun 2015",
"name": "Matthew McCall",
"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 a software package for interactive (via a shiny webapp) genomic analysis. By running R behind the scenes, this software addresses a common challenge in genomic data analysis -- the transition from simple initial analyses (typically performed by a novice user) and more complex later analyses (typically performed by an advanced user). When the initial analyses are not easily examined / reproduced, the advanced user often must start from scratch. The AGA software will hopefully address this issue. The title of the article is currently too broad -- the software is only able to handle Affymetrix expression arrays, Illumina 450k methylation arrays, and normalized RNA-seq gene counts. However, I trust that the authors will expand the functionality of the software to handle many other platforms and types of genomic data. My primary criticism of this work is that I was unable to successfully use the package. The package depends on a large number of other packages (shinyIncubator, googleVis, and heatmap among others). In particular, I was unable to install the heatmap package. Additional instructions on how to obtain / install all of the required dependencies should be added to the user manual.I also have a few minor criticisms of the article:In Figure 1, the gene names are not legible due to over-plotting, and the column names are truncated. The citation for the fRMA method is incorrect. The correct citation is:McCall MN, Bolstad BM, and Irizarry RA (2010). Frozen Robust Multi-Array Analysis (fRMA), Biostatistics, 11(2):242-253.",
"responses": [
{
"c_id": "1605",
"date": "21 Oct 2015",
"name": "Michael Considine",
"role": "Author Response",
"response": "1) The title of the article is currently too broad -- the software is only able to handle Affymetrix expression arrays, Illumina 450k methylation arrays, and normalized RNA-seq gene counts. However, I trust that the authors will expand the functionality of the software to handle many other platforms and types of genomic data.The title has been revised to \"AGA: Interactive pipeline for reproducible gene expression and DNA methylation data analyses\"2) My primary criticism of this work is that I was unable to successfully use the package. The package depends on a large number of other packages (shinyIncubator, googleVis, and heatmap among others). In particular, I was unable to install the heatmap package. Additional instructions on how to obtain / install all of the required dependencies should be added to the user manual.The number of packages is necessary for the diverse functionality and formatting, and to make the best possible application. We have added instructions to manually install the required packages on page 4 of the revised user’s manual. “In the event of difficulties installing libraries in R, copy lines 144 to 200 in the global.r script and enter them into your R console. Afterwards, run the command: sporelibs()” I also have a few minor criticisms of the article:3) In Figure 1, the gene names are not legible due to over-plotting, and the column names are truncatedWe have revised Figure 1 to reduce the number of genes so that the gene names are legible. The column names are set by the sample identifiers and must be truncated to facilitate visualization. We note in the revised caption to Figure 1 that: “We note that sample names are truncated in the heatmap, but users can reduce the lengths of sample names or ensure that sample identity can be determined by the final characters in the name to associate specific samples with the heatmap”.4) The citation for the fRMA method is incorrect. The correct citation is:By default, AGA implements RMA instead of fRMA. We have removed this citation from the revised manuscript and clarified this choice in by adding this second sentence of Initiation subsection of the revised manuscript: “For gene expression microarrays, AGA performs RMA normalization implemented in the Bioconductor package affy” and revising the fourth sentence of the Example subsection of the revised manuscript “We then click “Run the Analysis” to normalize the CEL files with RMA”"
}
]
}
] | 1
|
https://f1000research.com/articles/4-28
|
https://f1000research.com/articles/4-1092/v1
|
20 Oct 15
|
{
"type": "Review",
"title": "Epigenetic and microRNA regulation during osteoarthritis development",
"authors": [
"Di Chen",
"Jie Shen",
"Tianqian Hui",
"Jie Shen",
"Tianqian Hui"
],
"abstract": "Osteoarthritis (OA) is a common degenerative joint disease, the pathological mechanism of which is currently unknown. Genetic alteration is one of the key contributing factors for OA pathology. Recent evidence suggests that epigenetic and microRNA regulation of critical genes may contribute to OA development. In this article, we review the epigenetic and microRNA regulations of genes related to OA development. Potential therapeutic strategies may be developed on the basis of novel findings.",
"keywords": [
"Osteoarthritis",
"microRNA",
"epigenetics",
"miRNA",
"methylation",
"miR-140"
],
"content": "Introduction\n\nOsteoarthritis (OA) is the most common form of arthritis and is the leading cause of impaired mobility in the elderly1. It has been projected that more than 67 million people will be affected by OA in the US by 2030, resulting in an extremely high socioeconomic burden2,3. In recent years, the surgically induced destabilization of the medial meniscus (DMM) model4 and genetic mouse models5–12 have been developed to delineate the potential roles of affected genes in OA pathogenesis. However, a full understanding of the factors affecting the initiation and progression of the disease has not yet been revealed. Thus, there is no clinical diagnosis for early OA and no effective disease-modifying treatment for late-stage OA, except pain-relieving medication and surgical replacement of the damaged joints13–15. Compelling evidence has revealed that epigenetic and microRNA (miRNA) alterations occur in OA chondrocytes and in patients with OA, including several well-documented OA-related genes, indicating, to a certain extent, that epigenetic and miRNA regulation contributes to OA pathogenesis16–18. In this short review, we will summarize the current understanding of OA, speculate on the potential mechanism(s) of epigenetic and miRNA regulation underlying OA development and progression, and in this context propose potential therapeutic targets for the treatment of OA.\n\n\nPathogenesis of osteoarthritis\n\nOA is a degenerative joint disease with major clinical symptoms, including chronic pain, joint instability, stiffness, and radiographic joint space narrowing. During OA progression, articular chondrocytes undergo hypertrophy, leading to extracellular matrix (ECM) degradation and articular cartilage breakdown, followed by vascular invasion, subchondral bone sclerosis, and osteophyte formation eventually developing at the margins of the joint19–21. OA is a complex multi-factorial disease, and the effects of aging and obesity, mechanical influences, and environmental and genetic factors have been identified as major factors contributing to the initiation or progression (or both) of OA22,23. Because articular cartilage damage is the primary pathologic feature leading to the joint dysfunction, it has received much of the attention in OA studies. Normal articular cartilage emerges during the postnatal stage as a permanent tissue distinct from the growth plate. The articular cartilage tissue lining the surface of all diarthrodial joints is a smooth, hard, white tissue, which cushions and absorbs the shock between joints. Collagens and proteoglycans are the principal ECM molecules of articular cartilage24–27. Mutations of ECM-related factors, including, types II, IX and XI collagen, have been reported in human OA patients28–30. It has been established that articular chondrocytes are the cells responsible for maintaining joint cartilage homeostasis. Thus, dysregulation of articular chondrocytes is directly connected to the process of cartilage degradation in OA. An understanding of the phenotypic behavior of articular chondrocytes in homeostasis and disease has revealed several key environmental and genetic factors that impact OA development and progression.\n\n\nGenetic contributions to osteoarthritis\n\nA genetic predisposition to OA has been established for many years through several twin studies, segregation analyses, linkage analyses, and candidate gene association studies31–33. Although the genetics of OA are complex, the genetic contribution to OA is highly significant. It has been demonstrated that the heritability of OA may be as high as 40–65%, depending on the joint site and population studied34. In the past decade, the potential roles of genes and signaling pathways in OA pathogenesis have been demonstrated by ex vivo studies with tissue derived from OA patients and in vivo studies with surgically induced OA animal models as well as mouse genetic models. Transforming growth factor-beta (TGF-β), Wnt/β-catenin, Indian Hedgehog (Ihh), Notch, fibroblast growth factor (FGF), and hypoxia-inducible factor (HIF) pathways, by stimulating chondrocytes toward hypertrophy, have demonstrated the critical and unique roles of chondrocytes during OA development and progression in genetic mouse models5–7,9,10,35. These recent genetic findings further suggest that Runt-related transcription factor 2 (Runx2), Mmp13, and Adamts5 are common target genes involved in the above-mentioned signaling networks, disrupting the anabolic and catabolic balance in chondrocytes and eventually degrading the cartilage matrix by upregulation of matrix metalloproteinase (MMP) and a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) activity, which leads to degradation of type II collagen and aggrecan8,11,36–38. Although these studies have been important in determining the genetic components of OA, only a few OA-related genes have been identified by using human genetic and epidemiological approaches. More recent newer technologies, such as genome-wide association studies (GWASs), have been used to analyze large numbers of OA and control populations throughout the world in hopes of uncovering more genes associated with OA. To date, even these larger exploratory human genetic studies have produced very few genes important to the development and pathogenesis of human OA. Whereas some of the genes identified are important structural and ECM-related factors (Col2a1, Col9a1, and Col11a1) as well as critical signaling molecules in the Wnt (Sfrp3), bone morphogenetic protein (BMP) (Gdf5), and TGF-β (Smad3) signaling pathways, most have been previously implicated in OA or articular cartilage and joint maintenance by using mouse models of induced genetic alteration or surgically induced OA28–30,39–42. New single-nucleotide polymorphisms were identified in several genes, including GNL3, ASTN2, and CHST11, in recent genome-wide screen studies43, and these findings need to be further confirmed.\n\n\nEpigenetic alterations in osteoarthritis pathogenesis\n\nIn addition to GWAS analyses, growing evidence suggests that the gene expression profile can be largely regulated by epigenetic machinery that modulates local transcriptional activity and mRNA expression in chondrocytes44. In normal adult chondrocytes, like other somatic cells, the genomic arrangement and packaging are regulated by genetic and epigenetic mechanisms that provide instruction on how, where, and when genetic information should be used. In mammals, the major epigenetic regulatory mechanisms include DNA methylation and histone modification. miRNAs could be loosely defined as epigenetic factors and play important roles in OA45.\n\nDNA methylation is mediated by DNA methyltransferase (DNMT), which transfers the methyl group from the donor, methylated S-adenosyl-methionine (methyl-SAM), to DNA bases, particularly cytosine (CpG island). DNA methylation occurs in both the gene promoter region and gene bodies and regulates gene transcription46–48. Recent studies found that DNA methylation is dynamically regulated through a cyclic enzymatic cascade composed of cytosine methylation by DNMTs and demethylation by ten-eleven translocation methylcytosine-(TET) dioxygenases (TET1, 2, and 3)49. In mammals, there are three enzymatically active DNMTs, DNMT1, DNMT3a, and DNMT3b, and one related regulatory protein, DNMT3L48. DNMT1 is primarily a “maintenance” methyltransferase that recognizes the hemi-methylated DNA strand and preserves the methylation pattern throughout cell replication and division. The global knockout of the Dnmt1 gene is embryonically lethal at E10.5 because of a significant loss of global DNA methylation, suggesting that DNA methylation is essential for normal mammalian development50. In contrast, two de novo DNMTs, 3a and 3b, have tissue-specific expression patterns and create unique methylation signatures. Knockout mice with Dnmt3b deletion showed embryonic lethality between E11.5 and E15.5 as well as several skeletal defects, including growth impairment. However, loss of the Dnmt3b gene does not affect the entire genome methylation pattern51.\n\nIn recent decades, researchers have studied changes in the DNA methylation status of individual genes during OA development and progression and found that the promoter of Col10a1 appeared to be hypomethylated during chondrocyte hypertrophy and maturation followed by its upregulation52. Similarly, the CpG sites within the promoter area of a number of metalloproteinases, including MMP2, MMP9, MMP13, and ADAMTS4, showed decreased methylation profiles in OA compared to normal cartilage, correlating with elevated gene expression and resulting in ECM degradation53,54. Reduced CpG methylation was reported in the MMP13, IL-1β, and inducible nitric oxide synthase (iNOS) promoter in OA tissue which correlates with the increased MMP13, IL-1β, and iNOS expression in OA chondrocytes55,56. During the chondrocyte maturation process, changes in DNA methylation patterns were observed in several transcription factors, such as Sox9 and Runx257. Hypomethylation in promoter regions of those genes promoted gene transcription, which further activated downstream signaling molecules, including MMPs, and eventually stimulated chondrocytes toward hypertrophy and terminal maturation. Either hypomethylation or hypermethylation occurred in promoter regions within a subset of OA-specific genes, including ligands (e.g., BMP7 and IL-1β)58,59, receptors, transcription factors (e.g., Sox9 and Runx2)57, enzymes (e.g., MMPs and ADAMTS4/5)53,54, and ECM proteins (e.g., aggrecan, Col2a1, and Col10a1)52,60.\n\nRecent methylome screening data further confirmed that alterations in DNA methylation occurred in OA chondrocytes and that chondrocyte transcriptomes may be changed in OA patients, indicating that DNMTs influence OA susceptibility and severity by modulating pathways or signals leading to OA16–18,61,62. However, which DNMT factor or factors mediate these changes genome-wide remains largely unknown. In one of our ongoing experiments, we have found that DNMT3b, but not DNMT 1 or 3a, was highly expressed in articular chondrocytes, but its expression was significantly decreased in chondrocytes derived from patients with OA or from several OA mouse models, including the aging animal model, meniscal ligamentous injury (MLI) model, and obesity model (Shen et al., unpublished data). Recent reports demonstrated that TET1, 2, and 3 are present in human chondrocytes and that TET1 expression was significantly reduced by inflammatory factors, such as IL-1β or TNFα63. Recent studies have also revealed a significant increase in 5-hydroxymethylcytosine levels in OA chondrocytes because of TET1 downregulation64,65. Because DNA methylation is a reversible process, the role of the TET family members in OA development needs further investigation to better understand the regulation of DNA demethylation during OA development and progression.\n\nThe regulation of transcription factors on chondrocyte-specific genes through alterations of DNA methylation and histone modification has been reported in recent years. For example, it has been reported that methylation of the -110 bp CpG site in the Mmp13 promoter strongly correlates with the high Mmp13 expression in chondrocytes. This CpG site resides within a HIF consensus motif. The methylation of this site will decrease HIF-2α binding to the Mmp13 promoter55. AT-rich interactive domain 5b (Arid5b) is a newly identified transcriptional co-regulator of Sox9. Arid5b recruits Phf2, a histone lysine demethylase, to the promoter region of Sox9 target genes and stimulates H3K9me2 demethylation of these genes. In the promoters of chondrocyte marker genes, H3K9me2 levels are increased in Arid5b knockout chondrocytes66.\n\nWorking closely with DNA methylation, histone modification—including acetylation, phosphorylation, methylation, and ubiquitination—regulates gene expression by controlling the accessibility of the transcriptional machinery67,68. Recent studies demonstrated that histone acetylation and deacetylation are involved in OA pathogenesis by affecting chondrocyte anabolic and catabolic processes. Histone acetylation is mediated by histone acetyltransferases (HATs) and is a critical step in loosening the DNA structure, which allows regulatory factors to access the transcriptional machinery and the subsequent initiation of gene expression, whereas deacetylation is considered the termination or repression of gene expression69. Histone deacetylation is mediated by histone deacetylases (HDACs), including the classic HDAC and NAD+-dependent silent information regulator 2 (SIR2) families70,71. The use of large-scale analysis (ChIP-seq) of chondrocyte histone acetylation did not find global alterations in OA chondrocytes but did find changes in specific gene loci, encoding MMPs, ECM molecules, and inflammatory factors.\n\nIn patients with OA, elevated HDAC7 expression has been reported to contribute to cartilage degradation by inducing Mmp13 expression in OA cartilage. The inhibition of HDAC7 in vitro leads to suppression of inflammatory factor-induced Mmp13 expression72. The expressions of HDAC1 and HDAC2 are upregulated in OA synovial tissue as well, and this may lead to repression of Col2a1 expression in chondrocytes by interfering with the recruitment of Snail73,74. Therefore, HDAC inhibitors have been extensively studied in various OA models. Specific HDAC inhibitors can inhibit cytokine-induced MMP expression in chondrocytes to protect against proteoglycan loss and cartilage degradation75–77. HDAC inhibitors can also stimulate the expression of ECM components—such as Col2a1, cartilage oligomeric matrix protein (COMP), and aggrecan—in chondrocytes74,78. In the rabbit anterior cruciate ligament transection (ACLT) model, an HDAC inhibitor significantly decelerated injury-induced cartilage erosion, mainly due to reduced expression of MMPs and inflammatory cytokines, indicating that HDAC inhibitors may provide a potential treatment for OA79.\n\nIn the SIR2 family, SIRT1 has been extensively studied. SIRT1 is highly expressed in chondrocytes and its expression was found to be decreased in OA cartilage80,81. SIRT1 can promote expression of ECM genes, such as Col2a1, Col9a1, and COMP, possibly through deacetylation of Sox9, while inhibiting Col10a1 and Adamts582. SIRT1 also prevents apoptosis in chondrocytes by enhancing insulin-like growth factor (IGF) signaling to inactivate p53. The reduction of SIRT1 expression leads to an increase in chondrocyte apoptosis in OA cartilage83. Interestingly, the function of SIRT1 is closely linked to the inflammatory response and the hypoxic response as well, although SIRT1 has not been approved for use to treat OA. In a variety of tissues, SIRT1 initiates a gene-specific transcriptional repression program to terminate inflammatory response by deacetylating the p65 subunit of nuclear factor-kappa-B (NF-κB) and blocking NF-κB binding to the DNA elements84,85. SIRT1 can also directly deacetylate and activate HIF-2α, which is upregulated in OA cartilage, to promote MMP expression and eventually degrade the articular cartilage86,87.\n\nIn addition to histone acetylation, histone H3K4 methylation mediated by histone-lysine N-methyltransferase (HMT) was recently investigated. HMT expression level was found elevated in OA cartilage, which resulted in H3K4 methylation at the iNOS and COX-2 promoter areas and induction of gene expression88. Similarly, an age-dependent increase in H3K4me2 occurs in the nuclear factor of activated T cells 1 (Nfat1) promoter, which led to suppression of Nfat1 expression in adult articular chondrocytes and eventually developed OA-like phenotype in mice89,90. Increased demethylation mediated by histone demethylase LSD1 was also found in OA chondrocytes. Elevated LSD1 contributed to H3K9 demethylation in the microsomal prostaglandin E synthase 1 (mPGES-1) promoter and induction of gene expression in human OA chondrocyte91. Moreover, the architecture of histone acetylation and methylation in local genome can further guide the long-range chromatin interaction to regulate specific gene regulatory DNA elements92.\n\n\nMicroRNA regulation\n\nmiRNAs are endogenous non-coding RNAs and play important roles in negative regulation of RNA stability and protein expression93,94. Several miRNAs have been found to be more abundant in articular chondrocytes than in undifferentiated mesenchymal stem cells. The best example of this is miR-14095. miR-140 is found in an intron of the Wwp2 gene coding for WWP2 E3 ubiquitin ligase96. Deletion of miR-140 did not alter the expression level of Wwp2 in chondrocytes97. Analysis of the intronic sequence found two miR-140s: miR-140-5p and miR-140-3p98. The expression levels of miR-140-5p and -3p were both significantly reduced in OA chondrocytes98. During chondrocyte differentiation, miR-140 expression increased in parallel with Sox9 and Col2a1. However, in OA tissues, miR-140 expression is reduced and Adamts5 expression was upregulated95. In vitro treatment of chondrocytes with IL-1β suppresses miR-140 expression95. miR-140 is the only miRNA with a cartilage-specific expression pattern95,99. miR-140 deficiency accelerates chondrocyte differentiation into hypertrophic chondrocytes and inhibits differentiation of resting chondrocytes into columnar proliferating chondrocytes100. The reduction in miR-140 expression in OA cartilage may contribute to abnormal gene expression during OA development95. For example, miR-140 regulates the expression of histone deacetylase 4 (HDAC4), a co-repressor of Runx2 and myocyte-specific enhancer factor 2 (Mef2)101. miR-140 also targets Cxcl1299 and Smad3102, both of which are implicated in chondrocyte differentiation. In miR-140 null mice, OA-like changes were observed and characterized by proteoglycan loss and fibrillation of articular cartilage, probably due to increased Adamts5 expression103. This increased Adamts5 expression was reversed by transfection of ds-miR-140 into miR-140-deficient chondrocytes103. In addition, cartilage-specific miR-140-overexpressing transgenic mice had no abnormal skeletal phenotype during embryonic development but did show a protective effect in an antigen-induced arthritis model103. However, the upregulation of Adamts5 and Hdac4 expression in chondrocytes was not found in the other miR-140 knockout mouse model generated by Nakamura et al.97. Instead of upregulation of Hdac4 expression, miR-140 enhances HDAC4 function in chondrocytes100. miR-140 could interact with PTHrP-HDAC4 pathway to control chondrocyte differentiation. miR-140 deficiency and PTHrP or Hdac4 heterozygosity synergistically impair skeletal growth. Loss of miR-140 upregulates MEF2C expression. miR-140 negatively regulates p38 mitogen-activated protein kinase (MAPK) signaling, and inhibition of p38 MAPK signaling reduces MEF2C expression104. The functional role of miR-140 in cartilage homeostasis is also involved in the regulation of MMP13105. MMP13 is a well-known key player in cartilage biology and OA pathology. It has been reported that miR-140 is a negative feedback regulator of MMP13106. In addition, transfection with pre-miR-140 significantly decreased IGFBP-5 expression. In contrast, transfection with anti-miR-140 significantly increased IGFBP-5 expression107.\n\nSignificant progress has been made in recent years in OA research, and several OA mouse models, including genetic models and surgically induced OA models, have been developed and reported. One common feature of these animal models is upregulation of Runx25,9,36, leading to further increases in genes coding for matrix degradation enzymes, such as Mmp9, Mmp13, and Adamts5, because Runx2 is a key transcription factor regulating the transcription of these genes108–110. Key questions are how Runx2 is regulated and whether a therapeutic strategy can be developed by downregulation of Runx2 in OA cartilage.\n\nDuring skeletal development, Runx2 mRNA expression was detected in skeletal elements as early as E10.5 and E11.5; however, hypertrophic chondrocytes and primary ossification centers do not form until E14.5, although Runx2 is a key transcription factor driving chondrocyte hypertrophy111. These findings suggest that Runx2 protein expression is suppressed because of post-transcriptional regulation during early skeletal development since chondrocyte proliferation and expansion are needed at this stage. These findings also suggest that there is an endogenous negative regulatory mechanism for Runx2 protein expression.\n\nIn recent studies, we have examined potential miRNAs that may bind the 3′-non-coding region of the Runx2 gene and found that miR-204 and miR-211, two homologous miRNAs, bind Runx2 and regulate Runx2 expression in mesenchymal progenitor cells112. To further investigate the functions of these miRNAs in the regulation of Runx2 protein expression in articular chondrocytes and in cartilage homeostasis, chondrocyte-specific miR-204 and miR-211 transgenic mice and conditional knockout mice need to be generated and tested. In addition to miR-204 and miR-211, several other miRNAs have been reported to regulate Runx2 expression113. Their functions in OA development also need further investigation.\n\nThe role of miRNA regulation in OA development involves upstream regulation and downstream gene targeting. For example, it has been reported that IL-1β, an inflammatory cytokine, suppresses the expression of miR-140, which in turn causes upregulation of Adamts5, a target gene of miR-140, in chondrocytes95,103, so miR-140 could serve as a mediator during OA development. In addition, it has been reported that TGF-β/Smad3 regulates miR-140 expression in OA chondrocytes114. TGF-β signaling is one of the key signaling pathways in OA development and responds to mechanical loading. Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) is a novel post-transcriptional regulator of IL-6 expression and is targeted by miR-9. MCPIP-1 mRNA expression was low, but expression of miR-9 and IL-6 was high, in damaged OA cartilage. MCPIP-1 protein directly binds with IL-6 mRNA, and overexpression of wild-type MCPIP-1 destabilized the IL-6 mRNA. MCPIP-1 expression was altered by overexpression or inhibition of miR-9. These findings implicate miR-9-mediated suppression of MCPIP-1 in the pathogenesis of OA via upregulation of IL-6 expression in IL-1β-stimulated human OA chondrocytes115. These studies also suggest that miRNAs may serve as important mediators in OA, although they may not be able to trigger the OA occurrence.\n\n\nSummary\n\nAlthough OA is a multi-factorial disease, genetic factors may play a significant role in OA development and progression. Recent evidence suggests that epigenetic and miRNA regulation of genes related to OA development may contribute to OA pathology. To fully understand how mechanical instability and inflammation cause epigenetic and miRNA alteration, further leading to OA development and progression, more in-depth studies need to be conducted. These studies may lead to uncovering novel molecular targets for drug development to prevent and treat OA.\n\n\nAbbreviations\n\nADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; Arid5b, AT-rich interactive domain 5b; BMP, bone morphogenetic protein; COMP, cartilage oligomeric matrix protein; DNMT, DNA (cytosine-5)-methyltransferase; ECM, extracellular matrix; GWAS, genome-wide association study; HDAC, histone deacetylase; HIF, hypoxia-inducible factor; HMT, histone-lysine N-methyltransferase; IL, interleukin; IGF, insulin-like growth factor; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; MCPIP-1, monocyte chemoattractant protein-induced protein 1; Mef2, myocyte-specific enhancer factor 2; miRNA, microRNA; MMP, matrix metalloproteinase; Nfat1, nuclear factor of activated T cells 1; NF-κB, nuclear factor-kappa-B; OA, osteoarthritis; Runx2, Runt-related transcription factor 2; SIR2, silent information regulator 2; TET, ten-eleven translocation methylcytosine dioxygenase; TGF-β, transforming growth factor-beta.",
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PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDvir-Ginzberg M, Gagarina V, Lee EJ, et al.: Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase. J Biol Chem. 2008; 283(52): 36300–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFujita N, Matsushita T, Ishida K, et al.: Potential involvement of SIRT1 in the pathogenesis of osteoarthritis through the modulation of chondrocyte gene expressions. J Orthop Res. 2011; 29(4): 511–5. PubMed Abstract | Publisher Full Text\n\nGagarina V, Gabay O, Dvir-Ginzberg M, et al.: SirT1 enhances survival of human osteoarthritic chondrocytes by repressing protein tyrosine phosphatase 1B and activating the insulin-like growth factor receptor pathway. Arthritis Rheum. 2010; 62(5): 1383–92. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTakayama K, Ishida K, Matsushita T, et al.: SIRT1 regulation of apoptosis of human chondrocytes. Arthritis Rheum. 2009; 60(9): 2731–40. PubMed Abstract | Publisher Full Text\n\nYeung F, Hoberg JE, Ramsey CS, et al.: Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 2004; 23(12): 2369–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu TF, Yoza BK, El Gazzar M, et al.: NAD+-dependent SIRT1 deacetylase participates in epigenetic reprogramming during endotoxin tolerance. J Biol Chem. 2011; 286(11): 9856–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDioum EM, Chen R, Alexander MS, et al.: Regulation of hypoxia-inducible factor 2alpha signaling by the stress-responsive deacetylase sirtuin 1. Science. 2009; 324(5932): 1289–93. PubMed Abstract | Publisher Full Text\n\nYang S, Kim J, Ryu JH, et al.: Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction. Nat Med. 2010; 16(6): 687–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEl Mansouri FE, Chabane N, Zayed N, et al.: Contribution of H3K4 methylation by SET-1A to interleukin-1-induced cyclooxygenase 2 and inducible nitric oxide synthase expression in human osteoarthritis chondrocytes. Arthritis Rheum. 2011; 63(1): 168–79. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRodova M, Lu Q, Li Y, et al.: Nfat1 regulates adult articular chondrocyte function through its age-dependent expression mediated by epigenetic histone methylation. J Bone Miner Res. 2011; 26(8): 1974–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang J, Gardner BM, Lu Q, et al.: Transcription factor Nfat1 deficiency causes osteoarthritis through dysfunction of adult articular chondrocytes. J Pathol. 2009; 219(2): 163–72. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nEl Mansouri FE, Nebbaki SS, Kapoor M, et al.: Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes. Arthritis Res Ther. 2014; 16(3): R113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBartkuhn M, Renkawitz R: Long range chromatin interactions involved in gene regulation. Biochim Biophys Acta. 2008; 1783(11): 2161–6. PubMed Abstract | Publisher Full Text\n\nSato F, Tsuchiya S, Meltzer SJ, et al.: MicroRNAs and epigenetics. FEBS J. 2011; 278(10): 1598–609. PubMed Abstract | Publisher Full Text\n\nChuang JC, Jones PA: Epigenetics and microRNAs. Pediatr Res. 2007; 61(5 Pt 2): 24R–29R. PubMed Abstract | Publisher Full Text\n\nMiyaki S, Nakasa T, Otsuki S, et al.: MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses. Arthritis Rheum. 2009; 60(9): 2723–30. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nYang J, Qin S, Yi C, et al.: MiR-140 is co-expressed with Wwp2-C transcript and activated by Sox9 to target Sp1 in maintaining the chondrocyte proliferation. FEBS Lett. 2011; 585(19): 2992–7. PubMed Abstract | Publisher Full Text\n\nNakamura Y, Inloes JB, Katagiri T, et al.: Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling. Mol Cell Biol. 2011; 31(14): 3019–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwingler TE, Wheeler G, Carmont V, et al.: The expression and function of microRNAs in chondrogenesis and osteoarthritis. Arthritis Rheum. 2012; 64(6): 1909–19. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNicolas FE, Pais H, Schwach F, et al.: Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140. RNA. 2008; 14(12): 2513–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPapaioannou G, Inloes JB, Nakamura Y, et al.: let-7 and miR-140 microRNAs coordinately regulate skeletal development. Proc Natl Acad Sci U S A. 2013; 110(35): E3291–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTuddenham L, Wheeler G, Ntounia-Fousara S, et al.: The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett. 2006; 580(17): 4214–7. PubMed Abstract | Publisher Full Text\n\nPais H, Nicolas FE, Soond SM, et al.: Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level. RNA. 2010; 16(3): 489–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiyaki S, Sato T, Inoue A, et al.: MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev. 2010; 24(11): 1173–85. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPapaioannou G, Mirzamohammadi F, Lisse TS, et al.: MicroRNA-140 Provides Robustness to the Regulation of Hypertrophic Chondrocyte Differentiation by the PTHrP-HDAC4 Pathway. J Bone Miner Res. 2015; 30(6): 1044–52. PubMed Abstract | Publisher Full Text\n\nZhang R, Ma J, Yao J: Molecular mechanisms of the cartilage-specific microRNA-140 in osteoarthritis. Inflamm Res. 2013; 62(10): 871–7. PubMed Abstract | Publisher Full Text\n\nLiang ZJ, Zhuang H, Wang GX, et al.: MiRNA-140 is a negative feedback regulator of MMP-13 in IL-1β-stimulated human articular chondrocyte C28/I2 cells. Inflamm Res. 2012; 61(5): 503–9. PubMed Abstract | Publisher Full Text\n\nTardif G, Hum D, Pelletier JP, et al.: Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes. BMC Musculoskelet Disord. 2009; 10: 148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPei Y, Harvey A, Yu XP, et al.: Differential regulation of cytokine-induced MMP-1 and MMP-13 expression by p38 kinase inhibitors in human chondrosarcoma cells: potential role of Runx2 in mediating p38 effects. Osteoarthritis Cartilage. 2006; 14(8): 749–58. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nThirunavukkarasu K, Pei Y, Wei T: Characterization of the human ADAMTS-5 (aggrecanase-2) gene promoter. Mol Biol Rep. 2007; 34(4): 225–31. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTetsunaga T, Nishida K, Furumatsu T, et al.: Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells. Osteoarthritis Cartilage. 2011; 19(2): 222–32. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDucy P, Zhang R, Geoffroy V, et al.: Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 1997; 89(5): 747–54. PubMed Abstract | Publisher Full Text\n\nHuang J, Zhao L, Xing L, et al.: MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells. 2010; 28(2): 357–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeng Y, Wu S, Zhou H, et al.: Effects of a miR-31, Runx2, and Satb2 regulatory loop on the osteogenic differentiation of bone mesenchymal stem cells. Stem Cells Dev. 2013; 22(16): 2278–86. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTardif G, Pelletier JP, Fahmi H, et al.: NFAT3 and TGF-β/SMAD3 regulate the expression of miR-140 in osteoarthritis. Arthritis Res Ther. 2013; 15(6): R197. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMakki MS, Haseeb A, Haqqi TM: MicroRNA-9 promotion of interleukin-6 expression by inhibiting monocyte chemoattractant protein-induced protein 1 expression in interleukin-1β-stimulated human chondrocytes. Arthritis Rheumatol. 2015; 67(8): 2117–28. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "10882",
"date": "20 Oct 2015",
"name": "Kenneth B. Marcu",
"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",
"responses": []
},
{
"id": "10883",
"date": "20 Oct 2015",
"name": "Alexander Lichtler",
"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",
"responses": []
},
{
"id": "10884",
"date": "20 Oct 2015",
"name": "Tatsuya Kobayashi",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1092
|
https://f1000research.com/articles/4-1078/v1
|
16 Oct 15
|
{
"type": "Opinion Article",
"title": "Caveat emptor NICE: biased use of cost-effectiveness is inefficient and inequitable",
"authors": [
"Jack Dowie",
"Mette Kjer Kaltoft",
"Jesper Bo Nielsen",
"Glenn Salkeld",
"Mette Kjer Kaltoft",
"Jesper Bo Nielsen",
"Glenn Salkeld"
],
"abstract": "Concern with the threshold applied in cost-effectiveness analyses by bodies such as NICE distracts attention from their biased use of the principle. The bias results from the prior requirement that an intervention be effective (usually 'clinically effective') before its cost-effectiveness is considered. The underlying justification for the use of cost-effectiveness as a criterion, whatever the threshold adopted, is that decisions in a resource-constrained system have opportunity costs. Their existence rules out any restriction to those interventions that are 'incrementally cost-effective' at a chosen threshold and requires acceptance of those that are 'decrementally cost-effective' at the same threshold. Interventions that fall under the linear ICER line in the South-West quadrant of the cost-effectiveness plane are cost-effective because they create net health benefits, as do those in the North-East quadrant. If there is objection to the fact that they are cost-effective by reducing effectiveness as well as costs, it is possible to reject them, but only on policy grounds other than their failure to be cost-effective. Having established this, the paper considers and seeks to counter the arguments based on these other grounds. Most notably these include those proposing a different threshold in the South-West quadrant from the North-East one, i.e. propose a 'kinked ICER'. Another undesirable consequence of the biased use of cost-effectiveness is the failure to stimulate innovations that would increase overall health gain by being less effective in the condition concerned, but generate more benefits elsewhere. NICE can only reward innovations that cost more.",
"keywords": [
"cost-effectiveness",
"incremental cost-effectiveness",
"decremental cost-effectiveness",
"south-west quadrant"
],
"content": "Introduction\n\nThe publication of the Claxton report containing an estimate of the willingness to pay for an incremental Quality-Adjusted Life Year (QALY) implicit in the expenditure patterns of the NHS of England and Wales has refocused attention on the use by the National Institute for Care and Health Excellence (NICE) of cost-effectiveness as one criterion in its reimbursement decisions1,2. The suggestion that the empirical threshold for cost-effectiveness is about £13,000 (but probably lower), compared with the £20,000 to £30000 range acknowledged by NICE, assumes greater significance in the context of the growing adoption of the NICE model, or some version of it, in other jurisdictions facing the same challenges. The introduction and use of the formal and relatively transparent NICE process has undoubtedly been a major advance, compared with the situation in countries who are in official denial about the need to prioritise and do so with some transparency. The idea that any resource-constrained health or other public service can function efficiently and equitably - and hence ethically - without employing cost-effectiveness as a key principle we take to be absurd. How the principle - which would be better called opportunity cost-effectiveness - is implemented, is the only issue. There are numerous valid and important debates to be had on this, including the one that concerns us here.\n\nThe NICE advance has been bought at the price of biased use of the principle of cost-effectiveness and, as a corollary, biased support for innovative technologies. These biases are built into its legal obligations. NICE is, formally speaking, an independent ‘non-governmental public body’ whose remit comes from the Department of Health, which funds it. That remit is to appraise the clinical and cost-effectiveness of technology x within its licensed indication for treating disease y. To be considered in the scoping process for possible appraisal, the technology must be 'either new or an innovative modification of an existing technology with claimed benefits to patients or the NHS judged against the comparator(s).' The purpose of the NICE appraisal is to decide whether the new technology works well (is clinically effective) and good value for money (is cost-effective). At no stage of the scoping or appraisal process is an innovation that claims to be cost-effective and 'good value for money', but not 'clinically effective' in relation to the comparators, eligible for consideration. So there is no point in their being put forward. Formally, the ban on such innovations is imposed on NICE from above, but there has never been any indication that the organisation is other than in full agreement with it, and accordingly with the biased use of the cost-effectiveness principle involved in prior filtering by clinical effectiveness.\n\nThis is not an empirical issue. The major project by Claxton and colleagues has yielded important insights into the cost-effectiveness threshold implicit in the behaviour of the NHS, establishing the average cost of an extra QALY generated (conservatively put at £12,396), the number of QALYs likely to be forgone as a consequence of approving a more expensive technology, and where those QALYs are likely to be lost in its 23 broad programme budget categories. The authors claim that this explicit quantification of the scale of opportunity costs the NHS faces provides a basis for determining the appropriate threshold for NICE decisions, as well as those made centrally by the NHS and Department of Health.\n\nFor those concerned with the inadequacies of the QALY as an effectiveness measure, the report emphasises that the estimation methods can cope with other outcomes, such as patient-reported outcome measures (PROMs), subject to their being brought within the opportunity cost framework3. The methods can also be extended to allow weights to be attached to the type of health that is forgone.\n\nHowever, neither this impressive empirical progress, nor the subsequent debate4,5 impinge on the present argument concerning the biased application of the cost-effectiveness principle. This empirical advance will simply make it easier to establish the displacement consequences of new cost-effective innovations, wherever and however they occur. As has been the case since the founding of NICE, the report and discussion ignores the mammoth standing silently in the south-west corner of the policy room: the proper use of cost-effectiveness as a criterion.\n\nFrom its inception NICE has never adopted the principle of cost-effectiveness, only the censored version of it called incremental cost-effectiveness. The Claxton report accepts this corruption of the principle, the single peripheral mention of decremental cost-effectiveness being buried under the heading 'multiple thresholds' in an Appendix. As independent analysts, they might be expected to state, upfront in one sentence, that it is in the light of the NICE remit that they exclude from consideration any intervention which is cost-effective by being less effective, but less costly.\n\nThe objective in section 2 below is to end the sinister bifurcation of the single and unified cost-effectiveness principle. Separating incremental and decremental cost-effectiveness is as meaningful as separating right-handed and left-handed ambidexterity. It may be helpful for operational reasons to characterise the differing origins of cost-effectiveness, but the two cannot be separated for policy purposes without abandoning the principle.\n\nIn section 3 we present and seek to counter the main arguments against accepting and promoting innovations that fall in the South-West (SW) quadrant of the cost-effectiveness plane and under a linear Incremental Cost-Effectiveness Ratio (ICER).\n\nOne of the most powerful reasons for the individual citizen to favour a National Health Service will be its rationality from a Rawlsian perspective. Under great uncertainty (approximating a 'veil of ignorance') as to what diseases and conditions oneself, one's children, grandchildren and significant others will suffer from in the future, the greater the reason to support the consistent application of the principle of cost-effectiveness throughout the system. And hence the greater the reason for bodies making decisions within it to treat South-West innovations in exactly the same way as North-East ones, using the same threshold10.\n\n\nThe integrity of the cost-effectiveness principle\n\nWe believe we can achieve our aim quickly and simply, by taking the key diagram in Claxton, confined to the North-East (NE) quadrant of the cost-effectiveness plane, and extending it to include all its four quadrants (Figure 1). The original figure in Claxton implicitly acknowledges the existence of the SW quadrant by extending the dotted ICER, or threshold, line for a short distance into it, doing so without distortion or kink1,2. (A kinked threshold line, steeper in the SW quadrant than in the NE, is one of the main arguments considered in part 2.) We can leave the South-East and North-West quadrants empty, as having dominated solutions that make the argument here irrelevant. Any new technology in the SE quadrant should be adopted as cost-effective and, because it both costs less and is more effective, trumps any other intervention beneath the ICER line in either the NE or SW quadrants. (It will often be referred to as ‘cost-saving’ rather than ‘cost-effective’ by those whose attention is restricted to the eastern hemisphere.)\n\nEssentially we duplicate the Claxton diagram, rotate the duplicate through 180 degrees and place it in the SW quadrant. And relabel appropriately. For the text explanation accompanying the Claxton diagram and our translation of it for the SW quadrant, see the Appendix.\n\nAll we intend, and need, to show in this section, is that interventions NEA and SWA are equally cost-effective, both resulting in a Net Health Benefit increase of 1 QALY. NEA adds 2 and loses 1. SWA adds 3 and loses 2. Case made. QED.\n\nNEB (+2, -2) and SWB (-2, +2) are both neutral, involving equal gain and loss.\n\nNeither NEC (+2, -3) or SWC (-2, +1) is cost-effective, with loss exceeding gain.\n\nAny intervention below the dotted ICER line is cost-effective – assuming it is not dominated, i.e. there is not an intervention that is further south and at least as far to the east, or further east and at least as far to the south.\n\nIt is important to use consistent terminology throughout the plane. There is much talk about 'disinvestment' in the threshold debate, with the QALY lost by investing in NEA characterised as such. But SWA also represents an investment in new technology and the two QALYs lost as a result are the disinvestment resulting from this new investment.\n\nThis diagram, like any cost-effectiveness plane or analysis, assumes a particular threshold. But it should be clear that the slope of the ICER (whether the threshold is 13k, 20k or 30k), how much uncertainty surrounds any empirical calculation of it, and where displacement or disinvestment specifically occurs - are all irrelevant to the present argument. The diagram simply confirms that the principle of cost-effectiveness, justified either on efficiency or ethical grounds, requires its implementation in unbiased form, treating incremental and decremental origins as equally valid. If censoring is undertaken, it should be explicitly acknowledged as representing the abandonment of the cost-effectiveness principle and justification sought on other grounds.\n\nIt will be obvious that the lower the threshold, the smaller the area under the ICER line in the NE quadrant and the greater the area under it in the SW quadrant; hence the greater the scope for interventions to be developed within the latter. At the limit, if the threshold were approaching at, or approaching zero, all interventions in the SW quadrant would be cost-effective, and none in the NE.\n\nHaving rejected the principle of cost-effectiveness as the basis for ruling out SW innovations, what other grounds might be advanced against adopting or encouraging them?\n\n\nGo South West? The arguments and counter-arguments\n\n“SW interventions are simply wrong because they take away from them something people already have.”\n\nThe simplest argument against treating the SW and NE quadrants in the same way boils down to the rights-based objection that adopting a SW intervention would involve 'taking away' effectiveness (in this illustration, QALYs) from individuals who currently enjoy it. No benefits to others can justify this breach of rights, it is said. But this principle, even if it were to be agreed that current recipients would not be forced to move on to the less effective treatment because it is now the cost-effective one, lacks any justification when extended to those who acquire the same condition in the future. Having never enjoyed the effectiveness of the old treatment, they cannot have a right to it taken away from them. Those who become ill later cannot ethically be favoured, simply because they suffer from this disease or condition, rather than from some other one. The Rawlsian rationality of this social ethic, even from an individual perspective, is clear.\n\n‘… the rational Rawlsian patient – who does not yet know whether they will personally suffer from condition X, … or, instead, from any of the wide range of other possible conditions – should clearly favour the wider distribution of benefits that comes from applying the decision rule consistently in the SW as well as NE quadrant.’6 p.457\n\n“SW interventions will produce ill health which will require treatment and impose extra costs”\n\nGandjour7 argues that the experience of loss, or even anticipation of loss, can have negative health consequences of various sorts. Unfortunately apart from the individual focus of his example8, Gandjour fails to address the key issue regarding intervention for any ‘lossaversionitis’ resulting from the introduction of SW interventions. Consistency and equity demands that realistic interventions for lossaversionitis go into the cost-effectiveness analysis, along with all other interventions. So, while the illness created may be real, there is no guarantee it will be treated. Prevention of lossaversionitis may be the optimal strategy.\n\n“SW interventions should not occur unless it can be shown that there will be a net increase in health”\n\nSendi, Gafni and Birch’s challenge to the SW argument helps clarify an important point as to why we adhere to it and reject their alternative9. They point out that there is no guarantee that the amount of resources released by a specific SW intervention will result in a net increase in QALYs. This will occur only if the resources are diverted to an intervention that will achieve this and not every intervention below the ICER line will do so. Correct. But the inability to determine specifically where the resources are diverted from to fund a new intervention in the NE quadrant is also unknown. So fundamentally their objection is to the use of an 'overall subjective ICER threshold’ for the NE, not just the SW. Their alternative approach involves use of a ‘decision maker’s plane’, where a specific intervention replaces a specific intervention only if the effect on overall health gain is positive. This is simply not the real world of any national health service, let alone the NHS, as pointed out by Claxton and colleagues:\n\n‘NICE cannot be expected to reflect what is likely to be marked variation between local commissioners and providers in how they react to an effective reduction in their budget as a result of positive guidance. Given NICE’s remit, it is the expected health effects (in terms of length and QoL) of the average displacement within the current NHS (given existing budgets, productivity and the quality of local decisions) that is relevant to the estimate of the threshold.’2 p.8\n\nWe see no justification for imposing higher requirements of specificity regarding displacement on SW interventions than on NE ones.\n\n“Some SW interventions are acceptable, but only those under a (very) kinked ICER”\n\nSome see validity in the SW argument but wish to restrict its application. The main mechanism suggested is a 'kinked' ICER - a threshold line which is steeper in the SW quadrant than it is in the NE one10. The slope in the SW quadrant should reflect the ‘acceptable’ Willingness to Accept/Willingness to Pay (WTA/WTP) ratio. This will be greater than 1, hence the steeper slope. Along similar lines, Kent, et al. suggest establishing a Maximally Acceptable Difference (MAD) in an ‘acceptability trial’ for SW interventions11, the MAD being ‘a level of inferiority beyond which a new less expensive agent would no longer be attractive when compared to the best standard.’\n\nThe most frequent objection to the SW argument is that attitudes to loss and gain (WTA and WTP) are asymmetric, with WTA typically higher or much higher than WTP, because of 'loss aversion'. While income and other factors play some role, the dominant explanation offered for such loss aversion is the so-called ‘endowment effect'. 'We' regard losing a specified amount of what we already possess as proportionately worse than gaining that same amount and require greater compensation to accept the loss than we would pay for an equal size gain.\n\nNumerous empirical studies have confirmed loss aversion as descriptively true at the individual and aggregated individual level, so this is not in dispute. Nor is the fact that the WTA/WTP ratio varies from situation to situation. In an example particularly relevant for this paper\n\n… the farther a good is from being an ordinary private good, the higher the ratio…. Ratios are highest for health/safety and public/non-market goods, next highest for ordinary private… The closer the good comes to being actual money, the smaller the ratio12. pp.434–5\n\nGrutters et al.13 found that using a WTA and a WTP format for the cost attribute in a discrete choice experiment (on transferring elements of hearing aid provision from the medical sector to private hearing aid dispenser) elicited different preferences and monetary values . They concluded\n\nMost discrete choice experiments in health care use the concept of WTP, but WTA has also been used… to our knowledge, no study has paid explicit attention to when the cost attribute should be defined as a payment or a discount. The lack of clarity on how to address the disparity between WTA and WTP in discrete choice experiments probably results from the fact that before the present study, the disparity had not yet been examined…13 p.1118\n\nThe case for adopting a SW intervention becomes progressively stronger as the saving from the loss of a QALY increases. If there is a way one can save 60k rather than 30k by giving up a QALY, then the benefits generated elsewhere are doubled. But whether a SW intervention is cost-effective always depends on the ICER.\n\nIn a pharma-sponsored study Liew, et al. calculated that shifting patients from their atorvastatin to simvastatin would lead to a net cost saving of €131 per subject, but also a loss of 0.03 quality-adjusted life-years (QALYs) per subject14. These equated to a decremental cost-effectiveness ratio of €4,777 per QALY lost. The authors’ conclusion that ‘It would be cost effective to maintain patients on atorvastatin for primary prevention rather than switch them to simvastatin’ is valid, given the threshold is set above €4,777.\n\nIn an example relating to a new intervention for pain management, Soares and Dumville report a decremental ratio of £1,220, going on to show in a Cost Effectiveness Acceptability Curve analysis that this would be cost-effective only at very low thresholds15. The authors leave it ambiguous as to whether the decision rule (threshold) they rightly say is required in both NE and SE quadrants should be the same one. We maintain that the principle of cost-effectiveness requires that they be the same and that no logical or ethical case can be made for any kinked ICER in a public system16. We question the relevance of aggregated asymmetric individual preference results to group level policy making, in the context of a resource-constrained system committed to equitable efficiency. The fact that the Grutters study not only produced different ratios for 'gainers' and 'losers', but that the two sets of results also depended on how the cost attribute was framed, confirms to us that permitting this ratio to be other than 1 is unethical at a societal level. Searching for the conditions under which one or other framing should be used, which they contemplate, is inappropriate, since an equitable public policy requires an unbiased single estimate of WTP&A.\n\nEstablishing that single value becomes the research challenge. Whether it will result in a ICER near the current NE one is unknown, because stated community preferences have never been investigated under the appropriate, Rawlsian, conditions of complete uncertainty as to where the investment and disinvestment will fall, and hence complete uncertainty about the future personal implications for the respondent.\n\n“Prospect theory and psychic numbing are legitimate bases for public policy”\n\nDescriptive theories of decision making, such as prospect theory, claim that individuals do not maximise expected value or utility, instead treating probabilities as non-linear and having value functions that are concave for gains and convex for losses17. This may or may not be true at the individual level, but if is, to be used as the basis for rejecting SW policy interventions, transportation from the individual to society needs to be regarded as legitimate. In what Featherstonhaugh, Slovic and others refer to as ‘psychic numbing’ and the ‘collapse of compassion’, the value of a life-saving intervention emerges as being, in line with prospect theory, inversely proportional to the magnitude of the threat, rather than being determined by the absolute number of lives the intervention can save18,19.\n\nWe argue that the inability to relate emotionally to the loss of a relatively small amount of health by very large numbers, compared to the ability to relate to the gain of even a moderate amount for an identified individual – say one QALDay for 30,000 people compared with 1 QALY for one person - is to be treated as a problem to be addressed and overcome at the policy level, not to be automatically accommodated.\n\n\nDiscussion\n\nThe bias in relation to innovation is a corollary of the fundamental one. NICE is charged with objectives other than maximising the increase in public health and among its other obligations is to support innovation. But this turns out to be biased support, in that no support can be provided for the development of technologies that are simply cost-effective. These would include innovations which could improve population health by being less costly and less effective – such as SWA in Figure 1, or ones further to the east of the SW quadrant, including the ones that would fall under a kinked ICER, or meet the MAD test of Kent et al. No innovation in the SW quadrant can meet the filter test of clinical effectiveness administered prior to the test of cost-effectiveness. So while NICE has a remit to support the adoption of innovative new technologies, in practice the support is confined to those that will cost more.\n\nEckerman and Pekarsky have exposed the weaknesses of the current NICE procedures as contributions to improved allocative efficiency in the NHS6. Unless the disinvestment to fund a new technology occurs in the least cost-effective activity in the whole service, then allocative efficiency will not be improved as much as it could be, and indeed is quite likely to be reduced.\n\nThis is indisputable conceptually, but even more important, the existence of the missing knowledge of the actual shadow price would pose extreme difficulties for NICE. As Paulden and colleagues point out.\n\nThe use of thresholds based upon Eckermann and Pekarsky’s proposals by reimbursement bodies would likely result in fewer new technologies being adopted by public healthcare systems. To the extent that this might provide opportunity for resources to be reallocated into more efficient existing health services, this ought to be welcomed. Nevertheless, the implied consequence that technologies be rejected on the basis that there is a preferred option, but one that cannot be implemented, may be a bridge too far for most reimbursement bodies. This is particularly true for NICE, which has a remit, amongst other things, to support the adoption of innovative new technologies, and which operates in a political environment where the adoption of such a low threshold might be untenable.7 p.318\n\nPerhaps the supreme irony in this respect is that 'innovations' falling in the SW quadrant are in fact daily occurrences in most health services, though the denial of this reality, seen as necessary for political survival, persists. The problem is not merely that such SW innovations are disguised or denied - we see them as essential to the future of any National Health Service - but that they occur disproportionately in politically vulnerable areas of the service and with no consideration, even informal, of whether they were cost-effective at any threshold. For example, reducing the numbers of staff such as nurses, saves money at the expense of the effectiveness/quality of the service. The common pretence is that such a change falls in the SE quadrant, usually on its western border where no loss is suffered, few having the audacity to claim it actually increases effectiveness. This fools only those who wish to be fooled, who may or may not include the managers responsible, whose careers depend on delivering apparently SE changes within shrinking budgets.\n\nNone of this is in any way intended to discourage the search for and implementation of SE innovations. But the much publicised LEAN ones, which involve working smarter not harder, may well fall in the SW quadrant, as well as the SE, and still represent increased cost-effectiveness20.\n\nThere is, also ironically, an excellent example of NICE implementing a SW innovation in its own operations: its introduction of the cheaper Single Technology Appraisal, where the manufacturer is responsible for the analysis and an independent team is paid only to critique it, not conduct a full-scale Multi Technology Assessment using all appropriate comparators21. It seems politically unacceptable to admit that this is undoubtedly reducing the quality of the appraisal, even though the reduction could conceivably be relatively small and the cost saving large, thereby releasing resources for other uses - the essence of the SW argument.\n\nIt is not as if the key underlying issue is not well recognised by Claxton and colleagues22\n\nOne explanation for… ‘Acceptance creep’ (in the NICE appraisal process) is that the broad selection of stakeholders who contribute to the NICE process excludes a key constituency: those unidentified NHS patients who bear the true opportunity costs of NICE decisions. NICE undoubtedly faces extensive pressure from the direct beneficiaries of a positive recommendation, including manufacturers, the patients who might benefit and their clinicians. Indeed, these stakeholder groups have, quite appropriately, become an important part of the appraisal process. However, without institutional leadership to ensure balance, there is much less pressure to take full account of the likely impact on other NHS patients. The most recent evidence and the nature of the recent proposals suggests that NICE is not providing sufficient leadership and is failing to uphold this critical responsibility to all NHS patients.1. p.2\n\nThe evidence suggests that more harm than good is being done, but it is the unidentified and unrepresented NHS patients who bear the true (health) opportunity costs. Although finding reasons to approve new drugs is undoubtedly politically expedient, this cannot be ethically literate, because the interests of NHS patients, whether they are identifiable or not, are just as real and equally deserving of the type of care and compassion that can be offered by a collectively funded health care system. It is to be hoped that NICE will begin to place the unidentified NHS patients who bear the real opportunity costs at the heart of its deliberative process; especially as it reconsiders how other attributes of benefit might be taken into account.1 p.6\n\nThe question is whether they will acknowledge that their arguments require at least noting the elephant in the SW corner of the policy room, and suggesting that it cannot be ignored by those at the table if they wish to pursue cost-effectiveness in an unbiased way. The efforts to justify this censoring of cost-effectiveness, albeit well-intentioned in many cases, unfortunately coincide with the material interests of powerful stakeholders, commercial, professional and political, which are not always aligned with those of the citizens. Independent analysts need to ensure that they are not colluding, and, to avoid this accusation, should state explicitly that they have been told to not go SW.\n\n\nConclusions\n\nThe SW argument is simply that, given cost-effectiveness is the most important route to maximising group level health gain, not applying it logically and consistently in the SW as well as the NE quadrant is a clear breach of the opportunity cost-effectiveness principle and its underlying justification. While implementing the principle requires many lower-level and difficult decisions23, these must not be allowed to undermine the case for using it.\n\nIf one wants to reject cost-effectiveness as a principle, that is clearly possible. But distorting it, either by refusing to consider intervention in the SW quadrant or imposing different requirements (different threshold, or different demands regarding displacement impact) undermines the case for employing it at all, whether on efficiency or ethical grounds, or both. The task is to have SW innovations legitimated and discussed and evaluated as transparently as those in the NE.\n\nThe local and global consequence of rejecting the SW argument is that there is little or no incentive to develop interventions that are cost-effective by being cheaper but less effective - especially ones that would be considerably cheaper but only slightly less effective at the individual level. These would include many non-pharmacological interventions, including such things as health literacy promotion, decision support for medication adherence, or simple home care.\n\nIt is hard to convince people that making things better in one part of a system does not necessarily make them better overall, in fact often worse. So the ubiquitous mantra of ‘lowering costs without compromising quality’24 needs to be seen as part of the problem as well as part of the solution. There is a parallel to the ‘tragedy of the commons’ here25.\n\nThe healthy, selfish Rawlsian concerned only with themselves and their relatives should consider the opportunity costs of all policy decisions as if they were an anonymous other and therefore support unbiased application of the cost-effectiveness principle.\n\nCaveat emptor must be the message to potential NICE buyers, particularly in low or middle income countries26, but certainly not only in them.",
"appendix": "Author contributions\n\n\n\nJD, in collaboration with MKK, updated and developed his earlier published version of the SW argument. He drafted the paper, which was extensively revised in both content and organisation by MKK, as well as JBN, GS and himself. All authors approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMKK’s PhD study was funded by the Region of Southern Denmark, the University of Southern Denmark and The Health Foundation (Helsefonden). The contribution of GS was supported by the Screening and diagnostic Test Evaluation Program (STEP) funded by the National Health and Medical Research Council of Australia under program grant number 633003.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAppendix\n\nText adapted from 1. This text has been modified to refer to the NE quadrant of Figure 1 and includes additional labels: NHB, NEA, and NEB.\n\n“As [Figure 1] illustrates, CEA effectively becomes an analysis of net health benefits [(NHBs)]: does the health gain from the new intervention outweigh the health decrements associated with the displacement of existing services necessary to fund it? [Figure 1] shows the incremental costs and QALYs associated with a new intervention [NEA] relative to a comparator (the latter being shown at the origin). The new intervention generates 2 additional QALYs per patient and, at price P1, imposes an additional £20,000 per patient; the ICER is, therefore, £10,000 per QALY gained. At a threshold of £20,000 per QALY, the additional cost of £20,000 per patient translates into a decrement of 1 QALY (the distance between the y-axis and the threshold). This is because the threshold indicates the additional cost that needs to be imposed on the NHS budget in order to displace services that result in 1 QALY being forgone. Therefore, at that price, there is a net health gain of 1 QALY per patient (2 gained from the new intervention and 1 forgone through displacement). At a price of P2, the additional cost per patient of the new intervention [NEB] is £40,000 and the net health gain is 0: the 2 additional QALYs from the new intervention are the same as the QALYs forgone through displacement. At the highest price of P3, the adoption of the new intervention [NEC] would actually result in a net health decrement of 1 QALY as it generates fewer QALYs (2) than are forgone (3).” 1, pp. 3–4.\n\nText adapted from 1. This text has been modified to refer to the SW quadrant of Figure 1 and includes additional labels: NHB, SWA, and SWB.\n\n“As [Figure 1] illustrates CEA effectively becomes an analysis of net health benefits [(NHBs)]: does the health gain from the new intervention outweigh the health decrements associated with the displacement of existing services necessary to fund it? [Figure 1] shows the incremental costs and QALYs associated with a new intervention relative to a comparator (the latter being shown at the origin). The new intervention [SWA] generates3 additional QALYs per patient and, at [saving (negative price)] P1, [saves] £20,000 per patient; the ICER is, therefore, £10,000 per QALY gained. At a threshold of £20,000 per QALY, the [reduced] cost of £20,000 per patient translates into [an increment] of 1 QALY (the distance between the y-axis and the threshold). This is because the threshold indicates the [reduced] cost […] imposed on the NHS budget [as a result of the 2 QALYs] being forgone. Therefore, at that price, there is a net health gain of 1 QALY per patient (3 gained from the new intervention and 2 forgone through displacement). At a [saving (negative price)] of P2, the cost [reduction] per patient of the new intervention [SWB] is £40,000 and the net health gain is 0: the 2 additional QALYs from the new intervention are the same as the QALYs forgone through displacement. At the [lowest saving (negative price)] of P3, the adoption of the new intervention [SWC] would actually result in a net health decrement of 1 QALY as it generates fewer QALYs1 than are forgone (2).” 1, pp. 3–4.\n\n\nReferences\n\nClaxton K, Martin S, Soares M, et al.: Methods for the estimation of the National Institute for Health and Care Excellence cost-effectiveness threshold. Health Technol Assess. 2015; 19(14): 1–503, v–vi. PubMed Abstract | Publisher Full Text\n\nClaxton K, Martin S, Soares M, et al.: Methods for the Estimation of the NICE Cost Effectiveness Threshold. Final Report. CHE (York) Research Paper 81. 2013. Reference Source\n\nClaxton K: Three questions to ask when examining MCDA. ISPOR Values and Outcomes Spotlight. 2015; 18–20. Reference Source\n\nBarnsley P, Towse A, Schaffer SK, et al.: Critique of CHE Research Paper 81: Methods for the Estimation of the NICE Cost Effectiveness Threshold. Office of Health Economics Occasional Paper 13/01. 2013. Publisher Full Text\n\nClaxton K, Sculpher M: Response to the OHE critique of CHE Research paper 81. 1–8. Reference Source\n\nDowie J: Why cost-effectiveness should trump (clinical) effectiveness: the ethical economics of the South West quadrant. Health Econ. 2004; 13(5): 453–459. PubMed Abstract | Publisher Full Text\n\nGandjour A: Loss aversion and cost effectiveness of healthcare programmes. Pharmacoeconomics. 2008; 26(11): 895–898. PubMed Abstract | Publisher Full Text\n\nSeverens JL: Loss Aversion and Cost Effectiveness of healthcare programmes: Whose aversion counts anyway? Pharmacoeconomics. 2008; 26(11): 899–900. PubMed Abstract | Publisher Full Text\n\nSendi P, Gafni A, Birch S: Ethical economics and cost-effectiveness analysis: is it ethical to ignore opportunity costs? Expert Rev Pharmacoecon Outcomes Res. 2005; 5(6): 661–665. PubMed Abstract | Publisher Full Text\n\nO’ Brien BJ, Gertsen K, Willan AR, et al.: Is there a kink in consumers’ threshold value for cost-effectiveness in health care? Health Econ. 2002; 11(2): 175–180. PubMed Abstract | Publisher Full Text\n\nKent DM, Fendrick AM, Langa KM: New and dis-improved: on the evaluation and use of less effective, less expensive medical interventions. Med Decis Making. 2004; 24(3): 281–286. PubMed Abstract | Publisher Full Text\n\nHorowitz JK, Mcconnell KE: A review of WTA/WTP studies. J Environ Econ Manage. 2002; 44(3): 426– 447. Publisher Full Text\n\nGrutters JP, Kessels AG, Dirksen CD, et al.: Willingness to Accept versus Willingness to Pay in a discrete choice experiment. Value Health. 2008; 11(7): 1110–1119. PubMed Abstract | Publisher Full Text\n\nLiew D, Webb K, Marbaix S, et al.: Changes to the statin prescribing policy in Belgium: Potential impact in clinical and economic terms. Am J Cardiovasc Drugs. 2012; 12(4): 225–232. PubMed Abstract | Publisher Full Text\n\nSoares M, Dumville JC: Economic evaluation of healthcare technologies using primary research. Evid Based Nurs. 2008; 11(3): 67–71. PubMed Abstract | Publisher Full Text\n\nDowie J: No room for kinkiness in a public healthcare system. Pharmacoeconomics. 2005; 23(12): 1203–1205. PubMed Abstract | Publisher Full Text\n\nTversky A, Kahneman D: Advances in prospect theory: Cumulative representation of uncertainty. J Risk Uncertain. 1992; 5(4): 297–323. Publisher Full Text\n\nSlovic P, Zionts D, Woods AK, et al.: Psychic numbing and mass atrocity. New York University School of Law Working Paper 11–56. 2011. Reference Source\n\nFeatherstonhaugh D, Slovic P, Johnson SM, et al.: Insensitivity to the Value of Human Life: A Study of Psychophysical Numbing. J Risk Uncertain. 1997; 14(3): 283–300. Publisher Full Text\n\nSapountzis S, Kagioglou M: Applications of lean thinking: a briefing document. University of Salford. 2007. Reference Source\n\nBuxton MJ: Economic evaluation and decision making in the UK. Pharmacoeconomics. 2006; 24(11): 1133–1142. PubMed Abstract | Publisher Full Text\n\nClaxton K, Sculpher M, Palmer S, et al.: Causes for concern: is NICE failing to uphold its responsibilties to all NHS patients? Health Econ. 2015; 24(1): 1–7. PubMed Abstract | Publisher Full Text\n\nKind P: Cost-effectiveness analysis: a view into the abyss. Appl Health Econ Health Policy. 2015; 13(3): 269–71. PubMed Abstract | Publisher Full Text\n\nCosgrove T: Value-based health care is inevitable and that’s good. Harvard Business Review Blog. 2013. Reference Source\n\nHardin G: The tragedy of the commons. Science. 1968; 162(3859): 1243–1248. PubMed Abstract | Publisher Full Text\n\nAugustovski F, Alcaraz A, Caporale J, et al.: Institutionalizing health technology assessment for priority setting and health policy in Latin America: from regional endeavors to national experiences. Expert Rev Pharmacoecon Outcomes Res. 2015; 15(1): 9–12. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10845",
"date": "27 Oct 2015",
"name": "Jeffrey Braithwaite",
"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 times when health economics articles are dense and jargon laden. There are other times when health economics articles seem to say what common sense suggests anyway.This article is neither of these. It speaks with logic, authority and a great deal of wisdom about the importance of potential bias in the UK’s National Institute for Care and Health Excellence (NICE) and its approach to cost-effectiveness. The authors make the case that at the end of the day, “while NICE has a remit to support the adoption of innovative new technologies, in practice the support is confined to those that will cost more.” The authors want NICE and by extension anyone involved in this sort of appraisal not to omit to consider innovations and technologies that are simply cost-effective (as compared to those that are firstly determined to be clinically effective, and then assessed for cost-effectiveness).Without going into the detailed economic and conceptual arguments, which is of less interest to general readers, it is surely incumbent on policymakers to review this argument and take notice of it, or refute it. Either way, we should hear more about the potential of NICE to refine how it enables cost-effectiveness and innovation. Given the role NICE plays nationally and internationally, this is by no means a trivial request.",
"responses": []
},
{
"id": "17091",
"date": "19 Oct 2016",
"name": "Jes Søgaard",
"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 like the paper: “Caveat Emptor NICE: biased use of cost-effectiveness is inefficient and inequitable” by Jack Dowie et al and I have followed it on the way to this version. I think they have a logical point and they argue their case well. I cannot judge whether they miss some arguments against the SW case.\nI have two queries that the authors and/or editors might consider to be relevant for some thought.\nThe kinkiness issue is discussed at some length on page 5, but to me it is unclear whether it is an ethical and logical issue, as the authors conclude (referring to an earlier Dowie paper that I have not read) or whether it is an empirical issue i.e. whether kinkiness should reflect the WTP/WTA ratio “in society”, i.e. take loss aversion into account. In the analysis they state “We maintain that the principle of cost-effectiveness requires that they be the same and that no logical or ethical case can be made for any kinked ICER in a public system” and in the Discussion it seems that the authors accept the MAD principle which could imply kinked ICERs in the plane quadrants.\n\nThe other point is more practical – how would you administer SW cases. In the NE quadrant you document clinical efficacy and have some cost estimates to estimate ICERs, with cost data from typically secondary endpoints supplemented with observational cost data. In the SW quadrant, should you document cost savings, i.e. have costs as the primary end points for strong cost saving evidence? Should we/authorities encourage pharmaceutical and other industries to develop cost saving drugs and devices with inferior health effects? For example accept inferior generics – if cost savings are large enough? Or is the SW quadrant more relevant with the health providers cost saving ‘innovations’ when third party payers demand cost controls and budget cuts? What would the SW quadrant cases be?\nBesides these two queries I think the paper is ready to be indexed.",
"responses": [
{
"c_id": "2267",
"date": "02 Nov 2016",
"name": "Jack Dowie",
"role": "Author Response",
"response": "Interventions falling under a kinked ICER, or meeting the MAD criterion in the SW quadrant, are indeed SW interventions. But as a result they fall outside the ones NICE can consider as innovatory, which was the opening point in the Discussion. Our fundamental point is that the ethical justification of the use of CEA is the existence of opportunity costs in a resource-constrained national health service. It is both unethical and illogical (given the definition of CE) to apply this only to interventions that are incrementally cost-effective (fall under the chosen ICER threshold in the NE quadrant) but not to interventions which are decrementally cost-effective (fall under the same ICER threshold in the SW quadrant). There is therefore no ethical justification for a kinked ICER, or any other criterion (e.g. MAD) which partitions under-ICER SW interventions into those that are acceptable and not acceptable. Using the empirically observed difference between WTA and WTP infringes the ethical basis for the use of CEA. There are undoubtedly practical issues in calculating both the potential cost savings and loss of effectiveness for SW interventions, but we suggest these cannot trump the ethical case for tackling the task, anymore than they do in the NE quadrant. As to the sources of SW interventions, it is worth repeating that many cost saving changes are, in reality, SW interventions being presented as SE ones. However, the essence of the argument is that providers should indeed be seeking interventions that are inferior in effectiveness for a particular subgroup, but generate more benefits to the whole population for which they are responsible and accountable. Using CEA in a biased way is a purely political decision that has no scientific justification."
}
]
}
] | 1
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https://f1000research.com/articles/4-1078
|
https://f1000research.com/articles/2-179/v1
|
03 Sep 13
|
{
"type": "Research Article",
"title": "Hyperthermic intraperitoneal chemoperfusion with high dose oxaliplatin: Influence of perfusion temperature on postoperative outcome and survival",
"authors": [
"Johanna Verhulst"
],
"abstract": "Introduction: Hyperthermic intraperitoneal chemotherapy (HIPEC) is becoming a standard therapy in the treatment of peritoneal carcinomatosis (PC). Compared to systemic chemotherapy, HIPEC improves survival in patients with PC. This therapy has high morbidity rates (up to 41%). In vitro it has been demonstrated that hyperthermia has a toxic effect on malign cells. However, hyperthermia also affects normal tissue. To my knowledge, any additional effect of hyperthermia combined with chemotherapy has never been demonstrated in a clinical setting. In this study, the effects of hyperthermia on outcome and survival were analyzed.Methods: Patients with PC from any origin who were treated with HIPEC were included in this retrospective, non-randomized study. Data on patient characteristics, tumor characteristics, features of the surgery and postoperative complications were extracted from patient files. Models predicting time to removal of nasogastric tube (TRNT), post-operative major complications, the occurrence of anastomotic leaks and post-operative survival were built, using negative binomial regression, logistic regression or Cox proportional hazards regression as appropriate.Results: 138 patients treated with HIPEC were included. Maximal temperature during the operation was not statistically significantly associated with anastomotic leaks or post-operative major complications. Maximal temperature during the operation was negatively associated with post-operative survival (P=0.01).Conclusion: The results suggest that hyperthermia may negatively affect survival in patients who are treated with HIPEC for PC of various origins. This study has the classical limitations of a retrospective study. Therefore, randomized trials are required to confirm the results.",
"keywords": [
"The author of this article has informed F1000Research that the above study was set up in response to a request from her former supervisor to investigate the effects of perfusion temperature on post-operative morbidity and mortality after hyperthermic intraperitoneal chemoperfusion. The author has informed F1000Research that due to a disagreement over publication of these results with her former supervisor she resigned from her post."
],
"content": "Introduction\n\nPeritoneal carcinomatosis (PC) occurs in 5% of patients with colorectal carcinoma and in patients with FIGO (International Federation of Gynecology and Obstetrics) stage III and IV ovarian cancer1. PC can occur synchronous with the primary tumor or as a relapse (metachronous). Survival rates in patients with PC are rather poor. The median survival with standard chemotherapy is 50 and 23 months for PC of ovarian and colorectal origin, respectively2,3. When treated with hyperthermic intraperitoneal chemotherapy (HIPEC), survival rates of patients with PC of ovarian and colorectal origin increased to 66 and 30 months, respectively2,4.\n\nThere is an increasing interest in the use of locoregional antineoplastic drug therapy in patients with PC. The benefit of intraperitoneal chemotherapy arises from the existence of a peritoneal-plasma barrier. This barrier allows the local administration of higher doses of chemotherapy while minimizing systemic side effects5. Cytotoxic drugs penetrate only a few millimeters into tumor tissue6. To improve penetration, HIPEC is combined with cytoreductive surgery, where the tumor mass is decreased as much as possible before the administration of chemotherapy.\n\nOxaliplatin is recognized as a standard adjuvant treatment in colorectal cancer7. Promising results were also demonstrated in ovarian cancer, gastric cancer and malignant mesothelioma8–10. Oxaliplatin is rapidly absorbed intracellularly, as a result of its lipophilic structure11. Combined, these features make oxaliplatin a logical choice for local administration.\n\nHyperthermic perfusions are used because hyperthermia stimulates apoptosis in tumor cells12–14. Recently it was demonstrated that hyperthermia increases the peritoneal oxaliplatin concentration while reducing systemic absorption15. However hyperthermia also induces apoptosis in normal cells14 and affects the healing of anastomosis16–18. Furthermore, it was demonstrated in a rat model of PC of colorectal origin that hyperthermia did not increase survival compared to normothermic intraperitoneal treatment19. To my knowledge, any additional effect of hyperthermia combined with intraperitoneal chemotherapy compared to intraperitoneal chemotherapy alone has not been demonstrated.\n\nAlthough in a recent trial the preoperative level of functioning was reached three to six months after surgery20, the morbidity rates described in patients after HIPEC are rather high (up to 41%)21. The role of hyperthermia in the improved survival is not clear, and it is possible that the morbidity may be (partially) a result of the hyperthermia.\n\nThe aim of this study was to identify the impact of the temperature of the perfusate on post-operative ileus, major post-operative complications, the occurrence of anastomotic leaks and post-operative survival.\n\n\nMaterials and methods\n\nThis is a retrospective, non-randomized study. By the retrospective and anonymised nature of the study, no informed consent of the patients was required. The study included patients from one university hospital. All patients that presented with resectable PC from any origin were eligible for inclusion. Patients with primary PC were included, as well as patients with metachronous PC. Electronic patient files were reviewed and the following data were extracted: age at the time of the operation, gender, body mass index (BMI), duration of anesthesia, time to removal of nasogastric tube (TRNT, measured from the day of operation), duration of stay in intensive care unit (ICU), duration of total hospital stay, 30-days mortality, post-operative complications, maximal perfusate temperature (Tmax), and area under the temperature curve (AUC) as a measure of total temperature. Analyses of biochemistry and cell count were carried out on blood samples taken on the last day before and the first day after HIPEC. White blood cell count, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma-glutamyltransferase (γ-GT) were registered. TRNT was used as a measurement of the duration of post-operative ileus.\n\nThe temperature of the perfusate was measured in three locations: left and right in the upper abdomen, and in the pelvis. AUC was calculated separately for each registration location with a data summary model for repeated measures (baseline = 0) in Medcalc™ 12.5.0 (MedCalc Software, Acacialaan 22, B-8400 Ostend, Belgium.) The mean AUC over the three locations was used in this study. The unit of AUC is °C*minute.\n\nThe full dataset is provided in the accompanying Data File. Blanks in these tables represent missing data.\n\nPatients were placed in modified Lloyd Davies position and the upper body covered with a heating blanket (Bair Hugger, Arizant Healthcare Inc., Eden Prairie, MN, USA). Cytoreductive surgery aimed to remove all resectable implants of tumor while preserving the patient’s quality of life. Following verification of resectability and absence of undetected metastatic disease, the entire colon was mobilized starting from the ileocolic region working towards the left. The major omentum was removed en bloc with the affected colon whenever it was involved in the disease process. The spleen, or pancreatic tail were included in the specimen when affected by cancer. A peritonectomy of the diaphragm was performed according to a previously described method22. When required, the tendinous part of the diaphragm was partially resected. Following the resections in the upper abdomen, tumor tissue was removed from the pelvis. After that, the serosal surfaces covering the small bowel and mesentery were cleared from tumor tissue by a combination of tumorectomy, wedge resection, or segmental resection as required. At least 150 cm of small bowel had to be preserved. An open abdomen method was used for the administration of the intraperitoneal chemoperfusion, as described previously23. The skin was sutured to a retractor frame placed over the abdomen. A plastic hood was positioned over the frame in order to allow the evacuation of vapor escaping from the abdominal cavity. Two Tenkhoftype inflow drains and three multiperforated outflow drains connected to a roller pump were used for chemoperfusion. The drains were placed in the pelvis, right upper abdomen and left upper abdomen. A heat exchanger was placed along the drains in order to maintain the required temperature. Hypothermia (34°C) was maintained prior to the start of chemoperfusion. Central temperature was monitored with an esophageal temperature probe. Abdominal temperature was monitored by means of three thermocouple probes placed left and right in the upper abdomen, and in the pelvis.\n\nPrior to chemoperfusion, intravenous chemotherapy, consisting of folate 20 mg/m2 followed by a 5-fluorouracil 400 mg/m2 in 250 ml of saline perfusion over 1 hour, was administered to non-ovarian cancer patients according to standard procedures. Oxaliplatin (460 mg/m2) was added to the perfusion circuit when the abdominal temperature reached the set temperature. The duration of the chemoperfusion was 30 minutes. The abdominal cavity was not washed, in order to retain the efficacy of remaining drug. After the chemoperfusion, the abdomen was closed in layers.\n\nThe primary end-point of this study was overall survival. Overall survival was measured from the day of surgery till death. Patients who were alive at the last contact moment were censored at the date of last contact.\n\nThe secondary end-points were major complications, the occurrence of anastomotic leaks and TRNT. TRNT was a proxy variable for post-operative ileus. Univariate relations between Tmax or AUC and stay at ICU, total hospital stay, post-operative white blood cell count, AST, ALT, γ-GT and TRNT were explored by means of linear regression or negative binomial regression, as appropriate. Univariate relations between Tmax or AUC and overall survival were explored by means of Cox-regression.\n\nA negative binomial regression model predicting TRNT was built using Tmax, AUC, sex, age at the time of the operation, operation time, stay at ICU, post-operative white blood cell count, AST, ALT and γ-GT as independent variables. The parameters \"major complications\" and \"anastomotic leaks\" are binary variables; therefore a logistic regression model was built to predict these outcomes, including Tmax, AUC, sex, age at the time of the operation, BMI, post-operative white blood cell count, AST, ALT and γ-GT, operation time and number of anastomoses as independent variables. A Cox proportional hazards model predicting overall survival was built using Tmax, AUC, sex, age at the time of the operation, operation time, number of anastomoses, tumor type, stay at ICU, total hospital stay, post-operative white blood cell count, AST, ALT and γ-GT as independent variables. Backwards stepwise selection was used for model building. Statistical significance was assumed when P<0.05.\n\n\nResults\n\nFrom July 2005 until February 2011, 138 patients were treated with oxaliplatinbased HIPEC in a tertiary center. Demographic data are illustrated in Table 1. Mean age was 59 years, ranging from 17 to 82 years (Figure 1). Forty-four percent of the patients were males. Nearly 60% of the patients presented with PC originating from colorectal cancer. Ovarian cancer and pseudomyxoma peritoneii were the second and third most frequent cause of the PC, respectively.\n\nHistogram of frequencies of ages of the included patients.\n\nDetails of the surgery are illustrated in Table 2. The mean anesthesia time was nearly 10 h, ranging from 4 to 18 h and with a standard deviation of 2.8 h (Figure 2). On average, the maximal temperature was 40.5°C and the area under the temperature curve was 1340.63°C*minute (Figure 3 and Figure 4).\n\nHistogram of frequencies of the duration of anesthesia in the included patients.\n\nHistogram of frequencies of maximal temperature of the included patients.\n\nHistogram of frequencies of the AUC of the included patients.\n\nOutcome of surgery is summarized in Table 3. Two patients (1.4%) died within 30 days after the operation. Due to the small number of events the influence of AUC and maximal temperature on 30 days mortality could not be examined. Twenty-six patients needed reoperation. Reasons for reoperation were anastomotic leak (fourteen patients), intra-abdominal bleeding (five patients), perforation of the stomach (one patient), subobstruction (one patient), wound infection (one patient), bladder leak (one patient), and abdominal collection (three patients). In one patient, scald injuries were found during reoperation. Eighty-nine patients had at least one anastomosis, resulting in 155 anastomoses. Anastomotic leak occurred in sixteen patients. However, neither AUC nor Tmax was related to anastomotic leaks (P=0.68 and P=0.67, respectively) or major complications (P=0.50 and P=0.20, respectively).\n\nA logistic regression model assessing the relation between several predictors and the occurrence of anastomotic leaks was fitted (Table 4). Longer operation time, a high number of anastomoses and post-operative leukocyte count were associated with the occurrence of leaks. Two variables describing the temperature during the operation were included in the model: Tmax and AUC. Both were close to significance. However, the effects of these variables were going in opposite directions: increasing AUC was associated with the occurrence of leaks, while increasing Tmax was associated with no leaks.\n\nThe number of anastomoses and the total operation time were associated with the occurrence of major complications (Table 5). Tmax and AUC were not related to the occurrence of major complications.\n\nOn average, patients stayed 4 days in the ICU (median 2, range 1 to 87, Figure 5). The total hospital stay was 27 days on average (median 18, range 3 to 169, Figure 6). The relationship between stay at ICU and Tmax or AUC is illustrated in Figure 7 and Figure 8, respectively. From these figures, it seems that patients with an extremely long stay at the ICU were treated at higher temperatures. In terms of the total hospital stay, there were fewer outliers and the duration was more evenly spread as a function of Tmax and AUC (Figure 9 and Figure 10).\n\nHistogram of frequencies of stay at ICU in the included patients.\n\nHistogram of frequencies of duration of hospitalization in the included patients.\n\nScatterplot showing the relation between stay at ICU and Tmax.\n\nScatterplot showing the relation between stay at ICU and AUC.\n\nScatterplot showing the relation between total hospitalization duration and Tmax.\n\nScatterplot showing the relation between hospitalization duration and AUC.\n\nOn average, the nasogastric tube was removed after 7 days (median 5, range 0 to 77). The time to removal of the nasogastric tube seemed to remain constant with increasing temperature (Figure 11 and Figure 12). A model predicting the time to removal of nasogastric tube was fitted (Table 6). Four of the assessed variables turned out to be significantly related to TRNT: sex, Tmax, operation time and post-operative leukocyte count. On average, removal of the nasogastric tube was sooner after the operation in females than in males. With increasing maximal temperature, the expected TRNT decreased. Increasing post-operative leukocyte count and operation time was associated with an increased expected TRNT.\n\nScatterplot showing the relation between TRNT and Tmax.\n\nScatterplot showing the relation between TRNT and AUC.\n\nMedian survival was 23 and 27 months in patients with PC from colorectal and ovarian origin, respectively (Table 7). In univariate analysis Tmax was significantly associated with hazard of death (P=0.042), while AUC was not significantly associated to this hazard (P=1.117) (Table 8).\n\nA model predicting the hazard of death was built. Four of the considered variables turned out to be significantly related to this hazard: tumor type, sex, maximal temperature and operation time (Table 9). The expected hazard ratio for an increase of 1°C in maximal temperature was 1.6, with the other variables in the model kept at fixed values. The predicted survival curves from this model are presented in Figure 13 and Figure 14.\n\n1: males, 37°C; 2: males, 42°C; 3: females, 37°C; 4: females, 42°C. All at 579 minutes operation time (mean).\n\n1: 37°C; 2: 42°C. All at 579 minutes operation time (mean).\n\nThe following analysis was not initially planned. An additional model predicting the hazard of death was built to evaluate the whether or not blood concentration of sodium, potassium, glucose, lactate, and pO2 or pCO2 during the operation were significantly related to post-operative survival. The variables from the first model were included as well. Tumor type, operation time, sex and lactate concentration in the blood were significantly associated with hazard of death (Table 10). The predicted survival curves from this model are presented in Figure 15. Tmax was not significantly associated with the hazard of death after blood lactate concentration was included in the model. This is probably due to high correlation between Tmax and lactate concentration (Figure 16).\n\n1: males, 37°C; 2: males, 40°C; 3: males, 42°C; 4: females, 37°C; 5: females, 40°C; 6: females, 42°C. All at 579 minutes operation time (mean) and 9.9 lactate mmol/l (mean).\n\nScatterplot showing the relation between blood lactate concentration and Tmax.\n\n\nDiscussion\n\nIn selected patients, cytoreductive surgery combined with hyperthermic intraperitoneal chemoperfusion (HIPEC) results in a better survival compared with systemic chemotherapy24. Major complications occur frequently in the post-operative period. Hyperthermia is a possible risk factor for reduced anastomotic healing16–18. There is currently no evidence for better survival in patients treated with hyperthermic chemoperfusion compared to normothermic chemoperfusion. The aim of this study was to analyze the effects of hyperthermia on post-operative survival and on the post-operative complications.\n\nThe data presented here show a statistically significant association between increasing temperature during the perfusion and decreasing post-operative survival. More precisely, the expected hazard ratio is 1.6 times as large for an increase of 1 degree in the temperature of the perfusion.\n\nMaximal temperature was related to TRNT, with shorter TRNT for higher temperatures. The relation of temperature to the occurrence of anastomotic leaks was ambivalent. However, the results indicate the possibility of a negative effect of increasing temperatures on the occurrence of anastomotic leaks.\n\nSurvival analysis did show an inverse relation between Tmax and post-operative survival both in univariate and multivariate analyses. Previously, in an animal model of PC a negative relation between survival and high temperature was also suggested as well19.\n\nThe present study has several limitations. First it is not randomized. Hyperthermia is the standard for intraperitoneal chemoperfusion. The temperature was adjusted to the clinical status of the patients. Normothermic chemoperfusion was administered in patients with important comorbidity. This may cause a bias. However, survival is worse in patients treated with perfusate with a higher temperature although these were the patients with less comorbidity. Second, it is a retrospective study; therefore the data were not acquired in a standardized way which again is a possible source of biases.\n\nGiven these limitations, it is too early for a final conclusion on the relationship between hyperthermia and survival in the setting of HIPEC. However, the significantly worse survival in patients treated with hyperthermic intraperitoneal chemotherapy HIPEC raises important concerns about the safety of this method. Although, in selected patients, the results of HIPEC seems to be better than standard therapy2,4, treatment can possibly be further improved by reducing the temperature during the chemoperfusion to body temperature (i.e. 37–38°C). Future research on HIPEC should focus on two aspects of the treatment. First, randomized studies comparing normothermic to hyperthermic chemoperfusion are needed. Second, the causal mechanism of the worse survival in patients treated with hyperthermic chemoperfusion should be clarified.\n\n\nConsent\n\nAll data have been completely anonymised, without altering the scientific meaning of the analyses.",
"appendix": "Author contributions\n\n\n\nThe author collected all the data, did the statistical analyses and wrote the article.\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\nJones H 3rd, Ngan HY, Pecorelli S, et al.: FIGO staging classifications and clinical practice guidelines in the management of gynecologic cancers. FIGO committee on gynecologic oncology. Int J Gynaecol Obstet. 2000; 70(2): 209–262. PubMed Abstract | Publisher Full Text\n\nHuang HQ, Baergen R, Lele S, et al.: Intraperitoneal cisplatin and paclitaxel in ovarian cancer. N Eng J Med. 2006; 354(1): 34–43. PubMed Abstract | Publisher Full Text\n\nBrouquet A, Marchal F, Classe JM, et al.: Complete cytoreductive surgery plus intraperitoneal chemohyperthermia with oxaliplatin for peritoneal carcinomatosis of colorectal origin. J Clin Oncol. 2009; 27(5): 681–685. PubMed Abstract | Publisher Full Text\n\nBereder JM, Quenet F, Sideris L, et al.: Toward curative treatment of peritoneal carcinomatosis from nonovarian origin by cytoreductive surgery combined with perioperative intraperitoneal chemotherapy: a multi-institutional study of 1,290 patients. Cancer. 2010; 116(24): 5608–5618. PubMed Abstract | Publisher Full Text\n\nSugarbaker PH, Jacquet P: Peritoneal-plasma barrier. Cancer Treat Res. 1996; 82: 53–63. PubMed Abstract | Publisher Full Text\n\nCowan DSM, Egorin MJ, Tannock IF, et al.: Limited penetration of anticancer drugs through tumor tissue: A potential cause of resistance of solid tumors to chemotherapy. Clin Cancer Res. 2002; 8(3): 878–884. PubMed Abstract\n\nChau I, Cunningham D: Adjuvant therapy in colon cancer--what, when and how? Ann Oncol. 2006; 17(9): 1347–1359. PubMed Abstract | Publisher Full Text\n\nFerron G, Rigaud DB, Bourbouloux E, et al.: Oxaliplatin-based hyperthermic intraperitoneal chemotherapy in primary or recurrent epithelial ovarian cancer: a pilot study of 31 patients. J Surg Oncol. 2011; 103(1): 10–16. PubMed Abstract | Publisher Full Text\n\nIveson T, Nicolson M, Coxon F, et al.: Capecitabine and oxaliplatin for advanced esophagogastric cancer. N Eng J Med. 2008; 358(1): 36–46. PubMed Abstract | Publisher Full Text\n\nShea MT, Evans MT, Goonewardene TI, et al.: Phase II trial of vinorelbine and oxaliplatin as first line therapy in malignant pleural meothelioma. Lung Cancer. 2005; 47(2): 277–281. PubMed Abstract | Publisher Full Text\n\nMilano G, Lvi F, Massari C: Oxaliplatin: Pharmacokinetics and chronopharmacological aspects. Clin Pharmacokinet. 2000; 38(1): 1–21. PubMed Abstract\n\nMella O, Akslen L, Bruland O, et al.: Hyperthermia improves the antitumor effect of metronomic cyclophosphamide in a rat transplantable brain tumor. Radiother Oncol. 2008; 86(3): 435–442. PubMed Abstract | Publisher Full Text\n\nXu G, Ngai C, Fung K, et al.: Apoptotic and necrotic effects of tumor necrosis factor-alpha potentiated with hyperthermia on l929 and tumor necrosis factor-alpha-resistant l929. Int J Hyperthermia. 2010; 26(6): 556–564. PubMed Abstract | Publisher Full Text\n\nTamm M, Szilard J, Roth M, et al.: Mesothelioma cells escape heat stress by upregulating hsp40/hsp70 expression via mitogen-activated protein kinases. J Biomed Biotechnol. 2009; 2009: 451084. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSideris L, Pichette V, Drolet P, et al.: Rationale for heating oxaliplatin for the intraperitoneal treatment of peritoneal carcinomatosis: a study of the effect of heat on intraperitoneal oxaliplatin using a murine model. Ann Surg. 2011; 254(1): 138–144. PubMed Abstract | Publisher Full Text\n\nDecker M, Dimmler A, Hohenberger W, et al.: Hyperthermic intraperitoneal chemoperfusion (HIPEC) decrease wound strength of colonic anastomosis in a rat model. Int J Colorectal Dis. 2007; 22(8): 941–947. PubMed Abstract | Publisher Full Text\n\nMan BD, Lomme R, Boerman O, et al.: The effects of adjuvant experimental radioimmunotherapy and hyperthermic intraperitoneal chemotherapy on intestinal and abdominal healing after cytoreductive surgery for peritoneal carcinomatosis in the rat. Ann Surg Oncol. 2008; 15(11): 3299–3307. PubMed Abstract | Publisher Full Text\n\nKoga S, Shimizu T, Maeta M: Inuence of local hyperthermia on the healing of small intestinal anastomoses in the rat. Brit J Surg. 1991; 78(1): 57–59. PubMed Abstract | Publisher Full Text\n\nRutten HJT, Bleichrodt RP, de Hingh IHJT, et al.: Hyperthermia and intraperitoneal chemotherapy for the treatment of peritoneal carcinomatosis: an experimental study. Ann Surg. 2011; 254(1): 125–130. PubMed Abstract | Publisher Full Text\n\nShen P, Stewart JH, Levine EA, et al.: Survival and quality of life following cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy for peritoneal carcinomatosis of colonic origin. Ann Surg Oncol. 2011; 18(13): 3673–3679. PubMed Abstract | Publisher Full Text\n\nChua T, Morris D, Saxena A, et al.: Critical assesment of risk factors for complications after cytoreductive surgery and perioperative intraperitoneal chemotherapy for pseudomyxoma peritonei. Ann Surg Oncol. 2010; 17(5): 1291–1301. PubMed Abstract | Publisher Full Text\n\nSugarbaker P: Peritonectomy procedures. Ann Surg. 1995; 221(1): 29–42. PubMed Abstract | Free Full Text\n\nEdwards GD, Esquivel J, Sebbag G, et al.: Morbidity and mortality analysis of 200 treatments with cytoreductive surgery and hyperthermic intraoperative intraperitoneal chemotherapy using the coliseum technique. Ann Surg Oncol. 1999; 6(8): 790–796. PubMed Abstract\n\nYan TD, Savady R, Sugarbaker PH: Systematic review on the efficacy of cytoreductive surgery combined with perioperative intraperitoneal chemotherapy for peritoneal carcinomatosis from colorectal carcinoma. J Clin Oncol. 2006; 24(24): 4011–4019. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1679",
"date": "01 Oct 2013",
"name": "David L Morris",
"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 made me sit up and think. The finding of an adverse effect of Tmax during HIPEC on long term survival is certainly very challenging. The science of the effects of hyperthermic intraperitoneal chemoperfusion is far from solid, but we have achieved previously unachievable survival results by using it. If this article is correct, we are harming our patients by using heat. I do not have the best statistical brain in the world, but the number of patients is small and for example there are only 21/138 patients with a maximum temperature of <39.5°C and only 10/138 with a temperature >41.5°C. The model used in figures 13 and 14 may well be valid but when there are only one or two patients with a Tmax of 37°C it is not appropriate to plot a survival curve. The lactate data is interesting because it provides a clear illustration that a biologically important measure is being affected by temperature.This paper creates more questions than answers but is clearly a stimulus to others to examine their data.",
"responses": []
},
{
"id": "2235",
"date": "08 Nov 2013",
"name": "Pompiliu Piso",
"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 article is investigating the role of perfusion temperature on postoperative outcome and survival of patients with peritoneal carcinomatosis. The idea is interesting, however, the presented data does not support the conclusions.The analysis was retrospective, the time interval very long, monocentric with a bias regarding surgeons, used technique and chemotherapy regime (mixed patients with HIPEC alone or plus intravenous 5FU).The postoperative outcome is determined mainly by the extent of surgery and critical anastomosis and less by HIPEC itself. This has been shown by several published data. The TRNT is influenced by even more other factors. The causality relation between the maximal temperature and survival has not been demonstrated. Moreover, other significant prognostic factors e.g. peritoneal cancer index, completeness of cytoreduction, histologic subtype etc have not been evaluated. Out of any context, temperature may play in the statistical analysis a role but the clinical interpretation of the results should be very careful. For instance, who were the patients having maximal temperature during HIPEC: was this group having a high tumor load with a high Peritoneal Cancer Index?, a more difficult resection, were they co-morbid etc.In my opinion, this manuscript is confusing rather than helpful. We know from other hyperthermia studies that efficacy is affected by every °C – this sounds more reasonable to me.",
"responses": [
{
"c_id": "665",
"date": "13 Jan 2014",
"name": "Johanna Verhulst",
"role": "Author Response",
"response": "I am fully aware of the limitations due to the retrospective nature of the study. The intention of the study was to be a pilot study concerning the effects of hyperthermia in the context of HIPEC. The study does not claim to be able to draw any final conclusions on a causal relation between hyperthermia and post-operative survival. However, a negative correlation between those variables was observed. As it is not possible to make any final inference about this matter based on this study, further randomized studies should be conducted in order to confirm or disprove the results from this study.As mentioned in the article, the patients treated with lower temperatures, were the patients with higher co-morbidity. Nevertheless, these patients had a longer survival in the studied population. Therefore, it could be expected that in a randomized study, the negative effect would be even bigger.I would be grateful for references to articles studying the effects of hyperthermia on post-operative outcome."
}
]
},
{
"id": "3009",
"date": "08 Jan 2014",
"name": "Shigeki Kusamura",
"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 author evaluated the correlation of temperature during HIPEC with short-term surgical and long term oncologic outcomes in patients affected by several types of peritoneal carcinomatosis. The idea is interesting, the paper reasonably well-written, and the analysis conducted meticulously.The temperature was appraised using two parameters: Tmax and AUC.The first caveat of the paper is Tmax. If there is a possibility of tissue damage exerted by the heat, the negative effect depends not only on the level of temperature but also on the duration of the exposure to hyperthermic conditions. Under 43°C it is hard to observe significant tissue damage if the exposure does not last more than 30 minutes, according to experimental data. Therefore, in my opinion, this parameter is not suitable and unreliable to achieve the objectives of the study. The second weak point is the survival analysis. Important parameters such as PCI, completeness of cytoreduction, tumor grade, lymph node status (for colon cancer), and whether the tumor is primary or relapsing, the platinum sensitivity (for ovarian cancer) were not considered in the cox model. Moreover, in figure 13 the events occurred at the same time points from the surgery in all four subsets. Is that a coincidence? In the figure 14 groups 1 and 2 combined had 46 events (deaths). How could you explain if the total number of ovarian cases were 16 cases? The tables outlining logistic regression analysis could be better presented by changing the Estimates by Odds Ratio. Remember, OR=exp(B)=e estimate My advice is to reconsider the Tmax and survival analysis. Even though these parts contain the only significant correlations, I myself would take them out from the study. The correlations with short-term surgical outcome is sufficiently interesting to justify the publication.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-179
|
https://f1000research.com/articles/4-1075/v1
|
15 Oct 15
|
{
"type": "Research Article",
"title": "MinION Analysis and Reference Consortium: Phase 1 data release and analysis",
"authors": [
"Camilla L.C. Ip",
"Matthew Loose",
"John R. Tyson",
"Mariateresa de Cesare",
"Bonnie L. Brown",
"Miten Jain",
"Richard M. Leggett",
"David A. Eccles",
"Vadim Zalunin",
"John M. Urban",
"Paolo Piazza",
"Rory J. Bowden",
"Benedict Paten",
"Solomon Mwaigwisya",
"Elizabeth M. Batty",
"Jared T. Simpson",
"Terrance P. Snutch",
"Ewan Birney",
"David Buck",
"Sara Goodwin",
"Hans J. Jansen",
"Justin O'Grady",
"Hugh E. Olsen",
"MinION Analysis and Reference Consortium",
"Bonnie L. Brown",
"Miten Jain",
"Richard M. Leggett",
"David A. Eccles",
"Vadim Zalunin",
"John M. Urban",
"Paolo Piazza",
"Rory J. Bowden",
"Benedict Paten",
"Solomon Mwaigwisya",
"Elizabeth M. Batty",
"Jared T. Simpson",
"Terrance P. Snutch"
],
"abstract": "The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.",
"keywords": [
"MinION",
"nanopore sequencing",
"data release",
"long reads",
"minoTour",
"marginAlign",
"NanoOK",
"third-generation sequencing"
],
"content": "Introduction\n\nThe idea of using nanopores as biosensors was suggested by several groups starting in the 1990s (patent by Church et al., submitted 1995, published 1998; Kasianowicz et al., 1996). Investigators documented that ionic current passing through a nanopore depends on the identity of nucleic acid bases interacting with and transiting the nanopore (Akeson et al., 1999; Derrington et al., 2010; Manrao et al., 2011; Wallace et al., 2010). Nanopores were also found able to resolve the order of bases in nucleic acid molecules (Akeson et al., 1999; Bayley et al., 2006; Laszlo et al., 2014; Song et al., 1996). A final key step leading to sequencing was reduction of DNA translocation speed through the nanopore using enzymatic control (e.g., polymerase) to feed the nucleic acid strand to the pore, base-by-base, on a millisecond time scale (Cherf et al., 2012; Lieberman, 2010). Oxford Nanopore Technologies (https://www.nanoporetech.com) was founded in 2005 to translate these proof-of-concept studies into a commercial third-generation sequencing device. The announcement of the MinION, a device that can detect bases of a single-stranded DNA (ssDNA) molecule that passes through a nanopore with no theoretical limits on read length (except those introduced during sample preparation), was met with enthusiasm at the Advances in Genome Biology and Technology (AGBT) meeting in 2012 (Check Hayden, 2012; Eisenstein, 2012). Independent beta-testing of the MinION device began in April 2014 with the start of the MinION Access Programme (MAP) (https://www.nanoporetech.com/community/the-minion-access-programme) involving over 1,000 laboratories. The first publications appeared in late 2014 and early 2015 (Ammar et al., 2015; Ashton et al., 2015; Greninger et al., 2015; Jain et al., 2015; Karlsson et al., 2015; Kilianski et al., 2015; Laver et al., 2015; Loman et al., 2015; Mulley & Hargreaves, 2015; Quick et al., 2014; Urban et al., 2015; Wang et al., 2015) and these provided a first glimpse of the performance characteristics and limitations of the device at that time, as well as potential applications.\n\nThe MinION is the smallest high-throughput sequencing platform available to date: a 90g device, 10 cm in length, that is able to sequence individual molecules of DNA with a single-use flow cell. To enable sequencing of both strands, a library is constructed from double-stranded DNA (dsDNA) with a protocol similar to that used for short-read, second-generation platforms. The library preparation chemistries (SQK–MAP005 and SQK–MAP005.1) used in this study, contain two different adapters that are ligated to the DNA (Figure 1A). The first, the ‘leader adapter’, consists of two oligos with partial complementarity that form a Y-shaped structure once annealed. The second, the ‘hairpin adapter’, is a single oligo with internal complementarity to form a hairpin structure. Both adapters in the sequencing kit used for this study are preloaded with ‘motor proteins’ that mediate the movement of DNA through the pore. Another function of the adapters is to guide the DNA fragments to the vicinity of pores via binding to tethering oligos with affinity for the polymer membrane (Figure 1B). Sequencing begins at the single-stranded 5’ end of the leader adapter (Figure 1C). Once the complementary (double-stranded) region of the leader adapter is reached, the motor protein loaded onto the leader adapter unzips the dsDNA, allowing the first strand of the DNA fragment, the ‘template’, to be passed into the nanopore one base at a time, while the sensor measures changes in the ionic current. After reaching the hairpin adapter, an additional protein, the ‘hairpin protein’, allows the complementary strand of DNA to pass through the nanopore in a similar fashion. The current MinION flow cell has 512 channels, each connected to 4 wells which may each contain a nanometer-scale biological pore (nanopore) embedded in an electrically-resistant membrane bilayer (Figure 1D). Each channel provides data from one of the four wells at a time, the order of use defined by the allocation of wells to well-groups during an initial ‘mux scan’ (File S2 Glossary), allowing up to 512 independent DNA molecules to be sequenced simultaneously.\n\n(A) Suspended library molecules are concentrated near nanopores embedded in the membrane. A voltage applied across the membrane induces a current through the nanopores. (B) Schematic of a library molecule, showing dsDNA ligated to a leader adapter pre-loaded with a motor protein and a hairpin adapter pre-loaded with a hairpin protein, and the tethering oligos. (C) Sequencing starts from the 5’ end of the leader adapter. The motor protein unwinds the dsDNA allowing single-stranded DNA to pass through the pore. (D) A flow cell contains 512 channels (grey), each channel consisting of 4 wells (white). Each well contains a pore (blue) and a sensor. At any given time, the device is recording the data stream from the wells of the active well-group, in this example, g1. (E) Perturbation in the current across the nanopore is measured 3,000 times per second as ssDNA passes through the nanopore. (F) The ‘bulk data’ are segmented into discrete ‘events’ of similar consecutive measurements. The 5-mer corresponding to each event is inferred using a statistical model. (G) The 1D base-calls are inferred separately for the template and complement event signals. (H) Alignment of the 2D base-calls from the event signals from both, and the 1D base-calls are used to constrain the 2D base-calls.\n\nWhen a voltage is applied across the membrane, an ion current flows through the nanopore. The translocation of ssDNA through the nanopore causes a drop in the current that is characteristic of the bases in contact with the pore at that time (Figure 1E, Laszlo et al., 2014). A sensor measures the current in the nanopore several thousand times per second (at 3,000Hz in this study), the data streams are passed to the ASIC (application-specific integrated circuit) and the MinKNOW software. The raw current measurements are compressed into a sequence of ‘events’, each being a mean current value with an associated variance and duration (Figure 1F). The raw current measurements or the corresponding events, plotted over time, are referred to as a ‘squiggle plot’. The base-caller in use at this time modelled the characteristics of 45 (= 1,024) possible 5-mers and base-calling consisted of finding the optimal path (Figure 1G) through a Hidden Markov Model (HMM) of successive 5-mers using a Viterbi algorithm (http://www.bio-itworld.com/news/02/17/12/Oxford-strikes-first-in-DNA-sequencing-nanopore-wars.html). The 1D base-calls are inferred separately for the template and complement event signals (Figure 1G), the 2D base-calls from the event signals from both, and the 1D base-calls are used to constrain the 2D base-calls (Figure 1H).\n\nThe release of version R7+ flow cells by Oxford Nanopore to the MAP community provided highly positive feedback concerning both utility and quality of the MinION data. However, it became clear that groups were having different degrees of success with the MinION, with the possible influencing factors being difficult to infer from a single sequencing run. The MARC Phase 1 experiments were designed to assess the yield, accuracy, and reproducibility of MinION data by undertaking replicate experiments across multiple sites, with the intention of identifying technical factors important for consistently high performance. To this end, five laboratories initially sequenced the same Escherichia coli strain K-12 substrain MG1655, in duplicate, using a single shared protocol for culture, extraction of high-quality total genomic DNA, library preparation and sequencing (File S1). A laboratory E. coli strain was chosen as it has a single circular chromosome of 4.6 Mb that could be sequenced to sufficient depth in a single MinION run and a complete reference sequence is available (NCBI RefSeq NC_000913). The detailed protocol for sequencing double-stranded total genomic DNA was based on the standard protocol from ONT at the time the experiment was conceived. During the generation of the sequencing data for this work, referred to here as the Phase 1a experiments, updates to the ONT sequencing kit and protocol were made available (version MN005_1124_revC_02Mar2015, last modified 10 June 2015, https://wiki.nanoporetech.com/pages/viewpage.action?pageId=28246488). To ensure this study included data from these updates, we generated an equivalent dataset using the updated protocol, referred to here as the Phase 1b experiments.\n\nAn initial lack of tools for the analysis of data obliged the MAP community to develop a series of bioinformatics solutions for exploring the native FAST5 data (Table S2) produced by the MinION. Poretools (Loman & Quinlan, 2014, https://github.com/arq5x/poretools) and poRe (Watson et al., 2015, http://sourceforge.net/projects/rpore/) are packages for converting and visualising the raw data, whereas minoTour (http://minotour.github.io/minoTour) provides real-time analysis and control of a sequencing run and post-run analytics. NanoOK (Leggett et al., 2015, https://documentation.tgac.ac.uk/display/NANOOK/NanoOK) uses alignment-based methods to assess quality, yield, and accuracy of the data. New software packages such as marginAlign (Jain et al., 2015, https://github.com/benedictpaten/marginAlign), NanoCorr (Goodwin et al., 2015, http://schatzlab.cshl.edu/data/nanocorr/), Nanopolish (Loman et al., 2015, https://github.com/jts/nanopolish/) and PoreSeq (Szalay & Golovchenko, 2015, https://github.com/tszalay/poreseq) were developed to address the relatively high error rate of the raw data and allow genome assembly and error-correction from MinION reads. Some of these tools were used for the MARC Phase 1 data analyses.\n\nAt the time of this writing, around a dozen reports have emerged recounting utility of the MinION for de novo sequencing of viral, bacterial, and eukaryotic genomes. The MinION data from this study constitute the only resource, to date, of carefully replicated experiments across multiple laboratories that can be used to infer the volume, quality and reproducibility of data from the platform. At the time the Phase 1 experiments were run, extensive preliminary analysis revealed clear factors influencing site-to-site reproducibility and provided inspiration for future MARC experiments in which we will explore improvements to the MinION sequencing protocol.\n\n\nMaterials and methods\n\nEach group used the following protocols to obtain total genomic DNA from freshly grown cells, fragment the DNA, prepare libraries, and sequence the libraries using the MinION. The full methods are described in the supplementary information (File S1).\n\nTo remove variability that might be caused by freeze-thaw of genomic DNA and based on previous observations that fresh material gave better results, each group worked with freshly prepared total genomic DNA from E. coli str. K-12 substr. MG1655 purchased from DSMZ, Germany (https://www.dsmz.de, DSM No. 18039) on 21 January 2015. On arrival, the E. coli strain was rehydrated in LB broth. The rehydrated culture was used to inoculate ten replicate 10 mL LB broth tubes and one plate, all of which were incubated overnight at 37°C. Following incubation, the plate was examined to ensure the culture was pure. Broth cultures were centrifuged at 5,000 × g in a benchtop centrifuge to collect biomass for cryogenic bead tube (Protect, Lab M, Lancashire, UK) inoculation. Bead tubes were stored at -70°C until they were shipped, at room temperature, to four other laboratories (Table S1). Upon arrival, the bacterial culture was plated on LB agar, checked for viability and purity, and the bead tube stored at -80°C until the sample was ready for culture and extraction.\n\nAt each participating laboratory, DNA was extracted from approximately 4 × 109 log-phase cells using QIAGEN Genomic-tip 20/G according to the manufacturer’s instructions (QIAGEN, Valencia, California). A library was prepared the day after extraction using the Genomic DNA Sequencing Kit SQK–MAP005 according to the base protocol from Oxford Nanopore (version MN005_1123_revA_02Mar2015) with slight modifications from the MARC consortium (File S1).\n\nIn summary, genomic DNA (1 µg and 1.5 µg for the Phase 1a and 1b experiments, respectively) was fragmented using Covaris g-TUBE (Covaris, Ltd., Brighton, United Kingdom) to achieve a fragment distribution with a peak at ~10 Kb (3,300 × g). The sheared DNA was pretreated with PreCR Repair Mix (New England Biolabs, Ipswich, Massachusetts) to repair possible damage to the DNA that could interfere with the sequencing process: since the DNA passes through the pore as a single strand, the presence of a nick is of particular concern because it would prematurely terminate the sequencing of the molecule. To protect the DNA from further damage during the preparation of the library, vortexing was avoided and more gentle mixing approaches (i.e., pipetting, inverting, or gentle flicking) were used instead. After clean-up with 1× AMPure XP beads (Beckman Coulter, Brea, California) to remove PreCR reagents from the sample, the DNA was resuspended in fresh 10 mM Tris-HCl pH 8.5, and concentration and fragment size were assessed using the Qubit dsDNA BR assay (Life Technologies, Grand Island, New York) and the Agilent TapeStation where available (Agilent Technologies, Santa Clara, California). In Phase 1a, all remaining genomic DNA was used for the next stage while in Phase 1b, which started with 1.5 µg, 1 µg of the genomic DNA remaining at this point was used. For most libraries, an internal control DNA sequence (‘DNA CS’ from the SQK–MAP005 kit, corresponding to the last ~3,555 bases of Enterobacteria phage lambda, RefSeq NC_001416.1, with a single mutation G45352A) was added at this point. The DNA was then prepared using the NEBNext End Repair Module, cleaned with 1× AMPure beads, treated with the NEBNext dA-Tailing Module (New England Biolabs) and cleaned again with 1× AMPure beads prior to ligation.\n\nThe final ligation of adapter and hairpin was performed in Protein LoBind 1.5 ml tubes (to avoid loss of protein-loaded adapters) with Blunt/TA Ligase Master Mix (New England Biolabs) followed by a pulldown step using his-tag Dynabeads (Life Technologies). Extra care was taken to mix reagents during the ligation and following steps only through careful pipetting, so to avoid unnecessary contact of the ligated and protein-bound DNA with the tube walls.\n\nThe MinION device is controlled by the MinKNOW™ Software Agent on the connected computer. The Metrichor™ Desktop Agent manages the connection to the base-calling service in the cloud hosted by Metrichor. Installation of the most current version of software for both programs at the time of each experiment was strictly enforced. Thus, the software versions used to process different experiments were highly correlated with the date on which experiments were commenced. While the library was being prepared, the MinION device was made ready for sequencing. A new R7.3 flow cell, provided to the MARC Phase 1 laboratories from the same lot number, was fastened to the MinION device and the MinKNOW Platform QC recipe script was run to assess the number of pores in each channel available for sequencing. A minimum of 400 g1 channels (of a possible 512) was considered acceptable. At the end of the QC, the flow cell was primed/washed twice (File S1, steps 79–80) and the sequencing run started after loading the library (6 µl for Phase 1a runs or 12 µl for Phase 1b runs). Once the 48-hour sequencing recipe script had been initiated, the Metrichor Desktop Agent was started and the raw data files were automatically uploaded to the Metrichor cloud-based service for base-calling. To maximize the yield of higher quality sequence data from the device, an additional aliquot of stored library (that had been held at 4°C) was loaded at 24h to coincide with the pre-set g1-to-g2 pore switch to fresh wells with active pores.\n\nThe output of the MAP_48Hr_Sequencing_Run script is one FAST5 file per read. The FAST5 format (File S2) used by Oxford Nanopore is a variant of the HDF5 standard (File S2, https://www.hdfgroup.org) with a hierarchical internal structure designed to store the metadata associated with the sequencing of that read and the events (aggregated bulk current measurements) pre-processed by the MinION (Table S2). The data from each instance of the MAP_48Hr_Sequencing_Run script are allocated a ‘run number’ (referred to in this study as the ‘batch id’, File S2 Glossary), and within this batch, each read is produced by one of the 512 channels and numbered by a ‘file number’ starting from zero. The combination of experiment name, batch, channel and file number is sufficient to uniquely identify a read. During the Phase 1 experiments, a 128-bit numerical universally-unique identifier (UUID) (https://tools.ietf.org/html/draft-mealling-uuid-urn-03), represented as a 32-digit hexadecimal number, was introduced to the FAST5 format as an alternate unique identifier for each read.\n\nThe FAST5 file for each read is uploaded to the cloud base-calling service by the Metrichor agent, base-calls are inferred, the read is allocated to a ‘read class’ of either ‘pass’ or ‘fail’ based on the criteria used at the time (File S2). All the data in the raw FAST5 plus additional metadata and the base-calls themselves are packaged into a base-called FAST5 file (Table S2) with a more complex internal structure and downloaded to the ‘pass’ or ‘fail’ subfolder of the pre-specified ‘downloads’ directory on the client computer. At the time the Phase 1 experiments were performed and base-called, the read class could only be inferred from the directory in which it was deposited by Metrichor.\n\nThe base-called FAST5 files and associated metadata from each of the five labs and 20 experiments were collated on a server at the European Nucleotide Archive (ENA, http://www.ebi.ac.uk/ena) and run through a bespoke pipeline of pre-processing tools (Table S3). The ENA pipeline extracted the 2D base-calls from the base-called FAST5 files with poreTools version 0.5.1 (Loman & Quinlan, 2014), then aligned every read to the E. coli K-12 reference genome (NCBI RefSeq Accession NC_000913.1) using BWA-MEM version 0.7.12-41044 with the nanopore data parameters ‘-× ont2d’ (Li, 2013) and LAST version 460 (Kielbasa et al., 2011). Both the BWA-MEM and the LAST alignments were post-processed using marginAlign version 0.1 (Jain et al., 2015). Statistics on each of the four alignments were computed by SAMtools version 1.2 (Li et al., 2009), poreMap version 0.1.1 (https://github.com/camilla-ip/poremap), marginStats (Jain et al., 2015), and identity version 0.1 (https://github.com/enasequence/ONT). The number of target, control and unclassified reads produced during each experiment was inferred by mapping each 2D read to the E. coli and lambda reference sequences, then allocating each read to either target or control when there was a single significant alignment to the respective genome. The remaining reads were recorded as ‘unclassified’ if they mapped to both or neither of the possible references. A consensus sequence of the nanopore reads mapped to the appropriate E. coli reference was inferred by Nanopolish version 0.3.0 (Loman et al., 2015) and included with the analyses as part of the data release.\n\nAll base-called FAST5 files and the outputs of the ENA pipeline for the 20 experiments (Table S10) are available through ENA project PRJEB11008 (http://www.ebi.ac.uk/ena/data/view/PRJEB11008).\n\nIn this study, we describe the data that match the chronological order in which they were generated and processed, from raw events, to 1D, then 2D base-calls. We then explored accuracy, at each stage, quantifying the data produced under the standard MARC protocol and commenting on how variations from that protocol may have affected the data yield or accuracy. Preliminary analyses of the data relied on summaries and visualisations from the minoTour webserver (http://minotour.github.io/minoTour), reports generated by NanoOK version 0.54 (Leggett et al., 2015), and bespoke Python and R scripts. To explore variations over time, each read was allocated to the 15 minute interval in which the read commenced sequencing, the number of active pores (where an active pore was defined as one that was still producing reads), and the read counts were converted to number of reads per hour per active pore. Plots were generated by allocating the events from each read to the appropriate 15 min interval under the assumption that events are produced at a steady rate for each read. The percentage of the 512 active pores in each window was then computed, normalising event yield by the number of active pores to derive the event rate in events per hour per pore. The median read length in events was computed for the reads from each experiment commencing in each 15 min interval. Reads generated from the first 1h, between 24 and 25h, and the last 1h of the experiments were not shown as the flow cell characteristics determining the data generation rate were obscured by stochastic effects arising from the initiation, well switching, and low active pore numbers toward the end of each experiment. The default run script does not attempt to base-call reads with less than 200 or more than 230,000 events, the arbitrary limits originally introduced to limit the memory requirements of the base-caller. To reduce noise that would otherwise obscure the underlying degradation rate of the flow cell chemistry, reads outside the callable length range were excluded and although the ‘Basecaller XL’ workflow currently available can call reads with up to 1 million events, we did not attempt to base-call these extra long reads in this study. The final figures, tables, and supplementary material were based on summary statistics for every read from every experiment generated by poreQC version 0.2.10 (https://github.com/camilla-ip/poreqc) and poreMap version 0.1.1 (https://github.com/camilla-ip/poremap).\n\nThe spike-in of a control sample of known DNA is useful for calibrating the accuracy of data from an experiment, especially when a good reference sequence for the target sample is not available. Ideally, sufficient control sample reads would be obtained to perform these analyses, but not so many that the yield of target sample is significantly diminished. Thus, the proportion of reads that are from the target rather than the control sample is another metric that affects the usable yield of the MinION. The proportion of target and control reads in each sample was inferred by NanoOK, which mapped each 2D read to the E. coli and lambda reference sequences using BWA-MEM ‘-× ont2d’, and classified each read to the genome of the primary alignment, or reported the read as ‘unclassified’.\n\nTo quantify the error rate of reads produced by the MinION and explore the effect that different alignment methods, metrics and read types have on the values reported, we produced an error metric we refer to as ‘total percent error’ of a read; that is, the percentage of a read that is inaccurate due to miscalled bases, inserted bases in the read, and deleted bases that are missing from the read but present in the reference sequence. The intent of this approach was to circumvent alignment-dependent biases that may reduce the miscall rate at the expense of insertions and deletions (indels).\n\nSince the accuracy metrics are computed from alignments of base-calls to the appropriate reference and each alignment method used will produce slightly different estimates, we computed the total error, and the components, for four alignment strategies: initial alignment by BWA-MEM (parameters ‘-× ont2d’) or LAST (parameters ‘-s 2 -T 0 -Q 0 -a 1’, as recommended by Quick et al., 2014), followed by re-alignment with marginAlign (Jain et al., 2015), which uses expectation maximization to train an HMM and estimate Maximum Likelihood Estimation (MLE) parameters that are, in turn, used to infer higher confidence alignments guided by the AMAP objective function (Schwartz et al., 2007). The alignment-based calculations provided by minoTour, NanoOK and poreMap were based on BWA-MEM (parameters ‘-× ont2d’). Further data processing was performed by bespoke Python scripts and extracts of the data plotted using either bespoke R scripts or minoTour. For clarity, the data and algorithm used to derive each figure are described briefly at the appropriate point in the Results section.\n\nSequencing bias of the MinION was explored with the over- and under-represented 5-mer table produced by NanoOK. If a platform is capable of sequencing any DNA sequence, all possible 5-mers in the DNA should be proportionally represented in the data when counts are normalized for the distribution of all 5-mers in the genome. Thus, the most under-represented and over-represented 5-mers in the base-calls from the MinION may suggest limitations or biases of the nanopore sequencing process. The NanoOK tables were computed from a hash table of read k-mer counts generated by moving a sliding window of size 5 base-by-base over each FASTQ read and counting 5-mers. The relative abundance of each read k-mer was calculated by dividing the k-mer count by the total number of k-mers in all the reads. Similarly, a hash table of reference 5-mer counts was generated from the reference sequence. The most under-represented 5-mers were deemed to be those with the largest difference in relative abundance between the reads and reference and where the reference abundance was greater than the read abundance. The most over-represented 5-mers were deemed to be those with the largest difference in relative abundance between the reads and the reference and where the read abundance was greater than the reference abundance.\n\n\nResults\n\nA total of 20 experiments (individual flow cell runs) were performed in two stages (Phase 1a and 1b) by five laboratories. Experiments from Phase 1a and 1b used the SQK–MAP005 and SQK–MAP005.1 Genomic DNA Sequencing Kits, respectively, which required a template mass of 1 µg and 1.5 µg, and library volume of 6 µl and 12 µl, respectively. Each laboratory (Table S1) undertook two identical replicate experiments for each kit version. The 20 experiments are henceforth referred to as P1a-Lab1-R1 to P1b-Lab5-R2, following a ‘phase-lab-replicate’ format.\n\nThe Phase 1a and Phase 1b experiments started with an E. coli template DNA mass of 1 μg and 1.5 μg, respectively. A fraction was lost during each clean up step of the library preparation protocol so that after fragmentation, end repair, and dA-tailing, only 17% of the Phase 1a and 29% of the Phase 1b starting DNA was retained (Table S4). Measurements of the P1a-Lab4-R1 DNA size distribution revealed a peak at ~15 Kb that subsequently translated into a typical read-length distribution, suggesting that the read length achieved by the MinION closely resembles the length of the input DNA fragments.\n\nMost Phase 1a experiments deviated at least once from the standard protocol during the library preparation steps. Variations included starting with a higher DNA mass, the suspected addition of an incorrect concentration of fuel mix, skipping addition of the DNA CS (lambda phage control spike-in sample) DNA, and using a higher library volume (Table S5). Phase 1b experiments experienced less unplanned variation.\n\nThe number of active pores in each of the four well-groups was measured once during the Platform QC (-180 mV) (steps 52-53, File S1) before sequencing commenced and at the start of the 48h sequencing protocol (-140 mV) (step 87, File S1), and one of these measurements was recorded for each flow cell (Table S4). Although the numbers reported by the Platform QC are higher than mux numbers from the 48H script, possibly due to the different bias voltage used by the two scripts, either value gives a good indication of initial flow cell quality. The median number of active pores reported across the experiments was 484, 409, 262 and 78 for well-groups g1 to g4, respectively, which corresponds to 95, 80, 51 and 15% of the theoretical maximum of 512 each (Figure 2). The standard sequencing protocol only utilizes the first two well-groups during a run. Thus, although on average 60% of the 2,048 wells contained an active pore, only 44% of all pores in a typical flow cell were available for sequencing utilizing the standard sequencing protocol (Figure 2).\n\nThe distribution of the number of active pores (lower series) and the cumulative total (upper series) for well-groups 1 to 4 are shown for the 20 flow cells used in this study. The measurement for each experiment was made either during the Platform QC or at the beginning of the 48h script.\n\nAll Phase 1a and 1b experiments were performed over a period of about one month each, between 27 March and 27 April 2015, and between 15 and 21 April 2015 , respectively (Table S6). Comparison of the sequencing-related attributes stored in the FAST5 files (Table S2) confirmed that most parameters were identical among and between the Phase 1a and 1b experiments, the exceptions being minor variations in the versions of the MinKNOW and Metrichor Desktop Agents, the Oxford Nanopore sequencing protocol and the event detection software (Table 1, Table S7).\n\nOnce started, the MAP_48Hr_Sequencing_Run protocol performs a ‘Platform QC’ to allocate active pores to well-groups 1 through to 4, starts sequencing with the active pores in well-group 1, switches to use the active pores in well-group 2 at 24h, and automatically terminates at 48h. However, the sequencing protocol was aborted or restarted at least once for 5 of the Phase 1a and 3 of the Phase 1b experiments because: (i) the number of active pores and the data yield were so low that the user decided to discontinue the run without a restart (N=4); (ii) the sequencing computer crashed (N=1); or (iii) the hard drive filled up (N=3) (Table 2, Table S8). While the sequencer was being restarted, there was usually a period when it was idle, explaining differences between the total sequencing time and the total time over which the device was active (c.f. seq_duration_hrs and run_duration_hrs, Table S8). In addition, 6 of the 20 sequencing experiments were restarted at 48h (Table 2, Table S8) to test whether the device can continue to produce good data beyond the standard 48h script provided by Oxford Nanopore, but all such data were excluded from this analysis.\n\nAdherence to the standard wet-lab protocol for each batch, and the start number and well-group origins of reads produced in each experiment.\n\nDespite the existence of a detailed standard protocol, a number of method deviations were recorded arising variously from wet-lab omissions or errors, flow cell quality issues, and computer software and hardware issues (Table S5). Thus, we could not use all the data generated to infer the yield, accuracy, and variability produced by a MinION because of the variations among the 20 experiments (Table 2, Table S5). Eleven of the experiments (P1a: N=4; P1b: N=7) adhered precisely to the wet-lab component of the standard MARC protocol; the other 9 contained at least one variation, mostly due to uncontrollable factors (Table 2, Table S5). Therefore, data was restricted to reads generated during the first execution of the MAP_48Hr_Sequencing_Run script (held in common among experiments) and those generated under common, near-standard conditions. With this strategy, we avoided unusual data accumulation patterns resulting from experiment restarts, which results in well swapping via pore reselection (re-mux), while still taking advantage of all 20 experiments, even those that terminated before 48h due to computer failures or flow cell issues. Each start of the MAP_48Hr_Sequencing_Run protocol generates one batch of data, with up to ½ h being from flow cell calibration and mux pore selection, the next 24h being from the first well-group pores, and the remainder from the second well-group pores. We generated reads from the g1 and g2 well-group pores of the first start of 20 and 17 experiments, respectively. Of the 7 experiments that started the sequencing protocol for a second time, 7 generated data from the g1 well-group pores and 1 from the g2 well-group pores. Similarly, for the 3 experiments that had a third start, 3 experiments generated data from the g1 well-group pores and 1 from the g2 well-group pores (Table 2).\n\nAnecdotal reports from MAP participants have suggested that the temperature of the flow cell can affect the performance and data quality of the MinION. In our experiments, each flow cell operated at a characteristic temperature with only minor fluctuations over time. All flow cells had an ASIC temperature between 23.9 and 35.2°C (median 26.8°C) and a heat-sink temperature of 36.8 to 38.6°C (median 37.0°C). There was no correlation between the DNA input mass or fuel amount and the resulting operating temperature, and temperatures observed during Phases 1a and 1b were similar. The flow cells with the highest yields, P1a-Lab3-R1 and P1b-Lab4-R1, had ASIC temperatures that spanned the range observed (26.9°C and 35.2°C, respectively), suggesting that operating temperature does not tend to affect data yield.\n\nIf the deviations from the established protocol can be considered as corresponding to normal variation in use, examination of the total data produced by the 20 Phase 1 experiments provides an indication of the total yield that can be expected from the current platform. We found a high level of variability among the 20 experiments that was only partially attributable to protocol deviations: a median of 60,600 reads (inter-quartile range (IQR) of 38,000 to 74,000, max. 139,000) (Figure 3A,B) containing 650,000 events (IQR 434,000 to 750,000, max. 1.9 million) (Figure 3C,D). Very few (~0.2%) of the events were in reads that were not base-called by Metrichor because they were outside the pre-set callable length range of 200 events to 230,000 events.\n\nRead count as (A) raw counts and (B) a percentage. Event yield as (C) raw counts and (D) a percentage. The (E) entire distribution of callable read lengths and (F) a subset showing the lower part in more detail. The 6 experiments that adhered to the MARC protocol and sequenced for at least 46h are marked with a black dot. The upper callable threshold of 230,000 events is indicated by a red dashed line.\n\nThe median read lengths from the 20 experiments indicate most experiments had a broad distribution with a peak around 10,700 events and a long tail containing a very small number of reads that reached the upper limit of 230,000 events (Figure 3E,F). Typically, a median of 20% of the reads had a length of at least 21,000 events (Figure S1A), and 50% of the events were in reads of at least 13,600 events, 25% of the events were in reads of at least 29,000 events, and 5% in reads of at least 56,600 events (Figure S1B). The event generation rate was not constant during a sequencing run. Of the 9 experiments that ran for at least 46h, 67% of the events were produced in the first 24h (Figure 4A,B). Although a higher read count is associated with a higher event yield (Figure 5A), neither the number of reads nor the event yield was strongly correlated with the number of active g1 pores (Figure 5B,C), suggesting data yield is not solely dependent on the number of initial active pores. Although the experiments that followed the MARC wet-lab protocol precisely (blue triangles, Figure 5) had a higher event yield to read count and higher event yield to initial g1 pore count, the effect was not large and does not form a distinguishable cluster among the rest of the experiments.\n\n(A,B) Cumulative event yield. (C,D) Event yield per hour. (E,F) Percentage of the 512 pores that were active. (G,H) Event sequencing rate per pore. (I,J) Length of reads in events. The left plots show the values for each experiment, coloured by lab. The right plots show the values for each experiment more clearly. The DNA input mass for each experiment is provided in (B). Data collected during the first hour, the hour following the pore-group switch (24–25h) and the last hour (47–48h) are omitted for clarity.\n\nThe phase of the experiment is indicated by shape. The experiments that adhered to the MARC protocol for both the wet-lab and sequencing components are shown in blue.\n\nTo evaluate whether the variation could be due to deviations from the MARC protocol, we examined event data generated by the g1 pores of the first start of all 20 experiments, all of which ran for at least 23 hours. No significant relationship was found between the total read count, total event yield or event lengths and the input DNA mass (Pearson’s correlation coefficient, p=0.036, 0.221 and 0.149, respectively). Similarly, the Kruskal-Wallis test found no significant difference between the number of reads, total event yield, or median event lengths between the Phase 1a and 1b experiments (p=0.290, 0.151 and 0.482, respectively), the five labs (p=0.482, 0.159 and 0.263, respectively), or the 6 experiments that strictly adhered to the MARC protocol and the remainder of the experiments (p=0.909, 0.183, and 0.119, respectively).\n\nThe highest data yield was from experiment P1a-Lab3-R1, which commenced sequencing with the highest number of active g1 pores (506/512 = 98.8%) to produce over 138 thousand reads and almost 2 billion (1×109) events within the callable read length range (Table S4, Table S6). The library for this experiment contained a DNA input mass of 60 ng in 12 μL of PSM, which was less than the median of 70 ng across the 20 experiments (Table S6 Experiments). That the two experiments with the highest event yield (P1a-Lab3-R1 and P1b-Lab4-R1) used a lower mass of input DNA (60 ng and 9.1 ng, respectively), confirms that the amount of DNA loaded is greater than that required to keep the active pores adequately supplied with DNA molecules.\n\nExperiment P1a-Lab3-R2 was notable in that it was run for almost 62h, first for 48h using the standard sequencing script, then for an additional 8.1h and 4.8h with two starts of a modification of the MAP_48Hr_Sequencing_Run recipe script, MAP_2×8hrs_180_190_Sequencing_Run.py that performs a new allocation of wells to well-groups (re-mux) (File S2) well selection followed by 8h of sequencing at each of -180 mV and -190 mV, respectively (SQK–MAP005 script developed by John Tyson available to the MAP community at https://wiki.nanoporetech.com/x/tgLDAQ). During the extra 15h, the total accumulated yield increased by 8% (Table S6, Table S8), demonstrating that good flow cells can continue to produce significant amounts of data with the appropriate software.\n\nAll experiments demonstrated event accumulation rates that decreased for the first 24h, experienced a sharp increase at 24h following the pore group switch and library reload, then steadily decreased again until the run was terminated (Figure 4A). There was no obvious correlation between total yield and input DNA (Figure 4B), lab (Figure 4B), or phase (Figure S2). The flow cells commenced sequencing at 120–200 × 103 events h-1 (Figure 4G,H). Although the experiments generated between 0.2 and 1.2 billion events (Figure 4A), a typical run such as P1b-Lab2-R2 generated 47% of the data (367 million events) in the first quarter (12h) of the experiment and 69% of the data (544 million events) in the first half (24h) of the experiment (Figure 4B). The rate at which events accumulated over time in each experiment was similar (Figure 4), suggesting a shared mechanism. The decrease in event yield over time (Figure 4C,D) correlates with a decrease in the number of active pores (Figure 4E,F). However, the decreasing number of pores cannot be the sole determining factor as even when normalized for the number of active pores, the event yield still declined over time approximately linearly for the first 24h (with the exception of P1b-Lab4-R2), then less predictably for the next 24h (Figure 4G,H). The decrease in event length over time may be another contributing factor (Figure 4I,J), but the pore refill delay, or the time during which pores are idle, appears constant during a run (Figure S3I,J). The sequence of 5-mers inferred from a sequence of events may suggest that a base of the library molecule being sequenced has been skipped (e.g., a skip of 1 base may be inferred from a progression from AATGC to TGCCG) or that a base has been sequenced more than once (e.g., a stay may be inferred from consecutive 5-mers AATGC and AATGC). While we hypothesized that a decrease in events over time may be caused by an increase in skips and stays, we observed a decrease in the percentage of template skips (Figure S3A,B) but a lower and constant percentage of complement skips (Figure S3E,F), and an increase in template and complement stays over time (Figure S3C,D,G,H). In conjunction with 4h periodic effects in the plots (e.g., SI Figure 3B, P1a-Lab2-R1/R2), this suggests an increasing stay rate, possibly due to non-optimal bias voltage across the flow cell membrane, may be a contributing factor to the lower event rate observed during an experiment, and this phenomenon would benefit from further investigation. Another point to note is that the profiles of experiments produced at the same lab are more similar to each other than to experiments from other labs (Figure 4 and Figure S3, right side plots), suggesting lab effects or the MinION device may be contributing to the effect.\n\nBetween 63% and 99% (median 92%) of the reads were allocated to the target sample and most of the remainder to the control sample (Figure 6A). Two Phase 1a experiments omitted to include the control sample (P1a-Lab3-R1 and P1a-Lab3-R2) (Figure 6A). Phase 1b experiments P1b-Lab3-R1 and P1b-Lab3-R2 contained a larger proportion of reads (3.7% and 15.3%, respectively) that did not map to either the target or the control reference (Figure 6A), suggesting contamination. Taxonomic classification of all 2D reads using Kraken version 0.10.5-beta (Wood & Salzberg, 2014) found only two experiments with non-E. coli bacterial matches: P1b-Lab3-R1 had 2.3% of the reads classified as Pseudomonadales (probably Pseudomonas putida) and P1b-Lab3-R2 had 10.7% of reads as Pseudomonales (probably P. putida) and 2.2% as Burkholderiales (best match sp. P. delftia), species implicated in kit contamination (Salter et al., 2014) at percentages comparable to those inferred from the BWA-MEM alignments.\n\n(A) Proportion of target, control and unclassified 2D reads for each experiment. The read production rate (reads pore-1 h-1) for (B,C) target DNA, (E,F) control DNA, and (F,G) reads that could not be aligned uniquely either to the target or control reference sequence.\n\nWith the exception of outlier experiments from P1a-Lab4-R2 (that may have been run with extra initial fuel) and P1a-Lab5 (which, for reasons unknown, sequenced DNA at a higher rate than in other experiments), the proportion of target and control reads decreased at a similar rate, suggesting the platform was not biased towards either (Figure 6B–E). The increasing rate of unclassifiable reads over time (Figure 6F,G) likely reflects decreasing read quality over time.\n\nThe length of events and 2D base-calls of all target reads from all experiments had a linear relationship with a slope of 0.367 (ratio of 2.7 : 1) (Figure 7A). The median numbers of template, complement, 2D, and 2D ‘pass’ reads across the 20 experiments were 30,360, 25,370, 19,540 and 12,320 bases, respectively (Figure 7B); the median read lengths were 6,280, 5,940, 6,440 and 6,690 bases, respectively (Figure 7C); the median base yields were 167, 137, 115 and 74 million bases, respectively (Figure 7D); the median base yield of each type was 167, 138, 115 and 73 million bases, respectively; and the median of mean base quality of the base-calls of each type was 7.9, 7.9, 11.2 and 11.9, respectively (Figure 7E).\n\n(A) The relationship between event lengths and the length of 2D base-calls is linear, with a slope of 0.367 (ratio of 2.7 : 1). The distribution of (B) total number of reads, (C) read length; (D) total base yield; and (E) mean base quality of the target sample across the 20 experiments.\n\nNot only did the rate of read production decrease over time for all 1D and 2D reads (Figure S4A–D), all experiments also exhibited a declining trend in base quality over time (Figure 8 and Figure 9, Figure S4E). The template, complement, and 2D bases differed from the start of each sequencing run, having a mean base quality of about 2 units less after 24h of sequencing (Figure 8 and Figure 9). The increase in the rate of read production (Figure 4) at the 24h mux switch was accompanied by an increase in the base quality (Figure 8). Every 4h, there was a smaller-scale recapitulation of the decline followed by a return in base quality, most clearly seen in the P1b-Lab2 experiments (Figure 8 and Figure 9, Figure S4), coinciding with the -5 mV bias-voltage adjustment every 4h in the 48h sequencing protocol script (mux1 voltage sequence (mV): -140, -145, -150, -155, -160, -165; followed by mux2 voltage sequence (mV): -155, -160, -165, -170, -175, -180) to maintain a more uniform current flow.\n\nThe median base quality for template, complement, all 2D, and 2D pass bases in 15 minute intervals for target DNA reads. Statistics are inferred from data from the first start of each sequencing experiment. Data collected during the first hour, the hour following the pore-group switch (24–25h) and the last hour (47–48h) are omitted for clarity.\n\nThe mean base quality for 15 minute intervals for (A) all 2D reads and (B) 2D pass reads in each experiment.\n\nTo investigate the interplay between sequencing speed and base quality we determined the total time taken to sequence the template and complement bases per unit time per active channel. This provides a measure of the true mean rate that sequences were translocating through the pores. By incorporating the time for which active pores were not sequencing, an effective sequencing rate could be calculated. For a typical experiment, P1a-Lab2-R2, template and complement sequences were produced at a declining rate over the course of 24h. For both metrics, the rate at which template sequences translocate through the pore decreases more rapidly than the complement sequences (Figure S5A). Plotting the average occupancy rate of pores over time, alongside the number of active channels over time, demonstrates that active pores continued sequencing at similar rates until they become inactive, which happened at a relatively uniform rate during an experiment (Figure S5B). Thus, further investigation of how base quality (Figure 8), read accuracy (Figure 10) and the speed at which the DNA translocates through the pore (Figure S5) over time may suggest strategies for improving base-calling.\n\n(A) The total percentage error of each read, grouped by laboratory, for values computed from BWA-MEM alignments pre- and post-EM correction; (B) the median percentage error over time for alignments by BWA-MEM for each experiment; and (C) the median percentage error over time for alignments by BWA-MEM followed by EM correction for each experiment, showing the median total, miscall, insertion and deletion error for each 15 minute interval.\n\nA base-called FAST5 file is classified as ‘fail’ if: (i) base-calling failed; (ii) no 2D base-calls were inferred; or (iii) the 2D base-called read had a mean quality score ≤ 9. All other reads are classified as ‘pass’ and can be considered the ‘high-quality’ reads from the experiment. Although there was substantial variability in the proportion of 2D pass reads produced during the experiments, there was a clear decrease in median percentage of 2D pass reads from 85% to 20% over the course of the first 21h of the experiment (Figure 11). The drops in 2D pass yield coincide with the 4h bias-voltage adjustments (Figure 11), suggesting the reads produced during these transition periods do not have correctly calibrated base qualities.\n\nBoxplots showing the proportion of 2D pass reads started in each 15 minute interval were plotted for the 20 experiments (grey), and the median values connected with a black line.\n\nThe median total error of all 2D reads was 12% (Figure 10C, Figure S8A), with miscalls, insertions and deletions contributing 3%, 4% and 5%, respectively (Figure 10C). The 2D pass reads had a slightly lower total error of 10.5% (Figure 10A) and the 2D fail reads a much higher value of 20.7% (Figure 10A), based on the best alignment strategy attempted, of BWA-MEM followed by EM correction by marginAlign. The error estimated from alignments with BWA-MEM alone were significantly higher: a median total error of 15% for all 2D reads (Figure 10B), 11.6% for 2D pass reads and 22.6% for 2D fail reads (Figure 10A).\n\nThe application of a better alignment algorithm, in this case the EM correction implemented in marginAlign, had the effect of decreasing miscalls at the expense of a slight increase in insertions and a small increase in deletions, with the net decrease in the total error of 1.9% for 2D fail base-calls and 1.1% for 2D pass base-calls (Figure 10A). During an experiment, the total error inferred from BWA-MEM alignments increased during the first 24h of the experiment, dropped at the 24h re-mux and library reload, then increased again until the experiment was terminated (Figure 10). Use of a better alignment algorithm not only reduced the miscall, insertion, and deletion rates, but resulted in a more uniform profile of each error type during an experiment, and in particular, reduced the rate of increase of deletions during an experiment (Figure 10C). The 4h periodic effect observed previously in the base quality plots is also clearly evident in the error plots (Figure 10B).\n\nError rates inferred from the use of the BWA-MEM and LAST as the initial aligner were very similar; therefore, only the values based on BWA-MEM are described. The error estimates from BWA-MEM, pre- and post-EM alignment, were very similar for experimented from Phase 1a and 1b (Figure S6). Error estimates inferred from BWA-MEM alignments without EM correction showed that the error rate of the 1D template and complement base-calls were similar, and about twice that of the 2D base-calls; and the error of the base-calls from pass reads were always lower than for the fail reads of the same read type (Figure S7A). Similarly, the error estimates were similar for target and control base-calls across all laboratories (Figure S7B). The total percentage error of individual reads, and the miscall, insertion and deletion components, were almost constant over time, but interrupted by an increase in error for reads that were sequenced during the 4h bias-voltage adjustments (Figure S8).\n\nAccording to the metadata in the FAST5 data files (Table S2), the base quality Q is related to the probability of error p by the Phred scale formula Q = -1000log10(1-p). The linear relationship between the logarithm of percentage error and the mean base quality of 2D reads mapped with BWA-MEM confirms this relationship (Figure 12A), thus demonstrating that base quality is correlated with the accuracy of base-calls and can be used to filter reads of an unknown genome to the accuracy required for a particular analysis. We suspect the decrease of 10(-Q/1000)/TotalError over time, a value that should be the same for every read, was the result of decreasing mean read base qualities during an experiment and the 4h dips in the signal were due to the miscalculation of the mean base quality of reads that were being sequenced during a bias-voltage adjustment (Figure 12B).\n\n(A) The percentage error (on a log scale) plotted against the mean base quality of each 2D read. Reads from the Phase 1a and 1b experiments are distinguished by shape and the pass and fail read types by colour. The relationship between total error, and the miscall, insertion and deletion components, are shown separately. The linear regression line demonstrates that base quality and error are related by an exponential function. (B) The variation in 10(-Q/1000)/TotalError over time for each experiment. Although the value should be constant for all reads, the value declines over time. The characteristic unusual values occurring every 4h suggest that base quality is not as well correlated with accuracy for reads that were being sequenced during a bias-voltage adjustment.\n\nOne attribute that distinguishes nanopore sequencing from many next generation technologies is the possibility of acquiring base-calls that are over 10,000 bases long. Typically, 7.6%, 4.0%, 4.4%, and 3.6% of the reads had over 10,000 bases in the template, complement, 2D, and 2D ‘pass’ base-calls (Figure S1A). Similarly, 50% of reads had a length of at least 5,500, 5,600, 6,000 and 6,300 bases for the template, complement, 2D, and 2D ‘pass’ base-calls (Figure S1B). Generally, 5% of the reads had a length of at least 14.5, 13.0, 13.5 and 13.6 × 103 bases for the template, complement, 2D, and 2D ‘pass’ base-calls (Figure S1B). The longest template, complement, 2D, and 2D ‘pass’ base-calls observed in this study were 291.6, 300.5, 59.7 and 59.7 × 103 bases, respectively.\n\nThe median theoretical fold coverage of the target E. coli genome achieved by the 20 experiments was 25 for 2D reads (min=5.2, Q1=16.3, median=24.9, mean=29.0, Q3=36.5, max=78.5) and 16 if restricted to 2D ‘pass’ reads (min=1.7, Q1=11.3, median=15.9, mean=20.3, Q3=27.0, max=47.9). When the theoretical fold coverage of all 2D base-calls or just the 2D ‘pass’ base-calls was at least 20, 99.9% of the sites were called accurately by the majority consensus. A theoretical fold coverage of at least 60 was required to call 99.99% of the reference sites accurately from the majority consensus.\n\nThe GC content of 2D base-calls of the E. coli sample were very close to the actual value of 50.8% for all experiments, with some variation between the pass and fail base-calls (Figure 13A).\n\nThe distribution of (A) read GC content as a percentage; and (B) the length of the best perfect subsequences of target 2D pass and fail base-calls from each experiment.\n\nThe under-represented 5-mers for the 2D base-calls of the target and control samples suggest the nanopore sequencing technology has difficulty sequencing homopolymers (Table S9). Homopolymers and repeated bases were also prominent in the table of over-represented 5-mers, but the mechanism producing this phenomenon is not clear (Table S9).\n\nThe length of the longest perfect subsequence in the base-calls of each read is a measure of sequencing accuracy. The median length in 2D base-calls of the target sample was 50 and 90 for the fail and pass base-calls, respectively, across all experiments except P1a-Lab4-R2, which may have been run with a higher concentration of fuel mix (Figure 13B). However, the distribution for all experiments had a long tail, the longest exceeding 300 consecutive, perfect bases (Figure 13B).\n\n\nDiscussion\n\nThe overall objective of MARC is to provide a definitive description of the Oxford Nanopore Technologies sequencing platform through a flexible publication strategy that accommodates the rapid pace of nanopore sequencing technology development. In this first phase of the MARC collaboration, we generated 20 datasets at five laboratories on different continents for the same E. coli bacterial strain, with sufficient lab replicates to be able to quantify the data yield, quality, accuracy and reproducibility that can be expected from the MinION, flow cells, chemistry, software and protocols available in April 2015. We demonstrated that there was considerable variability in the quality of flow cells, but all flow cells that had a high number of active pores when they arrived at their destination laboratory produced data of comparable yield, quality and accuracy. This dataset, the largest replicate sequencing effort of its kind on nanopore sequencing to date, is published here to allow continued independent investigation by the broader scientific community and enable more rapid development of algorithms and software for these data.\n\nThe MARC Phase 1 experiments were designed to provide benchmark data that explored the relative contributions of instrument, flow cell, laboratory and user to the variation in MinION system performance observed by the MAP community. The experiments in this study (Table S6) followed a standard protocol based on that recommended by Oxford Nanopore at the time of the study, with clear choices made for the procedure to be followed when optional or open-ended steps existed. The protocol that we followed in this study (File S1 MARC protocol) was based on the standard SQK-MAP005 protocol provided by Oxford Nanopore (version MN005_1124_revC_02Mar2015, last modified 10 June 2015), the only amendment being the use of 12 µl of library in Phase 1b and annotations to make the protocol clearer.\n\nThe large number of replicates allowed us to make generalisations about the data yield and quality. Utilizing version R7.3 flow cells and SQK-MAP005 chemistry, a typical experiment yielded 115 million 2D bases in ~20,000 reads with a median protocol-specific shearing length of 6,500 bases and mean base quality of 11.2. When the 8 Kb shearing protocol was used, approximately 4.5% of the 2D reads had a length of at least 10,000 bases, with some having a length of over 50,000 bases. Up to 10% of the reads of an experiment were from the DNA CS control added during library preparation. About 32% of the reads from an experiment result in 2D reads from the target genome. The accuracy of base-calls decreases during the course of an experiment. However, the total error of individual 2D base-calls was ~12%, with miscalls, insertions and deletions contributing ~3%, ~4% and ~5%, respectively. A single experiment yielded sufficient 2D bases for ~25-fold coverage of the target E. coli genome. When restricted to 2D ‘pass’ reads, the yield decreased to ~12,000 reads containing 75 million bases with a read length distributed centred around 6,700 bases and a mean base quality of 11.9. A 2D base yield corresponding to at least 20-fold coverage of the target genome was required to correctly call 99.90% of the 4.6 Mb E. coli genome, and 60-fold coverage to correctly call 99.99% of the genome, from the majority consensus of mapped reads.\n\nAlthough the MARC standard protocol was documented in great detail, the quantity and quality of the output data varied due to many steps being sensitive to the quality of the materials and reagents used, stochastic variation in the application of the steps, accidental deviations from the protocol, and unexpected computer failures during a sequencing run. A large component of variability in MinION data quality was contingent on lab-specific behaviour. Although a number of minor deviations from the standard MARC protocol occurred, we found that the wet-lab method variations (e.g., DNA mass used to prepare a library, sheared length of DNA or the volume of library loaded on a flow cell) and occasional failures of computer software or hardware affected reproducibility but had minimal effects on data quality. The one notable exception was the amount of fuel mix, where a higher concentration of fuel mix loaded at the start of run P1a-Lab4-R2 was the most plausible explanation for the unusually high sequencing rate, shorter reads and poorer base qualities observed. According to Oxford Nanopore, a ‘fast mode’ enhancement will soon become available, including fine tuning of the event detection parameters to ensure that long read lengths are maintained upon addition of more fuel mix to increase speed.\n\nThe MinKNOW program, that uses sequencing protocol scripts to control the MinION device, was regularly upgraded during the study, as was the Metrichor agent that performed base-calling. In both instances, sequencing related parameters were similar during the period of our investigation. However, local forced restarts of the scripts were found to be the largest source of variation among the 20 runs, resulting in extreme variation in the length of the sequencing run, event yield and the event generation profiles. Restarts alter the specific pores being used for sequencing via mux selection and also disrupts a very prescribed bias-voltage profile required for an ideal ‘fresh’ flow cell to operate optimally through a 48h sequencing run. Alteration away from the ‘standard’ experimental conditions can therefore have a large impact on the performance of a flow cell, both positive and negative depending on parameters used, and confounds comparative analysis.\n\nThe performance of the MinION device itself was consistent. Each experiment ran at a characteristic temperature within an acceptable range that did not fluctuate during an experiment and no experiments experienced failures due to problems with the device. Although GC biases may be hard to detect through the sequencing of an E. coli strain with a mean GC content close to 50%, we did not observe a genome-wide GC bias in the 2D reads produced by this platform. Neither longer target nor shorter control library molecules were sequenced preferentially during the experiments, and the accuracy of target and control base-calls was very similar.\n\nThe most important determinant of data yield was the initial number of active pores in the flow cell. On delivery, ~60% of all the pores on the flow cell were usable and the best flow cells had ~95% and 80% active pores in the g1 and g2 well-groups at the commencement of an experiment. Active pores were sequencing for ~90% of their active time, with a uniform idle period between library molecules suggesting pores have consistent performance until they become inactive. The first hour of a run is generally predictive of total run yield. Flow cells that commenced sequencing with at least 400 of the maximum of 512 well-group g1 pores yielded optimal event yield profiles from high quality libraries.\n\nThe similarity of the 2D base quality profiles from the same lab suggest the base quality of an experiment may be dependent on the characteristic human or equipment-related sequencing conditions in a laboratory. But it is also possible that it may be due to the shipping procedure to that location. Thus, the reason for the decrease in base-call accuracy during an experiment is still not fully understood, but the large number of replicate experiments in this study, carried out in five laboratories on different continents, is the best available resource for exploring the possible mechanisms. The characteristic trend observed for all metrics of data quality produced by the current group of pores was a steady decrease over time, punctuated by a fluctuation every 4h coinciding with the pre-set bias-voltage adjustment. We hypothesize that variations in sequencing rate (measured in bases per second) were caused by decreasing flow cell performance over time that is not accounted for in the base-calling models. The adjustments in bias voltage every 4h appear to mitigate some of these effects, but the frequency of these adjustments do not track the changing state of the flow cell closely enough to result in uniform data quality during an entire experiment. This suggests the pre-programmed bias-voltage adjustments have been optimized for the library preparation protocol recommended for that flow cell chemistry, and the particular volumes of library and fuel expected during the sequencing run. As such, software or protocols that could maintain synchrony between these two aspects of the sequencing process may significantly improve the overall performance of the technology and confirm that re-calling bases of older experiments with new software is probably not advisable.\n\nThe addition of more library and fuel mix coincided with the switch from the use of the g1 to g2 pores, so it was not possible to tell which of the two factors was responsible for any changes in data yield or quality, or whether the lower overall performance in the second 24h period of the experiment may have been due to degradation of the DNA, adapters or motor proteins during 24h of storage. However, the increase in read production rate (Figure 4), and quality after the 24h mux switch suggest ‘fresh’ pores and/or sample produce higher quality data (Figure 8). Given that the base-calling algorithm is tuned to use normalized current profiles, ‘mid’-read bias-voltage changes would compromise this process and we hypothesize it causes a disproportionate decrease in the quality of the base-calls for a short transition period until complete reads are produced under the same ionic driving force. The similarity of the 2D base quality profiles from the same lab suggest the base quality of an experiment may be dependent on the characteristic human or equipment-related sequencing conditions in a laboratory (Figure 9). The two Phase 1a and 1b replicate experiments performed in Lab 2 and Lab 5 were run concurrently on different MinIONs while all other laboratories performed the replicate experiments sequentially (Table S8). The 2D plots for replicate experiments from these two labs are the only pairs of experiments that have a different rate of decrease, which suggests the MinION itself has some influence on the decrease in base quality over time (Figure 9A).\n\nFinding standard metrics for assessing the error of the long single-molecule reads was a challenge. Alignment-free approaches based on k-mer frequencies have lower accuracy for homopolymeric regions or those with a low or high GC content (Laehnemann et al., 2015). If a platform is capable of sequencing any DNA sequence, all possible 5-mers in the DNA should be proportionally represented in the data when counts are normalized for the distribution of all 5-mers in the genome. Thus, the most under-represented and over-represented 5-mers in the base-calls from the MinION may suggest limitations or biases of the nanopore sequencing process. Conversely, using alignment-based approaches, we have observed that stretches of 90 perfect bases in 2D ‘pass’ reads and 50 bases in 2D ‘fail’ reads were typical (Table S9), and that stretches of over 300 perfect bases were possible from the SQK-MAP005 chemistry. The accuracy (or error) values quoted in other studies have been difficult to compare because: (i) the precise values quoted are sensitive to the alignment method used to compare reads to the reference; (ii) there is a significant difference in the quality of the 2D ‘fail’ and ‘pass’ reads; and (iii) basing values on reads from both target and control DNA may affect the values if they have different GC contents. Quoting the percent identity of a read with respect to a reference can be misleading because an increase in the percent identity can be induced by a decreased rate of insertions or deletions. We found that the total error of 2D ‘fail’ and ‘pass’ reads was 23% and 12%, respectively, using the nanopore-tuned parameters for BWA-MEM, but re-alignment using an EM technique reduced the error to 21% and 12%, respectively. In fact, we expected error rates to differ between phases (due to different chemistries) and samples (due to different types of input DNA), but instead the only observed error rate differences were between the type of read (template/complement/2D) and whether or not it had been classified as pass or fail. Although the error of individual MinION reads is high compared to those from the more established short-read technologies, it has been demonstrated that these data are of sufficiently high-quality to infer full-length de novo assembly of the E. coli, Influenza virus, and Saccharomyces cerevisiae genomes (Goodwin et al., 2015; Loman et al., 2015; Quick et al., 2014; Wang et al., 2015).\n\nAlthough reported, the 1D reads were not fully explored and it is acknowledged that discounting these data likely underestimates error and reduces usable data. If there are regions of the target genome that only have coverage by template base-calls, the demonstrated correlation of mean base quality and accuracy could be used to select the more accurate 1D reads that exceed an appropriate base quality threshold.\n\nThe observations from this study suggest there are many ways in which the performance of the MinION platform could be improved. Clearer protocol steps, that describe software steps, could reduce mistakes and computer issues. Methods that deliver longer, intact library molecules to the flow cell would have a large impact on the length distribution of the resulting base-calls. Improved run scripts, that utilize the best available pore for each channel rather than relying on pre-defined well-groups, could dramatically increase data yield and quality. Improvements in base-call accuracy through finer-grained regulation of bias-voltage adjustments may be possible, but these would need to be accompanied by more accurate mean base qualities for reads that span voltage transitions. Yield of the target sequence could be improved by reducing the volume of the control sample in the library. Investigation of motifs that have no coverage in the 2D base-calls may suggest a means of alleviating these limitations. Development of base-calling algorithms that take into account the methylation profile of the target DNA could reduce the regions of the genome that are consistently unrecovered by the current technology. The lifetime of a flow cell is not limited to 48h, and this study demonstrates that significant amounts of additional data can be generated if sufficient active pores remain.\n\nThe data generated in this study are intended as a snapshot of the state of the MinION technology in April 2015. There are many other analyses that could have been presented here, but to release the datasets to the wider community rapidly, we have deliberately performed only preliminary analyses and hope the release of these datasets will inspire the development of software based on new algorithms that specifically address the unique properties of data from the MinION platform. We hope that more analyses will be performed on this dataset both by MARC members and others. During Phase 1 of the MARC collaboration, new minor versions of the flow cell chemistry and software were released, and the first ‘field’ runs of the new MinION Mk1 device with the new flow cells using SQK–MAP006 reagents and updated base-calling software based on 6-mers commenced in late September 2015. To provide a link between the data presented in this study and the MinION Mk1 data, MARC will conduct ‘bridging experiments’ to evaluate the differences in the data yield and accuracy and error profile, before embarking on the MARC Phase 2 experiments to identify protocol changes that improve the performance and extend potential applications of the platform.\n\n\nData availability\n\nThe raw and aligned nanopore reads, and files of statistics for each of the 20 experiments (Table S10) are available from the European Nucleotide Archive project PRJEB11008 (http://www.ebi.ac.uk/ena/data/view/PRJEB11008).",
"appendix": "Author contributions\n\n\n\nEB coordinated the study. EB, DB, JT, JOG, BB designed the study. MdC, PP, DB, SG, JOG, RML, SM, HJ, HEO and MJ designed the MARC protocol and performed the experiments. VZ, MJ, BP, CI, ML, RML collated the data for the group and ran bioinformatics pipelines over the data. CI, ML, RML, MJ, BP, EB, RB, LB and JT analysed the data. CI, RB, DB, EB, ML, RML, MJ, BP, HEO, PP, MdC, MS, JU, JOG, SG, JT, TPS, BB and DE drafted the manuscript. All authors participated in discussions relating to the generation and analysis of the data.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nCompeting interests\n\n\n\nEwan Birney is a paid consultant of Oxford Nanopore Technologies. All flow cells and library preparation kits were provided by Oxford Nanopore Technologies free of charge.\n\n\nGrant information\n\nThe research was supported by Wellcome Trust grant 090532/Z/09/Z (WTCHG); Rosetrees Trust grant A749 (JOG and SM, UEA); BBSRC grant BB/M020061/1 (ML); Canadian Institutes of Health Research #10677 (JT and TPS, UBC); BBSRC grant BB/J010375/1 (RML, TGAC); National Science Foundation awards DBI-1350041 and, IOS-1032105, and; National Institutes of Health award R01-HG006677 (MCS) Cancer Center Support Grant CA045508 (SG, CSHL) and funding from grant from T. and V. Stanley (SG, CSHL); NHGRI, USA award numbers HG007827 (Mark Akeson, UCSC) and U54HG007990 (BP, UCSC).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledegements\n\nWe thank Oxford Nanopore Technologies for the flow cells used in these experiments, promptly responding to questions, providing some of the graphics explaining the sequencing technology used in Figure 1, and reviewing the FAST5 data format description, the Glossary and the text; Gerton Lunter for providing the image in Figure 1 that relates the current measurements of the bulk data to events and 5-mers.\n\n\nSupplementary material\n\nSupplementary files\n\nFile S1. MARC protocol.\n\nFile S2. Glossary.\n\nSupplementary figures\n\n(A) The percentage of reads with a length greater than a specified read length. A boxplot of the percentage of reads was plotted for each read in multiples of 1000 until the read percentage dropped to 1%. Typically, 21% of the reads had a length of over 20,000 events, and 7.6%, 4.0%, 4.4% and 3.6% of the reads had over 10,000 bases in the template, complement, 2D and 2D ‘pass’ base-calls. (B) The length of reads containing a specified percentage of the data. Typically, 50% of the reads had a length of at least 13,600 events, and 5,500, 5,600, 6,000 and 6,300 bases for the template, complement, 2D and 2D ‘pass’ base-calls. Similarly, 5% of the reads had a length of at least 56,600 events, and 14,500, 13,000, 13,500 and 13,600 bases for the template, complement, 2D and 2D ‘pass’ base-calls.\n\nThe cumulative event yield was plotted for all reads from each experiment and coloured by the experimental phase. The yield for each phase has not affected the rate of event production over time or the total events produced. The total yield was not dependent on the input DNA mass (for input DNA mass, see SI Table 4). The re-mux and library reload at 24h is shown by a vertical dashed line.\n\n(A,B) Percentage of template events that are skips (i.e., event moves with a step length > 1). (C,D) Percentage of template events that are stays (i.e., event moves with a step length = 0). (E,F) Percentage of complement events that are skips (i.e., event moves with a step length > 1). (G,H) Percentage of complement events that are stays (i.e., event moves with a step length = 0). (I,J) Mean number of minutes that a pore is idle between sequencing instances. The number of skip and stay events was inferred for the template and complement strands of each reads and allocated to the 15 minute interval since the start of the experiment. The refill plot was based on values computed with poreQC version 0.2.10. The number of seconds the pore was idle before sequencing commenced was computed for each read, excluding the first read and any read which followed a read which did not result in a valid set of events, allocated to the 15 minute window in which the read commenced, grouped by experiment, and the median plotted for each experiment. Only data for the first sequencing script start is shown. The first hour, the hour following the pore-group switch and the last hour, are not shown for clarity. The 24h re-mux and library reload is shown by a vertical dashed line.\n\nEach row shows (A) read count, (B) read count per pore per hour, (C) base yield, (D) base yield per pore per hour, and (E) base quality for 1D template and complement reads, 2D reads and the 2D ‘pass’ reads. The values were inferred from the statistics computed by poreQC version 0.2.10 and poreMap version 0.1.1. Only data for the first sequencing script start is shown. The first hour, the hour following the pore-group switch and the last hour, are not shown for clarity. The 24h re-mux and library reload time is shown by a vertical dashed line.\n\n(A) Mean read sequencing rate of the template (light blue) and complement bespoke (green) strands, measured in bases per second, for each 15 minute interval for experiment P1a-Lab2-R2. The effective sequencing rate, computed as the total time taken to sequence bases the template and complement bases, per unit time, per active channel are shown for the template (orange) and complement (dark blue) for the same 15 minute intervals. In a typical experiment like P1a-Lab2-R2, template and complement sequences were produced at a declining rate over the course of 24h, and for both metrics, the rate at which template sequences translocate through the pore decreases more rapidly than the complement sequences. (B) The percentage of time that active pores were occupied (blue, left axis) and the number of active channels across the device (orange, right axis, maximum of 512), for 15 minute intervals during experiment P1a-Lab2-R2. Active pores continued to produce data at a similar rate until they became inactive, which happened at a relatively uniform rate during an experiment.\n\nThe pre- and post-EM percentage error for BWA-MEM alignments of target 2D base-calls, grouped by phase. There was little difference between the error rate of Phase 1a and 1b experiments.\n\nThe total error, and the contribution of miscalls, insertions and deletions for 2D base-calls of target reads for (A) template, complement and 2D base-calls split by the pass and fail classification, and (B) samples from the target or control DNA, grouped by laboratory. The percentage error was estimated from BWA-MEM alignments without EM correction, and thus, higher than the corresponding values in Figure 10. However, these values are sufficient to show that the error rate of the 1D template and complement base-calls are similar, and about twice that of the 2D base-calls. And the error of the base-calls from pass reads are always lower than for the fail reads of the same read type. Similarly, the error estimates were similar for target and control base-calls across all laboratories.\n\nThese data are the same as shown in Figure 10C, inferred from BWA-MEM alignments followed by EM correction, but separated by phase and lab to more clearly show the trends over time for each experiment. The total percentage error of individual reads, and the miscall, insertion and deletion components, were almost constant over time, but interrupted by an increase in error for reads that were sequenced during the 4h bias-voltage adjustments.\n\nSupplementary tables\n\nTable S1. Laboratories.\n\nTable S2. FAST5 format.\n\nTable S3. ENA pipeline.\n\nTable S4. Lab metadata.\n\nTable S5. Variations to MARC protocol.\n\nTable S6. Experiments.\n\nTable S7. Software parameters.\n\nTable S8. Batch metadata.\n\nTable S9. Under- and over-represented 5-mers in 2D base-calls.\n\nTable S10. ENA accessions.\n\n\nReferences\n\nAmmar R, Paton TA, Torti D, et al.: Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes [version 2; referees: 2 approved]. F1000Res. 2015; 4: 17. Publisher Full Text\n\nAkeson M, Branton D, Kasianowicz JJ, et al.: Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys J. 1999; 77(6): 3227–3233. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshton PM, Nair S, Dallman T, et al.: MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nat Biotechnol. 2015; 33(3): 296–300. PubMed Abstract | Publisher Full Text\n\nBayley H: Sequencing single molecules of DNA. Curr Opin Chem Biol. 2006; 10(6): 628–637. PubMed Abstract | Publisher Full Text\n\nCheck Hayden E: Nanopore genome sequencer makes its debut. Nat News. 2012. Publisher Full Text\n\nCherf GM, Lieberman KR, Rashid H, et al.: Automated Forward and Reverse Ratcheting of DNA in a Nanopore at 5-Å Precision. Nat Biotechnol. 2012; 30(4): 344–348. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChurch GM, Deamer DW, Branton D, et al.: Characterization of individual polymer molecules based on monomer-interface interactions. US patent # 5,795,782 (filed March 1995), 1998. Reference Source\n\nDerrington IM, Butler TZ, Collins MD, et al.: Nanopore DNA sequencing with MspA. Proc Natl Acad Sci USA. 2010; 107(37): 16060–16065. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEisenstein M: Oxford Nanopore announcement sets sequencing sector abuzz. Nat Biotechnol. 2012; 30(4): 295–296. PubMed Abstract | Publisher Full Text\n\nGoodwin S, Gurtowski J, Ethe-Sayers S, et al.: Oxford Nanopore sequencing, Hybrid Error Correction, and de novo assembly of a eukaryotic genome. bioRxiv. 2015. Publisher Full Text\n\nGreninger AL, Naccache SN, Federman S, et al.: Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Med. 2015; 7(1): 99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain M, Fiddes IT, Miga KH, et al.: Improved data analysis for the MinION nanopore sequencer. Nat Methods. 2015; 12(4): 351–356. PubMed Abstract | Publisher Full Text\n\nKarlsson E, Lärkeryd A, Sjödin A, et al.: Scaffolding of a bacterial genome using MinION nanopore sequencing. Sci Rep. 2015; 5: 11996. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKasianowicz JJ, Brandin E, Branton D, et al.: Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A. 1996; 93(24): 13770–13773. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKielbasa SM, Wan R, Sato K, et al.: Adaptive seeds tame genomic sequence comparison. Genome Res. 2011; 21(3): 487–493. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKilianski A, Haas JL, Corriveau EJ, et al.: Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer. Gigascience. 2015; 4: 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaehnemann D, Borkhardt A, McHardy AC, et al.: Denoising DNA deep sequencing data-high-throughput sequencing errors and their correction. Brief Bioinform. 2015; 1–26. PubMed Abstract | Publisher Full Text\n\nLaver T, Harrison J, O’Neill PA, et al.: Assessing the performance of the Oxford Nanopore Technologies MinION. Biomol Detect Quantif. 2015; 3: 1–8. Publisher Full Text\n\nLeggett RM, Heavens D, Caccamo M, et al.: NanoOK: Multi-reference alignment analysis of nanopore sequencing data, quality and error profiles. Bioinformatics. 2015; pii: btv540. 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\nLi H: Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv: 1303.3997. 2013. Reference Source\n\nLieberman KR, Cherf GM, Doody MJ, et al.: Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J Am Chem Soc. 2010; 132(50): 17961–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoman NJ, Quinlan AR: Poretools: a toolkit for analyzing nanopore sequence data. Bioinformatics. 2014; 30(23): 3399–3401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoman NJ, Quick J, Simpson JT: A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat Methods. 2015; 12(8): 733–735. PubMed Abstract | Publisher Full Text\n\nManrao EA, Derrington IM, Pavlenok M, et al.: Nucleotide discrimination with DNA immobilized in the MspA nanopore. PLoS One. 2011; 6(10): e25723. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaszlo AH, Derrington IM, Ross BC, et al.: Decoding long nanopore sequencing reads of natural DNA. Nat Biotechnol. 2014; 32(8): 829–833. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMulley JF, Hargreaves AD: Snake venom gland cDNA sequencing using the Oxford Nanopore MinION portable DNA sequencer. bioRxiv. 2015. Publisher Full Text\n\nQuick J, Quinlan AR, Loman NJ: A reference bacterial genome dataset generated on the MinIONTM portable single-molecule nanopore sequencer. Gigascience. 2014; 3: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalter SJ, Cox MJ, Turek EM, et al.: Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 2014; 12: 87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwartz AS, Pachter L: Multiple alignment by sequence annealing. Bioinformatics. 2007; 23(2): e24–e29. PubMed Abstract | Publisher Full Text\n\nSong B, Schneider GF, Xu Q, et al.: Atomic-scale electron-beam sculpting of near-defect-free graphene nanostructures. Nano Lett. 2011; 11(6): 2247–2250. PubMed Abstract | Publisher Full Text\n\nSzalay T, Golovchenko JA: De novo sequencing and variant calling with nanopores using PoreSeq. Nat Biotechnol. 2015. PubMed Abstract | Publisher Full Text\n\nTimp W, Comer J, Aksimentiev A: DNA base-calling from a nanopore using a Viterbi algorithm. Biophys J. 2012; 102(10): L37–L39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUrban JM, Bliss J, Lawrence CE, et al.: Sequencing ultra-long DNA molecules with the Oxford Nanopore MinION. bioRxiv. 2015. Publisher Full Text\n\nWallace EVB, Stoddart D, Heron AJ, et al.: Identification of epigenetic DNA modifications with a protein nanopore. Chem Commun (Camb). 2010; 46(43): 8195–8197. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang J, Moore NE, Deng YM, et al.: MinION nanopore sequencing of an influenza genome. Front Microbiol. 2015; 6: 766. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatson M, Thomson M, Risse J, et al.: poRe: an R package for the visualization and analysis of nanopore sequencing data. Bioinformatics. 2015; 31(1): 114–115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWood DE, Salzberg SL: Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014; 15(3): R46. 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}
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[
{
"id": "10824",
"date": "04 Nov 2015",
"name": "Michael Quail",
"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 supplied by the Minion Analysis and Reference Consortium is very thorough and a study that is worthy of indexation.It is however outdated as the technology is moving so fast and few of the findings (aside from the description of alignment tools) are likely to be of much practical use to users. That said this is a useful study that is very worthy of indexation and will gain widespread interest.I would recommend indexation (though it is already out there and has already been read by those interested) subject to the following revisions:The authors introduce nanopore sequencing at the start of the introduction and should mention Nanopore sequencing technologies and approaches other than ONT e.g Noblegen, Genia, INanoBio, etc. In paragraph 2 of the introduction the author say \"a library is constructed from double-stranded DNA (dsDNA) with a protocol similar to that used for short-read, second-generation platforms\" yet the library prep is more similar to that used by PacBio. Perhaps they should say \"a library is constructed from double-stranded DNA (dsDNA) with a protocol similar to that used for other NGS platforms\" At the end of paragraph 2 of the introduction the authors say \"Each channel provides data from one of the four wells at a time, the order of use defined by the allocation of wells to well-groups during an initial ‘mux scan’ (File S2 Glossary), allowing up to 512 independent DNA molecules to be sequenced simultaneously\". I'm not sure that someone who hasn't used a MinION would understand what the MuxScan is and that it's an algorithm choosing towards which of the 4 surrounding pores each channel should point. The text should be modified to explain this better. Paragraph 3 of introduction. Here the authors should make it clear that base calling doesn't take place on the computer connected to the MinION itself but can still be done while data is being acquired. Paragraph 3 of introduction. The authors claim that \"a single circular chromosome of 4.6 Mb that could be sequenced to sufficient depth in a single MinION run and a complete reference sequence is available\". This is only true if a good flowcell with sufficient active pores is obtained. This should be made clear and the authors should declare how many flowcells they or ONT screened in order to get enough flowcells with sufficient active pores. In figure 1 the authors should make it clear that the blue bar is the membrane. At the start of page 6 in the section \"Sequencer configuration and sequencing run conditions\" the authors say that a minimum of 400 g1 channels was considered acceptable. This was actually a rare event with the minION versions the authors describe but is more consistent now. The authors should note that this threshold isn't always met and is one of the major factors in variability in data yield. page 6, \"Data Analyses section\". The authors assume that that events are produced at a steady rate yet no evidence is given for this. As this is contrary to data given in ONT company presentation which show that dwell time per base is stochastic the authors need to show that this assumption is correct. page 6, \"Data Analyses section\". The authors say that they do not show reads generated during the first hour are not shown due to various effects. Yet almost a quarter of the reads are generated during this hour. If the authors are saying that reads during this period are substandard and not usable then they should say so. If they are usable then they should analyse them. page 7. results. The authors should say how many flowcells were tested and what % passed the 400 pore minimum threshold. Page 9. \"Total event yield\". The authors conclude \"suggesting data yield is not solely dependent on the number of initial active pores.\" They should include another possibility, the way ONT measure the number of active pores may not be accurate. Page 14. When talking about base quality the authors should state that this is a Q score. Page 15. \"Proportion of 2D pass and fail reads\". Users are interested in the overall proportion of passed reads not just the proportion during the first 21 hours. The overall proportion should be stated. Page 17. The authors stated that error estimates are similar for target and control base-calls. They should however point out that E.coli is a neutral GC genome similar to phiX and that other genomes with different base compositions have been reported to give a different error estimate to the lambda control. Page 17. \"Correlation between base quality and read accuracy\". Because there are so many events on figure 12 A it is impossible to establish if there is really a linear relationship, a gradient of colour intensifying where dots are overlapping would help the reader to see if it's really linear or just all over the place. page 17. The authors state \"A theoretical fold coverage of at least 60 was required to call 99.99% of the reference sites accurately from the majority consensus.\" This is 4 flowcells worth of sequence yet the authors claim a single flowcell to be sufficient in the introduction. page 17. In discussing \"Sequence motifs with lower accuracy\" the authors should also highlight the fact that homopolymers >5 cannot be resolved and are reported as 5 mers. page 19. In the discussion the authors say \"We demonstrated that there was considerable variability in the quality of flow cells\" Some figures would be useful here. page 19. In the discussion the authors say \"About 32% of the reads from an experiment result in 2D reads from the target genome.\" They should also state the percentage of 2D pass reads page 19. In the discussion the authors say \"When restricted to 2D ‘pass’ reads, the yield decreased to ~12,000 reads containing 75 million bases with a read length distributed centred around 6,700 bases and a mean base quality of 11.9.\" To put this in context with the previous sentence the authors should state the level of genome coverage that this achieved. page 20. Regarding fast mode. This is already available and giving superior quality data. Thus illustrating whether or not such a consortium can keep up other than perhaps on a blog? page 20. The authors say \"Although GC biases may be hard to detect through the sequencing of an E. coli strain with a mean GC content close to 50%, we did not observe a genome-wide GC bias in the 2D reads produced by this platform.\" In this context they should quote Goodwin et al. who report results from a non-base biased genome, Lver et al. Page 20. The authors say \"which suggests the MinION itself has some influence on the decrease in base quality over time\". Do they mean the minON or the flowcell. Page 21. The authors compare ONT error rates with short read technologies. They should also compare error rate and error profile with PacBio as this is also a long read technology and users would be more interested in that comparison.",
"responses": []
},
{
"id": "10821",
"date": "23 Nov 2015",
"name": "Nicholas J. Loman",
"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 produced by the MARC consortium is an impressively comprehensive study of run-level performance of the Oxford Nanopore MinION. The consortium have decided to focus on tightly defined parameters by sequencing a single bacterial strain, Escherichia coli K-12 MG1655, using a specific library preparation protocol and identical run parameters. Although some minor variability following the standard protocol between labs was observed, this is unlikely to affect the results.It therefore provides the most extensive view of platform performance variability to date, focusing on a specific combination of library preparation chemistry (SQK-MAP-005) and flow cell type (R7.3). Its strengths are therefore in the emphasis on platform reproducibility. There are a few extra things that could have been done to make the results more akin to user experiences, such as not pre-filtering flow cells for those with >400 group 1 pores, but this is not a significant issue, but it would be nice to see a spread of performance of all tested flow cells. The performance reported is in line with our own experiences.For me, the most interesting/useful elements of the paper were: the generally consistent results in terms of data quality achieved by different labs the extensive variability in throughput between flow cells which all give good QC (g1>400) values, suggesting that the QC stage is not a particularly reliable estimate of how well a flow cell will run Figure 1, which provides a useful summary figure (although please note caveats below) the close attention to detail to alignment methodologies the relationship between experiment run time, throughput and read quality, which suggest that a 48 hour workflow is not optimal, and pores should be remixed more frequentlyA frustration is that the underlying reasons for the variability in performance, with the exception of operational issues, are not really explained and therefore these results do not really help users plan how to mitigate the variability. This is not the authors fault but remains an issue for those planning experiments that require a particular yield.My major criticism is that the paper is over long and would have benefited from a good editor to try and reduce excessive verbiage. Sentences are often laborious and there is significant repetition throughout the manuscript. To pick on the first sentence of the manuscript: “the advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capacity embodies the next generation of large scale sequencing tools”. This is fairly garbled. What is “contextual sequencing capacity”. And why does it “embody the next generation of large scale sequencing tools” ? A few hours with a proof reader would do wonders. Greater use of active voice would improve readability. But it is up to the authors to decide whether they want to spend more time revising the manuscript in this manner.Generally I am happy with the manuscript to be approved as it is but I would suggest addressing a few technical points:Figure 1. I could not figure out panels G or H easily. I could not figure out the relationship between the k-mers relating to each strand, they did not obviously seem to match up or be reverse complements of each other. Panel H I also cannot figure out how the 2D consensus sequence relates to the 1D reads. For example, why is there an insertion in the 2D sequence which does not have a corresponding alignment in the 1D?In the section relating to the consensus sequences, the method seems to suggest that nanopolish was used to create a consensus sequence, using the reference sequence as the input alignment? I am not sure this is a particularly meaningful process. Accuracy measures like this (and any reference based alignment) are likely to be skewed by ‘reference attraction’, particularly given the alignment settings used - a pure de novo assembly may have been a more robust measurement. If that is too much work, the data could have been used (separately, or in combination) to polish the assembly from the nanopolish paper.I wish the analysis had not made so much of the (ad hoc) separation of 2D pass and fail reads. In reality reads are in a continuum of quality and the pass filter is simply defined by the Metrichor workflow, which is presumably version dependent (and can be turned off). At the least a definition of the pass filter would be useful, from figure 12 it looks like it relates to a Q value of around 10. A detailed treatment of the “usability” of 2D pass versus 2D fail reads is missing, but this is probably out of scope in the paper.One laboratory, Lab 3 reported high levels of Pseudomonas contamination. Although reagent contamination is a possibility, these very high levels are very unlikely to be due to vendor reagent contamination. Had this laboratory specifically handled P. putida in the recent past?Minor nitpicking points:Inconsistent use of trademarks e.g. MinION(TM), MinION in two contiguous sentences.AGBT 2012 presentation has been posted in the F1000 channel and can be referenced.“beta-testing” - is this formally defined or vernacular?It is not clear how much of the description of the chemistry is informed guessing, based on company materials or from unpublished communications, it would be nice to clarify what is a definitive statement and what is speculative. Along that line, I did not realise that tethers are on both strand, is that definitely true? 512 channels - this nomenclature is confusing, especially when compared to ‘wells’. Could this be clarified as to what a channel is?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1075
|
https://f1000research.com/articles/4-1073/v1
|
15 Oct 15
|
{
"type": "Systematic Review",
"title": "Leukocyte telomere length and hippocampus volume: a meta-analysis",
"authors": [
"Gustav Nilsonne",
"Sandra Tamm",
"Kristoffer N. T. Månsson",
"Torbjörn Åkerstedt",
"Mats Lekander",
"Sandra Tamm",
"Kristoffer N. T. Månsson",
"Torbjörn Åkerstedt",
"Mats Lekander"
],
"abstract": "Leukocyte telomere length has been shown to correlate to hippocampus volume, but effect estimates differ in magnitude and are not uniformly positive. This study aimed primarily to investigate the relationship between leukocyte telomere length and hippocampus gray matter volume by meta-analysis and secondarily to investigate possible effect moderators. Five studies were included with a total of 2107 participants, of which 1960 were contributed by one single influential study. A random-effects meta-analysis estimated the effect to r = 0.12 [95% CI -0.13, 0.37] in the presence of heterogeneity and a subjectively estimated moderate to high risk of bias. There was no evidence that apolipoprotein E (APOE) genotype was an effect moderator, nor that the ratio of leukocyte telomerase activity to telomere length was a better predictor than leukocyte telomere length for hippocampus volume. This meta-analysis, while not proving a positive relationship, also is not able to disprove the earlier finding of a positive correlation in the one large study included in analyses. We propose that a relationship between leukocyte telomere length and hippocamus volume may be mediated by transmigrating monocytes which differentiate into microglia in the brain parenchyma.",
"keywords": [
"Telomeres",
"Morphometry",
"Hippocampus",
"Microglia"
],
"content": "Introduction\n\nTelomeres are protective DNA-protein complexes at the ends of eukaryotic chromosomes1. In cells with limited proliferative capacity, telomeres shorten with each cell division2. Thus, telomere length in non-stem cells is a marker for biological aging. Germ cells, stem cells, and some cancer cells are able to maintain telomere length by expressing the enzyme telomerase reverse transcriptase1,2. Shorter leukocyte telomere length has been linked to adverse health outcomes including cancer3, cardiovascular disease4, and psychiatric disorders5, as well as poor self-rated health outcomes such as sleep quality and daytime functioning6. Recently, the association between leukocyte telomere length and brain morphology has been investigated by several research groups. The hippocampus has been a particular area of interest, possibly because it is a particularly plastic brain region, capable of neurogenesis7,8 and volume increases e.g. in spatial learning9,10, but also especially afflicted by atrophy in Alzheimer’s disease11. The relationship between leukocyte telomere length and hippocampus volume has however not been fully clarified by investigations to date.\n\nTwo possible moderators of a relationship between leukocyte telomere length and hippocampus volume have been proposed. Wikgren et al.12 proposed apolipoprotein E (APOE) genotype as a moderator, while Jacobs et al.13 proposed telomerase activity as a moderator, suggesting that the ratio of telomerase activity to telomere length is a better predictor than either measure alone.\n\nThis meta-analysis aimed primarily to estimate the correlation between leukocyte telomere length and hippocampus gray matter volume. Secondary aims were to investigate the possible moderating effect of APOE genotype on this correlation and to investigate whether the ratio of leukocyte telomerase to telomere length is a better predictor of hippocampus volume than either measure alone. We discuss possible mechanisms for the putative association between leukocyte telomere length and hippocampus volume and point to avenues for further inquiry.\n\n\nMethods\n\nPubMed (www.pubmed.gov) was searched on 2015-09-22 using the search string ”telomer* hippocampus” with no limitations by date nor language. Results were reviewed by title and abstract. Studies reporting data on the association between leukocyte telomere length and hippocampus volume in humans were included. Search results were reviewed independently by one investigator (GN) and by a committee of three investigators (KM, TÅ, and ML), reaching the same sample selection. Data were extracted by one investigator (GN) and checked for accuracy by another investigator (ST). The following variables were coded: number of participants and their sex distribution and age, observed correlation between leukocyte telomere length and hippocampus volume, analysis covariates, and whether apolipoprotein E (APOE) genotype or telomerase activity were also analysed. Figure 1 shows data inclusion. We analysed data with R version 3.2.014 with the metafor15, psych16, and pwr17 packages. A random-effects model was fitted due to heterogeneity between studies. A sensitivity analysis investigating the effect of excluding one study was performed post-hoc. The meta-analysis protocol was not predefined, and hence not registered. To compare telomere length, telomerase activity, and their ratio, data were estimated from published scatterplots using the freely available WebPlotDigitizer (http://arohatgi.info/WebPlotDigitizer/) version 3.818.\n\nOne study (Jacobs et al.13) reported results for right and left hippocampus separately. Partial r2 values for the relationship between hippocampus volume and leukocyte telomere length were however identical for both sides (r2 = 0.16) and this value was used. The same study reported data only for one subset of participants (APOE ∈4 non-carriers). All code and data needed to reproduce the analyses in this paper are available at 19, as is the list of PubMed search results.\n\n\nResults\n\nFive studies were included, of which two each contained two different participant cohorts, with a total of 2107 participants (Table 1). Several possible sources of heterogeneity were identified in sampling strategies and analysis methods. Grodstein et al.20 included a sub-group of patients with minimal cognitive impairment, and Wolkowitz et al.21 included a cohort with major depressive disorder. All studies determined relative average telomere length by PCR as the ratio of telomeres to single genes (T/S ratio). Two studies (Grodstein et al.20 and King et al.22) used the T/S ratio in statistical analyses, whereas the other three studies (Wikgren et al.12, Jacobs et al.13, and Wolkowitz et al.21) determined the average telomere base pair length and used that for statistical analyses. One study (King et al.22) log-transformed telomere length data prior to analysis in order to better approximate a normal distribution. Three studies (Grodstein et al.20, Jacobs et al.13, and Wikgren et al.12) did not use a log-transformation, and one study (Wolkowitz et al.21) reported that some variables were log-transformed but did not say whether telomere length was one of them.\n\nAPOE = Apolipoprotein E genotype, investigated (+) or not investigated (-); TA = telomerase activity, investigated (+) or not investigated (-); ∈3 & ∈4 = APOE alleles; MDD = Major Depressive Disorder; a = Age was not given for imaging subsample, age for whole healthy subsample substituted; b = Median and interquartile range.\n\nHippocampus volume was determined using magnetic resonance imaging in all included studies. King et al.22, Jacobs et al.13, and Wolkowitz et al.21 used FreeSurfer (http://freesurfer.net/) for automated parcellation. Wikgren et al.12 used manual tracing and did not specify whether the person performing the tracing was blind to other participant outcomes at the time. Grodstein et al.20 did not specify how hippocampus volumes were determined. Covariates for analyses differed between studies (Table 1). No study fractionated leukocytes nor analysed the relative contributions of different leukocyte subsets.\n\nA random-effects meta-analysis of all 7 datasets yielded a summary estimate of r = 0.12 [95% CI -0.13, 0.37], p = 0.35, Figure 2, indicating a positive direction of the relationship between telomere length and hippocampus volume, but failing to show a significant difference from 0. A test for heterogeneity was significant (τ2 = 0.086, I2 = 0.84, H2 = 6.25, Qdf=6 = 28.1, p < 0.0001), the Q-Q plot indicated deviation from normality, and the trim-and fill-method to test sensitivity for publication bias imputed two additional studies and yielded an estimate of r = 0.01 [95% CI -0.23, 0.25], p = 0.96, calling into question whether model assumptions were satisfied and suggesting that the effect estimate may be an overestimation due to publication bias. In spite of the small number of studies, we explored meta-regression as a means to address study heterogeneity, using mean age and fraction of female participants as independent variables. Neither was significantly associated (age: r2 = 0.00, p = 1.00; sex: r2 = 0.23, p = 0.14).\n\nThe observed deviation from normality was mostly due to the ∈4/∈4-positive cohort from Wikgren 201212 (Figure 3). In order to investigate the sensitivity of the meta-analysis to this particular study, the analysis was performed again without it. The meta-analytic estimate was now r = 0.23 [95% CI 0.05, 0.40], p = 0.01, without significant heterogeneity, although power to detect heterogeneity was now very limited (τ2 = 0.017, I2 = 0.48, H2 = 1.93, Qdf=4 = 7.9, p = 0.09), and with adequate normality as judged by inspection of the Q-Q plot (Figure 3). The trim-and fill-method to test sensitivity for publication bias imputed two additional studies and yielded an effect estimate of r = 0.12 [95% CI -0.07, 0.31], p = 0.22 (Figure 3).\n\nRisk of bias was assessed qualitatively and by inspection of funnel plots and Q-Q plots. Two of the included studies reported results only for subsets of participants, without indicating that these sub-group analyses were specified on beforehand. Grodstein et al.20 reported results only for healthy participants and participants with minimal cognitive impairment (MCI), leaving out 5 participants with dementia. Jacobs et al.13 reported results only for APOE ∈4 non-carriers, leaving out 19 APOE ∈4 carriers. Only one study (Wolkowitz et al.21) showed negative results. None of the studies reported blinding of experimenters carrying out laboratory nor statistical analyses. All studies except King et al.22 reported fewer than 30 participants. With 30 participants and using the effect estimate of r = 0.12 found in the main analysis, power would be 0.10. The largest effect estimate in this paper, observed in the sensitivity analysis, was r = 0.23, for which 30 participants would yield a power of 0.23. The observed low power among the included studies is not remarkable compared to the neuroscience field in general23. In combination with the preponderance of observed significant associations, it gives rise to a suspicion of bias. Funnel plots showed that the sample was right-skewed, although the small number of studies limits interpretation. The risk of bias was subjectively estimated to be moderate to high.\n\nDiamonds at bottom show estimates for a random-effects model including all studies and an estimate when excluding one study as a sensitivity analysis.\n\nA qualitative synthesis takes as point of departure the large study by King et al.22, since it contributes most of the participants (1960 of 2107) in this meta-analysis. This study was based on the Dallas Heart Study 2 cohort, a sample of 3401 participants examined in 2007–2009 and intended to be representative of the adult population of Dallas County, Texas. 2082 of these participants underwent magnetic resonance imaging and the final sample contained 1960 individuals. 48 brain regions were investigated using FreeSurfer. A positive direction of the association between leukocyte telomere length and brain volume was found for all 46 parenchymatous brain regions, whereas a negative direction of the association was found for the lateral and inferior lateral ventricles. Associations were significant at α = 0.05 in 27 regions after correcting for multiple comparisons using the false discovery rate method. The strongest association was found for the precuneus (r = 0.13), followed by the thalamus (r = 0.11), and then the hippocampus, lateral orbitofrontal cortex, inferior parietal cortex, posterior parietal cortex, inferior temporal cortex, and posterior cingulate cortex (all r = 0.10). Leukocyte telomere length predicted both total cerebral volume (r = 0.12) and total cortical volume (r = 0.11). The relationship between leukocyte telomere length and hippocampus volume was moderated by age and was strongest in older participants. Authors reported that only marginal effects were seen when adjusting for hypertension, obesity, diabetes mellitus, and smoking status. The final model adjusted for age, sex, and ethnicity. Hippocampal and cortical volumes were found in an overlapping sample to correlate to cognitive performance using the Montreal Cognitive Assessment24, supporting the validity of these volumetric measurements.\n\nFunnel plots show that the sample was right-skewed. Trim-and-fill analysis imputed two additional studies (unfilled circles) both in the full sample and in the reduced sensitivity analysis sample. Q-Q plots show that in the full sample, there was deviation from linearity due to one study. When this study was removed in the sensitivity analysis, linearity was acceptable.\n\nDemographic and clinical variables associated to leukocyte telomere length in the Dallas Heart Study 2 cohort have been reported in a previous paper including 3155 participants25. With telomere length categorized into tertiles, there was a negative correlation to monocyte fraction (short tertile: 6.88% (SD 2.21), middle tertile: 6.75% (SD 2.10), long tertile: 6.53% (SD 2.03), padj = 0.03), but not to other leukocyte fractions. Leukocyte telomere length also correlated positively to education and income, even when adjusting for age, gender and ethnicity. Since education and income were not analysed in King et al.22, they may represent unknown confounders or effect mediators.\n\nIn summary, the association between leukocyte telomere length and hippocampus volume found by King et al.22 gains credence from the large sample and the population-based sampling strategy. The consistent association found across parenchymatous brain regions further supports the finding of an effect in the hippocampus. The previously reported finding that leukocyte subsets were mostly stable across telomere length tertiles in the Dallas Heart Study 2 is reassuring, in that it suggests that apparent differences in telomere length were not due to differences in cell type composition, although such confounding cannot be fully ruled out by this analysis.\n\nThe other included studies besides King et al.22 all had samples for which results could be estimated of n < 30. While samples were small, reported effects were in the positive direction with the exception of one sub-group (from Wikgren et al.12), and estimated effects were larger than in King et al.22 with the exception of one further sub-group (from Wolkowitz et al.21). This pattern of results is expected in the presence of bias.\n\nQualitatively, the overall pattern of results favors a positive association between leukocyte telomere length and hippocampus volume.\n\nThe effect of APOE genotype on the relationship between telomere length and hippocampus volume, first proposed by Wikgren et al.12, has been subsequently investigated by King et al.22 and by Jacobs et al.13. Wikgren et al.12 reported a different effect magnitude in two groups (∈3/∈3 homozygotes: r = -0.52; ∈4 carriers: r = 0.13), but not an interaction test which would tell whether that difference was statistically significant. In contrast, Jacobs et al.13 reported a positive association (r = 0.40) only among ∈4-noncarriers. Jacobs et al.13 also did not test whether the difference between groups was statistically significant. King et al.22 reported no significant effect of APOE ∈2, ∈3 nor ∈4 genotype in 1409 participants for whom APOE data were available. It was not possible to investigate these results quantitatively as effect sizes were not reported. In summary, none of the included studies provide positive evidence for a moderating effect of APOE genotype on the association between telomere length and hippocampus volume.\n\nTwo studies with four datasets investigated leukocyte telomerase activity in addition to leukocyte telomere length as predictors for hippocampus volume. Jacobs et al.13 proposed that the leukocyte telomerase/telomere length ratio is a better predictor for hippocampus volume than either measure alone, but without formally testing whether the ratio yielded a higher correlation. We decided to investigate this proposition. Since it was necessary to perform a test for dependent correlations, and the correlations between telomere length and telomerase activity were not given, we extracted data from scatterplots presented in both papers and calculated the required correlations from the estimated data. Comparisons by inspection of original plots and plots of estimated data showed satisfactory data extraction accuracy and can be viewed at 19. Pairwise estimates of hippocampus volume correlated well (r’s = 0.99-1.00), further supporting successful data extraction.\n\nIn Jacobs et al.13, telomere length and telomerase activity did not correlate (left: r = -0.19 [95% CI -0.19, 0.53], p = 0.32; right: r = -0.14 [95% CI -0.49, 0.25], p = 0.48; Note that while these measures have no intrinsic laterality, data were extracted separately for the right and left sides, and the difference between sides is likely due to estimation error). The ratio of telomerase activity to telomere length was a better predictor for hippocampus volume than telomerase activity alone for the right side but not the left. On neither side was the ratio a better predictor than telomere length (Table 2). In Wolkowitz et al.21, telomere length and telomerase activity did not correlate (healthy controls: r = 0.09 [95% CI -0.44, 0.58], p = 0.75; patients with major depressive disorder: r = 0.14 [95% CI -0.34, 0.56], p = 0.57). The ratio of telomerase activity to telomere length was not a better predictor than telomerase activity nor telomere length in either group (Table 2). It is notable that correlations between telomerase activity and hippocampus volume differed in direction between the two studies (Table 2).\n\nData from Jacobs et al.13 were estimated from published scatterplots showing only the APOE ∈4-negative participant subset, separately for right and left hippocampus, and are adjusted for age. Further adjustments for education and BMI were not possible because data were not available. Data from Wolkowitz et al.21 were estimated from published scatterplots and are not adjusted for any covariates. Adjustments for age and sex were not possible because data were not available. TA = telomerase activity; TL = telomere length; HV = hippocampus volume; Ratio = ratio of telomere activity to telomere length; MDD = major depressive disorder; a = effect direction favors telomere length or activity over ratio.\n\n\nDiscussion\n\nWe investigated the correlation between leukocyte telomere length and hippocampus volume. In the main analysis, we found a correlation of r = 0.12 with considerable uncertainty and not significantly different from 0. Studies were heterogeneous and the exclusion, as a sensitivity analysis, of one study (Wikgren et al.12) shifted the estimate to r = 0.23, which was significantly different from 0. Trim-and-fill analyses to investigate publication bias yielded imputations which brought the estimated effect down to r = 0.01 in the full sample and r = 0.12 in the sample excluding one study, neither of which was significantly different from 0.\n\nA meta-analysis does not necessarily yield better estimates than a single high-powered study, mainly because a high-powered study can be expected to have low bias, and if smaller studies with more bias are included in a meta-analysis, then the resulting estimate may be more biased and hence less accurate than that of the single study26. Therefore, while this meta-analysis failed to corroborate a positive association, we argue that it does not provide compelling evidence to reject the positive estimate from the large study by King et al.22, which contributed 1960 out of 2107 participants.\n\nWe found no evidence for APOE genotype as an effect moderator between leukocyte telomere length and hippocampus volume. The ratio of telomerase activity to telomere length was a better predictor than telomerase activity on one side in Jacobs et al.13 but not in either subsample in Wolkowitz et al.21. We conclude that there is no evidence that APOE genotype moderates the relationship between leukocyte telomere length and hippocampus volume, while the evidence that the telomerase activity to telomere length ratio predicts hippocampus volume better than telomerase activity is inconclusive.\n\nAn important limitation of this meta-analysis is that none of the included studies fractionated leukocytes before analysis, meaning that any change in the leukocyte cell type composition may confound results. Hippocampal atrophy has been linked to outcomes that are also associated to changes in leukocyte subset composition, such as inflammation and sleep pattern changes27,28. Therefore, leukocyte cell type composition is a possibly important and uninvestigated confounder.\n\nFurthermore, this meta-analysis was limited by the small number of included studies, and by the relatively small sample sizes in all studies except one. Studies were heterogeneous and the risk of bias was subjectively judged to be moderate to high. Since none of the studies published their data in a format conducive to easy reuse, some analyses were precluded. Estimation of data from published scatterplots gives rise to measurement error, which, while probably small, could affect the results. Participant samples were predominantly middle-aged and all included more women than men, limiting external validity.\n\nDifferent models have been proposed to explain the relationship between leukocyte telomere length and hippocampus volume. King et al.22 have proposed, as one possibility among others, that longer telomeres are a surrogate marker for higher telomerase activity, causing greater proliferative capacity and more cell proliferation (Figure 4, model 1). Wikgren et al.12, on the other hand, have proposed that longer telomeres indicate fewer cell divisions have taken place, consistent with fewer cells and a smaller tissue volume (Figure 4, model 2). These two models make contradictory predictions, and if one accepts a positive correlation between leukocyte telomere volume and hippocampus volume, then a process according to model 2 cannot constitute a dominating contribution to the observed effect. The positive correlation between leukocyte telomerase activity and telomere length predicted by model 1 was however not demonstrated in the present datasets.\n\nA missing link in models 1 and 2 is the relationship between circulating leukocytes and brain parenchyma, as regards telomere length and cell turnover. Leukocyte telomere length represents an average over a large number of white blood cells of different types6,29. While peripheral leukocytes are a heterogeneous cell population, they share a histogenetic origin in the hematopoietic stem and progenitor cells of the bone marrow. By contrast, neurons and macroglia (oligodendrocytes, and astrocytes) all derive from neuronal stem cells. Neurogenesis continues to a limited extent in adulthood, especially in the hippocampus and subventricular zone7,8,30. Microglia, unlike neurons and macroglia, are derived from circulating mononuclear cells which originate in early fetal life from yolk sac hematopoietic stem cells, and later from mesodermally derived hematopoietic stem cells which in adults locate chiefly in the bone marrow47. Whether these two classes of hematopoietic stem cells have the same or different origin is not known31. There is controversy about the extent to which mononuclear cell transmigration and microglial differentiation continues in adult life. Studies using bone marrow transplantation in mice have found that 0 – 25% of microglia derived from the transplanted bone marrow after 4 – 15 weeks32–38. These findings have been interpreted as evidence both for and against a substantial contribution of bone marrow-derived cells to the microglial cell population in adulthood. In particular, it is not yet clear to what extent injury to the blood-brain barrier (induced, for example, by radiation) facilitates transmigration, and to what extent bone marrow-derived microglia remain in the brain after the resolution of local inflammation. To date, only one study has investigated the correlation between telomere length in leukocytes and in human postmortem brain tissue, and it found a moderate correlation of r = 0.42 in a study including 29 patients, all with Alzheimer’s disease39.\n\nExperiments using knock-out mouse models have shown that monocyte recruitment to the brain depends on monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2/small inducible cytokine A2 (MCP-1/CCL2) signalling trough the CCR2 receptor and that monocyte recruitment to the brain increases in peripheral inflammation40. MCP-1 knock-out mice had less microglial recruitment to the hippocampus and more neurogenesis following cranial irradiation41. Activated microglia can secrete neurotoxic factors including proinflammatory cytokines and reactive oxygen species (ROS), and microglial activation has been proposed as an important pathophysiological process in Alzheimer’s and Parkinson’s dementias27,42. Leukocyte telomere length has however not been conclusively linked to Alzheimer’s and Parkinson’s dementias in humans43–45. Zhou et al.46 found that telomerase inhibition caused impaired hippocampal neurogenesis, while overexpression of telomerase reverse transcriptase (TERT) led to increased hippocampal neurogenesis.\n\nIt is of course possible that an association between leukocyte telomere length and hippocampus volume could arise from factors determining both outcomes independently (Figure 5, model 3). King et al.22 speculate that chronic inflammation may be an important determinant. Wolkowitz et al.21 add that oxidative stress or endogenous hormonal regulation might influence telomerase activity and cell turnover in distant tissues such as leukocytes and brain. Lindqvist et al.5 similarly point to inflammation, oxidative stress, and cortisol signalling as possible mechanisms. None of these possible determinants are however clearly supported by empirical data at the present time. Model 3 can be seen as a reformulation of reasoning from these aforementioned papers. Alternatively, we propose speculatively that microglia are a mechanistic link between leukocyte telomere length and hippocampus volume (Figure 5, model 4). According to this model, hippocampus volume is affected by microglial telomere length by processes related to microglial proliferation and activation. Possibly, longer telomeres could prevent pathological activation of microglia which causes neuronal damage. This model is highly tentative, but it does have the important advantage of providing a possible mechanism by which leukocyte telomere length can affect the cell turnover kinetics of neurons and macroglia, even though they have completely different histogenesis. One observation consistent with the proposed model is that monocytes were the only leukocyte subset correlated to leukocyte telomere length in the Dallas Heart 2 study, even though the effect was small25. More research is needed to verify or falsify testable predictions arising from this model.\n\n\nConclusions\n\nThe high-powered study by King et al.22 estimated a positive correlation of r = 0.10 between leukocyte telomere length and hippocampus volume. The present analysis does not provide compelling evidence against this finding, while it also does not provide absolute evidence in favor. The main meta-analytic effect estimate was r = 0.12, with estimates ranging from r = 0.00 to r = 0.23 after trim-and-fill imputation and in sensitivity analyses. We found no support for a moderating effect of APOE genotype and inconclusive evidence for using the telomerase activity/telomere length ratio as a predictor for hippocampus volume instead of telomere length.\n\nMore research in this area is needed to answer several outstanding questions. Differential analyses of white blood cells will be important in order to verify that apparent differences in leukocyte telomere length are not in fact differences in leukocyte cell type composition. Studies comparing telomere lengths in peripheral blood cells and in neurons, macroglia, and microglia will help elucidate the possible mechanistic link due to monocyte-microglial differentiation. Longitudinal designs will allow investigation of whether telomere shortening precedes brain atrophy, or vice versa. Further study of the relationships between leukocyte telomere length, brain volume, and environmental, physiological, and behavioral determinants such as inflammation, sleep and endocrine signalling will help elucidate possible mechanistic factors. Multivariate predictive modelling will be useful to investigate putative diagnostic and/or prognostic properties of telomere length measures. Studies with large samples and open publication of data are greatly to be desired.\n\n\nData availability\n\nZENODO: Hippocampus-volume-telomere-length: Release for publication, doi: 10.5281/zenodo.3177119",
"appendix": "Author contributions\n\n\n\nConceived of study: GN. Collected data: GN. Analysed data: GN, ST. Interpreted results: GN, ST, KM, TÅ, ML. Drafted manuscript: GN. All authors read and approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nReferences\n\nBlackburn EH: Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett. 2005; 579(4): 859–862. PubMed Abstract | Publisher Full Text\n\nChan SR, Blackburn EH: Telomeres and telomerase. Philos Trans R Soc Lond B Biol Sci. 2004; 359(1441): 109–121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArtandi SE, DePinho RA: Telomeres and telomerase in cancer. Carcinogenesis. 2010; 31(1): 9–18. 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PubMed Abstract | Publisher Full Text\n\nPriller J, Flügel A, Wehner T, et al.: Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment. Nat Med. 2001; 7(12): 1356–1361. PubMed Abstract | Publisher Full Text\n\nMildner A, Schmidt H, Nitsche M, et al.: Microglia in the adult brain arise from Ly-6chiccr2+ monocytes only under defined host conditions. Nat Neurosci. 2007; 10(12): 1544–1553. PubMed Abstract | Publisher Full Text\n\nGinhoux F, Greter M, Leboeuf M, et al.: Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010; 330(6005): 841–845. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAjami B, Bennett JL, Krieger C, et al.: Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat Neurosci. 2011; 14(9): 1142–1149. PubMed Abstract | Publisher Full Text\n\nCapotondo A, Milazzo R, Politi LS, et al.: Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation. Proc Natl Acad Sci U S A. 2012; 109(37): 15018–15023. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurrell K, Hill RP, Zadeh G: High-resolution in-vivo analysis of normal brain response to cranial irradiation. PLoS One. 2012; 7(6): e38366. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOkonogi N, Nakamura K, Suzuki Y, et al.: Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue. J Radiat Res. 2014; 55(4): 713–719. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLukens JN, Van Deerlin V, Clark CM, et al.: Comparisons of telomere lengths in peripheral blood and cerebellum in Alzheimer’s disease. Alzheimers Dement. 2009; 5(6): 463–469. PubMed Abstract | Publisher Full Text | Free Full Text\n\nD’Mello C, Le T, Swain MG: Cerebral microglia recruit monocytes into the brain in response to tumor necrosis factoralpha signaling during peripheral organ inflammation. J Neurosc. 2009; 29(7): 2089–2102. PubMed Abstract | Publisher Full Text\n\nLee SW, Haditsch U, Cord BJ, et al.: Absence of CCL2 is sufficient to restore hippocampal neurogenesis following cranial irradiation. Brain Behav Immun. 2013; 30: 33–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLull ME, Block ML: Microglial activation and chronic neurodegeneration. Neurotherapeutics. 2010; 7(4): 354–365. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEitan E, Hutchison ER, Mattson MP: Telomere shortening in neurological disorders: an abundance of unanswered questions. Trends Neurosci. 2014; 37(5): 256–263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranco S, Blasco MA, Siedlak SL, et al.: Telomeres and telomerase in Alzheimer’s disease: epiphenomena or a new focus for therapeutic strategy? Alzheimers Dement. 2006; 2(3): 164–168. PubMed Abstract | Publisher Full Text\n\nThomas P, O’ Callaghan NJ, Fenech M: Telomere length in white blood cells, buccal cells and brain tissue and its variation with ageing and Alzheimer’s disease. Mech Ageing Dev. 2008; 129(4): 183–190. PubMed Abstract | Publisher Full Text\n\nZhou QG, Hu Y, Wu DL, et al.: Hippocampal telomerase is involved in the modulation of depressive behaviors. J Neurosci. 2011; 31(34): 12258–12269. PubMed Abstract | Publisher Full Text\n\nRezaie P, Male D: Colonisation of the developing human brain and spinal cord by microglia: a review. Microsc Res Tech. 1999; 45(6): 359–382. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10826",
"date": "02 Nov 2015",
"name": "Kevin S. King",
"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 current systematic review and meta-analysis of works evaluating the possible link between leukocyte telomere length and hippocampus volume is robust and thorough. It provides a useful quantitative analysis of the current research data and also evaluates potential theories on underlying mechanisms. My reviews are often more lengthy but I think this present work is comprehensive and I really do not think I have a lot to add. We were intrigued by existing studies but were worried about potential for bias in existing studies. A particular cause for concern was the small sample size of the studies and relatively modest size of the association being studied. This article gives concrete analysis to support this and determined the power of studies with a sample size of 30 to show an association was only 10%. As mentioned in this rather comprehensive work, some of the work stated that their findings were hypothesis generating and the associations tested were not stipulated at the outset but rather emerged in subgroup analysis. Thanks is still owed to these smaller studies for pioneering the ideas underlying this work. We were rather fortunate to have as a partner the Dallas Heart Study which made our work possible and are grateful for their support.",
"responses": []
},
{
"id": "10997",
"date": "24 Nov 2015",
"name": "Owen M. Wolkowitz",
"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 done study with appropriate analyses.It is timely and of interest.It is limited (as appropriately acknowledged by authors) by the small number of studies and the large impact of one study (King).There is a very good discussion of different theoretical models.It would be worth also highlighting in the abstract their analyses of telomerase itself, not just the TL/telomerase ratio.They present a secondary analysis without Wikgren. This was a post-hoc decision, but this is acknowledged, and the main findings of the meta-analysis include that study.The only real issue:There should have more discussion of the role of moderators (although this information was not available across all studies), e.g., age, sex, illness (medical and psychiatric), medications, education, SES, as well as MRI field strength, etc. Indeed, the authors reported that only marginal effects were seen when adjusting for hypertension, obesity, diabetes mellitus, and smoking status. This should be given more emphasis.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1073
|
https://f1000research.com/articles/4-897/v1
|
24 Sep 15
|
{
"type": "Research Article",
"title": "Impact of a structured review session on medical student psychiatry subject examination performance",
"authors": [
"Shan H. Siddiqi",
"Kevin J. Black",
"Fay Y. Womer",
"Kevin J. Black",
"Fay Y. Womer"
],
"abstract": "Introduction: The National Board of Medical Examiners (NBME) subject examinations are used as a standardized metric for performance in required clerkships for third-year medical students. While several medical schools have implemented a review session to help consolidate knowledge acquired during the clerkship, the effects of such an intervention are not yet well-established. One prior study reported an improvement in NBME psychiatry examination scores with a 1.5-hour review session, but this study was limited by a small sample size and the fact that attendance at the review session was optional, leading to likely selection bias. Methods: A 1.5-hour structured review session was conducted for medical students in the last week of each 4-week psychiatry clerkship between September 2014 and July 2015. Students were required to attend unless excused due to scheduling conflicts. Scores on the NBME psychiatry subject exam were compared with those of students taking the examination in the corresponding time period in each of the previous two academic years. Results: 83 students took the exam during the experimental period, while 176 took the exam during the control period. Statistically significant improvements were found in mean score (p=0.03), mean for the two lowest scores in each group (p<0.0007), and percentage of students scoring 70 or less (p=0.03). Percentage of students achieving the maximum possible score (99) was higher in the experimental group, but did not reach significance (p=0.06). Conclusions: An end-of-clerkship review session led to increased mean scores on the NBME psychiatry subject examination, particularly for students at the lower end of the score range. Future research should investigate the impact of such an intervention in other specialties and other institutions.",
"keywords": [
"Medical education",
"NBME",
"shelf",
"review session",
"medical students",
"academic psychiatry",
"clinical clerkship"
],
"content": "Background\n\nThe National Board of Medical Examiners (NBME) subject examinations are widely used in North America as a means of assessing overall performance and potential need for remediation in required third-year medical student clerkships; their utility is rooted in the fact that they provide a standardized and objective measure of knowledge acquired during the clerkship1. While the utility of NBME examinations for internal evaluation of students has been questioned2, this notion is challenged by the findings that performance on these examinations is correlated with other measures of a medical student’s knowledge base3,4, suggesting that higher scores are associated with improved overall educational outcomes. Furthermore, these scores are also correlated with a student’s eventual performance on the United States Medical Licensing Exam (USMLE) Step 2CK, which is a critical component of evaluation for residency selection5.\n\nHowever, strategies for preparing students for these examinations remain inconsistent6. This process is particularly challenging for the psychiatry subject examination (PSE), in which performance has been found to be more strongly associated with interpersonal skills than with subjective faculty evaluations of a student’s medical knowledge and clinical skills7, although alternate measures of student performance (including faculty evaluations and standardized patient encounters) are still correlated with PSE scores8.\n\nThe impact of structured teaching on PSE scores has garnered some attention in the literature. Prior studies have demonstrated a significant improvement with a series of eight resident-led tutorials9 and with a complete curriculum overhaul with a goal of improving scores10. A single end-of-clerkship review session for the subject examination has also demonstrated an increase in scores, but this study was limited by a relatively small sample size, which limited the range of outcomes that could be effectively measured, and by potential selection bias, since attendance at the session was not mandatory11. We investigated the impact of a single review session with a larger sample size and with mandatory attendance.\n\n\nMethods\n\nThe study retrospectively investigated scores on the PSE after implementation of a review session covering a general overview of adult psychiatry with a focus on topics that are critical for medical students to understand. The review session was conducted less than 1 week before students were required to take the PSE. Students were required to attend, but were excused in the event of a conflict with their rotation schedules. Data were analyzed retrospectively based on de-identified scores provided by the NBME. This study was deemed exempt from review by the institutional review board at Washington University in St. Louis, which determined that consent from individual students was not required and students need not be notified because data were de-identified prior to retrospective analysis. The Associate Dean for Education at Washington University School of Medicine also approved the retrospective review of de-identified scores.\n\nThe review session was based on an interactive case-based discussion of evaluation and management of common psychiatric problems, with a focus on topics that are commonly misunderstood by medical students. The session was designed and conducted by a resident physician (SHS) with prior experience in developing study materials for various standardized examinations, including the PSE. Cases demonstrated hypothetical patients with mania, depression, psychosis, substance abuse, anxiety/panic, eating disorders, personality disorders, somatoform disorders, and psychotropic medication toxicity. Additional non-case-based discussions were included to differentiate the types of dementia and understand legal/ethical issues in psychiatry. Child psychiatry topics were not included because the clerkship already included a separate lecture on child psychiatry during the same week. Detailed psychopharmacology was also not included due to time constraints; instead, students were advised to independently review mechanisms, indications, and toxicity profiles of the different classes of psychotropic medications.\n\nThe experimental group consisted of nine groups of students completing their psychiatry clerkships between September 2014 and July 2015. The control group consisted of students completing the examinations during the corresponding time periods in the previous two academic years; the other months in previous years were not included to avoid confounding due to the tendency of scores to increase as the academic year progresses. No other changes were made to the students’ lecture schedules.\n\nStatistical analyses were completed using R version 3.2.0 using individual de-identified scores that are provided by the NBME in paper form. Mean scores for the full September to July period were compared between the experimental group and the control group via two-tailed paired t-test. In order to evaluate the effects on students with weaker knowledge base, a paired t-test was also used to compare means for all students who achieved lowest two scores in each 4-week clerkship block between the experimental group and the control group. A one-tailed Z-test for proportions was used to compare the fraction of students scoring 99 (the maximum possible score) and the fraction of students scoring 70 or less (typically corresponding approximately to the 10th percentile in the national sample; our school considers this a failing exam score that must be remediated to earn credit for the psychiatry clerkship).\n\n\nResults\n\nEighty-three students took the exam during the experimental period, while 175 took the exam during the control period. Statistically significant improvements were found in the mean score, the two lowest scores in each group, and the fraction of students scoring 70 or less. Improvement in fraction of students achieving the maximum possible score (99) did not reach significance (p = 0.06). These results are summarized in Table 1.\n\n\nDiscussion\n\nImplementation of a mandatory end-of-clerkship review session was associated with improvements in mean scores on the PSE, particularly for students whose scores were in the lower range. While similar improvements have been suggested in the past11, this study reproduces these findings with a larger sample size, thereby allowing analysis of performance in different scoring ranges. This study also demonstrated a significant effect of the intervention despite higher baseline scores in this sample (mean baseline scaled score 85.3, compared to 77.2 in the previous study). Furthermore, attendance at the review session in this study was mandatory, thereby controlling for the selection bias introduced by the possibility that students choosing to attend a voluntary review session may have been more motivated at baseline.\n\nDue to the retrospective nature of the analysis and lack of randomization, this study is subject to several limitations. Performance was compared between different academic years, so inter-class differences unrelated to the intervention may have confounded the results. Furthermore, while the review sessions followed a standardized format, we do not know how reproducible they may be in other academic settings.\n\nThis study did not investigate whether the improvement in students’ PSE performance translated to improvements in clinical skills. However, a recent large meta-analysis showed that clerkship grades (which usually incorporate NBME subject examination scores1) and USMLE Step 2CK scores (which are correlated with NBME subject examination scores) predict a resident’s performance on both objective and subjective evaluations12. Further research is needed in order to determine whether an end-of-clerkship review session translates to improvements in other measures of a student’s clinical skills and knowledge.\n\nOverall, these results provide further support for the notion that a single end-of-clerkship review session improves scores on the NBME psychiatry subject examination, even when eliminating selection bias by making the review session mandatory. Future studies should be geared at reproducing these findings in other specialties and standardizing the course for improved generalizability.\n\n\nData availability\n\nF1000Research: Dataset 1. Medical student scores on the psychiatry NBME subject examination before and after institution of a mandatory review session, 10.5256/f1000research.7091.d102704",
"appendix": "Author contributions\n\n\n\nCourse design and implementation, collecting data, literature review, writing: SHS. Conceptualization, development/support of overall medical student curriculum, reviewing/editing the manuscript: KJB, FYW.\n\n\nCompeting interests\n\n\n\nDr. Siddiqi writes practice questions for medical student exams for ExamGuru, which was not involved in the production of this work. The authors disclose no other competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors thank Tammie Repko for administrative support with implementing the review sessions and collating the data. We also thank Dr. Brendan O’Connor for contributions to academic coordination of the medical student psychiatry rotation.\n\n\nReferences\n\nLevine RE, Carlson DL, Rosenthal RH, et al.: Usage of the National Board of Medical Examiners Subject Test in Psychiatry by U.S. and Canadian clerkships. Acad Psychiatry. 2005; 29(1): 52–7. PubMed Abstract | Publisher Full Text\n\nHoffman KI: The USMLE, the NBME subject examinations, and assessment of individual academic achievement. Acad Med. 1993; 68(10): 740–7. PubMed Abstract | Publisher Full Text\n\nHemmer PA, Grau T, Pangaro LN: Assessing the effectiveness of combining evaluation methods for the early identification of students with inadequate knowledge during a clerkship. Med Teach. 2001; 23(6): 580–584. PubMed Abstract | Publisher Full Text\n\nGriffith CH 3rd, Wilson JF: The association of student examination performance with faculty and resident ratings using a modified RIME process. J Gen Intern Med. 2008; 23(7): 1020–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZahn CM, Saguil A, Artino AR Jr, et al.: Correlation of National Board of Medical Examiners scores with United States Medical Licensing Examination Step 1 And Step 2 scores. Acad Med. 2012; 87(10): 1348–54. PubMed Abstract | Publisher Full Text\n\nTorre D, Papp K, Elnicki M, et al.: Clerkship directors' practices with respect to preparing students for and using the National Board of Medical Examiners Subject Exam in medicine: results of a United States and Canadian Survey. Acad Med. 2009; 84(7): 867–71. PubMed Abstract | Publisher Full Text\n\nRamchandani D: Grading medical students in a psychiatry clerkship: correlation with the NBME subject examination scores and its implications. Acad Psychiatry. 2011; 35(5): 322–4. PubMed Abstract | Publisher Full Text\n\nMcLay RN, Rodenhauser P, Anderson DS, et al.: Simulating a full-length psychiatric interview with a complex patient: an OSCE for medical students. Acad Psychiatry. 2002; 26(3): 162–7. PubMed Abstract | Publisher Full Text\n\nMcKean AJ, Palmer BA: Psychiatry resident-led tutorials increase medical student knowledge and improve national board of medical examiners shelf exam scores. Acad Psychiatry. 2015; 39(3): 309–11. PubMed Abstract | Publisher Full Text\n\nSpollen J, Cluver J: An approach to improving psychiatry NBME and USMLE performance. Acad Psychiatry. 2013; 37(3): 207–10. PubMed Abstract | Publisher Full Text\n\nSidhu SS, Chandra RM, Wang L, et al.: The effect of an end-of-clerkship review session on NBME psychiatry subject exam scores. Acad Psychiatry. 2012; 36(3): 226–8. PubMed Abstract | Publisher Full Text\n\nKenny S, McInnes M, Singh V: Associations between residency selection strategies and doctor performance: a meta-analysis. Med Educ. 2013; 47(8): 790–800. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10461",
"date": "02 Oct 2015",
"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 article reports on a study of the impact of a structured review session at the end of a 4-week clerkship for third year medical students on psychiatry examination performance, over a 10-month period. Examination scores are compared with historical controls over the same period of time in the previous 2 years. Significant improvements were reported for overall scores and the lowest scoring students. The intervention appeared to be of value. The authors recommend further research for other medical specialties and in other institutions. This appears justified. The differences in scores were small but significant.No ethical approval was required for the present small study but may be needed if more intricate study designs are used, including randomisation of larger numbers possibly through the use of cluster randomisation. The authors could provide some suggestions. They address the limitations in the present study well.Some small corrections:The abstract in its introduction refers to \"One prior study\" and its finding - this could be written in more general terms....In the background (p3), 2nd para, line 1: ...for THESE examinations.... - not clear which examinationsMethods (p3), right-hand column, 3rd para down, line 1: ...USING...USING - replace 1st with 'WITH'; line 2: ...that WERE provided by...Discussion (p4), right-hand column, top of text: ...these results provide [further] support...ie delete 'further'",
"responses": []
},
{
"id": "10688",
"date": "06 Oct 2015",
"name": "Andrew 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\nIn many institutions, the National Board of Medical Examiners (NBME) subject examinations are used as a performance metric for medical students and this study was conducted to see if a structured review session might improve scores on the exam.The authors used a mandatory 1.5-hour structured review session in the last week of each 4-week psychiatry clerkship (September 2014 and July 2015) and the primary outcome measure was a comparison of scores on the NBME psychiatry subject exam before and after the interventional period. The sample size was reasonable for a study of this type with 83 students in the experimental period and 176 in the historical control period. As might be expected, there were statistically significant improvements in mean score (p=0.03), mean for the two lowest scores in each group (p<0.0007), and percentage of students scoring 70 or less (p=0.03) in the experimental, interventional arm. Interestingly, the percentage of students achieving the maximum possible score (99) was higher in the experimental group, but did not reach significance (p=0.06). The authors concluded that \"an end-of-clerkship review session led to increased mean scores on the NBME psychiatry subject examination, particularly for students at the lower end of the score range.\"These results suggest what is intuitively known but deserves emphasis: 1) structured, scheduled, mandatory, review is helpful in improving performance on standardized testing of the same or similar core content; 2) timeliness and proximity of the review to the testing may improve performance in the short term but this study (and many similar study designs) do not speak to long term retention and sustainability; and 3) multiple barriers exist in the status quo that might limit implementation or generalizability including the usual suspects (time, money, resources).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-897
|
https://f1000research.com/articles/4-1062/v1
|
13 Oct 15
|
{
"type": "Research Article",
"title": "ve-SEQ: Robust, unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly diverse pathogens",
"authors": [
"David Bonsall",
"M. Azim Ansari",
"Camilla Ip",
"Amy Trebes",
"Anthony Brown",
"Paul Klenerman",
"David Buck",
"STOP-HCV Consortium",
"Paolo Piazza",
"Eleanor Barnes",
"Rory Bowden",
"David Bonsall",
"M. Azim Ansari",
"Camilla Ip",
"Amy Trebes",
"Anthony Brown",
"Paul Klenerman",
"David Buck",
"Paolo Piazza",
"Eleanor Barnes"
],
"abstract": "The routine availability of high-depth virus sequence data would allow the sensitive detection of resistance-associated variants that can jeopardize HIV or hepatitis C virus (HCV) treatment. We introduce ve-SEQ, a high-throughput method for sequence-specific enrichment and characterization of whole-virus genomes at up to 20% divergence from a reference sequence and 1,000-fold greater sensitivity than direct sequencing. The extreme genetic diversity of HCV led us to implement an algorithm for the efficient design of panels of oligonucleotide probes to capture any sequence among a defined set of targets without detectable bias. ve-SEQ enables efficient detection and sequencing of any HCV genome, including mixtures and intra-host variants, in a single experiment, with greater tolerance of sequence diversity than standard amplification methods and greater sensitivity than metagenomic sequencing, features that are directly applicable to other pathogens or arbitrary groups of target organisms, allowing the combination of sensitive detection with sequencing in many settings.",
"keywords": [
"Virus genome sequencing",
"Sequence capture and enrichment",
"Anti-viral resistance",
"Hepatitis C virus"
],
"content": "Introduction and background\n\nWith a world-wide prevalence estimated at 2.8%1,2 hepatitis C virus (HCV) poses a global health challenge unrivalled by any curable viral infection. In recent years, direct-acting antiviral (DAA) combination therapies have substantially improved outcomes, but fundamental barriers to eradication remain, including reduced efficacy against genotype 3 infections3,4 and a cost of modern treatments that is out of reach of even middle-income countries. Newer DAAs such as those targeting HCV’s polymerase and NS5a proteins augment protease inhibitors5, but genotype-limited efficacy and the possibility of resistance mean that HCV genotyping and periodic monitoring of viral load (VL) will remain important in the selection and monitoring of DAA therapies.\n\nResistance testing by PCR and sequencing of relevant genes is routinely used before initiation of HIV treatment and after its virological failure6. Similar testing in HCV is an exciting prospect, with potential benefits in efficacy and cost. With some notable exceptions, resistance-associated variant (RAV) status at baseline has not been shown to be strongly predictive of treatment success (https://www.nice.org.uk/guidance/ta331), however the role of resistance testing in informing choice and timing of therapy after HCV treatment failure is an active area of clinical research (e.g. HCV-TARGET7). It is clear from clinical trials in which RAVs were assessed via amplicon sequencing that the relevance of particular mutations depends on both the drug in question and the genetic background of the virus, and attempts have been made to summarise these data as more drugs enter clinical practice8.\n\nAs more data is acquired through phase 4, post-marketing studies, our ability to predict treatment success from viral genetic information is likely to improve, leading to higher cure rates across a greater variety of antiviral agents, with potential long-term benefits in treatment cost. However, several questions remain unanswered, including the relevance of variants detected at low frequency within the viral quasispecies and the impact of combinations of mutations on viral fitness, drug susceptibility and the genetic barrier to resistance. To date, these questions have escaped formal investigation owing to the technological challenges in obtaining whole-genome HCV sequences. A complete evaluation of prospective RAV characterization in guiding therapeutic options requires a comprehensive method for high-sensitivity variant detection, for which the development of efficient, unbiased, and cost-effective whole-genome sequencing methods seems a key requirement. Recent advances in genotype-agnostic whole-genome sequencing of HCV have been promising9, but there is still room for improvement in sensitivity, throughput and cost.\n\nHCV strains fall into seven recognized genotypes which differ from each other at an average of 30–35% of nucleotide sites across the ~9650 nt genome10, which is divided into highly conserved and extremely diverse regions of sequence. Genotypes are classified into approximately 67 subtypes, which differ at up to approximately 15% of nucleotide sites and include the globally distributed subtypes 1a, 1b, 2a, and 3a10. Available methods for the characterization of genetically diverse viruses such as HCV in clinical samples present several technical challenges. Amplification of reverse-transcribed virus RNA by PCR relies on a close match between primers and relatively conserved regions of the target, including an absolute match at the 3’ end of each primer, necessitating the design of multiple, genotype-specific sets of overlapping amplicons to recover complete genome sequences. In practical terms, PCR-based whole genome sequencing for HCV is complex and prone to technical failure, requiring a genotyping stage for primer selection, followed by genotype-specific amplification of several fragments and sequencing11,12, typically using a next-generation platform such as Illumina. The results can include high-depth coverage of the identified genotype, useful for the identification of known drug-related and immune escape variants, but the technique is less appropriate for the detection of low-frequency co-infections, uncovering novel diversity, or high-throughput analysis.\n\nAn alternative approach, and the starting point of this research, is a method termed virus RNA-seq13, which efficiently obtains direct “metagenomic” sequence data in the form of Illumina sequence reads from clinical material such as plasma14 and which we used recently to identify a genotype 4 – genotype 1 chimeric isolate from a patient in Cameroon15. Virus RNA-seq is demonstrably unbiased with respect to the detection of any virus genotype, but relatively insensitive and costly for the recovery of whole virus genomes, even with modern sequencing technologies, because in many cases >99% of all sequence data generated derives from the host and is discarded9,13.\n\nStrategies to deplete host-derived nucleic acids in virus metagenomic whole-genome sequencing have been applied successfully but are intrinsically limited in their effectiveness by the often-variable characteristics of the input sample. Using DNAase digestion of plasma before reverse transcription-based RNA amplification and a modified low-input Illumina library preparation, HCV-specific read proportions of 1.5%–47.7% have been reported9, for samples with relatively high VLs (>1.8 105 IU/ml), sequenced in small multiplexes of eight samples per Illumina MiSeq run. Oligonucleotide-targeted RNAse H digestion of host rRNA has been used to improve the yield of Lassa and Ebola virus sequences but virus-specific sequencing efficiency remains close to 1%16. More promisingly, enrichment using biotinylated probes that target viral sequences has significantly improved sensitivity and efficiency of herpesvirus17, Lassa virus16 and Mycobacteria tuberculosis18 sequencing.\n\nThe ideal methodology for one-step, high-throughput clinical virus sequencing would combine the benefits of high-throughput sequencing with the sensitivity of PCR, while avoiding the pitfalls of PCR-based amplification and the inefficiencies of RNA-seq based metagenomic approaches. We report a comprehensive approach to virus-specific, genotype-agnostic, probe-based enrichment and sequencing of whole HCV genomes at a depth sufficient to call minor variants without bias and at a cost compatible with routine clinical HCV genotyping, that in principle can also be applied to other pathogens.\n\n\nMaterials and methods\n\nSamples for optimization of sequencing methods were acquired from HCV Research UK (http://www.hcvresearchuk.org/), whose clinical samples were used with informed consent, conforming to the ethical guidelines of the 1975 Declaration of Helsinki. Study protocols were approved by the NRES Committee East Midlands, Derby (Ethics reference 11/EM/0323). Samples for resistance testing were obtained from patients enrolled and consented as part of the OxBRC Prospective Cohort Study in Hepatitis C (Ethics reference 09/H0604/20) at the Oxford University Hospitals NHS Trust.\n\nPatient plasma was collected from EDTA blood tubes by centrifugation for 10 minutes at 600g in a Heraeus Megafuge, and stored at -80°C. RNA was isolated from 500µl plasma volumes using the NucliSENS magnetic extraction system (bioMerieux) and collected in 30µl of kit elution buffer for storage in aliquots at -80°C.\n\nLibraries were prepared for Illumina sequencing using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (New England Biolabs) with 5µl sample (maximum 10ng total RNA) and previously published modifications of the manufacturer’s guidelines (v2.0)13, briefly: fragmentation for 5 or 12 minutes at 94°C, omission of Actinomycin D at first-strand reverse transcription, library amplification for 15–18 PCR cycles using custom indexed primers19 and post-PCR clean-up with 0.85× volume Ampure XP (Beckman Coulter).\n\nLibraries were quantified using Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen) and analysed using Agilent TapeStation with D1K High Sensitivity kit (Agilent) for equimolar pooling, then re-normalized by qPCR using the KAPA SYBR® FAST qPCR Kit (Kapa Biosystems) for sequencing. Metagenomic virus RNA-Seq libraries were sequenced with 100b paired-end reads on the Illumina HiSeq 2500 with v3 Rapid chemistry.\n\nA 500ng aliquot of the pooled library was enriched using the xGen® Lockdown® protocol from IDT (Rapid Protocol for DNA Probe Hybridization and Target Capture Using an Illumina TruSeq® or Ion Torrent® Library (v1.0), Integrated DNA Technologies) with equimolar-pooled 120nt DNA oligonucleotide probes (IDT) followed by a 12-cycle, modified, on-bead, post-enrichment PCR re-amplification. The cleaned post-enrichment ve-Seq library was normalized with the aid of qPCR and sequenced with 100b paired-end reads on a single run of the Illumina MiSeq using v2 chemistry.\n\nDe-multiplexed sequence read-pairs were trimmed of low-quality bases using QUASR v7.0120 and adapter sequences with CutAdapt version 1.7.121 and subsequently discarded if either read had less than 50b remaining sequence or if both reads matched the human reference sequence using Bowtie version 2.2.422. The remaining read pool was screened against a BLASTn database containing all 165 ICTV (International Committee on the Taxonomy of Viruses) HCV genomes (http://talk.ictvonline.org/ictv_wikis/m/files_flavi/default.aspx) both to choose an appropriate reference and to select those reads which formed a majority population for de novo assembly with Vicuna v1.323 and finishing with V-FAT v1.0 (http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/v-fat). Reads were mapped back to the assembly using Mosaik v2.2.2824, variants were called by V-Phaser v2.025 and intra-host diversity was explored with V-Profiler v1.026.\n\n\nResults\n\nWe first evaluated the performance of a conventional, “metagenomic” approach to virus whole-genome sequencing13. Indexed sequencing libraries were constructed in duplicate from plasma RNA of 29 subjects infected with diverse HCV subtypes (1a, 1b, 2a, 2b, 3a, 4a and 4d) and a 3.5-log range of VLs (2,200–4.9 million IU/mL; 1 IU = 2.7 copies on the instrument we use) and sequenced on a single Illumina HiSeq 2500 Rapid run, producing a median of 8.0 million reads per sample (range 6.0–24.9 million), of which 0.37% originated from HCV (range 0.03%–2.8%) (Supplementary Table S1). There was a linear relationship between HCV VL and the yield of HCV reads with high mapping quality (Figure 1). Mapping the HCV reads for each sample to the closest available reference (either a database reference or a de novo assembly of the same reads) produced patterns of peaks and troughs in sequence coverage along the genome that showed some similarity between samples of different subtypes and were highly reproducible between library and sequencing technical replicates; we therefore infer patterns of coverage are caused mainly by genomic features such as secondary structure and melting temperature27.\n\nThe yield of reads that map to any HCV genome and the probability of successful de novo assembly of a complete genome sequence both depend on viral load (VL). Samples were prepared as replicate libraries that were sequenced simultaneously with consistent yield. Blue circles: successful de novo assembly (>90% complete genome length recovered); red circles: incomplete genome assembly. a. With standard mapping criteria, up to 2.8% of reads match HCV and a background 0.02–0.1% of low-complexity human-derived sequences overwhelms the HCV signal in low-VL samples. Linear trend is plotted for samples with VL > 105 IU/ml. b. Under stringent mapping criteria (mapping Q > 40), lower complexity human and HCV reads are excluded and yield is proportional to VL (slope of linear trend in log-log space not significantly different from 1) across the VL range.\n\nIn its standard form, metagenomic sequencing of a batch of up to 96 samples costs <£100 per sample. In this experiment, a VL of approximately 2×105 IU/mL was sufficient to attain a mean read depth across the genome of ~30 and a high probability of successful de novo assembly, but higher read depths are necessary for precise characterization of minor variants. Results are better with high-VL samples, and measures to increase library complexity and improve release of virus during RNA isolation may improve variant-calling sensitivity, but the low efficiency of metagenomic sequencing poses a fundamental problem.\n\nWhen the sequence of interest comprises only a small fraction of the starting material, probe-based sequence capture, as used in exome sequencing, can dramatically increase sequencing efficiency17,28. Anticipating the challenge posed by the extreme diversity of HCV, we drew on a representative genome sequence from each of four common genotypes (1a, 2b, 3a and 4a) to construct a combined panel of biotinylated DNA oligonucleotides (xGen® Lockdown® probes, IDT) comprising four sets of 155–157 probes, each a 120 nt sequence fragment overlapping the next by 60 nt, and excluding the 3’ poly-(U) tract to avoid enrichment of low-complexity non-HCV sequences.\n\nWe enriched the previously-sequenced pool of libraries for HCV sequences by solution hybridization with the 4-genotype probe panel and sequenced it on the Illumina MiSeq platform. This yielded a greater-than 16 × increase in the total number of HCV reads produced, even with an output of ~14 × fewer reads than the previous metagenomic sequencing on the higher-output HiSeq (Supplementary Table S1). HCV sequence content reached 86% in the enriched pool (range 1–98% among samples), equivalent to a median 1,660 (range 10–75,700) genomic average read depth or >103-fold enrichment for samples with mid-range VL (Supplementary Figure S1); and hit saturation point (near-100% HCV reads) for samples with higher starting HCV content. Although probe panels can be expensive to synthesize, they can be used for many (hundreds of) pooled captures, so the lower sequencing costs in ve-SEQ more than account for the extra costs of the enrichment step.\n\nWe used a single-genome, subtype 1a subset of the 4-genotype probe panel to investigate the effect of varying probe-target sequence identity on ve-SEQ enrichment success (Figure 2). When a sample is enriched with probes derived from that sample’s consensus sequence, there is no detectable bias in read depth with genomic position (i.e. coverage across the genome for enriched data follows a pattern almost identical to unenriched data, albeit at much higher read depth). When a non-identical sample of the same subtype is enriched, coverage patterns coincide, but are not identical. When a sample from the same genotype but a different subtype to the probe panel is enriched, large sections of the genome are adequately sequenced, but the most divergent regions are covered poorly and whole-genome assembly fails for samples with low viral load (Supplementary Table S1). When the sample and the enrichment probe set come from different genotypes, only the most conserved parts of the genome are adequately represented with ve-SEQ data and read depth is essentially zero for divergent regions.\n\nRead depth across the genome before (blue, left axis) and after (red, right axis) enrichment with a single-sequence subtype 1a probe set. a. The HCV genome comprises 5’ and 3’ untranslated regions (UTRs) and a large central segment encoding a single polyprotein that is cleaved into ten proteins. b. A subtype 1a sample enriched with probes derived from its own consensus sequence yields coverage patterns across the genome essentially identical to metagenomic sequencing. c. A distinct subtype 1a sample produces highly similar but non-identical patterns of pre- and post-enrichment genomic coverage. d. A subtype 1b sample yields low read depths at loci that are relatively divergent from the 1a probe sequence (E1, E2, NS2 and NS5a). e. Sequence capture of a sample from a different genotype, 3a, is poor across large segments of the genome. f. Heat map representing average diversity (calculated as Shannon entropy) among 165 HCV reference genomes. Nucleotide diversity varies dramatically across the genome and tracks drops in enrichment efficiency between phylogenetically distinct probe-target combinations.\n\nIn order to rationalize our approach to probe choice and enable the design of an efficient, comprehensive HCV enrichment probe set, we analysed the relationship between probe-target similarity and the relative efficiency of enrichment (Figure 3). Noting a strong inflection point, we deduced that a minimum 80% identity between a 120 nt segment of sample sequence and its closest matching probe was sufficient to ensure near-maximal enrichment, assuming that each sequencing library molecule interacted with a single probe molecule and ignoring the potential effects of bridging capture (i.e. successful sequencing of a poorly matching fragment effected by hybridization of an adjacent target sequence on the same library molecule to a better-matching probe). The 20% divergence cutoff for successful enrichment falls between the mean inter-subtype (<15%) and inter-genotype (30–35%) divergence levels, explaining why enrichment with a subtype-mismatched probe set leads to only localized bias, while genotype-mismatch results in failure across most of the genome. It also follows from this analysis that when enrichment is performing well, there should be no detectable bias in the representation of single nucleotide variant alleles such as RAVs.\n\nA set of 10 HCV samples with highest VL was sequenced before and after enrichment with a single-genome, subtype 1a probe set, and for each sample the relative read depth for each probe window was plotted against the maximum identity between target and any probe. Read depth ratio was normalized by giving the most efficiently enriched probe position (in the highly conserved 5’ UTR) a value of 1. Maximal enrichment is observed where probe-target identity exceeds approximately 80% and enrichment decreases dramatically as identity falls below 80%.\n\nAs is evident from the previous section, a probe panel based on just four subtype-representative sequences cannot perfectly capture HCV global diversity. Exploiting the observation that some regions of the HCV genome (e.g. the 5’UTR) are well-enough conserved to not require multiple probe sets, together with the 20% divergence cutoff for efficient capture, we implemented an algorithm for efficient probe set design that would facilitate a comprehensive HCV enrichment panel as well as, in principle, efficient probe sets for other organisms.\n\nWe started with the 4-genotype probe panel and added extra probes to improve coverage for already-included subtypes 1a, 2b, 3a and 4a as well as the extra subtypes 1b, 2a, 2c, 5a and 6a, using a database of 482 reference whole-genome sequences. First we calculated a consensus sequence for each subtype. Then, starting with the existing probe set and the first genome in the most common subtype (1b), we identified genomic regions with less than 80% identity to any of the probes already in the panel. For each such region the subtype consensus sequence was considered as a potential probe but only used if it was ≥80% identical to the genomic sequence it replaced; otherwise the genomic sequence fragment was added as a new probe. The process was repeated for each 1b reference sequence and then similarly for each subtype.\n\nIn contrast to the naïve design of probe sets with the standard IDT approach that requires 155–157 probes per HCV target genome, we were able to augment our 4-genome probe panel to represent the known diversity of nine subtypes spanning six of the seven recognized genotypes with only another 491 probes (1,116 total). Our algorithm substantially and automatically reduces redundancy: a completely naïve approach that simply encoded every genome in the reference set, without accounting for similarity between genomes, would have dictated a prohibitively expensive set of ~75,000 probes. In contrast, if we had instead started from scratch, we estimate that our simple algorithm could have produced an equally effective combined panel for nine subtypes with as few as 955 probes. In informal testing, a typical sample from the newly added subtype 1b achieved near-zero bias even though its exact sequence was not encoded in the probe set but was instead covered by reference to recorded sequence diversity (Supplementary Figure S2), and a sample from subtype 4d, not included in the revised probe set, achieved adequate although imperfect enrichment (Supplementary Figure S3), consistent with previous subtype-mismatched captures. Although feasible and relatively inexpensive, we have deferred the addition of probes for remaining rare subtypes.\n\nTo explore the potential utility of high-depth RAV data in predicting the clinical effectiveness of HCV treatment, we used ve-SEQ to analyse retrospectively plasma samples collected from 33 genotype 1-infected patients before NS3-targeting DAA therapy with Boceprevir (14 patients) or Telaprevir (19 patients) (Supplementary Table S2). We obtained whole-genome sequences for all samples, with a mean read depth of 4600 across the NS3 gene. We first confirmed that our sequence data (28 subtype 1a and 5 subtype 1b) matched clinical subtyping data where the latter was available.\n\nMutations in the NS3 gene, denoted T54S and V55I, were detected in patient P23, in whom Boceprevir treatment failed to suppress HCV. Only one other patient had relevant baseline resistance: P6 possessed a single T54S mutation, yet cleared infection with 48 weeks of BCP. Additionally, Simeprevir RAVs Q80K/R were detected in five patients with genotype 1a virus, consistent with the reported prevalence of these mutations in PI-naïve patients29. Variants associated with NS5A inhibitor resistance were detected in 11 patients, including nine with combinations of two or more RAVs, previously associated with higher relapse rates than Lidipesvir/Sofosbuvir30.\n\nIn samples taken after treatment cessation, five patients carried both V36M and R155K NS3 variants, associated with drug resistance but also reduced virus fitness in the absence of treatment31,32, including three patients illustrated in Figure 4. RAVs V36M and R155K were each detected independently of the other (in P30 and P33, respectively) and virus sampled in P27 during treatment revealed approximately 2-fold more V36M variants than R155K, confirming that V36M alone was sufficient to confer resistance on individual genomes. Telaprevir had failed to suppress virus in subject P24 by week 4 when V36M and R155K variants circulated in approximately half of virus. It is therefore not surprising that a subsequent treatment attempt also failed, providing a real-world clinical example of where sequencing might have prevented futile retreatment. Six weeks after the second treatment attempt had failed, the R155K mutation had reverted to the wild-type arginine residue in all sequence reads. Partial reversion was also observed in P18, although in this instance, reversion of V36M occurred some 20 or more weeks after the cessation of treatment and R155K was still present in 100% of variants 1 year later.\n\nVL and RAV status for three patients who failed to achieve sustained virological response after Telaprevir-based therapy. Grey shading: duration of therapy (weeks starting at time 0); squares: VL measurements; inverted triangles: samples sequenced using the comprehensive probe panel (open: no Telaprevir RAVs detected, black: RAVs and supporting read proportions, where <100%).\n\n\nDiscussion\n\nOur ve-SEQ method provides improvements over other approaches currently used for rapid, high-throughput, high-sensitivity characterization of complete virus sequences from clinical samples. These advantages include sequencing efficiency for low-VL samples not available from metagenomic approaches9 and robustness to extreme sequence diversity such as that found in HCV that is not available from PCR-based methods8. Our approach is similar to published methods16–18,28 but benefits from low enrichment costs and defined performance that come from efficient probe design and non-proprietary, high throughput sample processing.\n\nIn this study, treatment-naïve individuals carried RAVs to NS3 and NS5A inhibitors and emerging resistance was shown to persist 1 year after treatment failure, which stands to complicate empiric selection and timing of HCV treatment, particularly in previously treated patients. Stratification by viral genotype is currently the best strategy for successful treatment; ve-SEQ performs as well as current routine subtyping techniques at comparable cost while additionally offering high-depth, high-throughput and unbiased detection of RAVs, enabling future large-scale evaluation of resistance testing in clinical studies and offering the possibility of replacing current practice with a single highly informative test. Our preliminary analyses reveal cases in which such data may be clinically useful, and the cost of the test compared with that of a failed DAA treatment (e.g. ~£40K for HARVONI®, https://www.nice.org.uk/guidance/gid-tag484) suggests potential for ve-SEQ to be cost-effective in a clinical setting.\n\nOur general approach also has clear application in the detection and sequencing in a single protocol of other pathogens – none is as diverse as HCV – including the potential for multi-pathogen, sub-genomic panels that might replace multiplex PCR-based screening and diagnostic techniques with more comprehensive, higher resolution data at comparable sensitivity33. ve-SEQ works at high-throughput scales, with a standard, plate-based format that makes it affordable and comparable in overall cost to less informative assays. To avoid turnaround delays while maintaining efficiency for routine use, in principle the HCV assay could be combined with assays for other pathogens, and plasma RNA-seq libraries could be pooled with RNA- and DNA-originating libraries from other sample types, for a routine test run on sequencing platforms like the Illumina MiSeq, that are becoming more generally available in large-hospital diagnostic labs. The more a pool of libraries is enriched, the more individual library complexity (broadly, the number of starting molecules of HCV included) becomes important: since the ve-SEQ approach can be used with any library methodology we have now turned our attention to ways of optimizing the yield of HCV in plasma RNA, increasing the amount of library input material and improving library efficiency.\n\nThe robustness of probe-based enrichment provides a practical alternative to PCR and similar amplification-based approaches that require a close match between primer and target. We envisage that enrichment could provide almost-hypothesis-free detection for all plausibly present pathogens in clinical samples, both for low-diversity target genomes in which a single representative probe set is sufficient, and by using algorithms such as the one we implement here to efficiently capture more diverse pathogens. Because less sequencing effort is required, the overall cost of an enrichment-based protocol is lower than that of a no-enrichment approach and achieves a greater yield of useful data, more efficiently and robustly than PCR.\n\n\nData availability\n\nSequence data, filtered to remove human reads, is available from the European Nucleotide Archive (ENA) under accession PRJEB9338.",
"appendix": "Author contributions\n\n\n\nDavid Bonsall contributed to study design, analysis and writing of the paper. Azim Ansari contributed to methods development, analysis and writing of the paper. Camilla Ip analysed data and assisted in writing the paper. Amy Trebes developed methodology and performed experimental work. Anthony Brown performed experimental work. Paul Klenerman advised on study design and writing of the paper. David Buck contributed to study design. Paolo Piazza developed experimental methodology and contributed to study design and data production, Eleanor Barnes co-directed the research and contributed to study design and writing of the paper, Rory Bowden co-directed the research and contributed to study design, analysis and writing of the paper. All authors have read and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe STOP-HCV consortium is funded by a grant from the Medical Research Council. This research was supported by Core funding to the Wellcome Trust Centre for Human Genetics provided by the Wellcome Trust (090532/Z/09/Z), AA is funded by the Oxford Martin School, PK is funded by the Oxford Martin School, NIHR Biomedical Research Centre, Oxford, by the Wellcome Trust (091663MA) and NIH (U19AI082630) and EB is funded by the MRC as an MRC Senior Clinical Fellow, and by the Oxford Martin School and NIHR Biomedical Research Centre, Oxford.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors wish to acknowledge the role of the HCV Research UK Biobank in collecting and making available samples and data used in this publication. The authors acknowledge the support of research nurses Denise O'Donnell, Elizabeth Stafford and Mark Ainsworth and technical advice from Nick Downey at Integrated DNA Technologies (IDT). Paolo Piazza, Eleanor Barnes & Rory Bowden are joint senior authors.\n\n\nSupplementary materials\n\nSupplementary Figure S1. Relationship between VL and HCV enrichment ratio.\n\nAliquots of the same library pool were sequenced before and after enrichment with a 4-genome (subtypes 1a, 2b, 3a and 4a) enrichment probe set and the ratio of post-enrichment to metagenomic HCV yield (% HCV-matching reads) was plotted against VL. Under stringent mapping criteria (mapping Q > 40) that exclude low-complexity human and HCV sequences, enrichment exceeds 103 × for low- to medium-VL samples. For high-VL samples where post-enrichment yield approaches saturation (100%), the achievable enrichment ratio decreases. For such samples, metagenomic sequencing yield is often around 1% in any case.\n\nClick here to access the data.\n\nSupplementary Figure S2. Enrichment of a 1b sample.\n\nRead depth across the genome before (blue, left axis) and after (red, right axis) enrichment of a subtype 1b sample with each of the three probesets.\n\na. 1a probeset: moderate coverage with genotype- but not subtype-specific probes.\n\nb. 1a, 2b, 3a and 4a pooled probeset: similar levels of enrichment to (a) with a probeset that includes single genomes of four genotypes but still does not include 1b.\n\nc. 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a and 6a pooled probeset: almost-unbiased enrichment with a probeset of nine subtypes, including optimized coverage of 1b but not an exact match between probes and the consensus sequence of this sample.\n\nClick here to access the data.\n\nSupplementary Figure S3. Enrichment of a 4d sample.\n\nRead depth across the genome before (blue, left axis) and after (red, right axis) enrichment of a subtype 4d sample with each of the three probesets.\n\na. 1a probeset: poor coverage with no genotype-specific probes\n\nb. 1a, 2b, 3a and 4a pooled probeset: reduced bias in enrichment with genotype- but not subtype-matching probes\n\nc. 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a and 6a pooled probeset: similar levels of enrichment to (b) with a probeset that includes nine subtypes but still does not include 4d.\n\nClick here to access the data.\n\nSupplementary tables S1 and S2.\n\nSupplementary tables S1: HCV sequencing statistics and S2: Pre- and post-treatment resistance detected by ve-SEQ\n\nClick here to access the data.\n\n\nReferences\n\nMohd Hanafiah K, Groeger J, Flaxman AD, et al.: Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology. 2013; 57(4): 1333–1342. 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[
{
"id": "10798",
"date": "26 Oct 2015",
"name": "Fabio Luciani",
"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 original research work the authors have developed a novel method for detection and sequencing of HCV genomes from clinical samples adopting a DNA probe approach.Similar methods have been developed previously for targeted genomes. This approach has been adapted for HCV and has great potential to be applied to other RNA viruses. Overall, in my opinion, this paper is an interesting and novel application which resolve few long standing issues with identification and sequencing of complex viral populations from clinical samples. It would be very helpful for the readers if the authors would address the comments below.It would be helpful to have more details on how the first set of probes were chosen. The authors state that these were 155-157 probes, each of length 120t, which roughly equate to 2 full HCV genomes. Can the authors describe what exactly was the algorithm to identify those fragments from the total genomes considered?It is clear that the second set for the rare GT were constructed with 80% dissimilarities from the first set. The critical message is that this approach seems to break the barrier of sequencing very low viral loads in an unbiased approach. I found this a very important result. It is however clear from the data that the attempt is not fully successful as only partial genomes are obtained. Maybe some more clear statements highlighting where we are up to with this method and what can be done to improve. I would recommend to have Supp Figure 1 in the main text as this is a rather interesting result showing that there is a better enrichment for low viral loads. I don't fully agree with the authors with the conclusions.This method is still not reliable in terms of detecting near-full length genome at low viral load, and therefore the classical PCR-primer genotype specific primers are needed.Rather, I would encourage the authors to discuss more the implication of such an approach (and improved ones into the future) for sequencing more complex scenarios, such as recombination, multiple infections, reinfections, superinfection etc. A comment that I hope will generate some feedbacksDAA treatment are much better than those considered in this manuscript. HARVONY and GS-5816 are breaking the barrier of 95% SVR pan genotype. This is the first time in history of antiviral therapy of such a limited drug resistance.I think the proposed method will have higher chances to be applied in other settings (as mentioned before to study complex genomic rearrangements)Maybe worth thinking about this. Finally, this work made me also wonder on what limitations still exist that this method does not assess. It would be interesting to mention that for understanding viral evolution, including drug resistance, there is need to identify compensatory mutations and epistatic interactions, which may occur between viral mutations that are far apart in the genome.This is a problem of haplotype reconstruction which has been proven to be very difficult to solve if the staring points are short reads. Thank you for the opportunity to comment on such a novel and interesting work.",
"responses": []
},
{
"id": "12385",
"date": "01 Apr 2016",
"name": "Nicholas J. Loman",
"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 potentially important protocol for sequencing viral genomes using an adapted nucleic acid bead capture method. The results are impressive and I think the technique is likely to be useful to many who are undertaking viral sequencing.The article is written well and I believe acceptable for indexing in its current version.I have a number of suggestions that may improve the utility of the article to readers:I would appreciate a longer description of the NEBNext protocol - I assume this is first strand cDNA synthesis using random hexamers, but this would be useful to spell out. The main innovation is the use of IDT xGen Lockdown protocol, but the protocol is not described in much detail. I would appreciate a flowchart or textual description of the protocol, because I would like to know how long it takes, what steps are involved and what equipment is needed to carry it out.The costs of the per-sample sequencing is given, I would like to see this broken down by component. I have a major issue with Figure 2 as presented, due to the use of multiple Y-axes which I think makes it very very hard to interpret. Please split this out into panels with enrichment and unenriched data presented separately. Also please decide on consistent use of scientific notation or regular numbers (I prefer the latter). In fact rather than reporting read depth it would be more informative to report as a fraction of the number of reads from that barcode. I'm afraid I can't get on with the \"ve-Seq\" name, because I read it like \"negative-Seq\" each time! The authors might consider a more informative and easier to communicate name. The authors may consider citing some of the recently published pan-viral capture papers such as VirCAP-Seq 1 and relating ve-Seq to this technique.",
"responses": []
},
{
"id": "12905",
"date": "18 Apr 2016",
"name": "Tanya L Applegate",
"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\nBonsall et al. describe an improved metagenomic approach for sequencing HCV, which is adequately described by the title “-ve-SEQ: Robust,unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly divers pathogens”. The article provides proof-of principle data to the detection of known HCV associated resistance associated variants in a small number of subjects across a range of genotypes. This is a valuable addition to the limited repertoire of sequencing methods available for full HCV genome sequencing.The article is clearly written, the abstract provides an accurate summary of the article and the overall conclusions are justified on the basis of the results. However, considering this manuscript describes a method, the paper does need more detail regarding the description of methods (including additional information outlining the probe design) and results would allow the reader to reproduce the data, draw their own conclusion and add value to this paper. Additional specific comments are listed below for the authors consideration. Introduction:First paragraph – the authors might wish to consider adding ‘reinfection’ as major challenge to controlling the HCV epidemic. Many of the at-risk populations become re-infected with HCV which will limit long-term DAA success and also necessitate good robust sequencing techniques that distinguish reinfection from relapse. 4th paragraph – the authors may wish to consider the recent publication by Bull et al. BMC Genomics 2016 in this discussion. It offers a slight improvement to some of the other amplification based methods, including detection of co-infection, but as correctly stated but the authors is still, as are all targeted based amplification methods, biased by primer design. Typo in the following statement “with relatively high VLs (>1.8 105 IU/ml) ” need an ‘x’.Methods:Given that this is a methods paper, the methods lack sufficient detail and the probe design is vague. For example, how was the reference sequence for each genotype selected? Is this a prototype strain, in which case genbank ID should be provided or a constructed genotype consensus sequence? I assume that each set in the 4 sets of probes represents a specific genotype. Samples selection: Could the authors provide more detail on the criteria by which a \"representative\" 1a sequence was selected? The authors show a single comparison of how the 1a probe set compares with the other subtypes / genotypes but it is hard to estimate how this might perform in the real world. The authors mention \"informal testing of a typical sample\", but more information is required to the support claims in the paper. Limit of detection: The authors should define the level of threshold called as “no resistance”. What was the lowest percentage threshold considered reliable to call variants? What is the minimum viral load at which this quantification is reliable?Results:Sequence success: Could the authors please clarify that only 29 samples were tested and all 29 were successfully sequenced. Cost breakdown: In regards to the statement “n its standard form, metagenomic sequencing of a batch of up to 96 samples costs <£100 per sample.” As the reduced cost re. rationale probe design is discussed as one of the main advantages of this method, could the authors please clarify exactly what is included in the ‘standard metagenomics cost’ and provide a disaggregation of the costs. Ie., is it just the cost of sequencing 96 samples on the HiSeq or did they also include library prep costs in that cost estimate. Is there an estimate of the number of samples that would be required to make this a cost-effective compared in comparison to bulk sequencing the NS3-NS5B regions? Figure 2 needs a key to describe the heat map (does Yellow = higher entropy) and more detailed description of how genetically\" distinct\" genotype 1a is in panel c. Probe design: Did the authors in their probe design consider or attempt to target the relatively conserved sequence after the poly U/C tract at the 3’ end of the genome? Perhaps it is too short or lack of reliable sequence for probe design? For while the 3’UTR is unlikely to be of interest for RAV analysis it has been proposed to be important in viral pathogenesis and induction of innate immunity and the exclusion in obtaining 3’UTR sequence in this method does present a small limitation for a subset of viral diversity studies. The authors mention \"higher read depths\" are required for precise characterisation of minor variants, could the authors describe the minimum number of reads that would be required for this? The authors refer to a database from which they got their 482 reference whole-genome sequences from which they designed their probes? What is this database and is it publicly available? In supplementary table 1 what does probe panel “G123456” mean? According to methods probes were only designed on Gt 1,2,3 and 4….. Ok now that I have continued reading the results I now understand as the design of G123456 probes is described in the results on page 6. I suggest that a section outlining the expanded probe design be added to the methods section so that there is not confusion in understanding the tables. This expanded probe design offers much more potential than the original probe design and needs highlighting. Figure 4: - Figure 4 labeling and description in the text needs to be improved to allow the reader to follow which subject is being described. Figure 4a: was reinfection with a different variant ruled out? For example what was the genetic distance of the consensus variants between pre-treatment and relapse? What percentage where the RAVs present after treatment? The authors indicate NS5A variants were found, but don't provide any more data (what RAVS, what level they were found). Sup figures 2 and 3: results look great but it would help for comparison of the different probe panels if the right y-axis was put on the same scale, as has been done for the left y-axis. This is unlikely to be possible with figure 2 as there is an order of magnitude difference between the plots. For the RAV analysis which probe set was used?Discussion:First sentence – the reviewers agree that the method is a valuable improvement in comparison to other metagenomic approaches but it still does have some limitations (and some advantages as already discussed in intro) when compared to amplicon approaches and these should be acknowledged and discussed. Specifically, sequencing samples with low viral loads, and the detection of low frequency RAVs is currently more sensitive with targeted amplicon approaches. Sensitivity: There is a potential sensitivity issue that has not been addressed with this assay in regards to RAVs as mentioned in the results section. Unless the authors add data to show high sensitivity then this should be discussed in paragraph 2 of the discussion. Minor point, Paragraph 3 – “none is as diverse as HCV” – I would change this to “few are as diverse as HCV”. It is debatable depending on your classification but the Enteroviruses are an extremely diverse group…",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1062
|
https://f1000research.com/articles/4-1050/v1
|
12 Oct 15
|
{
"type": "Review",
"title": "Biomarkers and recent advances in the management and therapy of sickle cell disease",
"authors": [
"Marilyn J. Telen"
],
"abstract": "Although production of hemoglobin S, the genetic defect that causes sickle cell disease (SCD), directly affects only red blood cells, the manifestations of SCD are pervasive, and almost every cell type and organ system in the body can be involved. Today, the vast majority of patients with SCD who receive modern health care reach adulthood thanks to vaccine prophylaxis and improvements in supportive care, including transfusion. However, once patients reach adulthood, they commonly experience recurrent painful vaso-occlusive crises and frequently have widespread end-organ damage and severely shortened life expectancies. Over the last several decades, research has elucidated many of the mechanisms whereby abnormal red blood cells produce such ubiquitous organ damage. With these discoveries have come new ways to measure disease activity. In addition, new pharmaceutical interventions are now being developed to address what has been learned about disease mechanisms.",
"keywords": [
"sickle cell",
"anemia",
"biomarkers",
"red blood cells"
],
"content": "Introduction and context\n\nAlthough it has long been straightforward to define sickle cell disease (SCD) and its subtypes through biochemical and genetic analyses of hemoglobin and its encoding genes, understanding the pathophysiologic mechanisms leading to the disease’s protean manifestations has been more challenging. After decades of research, a great deal has been learned about the many pathways and processes affected downstream by the hemoglobin S (HbS) mutation, and, finally, new therapeutic approaches targeting these mechanisms are being developed. Nevertheless, a lack of even a basic ability to document and follow the processes leading to vaso-occlusive episodes persists. At this time, both the diagnosis of vaso-occlusion and the definition of its resolution rely exclusively on patient reports of pain.\n\nThe difficulty in obtaining objectively measurable biomarkers of disease activity and variability hampers many aspects of the management of SCD, including (1) diagnosis of acute vaso-occlusion, acute chest syndrome, and other sequelae of SCD; (2) understanding of the inciting events leading to vaso-occlusion and its complications in SCD; (3) personalized medicine through identification of particularly suitable or unsuitable candidates or events for targeted disease therapies; and (4) documentation of new drug effects and mechanisms of action.\n\nDespite the fact that the abnormal hemoglobin that defines SCD is expressed only in erythrocytes, all blood cells as well as soluble blood elements and most other organ systems are affected by SCD. Patients with SCD have, at baseline, elevated leukocyte and platelet counts, abnormally increased leukocyte and platelet activation, abnormal activation of coagulation pathways, increased expression of multiple inflammatory markers, increased expression of soluble markers of endothelial activation and injury, and increased markers associated with a broad range of end-organ damage.\n\nMany such “biomarkers” have been studied either in “steady-state” SCD or during acute vaso-occlusive episodes, and some have been shown to correlate with long-term survival. In addition, because vaso-occlusion may arise from a variety of inciting events (e.g., infection and physiologic stress), the mechanisms of vaso-occlusion may be variable among patients and vaso-occlusive events. Identification of biomarkers specific for processes contributing to vaso-occlusion could help determine which drugs might be most beneficial and might also elucidate the mechanism of action of new therapeutic agents. Biomarkers might also help determine when new therapeutic agents might be useful as prophylactic therapy to prevent progressive end-organ damage.\n\nThe past few decades, during which both mechanisms of vaso-occlusion and biomarkers have been identified, have also brought the dawn of targeted therapies for vaso-occlusion. Although none has yet to be proven useful or become US Food and Drug Administration (FDA)-approved since approval of hydroxyurea (HU) for the prevention of vaso-occlusion and acute chest syndrome, several promising targeted agents are in various stages of clinical trials. Many such studies are also examining biomarkers, as they are affected by both vaso-occlusion and the new potential therapeutic agents. Thus, it is reasonable to expect that progress in therapeutics and in the discovery of useful biomarkers will go hand-in-hand in the future, leading potentially to both targeted and personalized therapy for SCD.\n\n\nPathophysiology and biomarkers\n\nEarly studies of the natural history of SCD, largely in children and young adults, identified several markers of disease severity and poorer overall survival1,2. Frequency of vaso-occlusive episodes was a marker of poorer survival in patients with sickle cell anemia (homozygous for HbS) who were more than 20 years old2. High rates of vaso-occlusive episodes were also associated with higher hematocrit and lower fetal hemoglobin levels in that study. A second study of largely the same patient population further revealed that acute chest syndrome, renal failure, seizures, a high baseline white cell count (>15,000 cells/mm2), and low fetal hemoglobin were associated with an increased risk of early death; early mortality was again shown to be more frequent among the most symptomatic patients1. Finally, a more recent analysis of the cohort enrolled as newborns in the same study showed that more pronounced reticulocytosis increased the risk of stroke and mortality during childhood3.\n\nMore recent studies of factors associated with mortality in SCD have presumably reflected the era of HU therapy, and some have studied factors related to survival in different resource settings. Recent studies4–6 of adults have shown that more frequent episodes of vaso-occlusion remain a marker of increased mortality4,5, and sickle nephropathy is also a significant risk factor4–6. In addition, the presence of an elevated tricuspid regurgitant jet velocity (TRV) (≥2.5 m/sec), with or without catheterization-proven pulmonary arterial hypertension, is a very significant risk factor for accelerated mortality4–8. A history of cumulative end-organ damage and stroke was also associated with earlier mortality5.\n\nInterest in pulmonary hypertension and SCD has also spurred investigation of markers thought to be related to endothelial damage and inflammation. Of these, vascular cell adhesion molecule 1 (VCAM-1) levels have been reproducibly associated with survival5,9.\n\nHemoglobin F (HbF) levels are often measured clinically, and several studies have supported the hypothesis that higher HbF levels lead to less severe SCD. HbF has higher oxygen affinity than HbA or HbS, and sickle red cells containing more HbF survive longer in the circulation10. Moreover, elevation of HbF is a key (though not the only) factor in the salutary effects of HU on SCD severity11.\n\nSickle red cells are abnormally adherent to many substrates, including endothelial cells12, leukocytes13, platelets14, and extracellular matrix molecules such as laminin13,15 and thrombospondin16,17. Hebbel and colleagues found evidence decades ago that patients with more adherent cells were more likely to suffer vaso-occlusive episodes18. However, while such adhesion almost certainly contributes to vaso-occlusive pathogenesis, measuring adhesion either ex vivo or in vivo remains difficult. Thus, assays of cell adhesion are often used for research purposes but have not been extensively explored as markers of disease.\n\nNevertheless, one measurable outcome of adhesion is the formation of circulating heterocellular aggregates that can be measured by flow cytometry, now a part of most clinical hospital laboratories. Sickle red cells, as well as leukocytes from patients with SCD, can be found in circulating aggregates involving both each other as well as platelets14,19. How such measures related to clinical status and outcomes in SCD remains to be better defined.\n\nSCD is accompanied by a broad array of inflammatory processes. At steady state, in the absence of symptomatic vaso-occlusion, patients with SCD have increased numbers of activated leukocytes20, activated platelets, and formation of multicellular aggregates.\n\nIn addition, patients with SCD may have elevations of multiple inflammatory cytokines (Table 1), both in steady state as well as during vaso-occlusive events. Although not all studies demonstrate concordant findings, among the cytokines consistently found to be both elevated at steady state and then further elevated during vaso-occlusive events are interleukin-10 (IL-10), macrophage inflammatory protein 1α (MIP-1α), placenta growth factor (PlGF), prostaglandin E2 (PGE2), and soluble CD40 ligand (sCD40L). Current investigations are focusing on how these cytokines can contribute to the pathophysiology of vaso-occlusion.\n\nCoagulation pathways are broadly activated in patients with SCD21. Thus, SCD is considered a “hypercoagulable state”, and indeed there is a higher prevalence of pregnancy-related thrombosis and pulmonary emboli in patients with SCD than in age-matched African-American controls22,23. Clinically, levels of D-dimer are often chronically elevated and increase further during vaso-occlusive events24,25. Other biomarkers of activated coagulation, such as plasma levels of prothrombin fragment 1.2 (F1.2), thrombin-antithrombin (TAT) complexes, plasmin-antiplasmin complexes, and fibrinopeptide A, are also elevated in SCD. There is at least some evidence that the degree of elevation of D-dimer levels is predictive of the frequency of vaso-occlusive episodes26. Furthermore, the hypothesis that abnormal SCD red cells, and specifically those with increased phosphatidylserine (PS) exposure at their surfaces, are responsible for activation of coagulation is supported by the demonstration that the number of PS-positive sickle red cells is related to the degree of elevation of D-dimer, F1.2, and plasmin-antiplasmin complex levels27,28.\n\nInvestigators have also shown that there are elevated levels of tissue factor in the circulation in SCD29–31 and that platelets are also activated in greater numbers32,33. Blood from patients with SCD also contains increased levels of microparticles derived from multiple cell types, including red cells, platelets, leukocytes, and endothelial cells. Tissue factor-expressing microparticles appear to be derived primarily from monocytes and endothelial cells34. Finally, recent evidence suggests that free plasma iron may also contribute to activated coagulation in SCD35.\n\nOxidant damage appears to occur at an accelerated rate in SCD, both within the red cell as well as in other tissues. Hemolysis results in the release of hemoglobin, which itself is a powerful oxidant. In addition, several investigators have reported reduced anti-oxidant compounds both within sickle red cells as well as in plasma. Plasma lipid peroxidation is higher in SCD patients than controls, and red cell content of glutathione reductase and superoxide dismutase is lower in sickle erythrocytes36. Depletion of glutathione has been associated with elevated TRV, itself a biomarker for pulmonary hypertension and early mortality37. Finally, some evidence suggests that reduction in expression of genes responsible for the synthesis of anti-oxidant compounds may also contribute to worsening anemia in SCD38. HU has been found to reduce markers of oxidative stress in SCD39,40.\n\nStroke is a common and potentially devastating problem in SCD, and strokes start to occur in very young children41. Once a stroke occurs, patients are at great risk for recurrent strokes, which can be largely prevented by chronic transfusion but have a high frequency of recurrence without continuing transfusion42. Transcranial Doppler (TCD) measurements have been known to provide a good measurement of the risk of stroke in children with SCD since 1992, when children with abnormal TCDs were shown to be at 44 times the risk for stroke than children with normal TCDs43. The Stroke Prevention Trial in Sickle Cell Anemia (STOP) showed that regular transfusion could reduce the risk of a first stroke by 92%, compared with non-transfused children with abnormal TCDs, who had about a 10% incidence of stroke annually44. However, a subsequent study of children who had already undergone at least 30 months of transfusion for abnormal TCDs failed to show that such transfusion could be safely stopped45. Thus, while TCDs are now standard of practice in pediatric care of SCD, in order to identify children at risk for primary strokes, they do not allow avoidance of transfusion for the relatively large number of children who would not ultimately have a stroke, despite their abnormal TCDs. Efforts to better define the at-risk population, such as through identification of genetic risk factors or other biomarkers, have not yet defined a solution to this problem.\n\nAcute chest syndrome (ACS) is one of the most feared complications of vaso-occlusive episodes and is highly associated with mortality. Secretory phospholipase A(2) (sPLA(2)) levels become quite elevated in about 80% of patients with ACS46,47 and often become quite elevated shortly before patients become symptomatic47,48. Thrombospondin-1 levels have also been reported to become markedly increased in ACS49, as have levels of pentraxin-350. C-reactive protein has been reported to parallel sPLA(2) in the context of vaso-occlusion and ACS51. At least one study has attempted to prevent ACS with transfusion in patients with high sPLA(2) levels52, but a definitive study has not been conducted.\n\nPulmonary hypertension has become recognized as a major risk factor for death in adults with HbSS and HbSβ0 thalassemia8. However, the most widely available screening test for pulmonary hypertension—echocardiography—does not reliably reflect pulmonary arterial pressures, as measured by right heart catheterization. In fact, right heart catheterization may confirm pulmonary hypertension in only 10% to 25% of patients with elevated TRV53,54, giving echocardiography a positive predictive value of only 25% to 32%55. Nevertheless, a TRV of over 2.5 to 3 m/sec has been an indicator of risk for mortality in several studies6,7,56 despite its lack of reliability as an indicator of catheterization-measurable pulmonary arterial hypertension6. Thus, the recent National Institutes of Health guidelines for sickle cell anemia management did not recommend for or against routine echocardiographic screening for pulmonary hypertension in asymptomatic adults with SCD57. In addition, several studies have shown that elevated pro-brain natriuretic peptide (pro-BNP) is associated with a high risk of mortality, especially in combination with a TRV of at least 3 m/sec6. VCAM-1, a marker of endothelial activation and damage, is also present at higher levels in SCD patients with pulmonary hypertension58. VCAM-1 is also reproducibly associated with poorer survival5,9.\n\nAlthough the biomarkers discussed above do not represent the totality of biomarkers explored and found to be possibly informative in the context of SCD, they do point to the panoply of pathophysiologic mechanisms now appreciated as active in SCD. Adhesion, inflammation, coagulation, and oxidative damage are likely the most important, though not the only, contributors to the development of vaso-occlusion and organ damage in this disease. Moreover, we do not completely understand how the processes implicated by the evidence of biomarkers produce the varied physiologic events we observe. For example, SCD involves both large-vessel events (e.g., strokes) as well as occlusion of microvascular structures, which is believed to be involved in typical painful vaso-occlusive events. Nevertheless, current efforts to develop new therapies for SCD are concentrating on the processes reflected by the biomarkers discussed above. In the future, biomarkers may assist us in personalizing treatment according to the predominant mechanisms involved in a particular disease sequela or event.\n\n\nNew and targeted drugs in development\n\nSince SCD was recognized and defined in the early 20th century, survival and quality of life have until recently improved primarily because of advances in supportive care, including penicillin prophylaxis, routine immunizations, transfusion for stroke and acute chest syndrome, and hydration and narcotic therapy for vaso-occlusive episodes. This changed after the Multicenter Study of Hydroxyurea59, which showed that HU reduced the frequency of both vaso-occlusive episodes and acute chest syndrome while increasing both total hemoglobin levels and hemoglobin F percentages and decreasing neutrophil counts. However, the study was not designed to, and did not, determine the mechanism whereby HU reduced vaso-occlusive and acute chest syndrome events. In this setting, the FDA approved the drug for use in adults with SCD in 1998. Subsequent studies have confirmed that the drug is cost-effective60 and improves long-term survival61–63. The drug has also proven to be safe and effective in children64,65. Nevertheless, the consensus remains that the drug is underutilized in both children and adults (http://consensus.nih.gov/2008/sicklecellstatement.htm). One recent study suggested that, outside a center dedicated to treating SCD, only about 25% of patients meeting the original study criteria actually receive HU66. Moreover, the mechanisms whereby HU has its beneficial effects remain only partly understood.\n\nNot only have new therapies for SCD not arrived since HU gained FDA approval in 1998, but truly curative therapies have been difficult to achieve (Table 2). Hematopoietic stem cell transplantation and gene therapy offer the best chances for cure but each presents numerous challenges that have been difficult to overcome. Although there were initial successes in young pediatric patients, successful hematopoietic stem cell transplantation with good survival and tolerable graft-versus-host disease in adults proved much harder to achieve. Nevertheless, such transplants can now be confidently undertaken67–69, and many ongoing clinical trials are looking at ways to improve both engraftment and the availability of donors.\n\nCO, carbon monoxide; Hb, hemoglobin; HbF, hemoglobin F; HbS, hemoglobin S; HU, hydroxyurea; iNKT, invariant natural killer T; NO, nitric oxide; SCD, sickle cell disease; ULVWF, ultra-large von Willebrand factor; VOC, vaso-occlusive crisis; VWF, von Willebrand factor.\n\nGene therapy is an even more challenging but ultimately less toxic approach to achieving curative therapy for SCD. The general approach has been to develop methods for inserting into autologous hematopoietic stem cells either a gene encoding normal β-globin or a globin chain with anti-sickling properties, such as γ-globin (that leads to production of HbF). Another approach that has been explored involves methodologies for “correcting” the faulty β-globin gene. These approaches, however, either are still in development or are in very early clinical trials.\n\nOne of the most attractive therapeutic targets in SCD is cell adhesion. Although SCD severity was first linked to the degree of red cell adhesion exhibited by patients’ red cells, it is now apparent that leukocyte and platelet activation and adhesion also contribute to pathophysiology. Therefore, cell adhesion has become a primary target for the development of new therapeutic approaches. In general, such approaches may involve inhibition of cell-cell interactions generally, specific inhibition of adhesion receptors, or interference with the signaling mechanisms that lead to activation of adhesion receptors.\n\nNon-specific inhibition of cell adhesion by poloxamer-188 was studied in a multicenter randomized phase III trial involving both children and adults to determine its effect on duration of painful vaso-occlusive episodes. Although the results were statistically significant, differences in time to resolution of painful episodes were small but were slightly greater in children70. Another phase III study of this drug, using a somewhat different study design, is now under way (NCT01737814, ClinicalTrials.gov).\n\nAnother anti-adhesion therapeutic has been developed to address adhesive interactions involving primarily E-selectin. This drug (GMI-1070, now known as rivipansel) was quite successful in abrogating vaso-occlusion in sickle mice71. In a subsequent phase I study of SCD patients in steady state, rivipansel was well tolerated and appeared to improve blood flow in a subset of patients. Perhaps most interesting, however, was the drug’s effect on biomarkers: the drug was associated with significant decreases in biomarkers of endothelial activation, including sE-selectin, sP-selectin, and soluble intercellular adhesion molecule-1 (sICAM). Markers of leukocyte activation and coagulation were also decreased72. A phase II study of the drug in patients experiencing painful vaso-occlusive episodes showed large and consistent decreases in all measures of time to crisis resolution, although these were not statistically significant. Moreover, opiate usage was markedly and statistically significantly decreased with drug versus placebo73. A phase III study is expected to be under way shortly (NCT02187003, ClinicalTrials.gov).\n\nOther drugs, both new and old, also have potential anti-adhesive effects that could be useful in SCD. P-selectin is known to contribute to adhesion of both sickle red cells and leukocytes to endothelial cells74–76, and heparins have both anti-P-selectin as well as anticoagulation effects75. In fact, heparin blocks P-selectin-mediated adhesion at levels considerably lower than those needed for anticoagulation77. A small phase 2 study of pentosan polysulfate sodium has shown promising results, in that a single oral dose improved microvascular blood flow, and repeated daily doses were associated with decreased plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1)77. Another compound chemically related to low-molecular-weight heparin (LMWH), sevuparin (Dilaforette), has shown promising results in vitro and in vivo in a mouse model of vaso-occlusion78, and plans are under way to study the drug in SCD. Another P-selectin-targeted drug, SelG1 (Selexys Pharmaceuticals), is currently in clinical trial for use as a prophylactic agent to prevent vaso-occlusive crises (NCT01895361, ClinicalTrials.gov).\n\nFinally, several studies in animals and patients have addressed the possibility that downregulation of signaling pathways may decrease cell adhesion. Several red cell adhesion receptors, including the BCAM/Lu receptor for laminin79 and the ICAM-4 receptor for integrins80,81, are activated downstream of β-adrenergic receptor signaling pathways. Animal studies and a phase 1 trial of propranolol showed that propranolol decreased sickle red cell adhesion measured in vitro and decreased vaso-occlusion in mice in vivo81,82. In addition, the ERK signaling pathway appears to be involved in sickle red cell adhesion83,84, and the ability to affect this pathway via MEK inhibition is now being explored85.\n\nGiven the abundant data that coagulation pathways are abnormally activated in SCD, early studies explored the possibility that anticoagulation might have a beneficial effect in SCD, but most of those studies were too small or time-limited to be definitive. Using acenocoumarol, one study showed that achieving an international normalized ratio (INR) of 1.64 (range of 1.18–2.2) was associated with normalization of the F1 + 2 level and therefore concluded that low-intensity oral anticoagulation could normalize the hypercoagulability in SCD86. Newer studies have again approached the potential usefulness of anticoagulation in SCD. A randomized double-blind clinical trial of an LMWH, tinzaparin, versus placebo was conducted during the management of acute painful vaso-occlusive episodes. This 253-patient study administered tinzaparin subcutaneously at 175 IU/kg once daily, along with usual supportive care and analgesia. Although the endpoints and criteria for discharge were different from those usually used in the United States and other Western countries, analysis demonstrated a statistically significant reduction in several measures of time to resolution87. Another double-blind prospective study randomized SCD patients hospitalized for pain episodes to receive prophylactic LMWH (dalteparin 5,000 IU subcutaneously daily) or placebo. Although this study did not meet its target enrollment, the group receiving dalteparin had a greater decrease in pain scores at day 3 than did the placebo group (NCT01419977, ClinicalTrials.gov), although these results are unpublished to date. Another study used low-dose warfarin during vaso-occlusive crisis and studied D-dimer levels as their primary endpoint. They found that patients on warfarin had significantly lower D-dimer levels than patients not receiving the drug88; however, effects on clinical endpoints, such as time to resolution of painful episode, were not reported. Finally, another study of acenocoumarol showed that treatment to INR values of 1.6 to 2 failed to lower the plasma levels of endothelial activation markers in SCD89, raising questions about the likely clinical utility of anticoagulation to prevent SCD-related vascular events. Nevertheless, studies of the newer direct factor X inhibitors (apixaban and rivaroxaban) are currently planned or ongoing (NCT02179177 and NCT02072668, respectively, ClinicalTrials.gov). In addition, a study is under way to determine the feasibility of performing a larger multicenter phase III trial to assess the effects of unfractionated heparin in acute chest syndrome (NCT02098993, ClinicalTrials.gov).\n\nAnti-platelet agents have also received attention and continue to be studied in the context of SCD. Eptifibatide is an anti-platelet agent that binds to the αIIbβIII integrin on platelets and decreased in platelet aggregation and sCD40L levels in patients with SCD in a phase I study90. sCD40L is a pro-inflammatory cytokine released by platelets and chronically elevated in SCD plasma91. However, use of eptifibatide in a small pilot study showed that eptifibatide did not improve the times to crisis resolution or hospital discharge92. Another anti-platelet agent being studied in SCD is prasugrel. Recently, a multicenter phase 2 study of prasugrel versus placebo in adult patients with SCD showed that the drug could be safely used, and although it did not achieve statistically significant reductions in pain scores, it did reduce both platelet surface P-selectin and plasma soluble P-selectin levels, compared with placebo93.\n\nInflammatory pathways in SCD are both the result of red and white cell adhesion to endothelial cells and to each other as well as promoters of such adhesion. In addition, transient vaso-occlusion leads to hypoxia/reperfusion injury with a robust inflammatory component. Therefore, targeting inflammatory pathways is a rational approach to treating or trying to prevent vaso-occlusion in SCD. Corticosteroids have been investigated but with mixed results. Newer therapies are now addressing the role of invariant natural killer T (iNKT) cells, which are known to play an important role in ischemia/reperfusion injury and which are increased in both number and activity in SCD94. In sickle mice, inhibition of iNKT cell activity with regadenoson, an adenosine A2A receptor agonist, led to a reduction in pulmonary inflammation and injury94. A phase 1 study in patients also showed promising results, including reduction in phospho-NF-kappa-B p65 activation in iNKT cells, compared with pretreatment baseline during vaso-occlusion95. The phase 2 study of regadenoson for treatment of vaso-occlusion is ongoing (NCT01788631, ClinicalTrials.gov). Another drug targeting iNKT cells is NKTT 120, a humanized monoclonal antibody against iNKT cells. After an initial study indicating safety, further ascending-dose phase 1 studies are being conducted to evaluate the safety, pharmacokinetics, pharmacodynamics, and biologic activity of the drug (NCT01783691, ClinicalTrials.gov).\n\nAlthough the salutary effect of HU on SCD may be due to a more complex mechanism than its ability to raise HbF levels in about 50% of patients, it is also clear that not everyone responds to HU either clinically or with appreciably higher HbF levels. Therefore, other ways to increase HbF continue to be explored. Early studies showed that decitabine could substantially increase HbF levels in HU-non-responders96 and that such increased HbF levels were sustainable with repeated intermittent treatment97. Decitabine continues to be studied, both alone (NCT01375608, ClinicalTrials.gov) and in combination with tetrahydrouridine, a competitive inhibitor of cytidine deaminase that is being studied in an effort to improve oral bioavailability of decitabine98 (NCT01685515, ClinicalTrials.gov). Other agents that are being studied for their effects on HbF levels include panobinostat, vorinostat, pomalidomide, arginine butyrate, and HQK-1001 (2,2-dimethylbutyrate)99–101. However, in a phase 2 study, HQK-1001 was associated with only a modest HbF response and a paradoxical increase in vaso-occlusive episodes101.\n\n\nImplications for the future\n\nThe complexity of the pathogenesis of SCD has resulted in a plethora of potential druggable targets in our effort to ameliorate the disease’s sequelae. However, this same complexity has also prevented us from knowing which targets are optimal ones. Biomarkers may be helpful in patient selection for both research studies or therapy, but proof that changes in biomarkers are associated with clinical improvement remains elusive in most instances. Therefore, until we achieve wide availability of curative therapies through either hematopoietic stem cell transplantation or gene therapy, we need to continue our search for therapies that can be provided either to prevent or treat acute disease manifestations, such as vaso-occlusive pain, and chronic organ damage, such as sickle cell nephropathy and pulmonary hypertension. These goals, however, are still not quite within reach.",
"appendix": "Competing interests\n\n\n\nThe author has received research support from GlycoMimetics (maker of GMI-1070) and Dilaforette (maker of sevuparin). She currently serves on the steering committee for the ongoing phase 3 trial of rivipansel sponsored by Pfizer.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPlatt OS, Brambilla DJ, Rosse WF, et al.: Mortality in sickle cell disease. Life expectancy and risk factors for early death. New Eng J Med. 1994; 330(23): 1639–1644. PubMed Abstract | Publisher Full Text\n\nPlatt OS, Thorington BD, Brambilla DJ, et al.: Pain in sickle cell disease. Rates and risk factors. New Eng J Med. 1991; 325(1): 11–16. PubMed Abstract | Publisher Full Text\n\nMeier ER, Wright EC, Miller JL: Reticulocytosis and anemia are associated with an increased risk of death and stroke in the newborn cohort of the Cooperative Study of Sickle Cell Disease. 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Thromb Haemost. 2007; 98(2): 392–396. PubMed Abstract | Publisher Full Text\n\nAhmed S, Siddiqui AK, Iqbal U, et al.: Effect of low-dose warfarin on D-dimer levels during sickle cell vaso-occlusive crisis: a brief report. Eur J Haematol. 2004; 72(3): 213–216. PubMed Abstract | Publisher Full Text\n\nSchnog JB, Mac Gillavry MR, Rojer RA, et al.: No effect of acenocoumarol therapy on levels of endothelial activation markers in sickle cell disease. Am J Hematol. 2002; 71(1): 53–55. PubMed Abstract | Publisher Full Text\n\nLee SP, Ataga KI, Zayed M, et al.: Phase I study of eptifibatide in patients with sickle cell anaemia. Br J Haematol. 2007; 139(4): 612–620. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLee SP, Ataga KI, Orringer EP, et al.: Biologically active CD40 ligand is elevated in sickle cell anemia: potential role for platelet-mediated inflammation. Arterioscler Thromb Vasc Biol. 2006; 26(7): 1626–1631. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDesai PC, Brittain JE, Jones SK, et al.: A pilot study of eptifibatide for treatment of acute pain episodes in sickle cell disease. Thromb Res. 2013; 132(3): 341–345. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWun T, Soulieres D, Frelinger AL, et al.: A double-blind, randomized, multicenter phase 2 study of prasugrel versus placebo in adult patients with sickle cell disease. J Hematol Oncol. 2013; 6: 17. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nField JJ, Nathan DG, Linden J: Targeting iNKT cells for the treatment of sickle cell disease. Clin Immunol. 2011; 140(2): 177–183. PubMed Abstract | Publisher Full Text | Free Full Text\n\nField JJ, Lin G, Okam MM, et al.: Sickle cell vaso-occlusion causes activation of iNKT cells that is decreased by the adenosine A2A receptor agonist regadenoson. Blood. 2013; 121(17): 3329–3334. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKoshy M, Dorn L, Bressler L, et al.: 2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia. Blood. 2000; 96(7): 2379–2384. PubMed Abstract\n\nDeSimone J, Koshy M, Dorn L, et al.: Maintenance of elevated fetal hemoglobin levels by decitabine during dose interval treatment of sickle cell anemia. Blood. 2002; 99(11): 3905–3908. PubMed Abstract | Publisher Full Text\n\nTerse P, Engelke K, Chan K, et al.: Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014; 33(2): 75–85. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKutlar A, Ataga K, Reid M, et al.: A phase 1/2 trial of HQK-1001, an oral fetal globin inducer, in sickle cell disease. Am J Hematol. 2012; 87(11): 1017–1021. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKutlar A, Reid ME, Inati A, et al.: A dose-escalation phase IIa study of 2,2-dimethylbutyrate (HQK-1001), an oral fetal globin inducer, in sickle cell disease. Am J Hematol. 2013; 88(11): E255–260. PubMed Abstract | Publisher Full Text\n\nReid ME, El Beshlawy A, Inati A, et al.: A double-blind, placebo-controlled phase II study of the efficacy and safety of 2,2-dimethylbutyrate (HQK-1001), an oral fetal globin inducer, in sickle cell disease. Am J Hematol. 2014; 89(7): 709–713. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHibbert JM, Hsu LL, Bhathena SJ, et al.: Proinflammatory cytokines and the hypermetabolism of children with sickle cell disease. Exp Biol Med (Maywood). 2005; 230(1): 68–74. PubMed Abstract | Free Full Text\n\nSinghal A, Doherty JF, Raynes JG, et al.: Is there an acute-phase response in steady-state sickle cell disease? Lancet. 1993; 341(8846): 651–653. PubMed Abstract | Publisher Full Text\n\nQari MH, Dier U, Mousa SA: Biomarkers of inflammation, growth factor, and coagulation activation in patients with sickle cell disease. Clin Appl Thromb Hemost. 2012; 18(2): 195–200. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPathare A, Al Kindi S, Alnaqdy AA, et al.: Cytokine profile of sickle cell disease in Oman. Am J Hematol. 2004; 77(4): 323–328. PubMed Abstract | Publisher Full Text\n\nFrancis RB Jr, Haywood LJ: Elevated immunoreactive tumor necrosis factor and interleukin-1 in sickle cell disease. J Natl Med Assoc. 1992; 84(7): 611–615. PubMed Abstract | Free Full Text\n\nGraido-Gonzalez E, Doherty JC, Bergreen EW, et al.: Plasma endothelin-1, cytokine, and prostaglandin E2 levels in sickle cell disease and acute vaso-occlusive sickle crisis. Blood. 1998; 92(7): 2551–2555. PubMed Abstract\n\nMichaels LA, Ohene-Frempong K, Zhao H, et al.: Serum levels of substance P are elevated in patients with sickle cell disease and increase further during vaso-occlusive crisis. Blood. 1998; 92(9): 3148–3151. PubMed Abstract\n\nLanaro C, Franco-Penteado CF, Albuqueque DM, et al.: Altered levels of cytokines and inflammatory mediators in plasma and leukocytes of sickle cell anemia patients and effects of hydroxyurea therapy. J Leukoc Biol. 2009; 85(2): 235–242. PubMed Abstract | Publisher Full Text\n\nBrittain JE, Hulkower B, Jones SK, et al.: Placenta growth factor in sickle cell disease: association with hemolysis and inflammation. Blood. 2010; 115(10): 2014–2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHagag AA, Elmashad G, Abd El-Lateef AE: Clinical significance of assessment of thrombospondin and placenta growth factor levels in patients with sickle cell anemia: two centers Egyptian studies. Mediterr J Hematol Infect Dis. 2014; 6(1): e2014044. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSundaram N, Tailor A, Mendelsohn L, et al.: High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension. Blood. 2010; 116(1): 109–112. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPerelman N, Selvaraj SK, Batra S, et al.: Placenta growth factor activates monocytes and correlates with sickle cell disease severity. Blood. 2003; 102(4): 1506–1514. PubMed Abstract | Publisher Full Text\n\nAslan M, Celmeli G, Özcan F, et al.: LC-MS/MS analysis of plasma polyunsaturated fatty acids in patients with homozygous sickle cell disease. Clin Exp Med. 2015; 15(3): 397–403. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGarrido VT, Proença-Ferreira R, Dominical VM, et al.: Elevated plasma levels and platelet-associated expression of the pro-thrombotic and pro-inflammatory protein, TNFSF14 (LIGHT), in sickle cell disease. Br J Haematol. 2012; 158(4): 788–797. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "10774",
"date": "12 Oct 2015",
"name": "James G. Taylor VI",
"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",
"responses": []
},
{
"id": "10775",
"date": "12 Oct 2015",
"name": "Emily Meier",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1050
|
https://f1000research.com/articles/4-1049/v1
|
12 Oct 15
|
{
"type": "Case Report",
"title": "Case Report: Management of idiopathic chylothorax",
"authors": [
"Maher Abouda",
"Yangui Ferdaous",
"Miriam Triki",
"Mehdi Charfi",
"Mohamed Ridha Charfi",
"Yangui Ferdaous",
"Miriam Triki",
"Mehdi Charfi",
"Mohamed Ridha Charfi"
],
"abstract": "Chylothorax is characterized by the presence of chyle in the pleural space and results from lesion or obstruction of the thoracic duct. We present two cases of non-traumatic, idiopathic chylothorax in two females that were treated differently. The first is a 42 year old female who presented with a symptomatic right chylothorax. Treatment by a low-fat diet supplemented with medium chain triglyceride and evacuation of the pleural fluid was sufficient. The second patient is a 25 year old female admitted for a bilateral chylothorax. Despite optimal medical therapy, chylothorax continued to persist. Finally thoracic duct ligation was performed, which resulted in resolution of the effusion. These two cases illustrate that the management of idiopathic chylothorax can be surgical or nonsurgical.",
"keywords": [
"Pleural diseases",
"Idiopathic chylothorax",
"thoracic duct",
"chyle",
"surgery",
"diet"
],
"content": "Introduction\n\nChylothorax is characterized by the presence of chyle, which is rich in triglycerides and chylomicrons in the pleural space resulting from damage of the thoracic duct1,2. In the etiology of chylothorax, traumas and malignancies are the first two leading causes2. Idiopathic chylothorax in adults is a rare clinical condition that can be retained only after ruling out all other causes of chylothorax3. The management of idiopathic chylothorax can be surgical or nonsurgical4. We report two cases of spontaneous chylothorax in adults, that were treated differently.\n\n\nCase report 1\n\nA previously healthy, 42 year old woman who was a non-smoker, presented with two days history of right chest pain. She was first evaluated in the consultation. At the chest physical examination, dullness to percussion with decreased breath sound over the right lung base was noted. There was no history of trauma, recent infection, fever or weight loss. Posteroanterior (Figure 1) and lateral chest radiographs demonstrated right pleural fluid collection. Routine laboratory studies (including complete blood count, erythrocyte sedimentation rate, coagulation, liver and renal function tests, and lipid profile) were within normal limits. Thoracentesis yielded a creamy-milky like fluid (Figure 2), with protein levels 86 g/l, triglycerides 2253 mg/dl, cholesterol 124 mg/dl and white blood cell count 78/mm3 (30 percent polymorphonuclear leukocytes and 70 percent lymphocytes) confirming the laboratory testing profile of chylothorax. A bronchoscopy was performed and shows a normal bronchial tree. The bacteriologic and cytologic analyses of the endobronchial aspiration fluid were normal. Complementary laboratory studies such as antinuclear antibody and viral serology, were negative. A computed tomography (CT) scan of the chest and abdomen was normal and shows no evidence of tumor or lymphadenopathies. Treatment consisted of relative rest and a medium-chain triglyceride diet and evacuation of the pleural fluid by multiple thoracentesis. In few days, the effusion had completely resolved, and at one year, the patient remained asymptomatic.\n\n\nCase report 2\n\nA 25 year old woman, non-smoker, was admitted for a bilateral pleural effusion fortuity discovered by a chest radiography (Figure 3). The effusion was predominant in the right side. There was no history of trauma, recent pulmonary infection, or weight loss. Routine laboratory studies (including complete blood count, erythrocyte sedimentation rate, coagulation, liver and renal function tests, and lipid profile) were within normal limits. Right thoracentesis yielded a red-milky like fluid, with protein levels 56 g/l, triglycerides 3200 mg/dl, cholesterol 140 mg/dl, white blood cell count 165/mm3 (22 percent polymorphonuclear leukocytes and 78 percent lymphocytes) and numerous red blood cells confirming the laboratory testing profile of chylothorax. All explorations such as bronchoscopy, bacteriologic and cytologic analyses, antinuclear antibody and viral serology were negative. A CT scan of the chest and abdomen was normal and shows no evidence of tumor or lymphadenopathies. At first a non-surgical therapy was tried, including a modified diet, chest tube insertion and total parenteral nutrition. Yet, despite optimal medical therapy, the effusion worsened with appearance of ascites. A second CT scan of the chest shows the accentuation of the effusion, particularly in the left side (Figure 4). Surgical thoracic duct ligation at the level of the seventh dorsal vertebra was performed. The resolution of the effusion was obtained after two weeks. At two years, the patient remained asymptomatic.\n\n\nDiscussion\n\nChylothorax is a relatively uncommon cause of pleural fluid collection, causing 3 percent of pleural effusions2. The pleural fluid in chylothorax is rich in triglycerides and chylomicrons. This fluid is the chyle from the intestine, liver and abdominal wall, that flows in the pleural space after a rupture, laceration or obstruction of the thoracic duct5. Damage of the thoracic duct can occur after thoracic trauma or thoracic surgery (49 percent), or in various other medical conditions (43 percent) such as: central venous catheterization, extensive venous thrombosis in the neck, compression by mediastinal lymphadenopathy such as in non-Hodgkin’s lymphoma2,6. In about 6.4 percent of cases no reason can be found for the chylous pleural effusion2. Most of these idiopathic chylothoraces are thought to be related to minor trauma. In our cases, no trauma was reported by the patient. Chylothorax presents as a non-infectious pleural effusion causing non-specific symptoms such as cough, asthenia or abdominal pain, and sometimes respiratory distress1–6. Chylothorax is easily diagnosed by thoracocentisis. Biochemical and other analyses of the pleural fluid are the most important diagnostic step. The pleural fluid appearance is generally milkly white or whitish. Pleural fluid triglyceride levels have been used in diagnosing chylothorax. Level of pleural fluid triglyceride must be >1.10 mg/dl. Presence of chylomicrons, low cholesterol level, and elevated percentage of lymphocyte are necessary for the diagnosis of chylothorax1,6. In non-traumatic or non-chirurgical cases, CT abdomen and thorax scans should be performed to rule out association with malignancy or tuberculosis2,3. This may demonstrate evidence of tumor or lymphadenopathy. Lymphangiography may be used to demonstrate the side of leakage or blockage6. In our hospital lymphography is not performed. In countries with relatively high incidence of tuberculosis, like in North Africa, sputum acid fast bacillus smear and culture may be helpful in etiologic diagnosis. In some cases laboratory studies such as antinuclear antibody and viral serology must be performed, especially in women7. Treatment is controversial and can be either conservative or surgical. Conservative treatment includes the use of a low-fat diet supplemented with medium chain triglycerides (MCT) and/or total parenteral nutrition (TPN)4. In our first case, a low-fat diet without long chain triglycerides was maintained to limit lymph flow with good evolution and regression of the chylothorax. Octreotide, a somatostatin analogue, may be used for the management of patients with refractory chylothorax8. The use of intrapleural streptokinase is not established but can be proposed for patients with idiopathic chylothorax who failed conservative therapy but refused surgery10. Drainage of the effusion by thoracocentesis or chest tube insertion is necessary in cases of significant respiratory distress or important effusion4,6. In case of therapeutic failure, surgery is required. Surgery may act on the leak itself: ligation of the thoracic duct, sutures of the leaking collaterals, or various other procedures4,6,11. Invasive chirurgical treatment with low efficacy such as pleuroperitoneal shunt or pleurodesis is indicated in recurrent chylothorax or in extensive lesion12.\n\n\nConclusion\n\nThe pleural fluid in chylothorax is the chyle from the intestines, liver and abdominal wall that flows in the pleural space after a rupture, laceration or obstruction of the thoracic duct and diagnosis is easily made by Thoracentesis. In about 6.4 percent of chylothoax no reason can be found for the pleural effusion. In such cases Treatment is controversial and can be either conservative or surgical.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and images was obtained from the patients.",
"appendix": "Author contributions\n\n\n\nAM, YF and TM carried out the cases. CM contributed to the selection of images. AM and YF contributed to 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHillerdal G: Chylothorax and pseudochylothorax. Eur Respir J. 1997; 10(5): 1157–62. PubMed Abstract | Publisher Full Text\n\nDoerr CH, Allen MS, Nichols FC 3rd, et al.: Etiology of chylothorax in 203 patients. Mayo Clin Proc. 2005; 80(7): 867–870. PubMed Abstract | Publisher Full Text\n\nRomero S: Nontraumatic chylothorax. Curr Opin Pulm Med. 2000; 6(4): 287–91. PubMed Abstract | Publisher Full Text\n\nEpaud R, Dubern B, Larroquet M, et al.: Therapeutic strategies for idiopathic chylothorax. J Pediatr Surg. 2008; 43(3): 461–465. PubMed Abstract | Publisher Full Text\n\nSteinemann DC, Dindo D, Clavien PA, et al.: Atraumatic chylous ascites: systematic review on symptoms and causes. J Am Coll Surg. 2011; 212(5): 899–905.e1–4. PubMed Abstract | Publisher Full Text\n\nBüttiker V, Fanconi S, Burger R: Chylothorax in children: guidelines for diagnosis and management. Chest. 1999; 116(3): 682–7. PubMed Abstract | Publisher Full Text\n\nManzella DJ, Dettori PN, Hertimian ML, et al.: Chylous ascites and chylothorax as presentation of a systemic progression of discoid lupus. J Clin Rheumatol. 2013; 19(2): 87–89. PubMed Abstract | Publisher Full Text\n\nHashim SA, Roholt HB, Babayan VK, et al.: Treatment of chyluria and chylothorax with medium-chain triglyceride. N Engl J Med. 1964; 270: 756–761. PubMed Abstract | Publisher Full Text\n\nRoehr CC, Jung A, Proquitté H, et al.: Somatostatin or octreotide as treatment options for chylothorax in young children: a systematic review. Intensive Care Med. 2006; 32(5): 650–657. PubMed Abstract | Publisher Full Text\n\nKuan YC, How SH, Ng TH, et al.: Intrapleural streptokinase for the treatment of chylothorax. Respir Care. 2011; 56(12): 1953–1955. PubMed Abstract | Publisher Full Text\n\nCevese PG, Vecchioni R, D’Amico DF, et al.: Postoperative chylothorax. Six cases in 2,500 operations, with a survey of the world literature. J Thorac Cardiovasc Surg. 1975; 69(6): 966–971. PubMed Abstract\n\nMurphy MC, Newman BM, Rodgers BM: Pleuroperitoneal shunts in the management of persistent chylothorax. Ann Thorac Surg. 1989; 48(2): 195–200. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "14560",
"date": "24 Jun 2016",
"name": "Mohammed Atie",
"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\nIt is crucial when a chylothorax is being either medically or surgically treated to state the volumes of chyle that are produced on a daily basis. This dictates further management. The authors fail to provide that.\n\nThe thoracic duct passes from the cysterna chyli directly into the right pleural space. Anatomically, there is no communication between the right pleural space and the left pleural space. While a left sided chylothorax is possible after surgical intervention due to disruption of the left pleura, idiopathic chylothrorax is normally right sided. The authors fail to provide an explanation of how chyle from the thoracic duct’s main trunk tracked into the left chest.\n\nSupposing that the leak into the left chest was from an aberrant or side branch that originated at point below the right pleural space, how did the authors identify that? Locating the thoracic duct main trunk is feasible surgically, but locating a side branch proves impossible surgically even with prior localisation by means of lymphangiography. I presume that the thoracic duct ligation was done blindly at the level of the hiatus.\n\nThe authors highlight two methods for the management of chylothorax. These methods have been well established and the report itself does not add any new material to what is already known from the literature.",
"responses": []
},
{
"id": "14897",
"date": "11 Jul 2016",
"name": "Ahmed Abdelghani",
"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 tow cases of pleural effusion as an initial presentation of the thoracic duct lesion. While the diagnosis of chylothorax is easy, the idiopathic origin of the affection can be retained only after ruling out all other causes of chylothorax. The chylothorax seem to be idiopathic in the two cases, however the authors do not explain how idiopathic chylothorax can be located at left side.\nThis article points out the difficulty of the treatment which can be either conservative or surgical.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1049
|
https://f1000research.com/articles/2-172/v1
|
13 Aug 13
|
{
"type": "Research Article",
"title": "Characterization of known protein complexes using k-connectivity and other topological measures",
"authors": [
"Suzanne R Gallagher",
"Debra S Goldberg"
],
"abstract": "Many protein complexes are densely packed, so proteins within complexes often interact with several other proteins in the complex. Steric constraints prevent most proteins from simultaneously binding more than a handful of other proteins, regardless of the number of proteins in the complex. Because of this, as complex size increases, several measures of the complex decrease within protein-protein interaction networks. However, k-connectivity, the number of vertices or edges that need to be removed in order to disconnect a graph, may be consistently high for protein complexes. The property of k-connectivity has been little used previously in the investigation of protein-protein interactions. To understand the discriminative power of k-connectivity and other topological measures for identifying unknown protein complexes, we characterized these properties in known Saccharomyces cerevisiae protein complexes in networks generated both from highly accurate X-ray crystallography experiments which give an accurate model of each complex, and also as the complexes appear in high-throughput yeast 2-hybrid studies in which new complexes may be discovered. We also computed these properties for appropriate random subgraphs. We found that clustering coefficient, mutual clustering coefficient, and k-connectivity are better indicators of known protein complexes than edge density, degree, or betweenness. This suggests new directions for future protein complex-finding algorithms.",
"keywords": [
"Proteins are a critical unit in biology. Rather than performing their function alone",
"many proteins form protein complexes",
"groups of proteins that bind together to perform a specific task. Some of these complexes",
"such as the proteasome",
"are well-characterized",
"but others are not. In addition",
"it is hypothesized that there are many protein complexes in the cell that have not yet been identified. Complexes play an important role in the function of the cell",
"and by discovering new complexes and learning more about their composition and structure",
"we can gain insights into cellular biology."
],
"content": "Background\n\nProteins are a critical unit in biology. Rather than performing their function alone, many proteins form protein complexes, groups of proteins that bind together to perform a specific task. Some of these complexes, such as the proteasome, are well-characterized, but others are not. In addition, it is hypothesized that there are many protein complexes in the cell that have not yet been identified. Complexes play an important role in the function of the cell, and by discovering new complexes and learning more about their composition and structure, we can gain insights into cellular biology.\n\nEver since high-throughput protein-protein interaction data sets have become widely available, scientists have used the interaction data to create graphs called protein-protein interaction (PPI) networks. The vertices in PPI networks represent proteins, and there is an edge between two vertices if the corresponding proteins interact. These graphs are not perfect models of protein interaction in an organism since the experiments that produced the edges are error-prone and contain both false positives and false negatives. Despite these errors, however, they are useful tools for studying the proteome of an organism.\n\nOne use for PPI networks is to predict unknown protein complexes from the interaction data. Previous algorithms have used several different properties to find complexes. By far the most common property has been edge density, the fraction of pairs of nodes (possible edges) that have an edge connecting them1–7. Most edge density algorithms search for subgraphs with edge density above a certain threshold1–4. Other properties that have been used include clustering coefficient7, degree statistics7,8, maximum flow9, and path length10,11. Biological networks have also been examined using the properties of mutual clustering coefficient12,13 and betweenness centrality14–17.\n\nThe k-connectivity of a graph is a measure of how many distinct paths exist between any pair of vertices. A graph or subgraph is k-connected if there are k disjoint paths between every pair of nodes, or equivalently, if the removal of at least k vertices or edges from the graph are required in order to disconnect it. We believe that a high k-connectivity may be more indicative of a protein complex than other measures, and can serve to identify protein complexes even with low edge density. If each protein in the complex binds to some number of adjacent proteins, then as the number of proteins in the complex increases, the edge density will decrease because the maximum number of proteins that a single protein can bind to is limited by steric constraints. The k-connectivity, however, will stay roughly constant as long as each protein remains bound to roughly the same number of neighbors. Also, k-connectivity implies a certain degree of stability, and a complex with a high k-connectivity might be able to retain its structure and even partial function in the event of a mutation that caused an interaction to be lost or for a certain protein to be missing altogether.\n\nk-connectivity has only rarely been used in connection with finding protein complexes. Habibi et al.18 found that, in mass spectrometry data, k-connectivity was a better indicator of protein complexes than edge density. Hartuv and Shamir19 looked for connected subgraphs of n proteins that are n/2-connected; however, because their stopping condition is a function of the number of proteins in the subgraph, this is closer to a measure of edge density than k-connectivity.\n\nIn order to test the hypothesis that k-connectivity is a useful indicator of complexes in pairwise interaction data, we examined known complexes in the iPFam20 and MIPS21 databases. For each of these known complexes, we computed k-connectivity as well as various other topological properties, with a particular focus on those used in previous complex-finding algorithms: edge density, degree statistics, clustering coefficient, mutual clustering coefficient, number of triangles and 4-cycles, and betweenness centrality. We calculated these statistics in protein interaction graphs representing complexes. For each complex we used interactions determined by low-throughput X-ray crystallography data, where available, as well as high throughput yeast 2-hybrid (Y2H) studies. Finally, in addition to surveying these topological measures in complexes, we compared them to those of random complex-like subgraphs, which we call pseudocomplexes, pulled from the PPI network. This allows us to assess the utility of each of these statistics for discovering unknown protein complexes.\n\n\nMethods\n\nWe obtained details about protein complexes in Saccharomyces cerevisiae from two different sources. The first source was iPFam, where we were able to obtain data about protein complexes as well as which proteins interact within the complex20. These interactions were determined via X-ray crystallography, which, while not perfectly accurate, should be considered highly reliable. Unfortunately, only 13 complexes with at least three distinct proteins were included in this database. The second source of data on known complexes was the MIPS database21. The MIPS database is far more extensive, but only contains the proteins present in the complex, not the interactions that occur within the complex.\n\nWe obtained pairwise Y2H interaction data from Biogrid and created an interaction graph using a composite of all Y2H studies in yeast available on Biogrid22. We did not include data from high-throughput affinity purification-mass spectrometry experiments, as did Habibi et al.18, because these experiments are biased towards protein complex interactions, and we sought to understand the properties of protein complexes and how these differ from a random background. To discover new protein complexes, it is appropriate to use all available data, as in the Habibi et al. study, but this was not our purpose. In addition, we wished to avoid complications from representing mass spectrometry interactions, which are not intrinsically binary, in a binary graph. We used high-throughput Y2H interactions exclusively because they are intrinsically binary, and do not suffer from known biases towards interactions within protein complexes. For similar reasons, we did not use the PCA binary interactions from Tarassov23 because that study used known complexes to filter the results and therefore would be biased in favor of known complexes.\n\nThe high-throughput Y2H data set, however, has a high error rate and includes both false positives (proteins that don't interact but have been reported to interact in one or more studies) and false negatives (proteins that do interact but whose interaction has not been reported in a Y2H study). We considered using the Y2H Union subset of interactions24, a subset of the interactions with higher confidence, but there aren't enough interactions in this data set between proteins in the same complex to give us meaningful results; only 25 of the 154 complexes in MIPS induced a connected graph, and of those 25, only 4 had more than 3 proteins in the data. This was not enough data to give a meaningful picture of complexes, so we decided it was better to accept the lower quality but higher number of interactions from the composite data set. It is worthwhile to discover metrics that would allow us to find protein complexes in the abundantly available data. We therefore decided to accept a lower specificity and a higher number of false positives in order to increase the sensitivity.\n\nIn order to avoid confusion, for the remainder of the paper, we will refer to the entire collection of proteins and interactions determined by Y2H interactions as the \"network\". The collection of proteins and interactions in a complex will be a \"graph\" while a subset of those interactions will be a \"subgraph\".\n\nFor the complexes from iPFam, we looked at both the interactions determined by the X-ray crystallography on isolated complexes and also the graph induced in the Y2H network by the proteins determined to be in the complex and all Y2H edges amongst these proteins. See Figure 1. The X-ray crystallography data set gives us an idea of how complexes might look in a complete and accurate interaction network, while the Y2H data set gives us an idea of how complexes look in our real error-prone data. For the complexes from MIPS, we were only able to look at the induced graphs from the Y2H data. The code used for calculating the statistics of protein complexes can be found at https://github.com/suzanneg/complex-stats.\n\nThe graph in the middle represents the interactions from the isolated complex (from iPFam), while the graph on the right contains the same proteins but gets its edges from the Y2H network (from Biogrid).\n\nWe assessed the following graph measures:\n\nEdge and vertex k-connectivity: Measures of the number of distinct paths between any pair of vertices. A graph or subgraph is k-edge-connected (k-vertex-connected) if between every pair of nodes there are at least k edge-disjoint (intermediate vertex disjoint) paths. Equivalently, any k–1 edges (vertices) can be removed from the graph without disconnecting it. In the remainder of this paper, k-connectivity refers to vertex k-connectivity.\n\nEdge density: The number of interactions (edges) divided by the number of possible interactions (pairs of vertices).\n\nDegree statistics: The maximum, minimum, and mean degrees for each graph, along with the standard deviation of the mean. In order to compare these statistics between complexes with differing numbers of proteins, we normalize by dividing the degree statistics by the number of vertices in the graph.\n\nClustering Coefficient (CC): A measure of how many of a vertex's neighbors are neighbors of each other. Over a graph or subgraph, clustering coefficient is defined as 3 times the number of triangles divided by the number of length 2 paths.\n\nMutual Clustering Coefficient (MCC): For a pair of vertices, the percentage of their neighbors that they share. There are several different ways of defining the mutual clustering coefficient between two vertices, but for our purposes, we define it as the number of shared neighbors divided by the minimum degree (number of neighbors) of the two vertices. This method was the best of the ratio methods from Goldberg and Roth13 for assessing confidence in PPI networks. We calculate the MCC between all pairs of vertices in a complex, and as with degree, we report the maximum, minimum, mean, and standard deviation.\n\nMotifs: Particular subgraphs in each complex. We were interested in the number of triangles and 4-cycles.\n\nBetweenness centrality: For a vertex, the number of shortest paths between all other pairs of vertices that contain that vertex. Again, we report the maximum, minimum, mean, and standard deviation. As with the degree statistics, we normalize by dividing by the number of vertices in the graph. Because complexes are expected to be well-connected, we expect betweenness values to be small.\n\nFor each graph of a complex, we looked at three subgraphs: 1) the original graph, which includes vertices representing all proteins in the complex; 2) a \"haircut\" subgraph, where we recursively eliminate all vertices of degree 1 or less, ensuring the subgraph has a minimum degree of 2 (this is the same as the haircut part of the algorithm of Bader and Hogue7); and 3) the subgraph that is k-connected for the highest value of k, which we call the most highly connected subgraph (MHCS).\n\nWe look at these additional subgraphs because we believe that several properties will be more discernible in these subgraphs, so that these subgraphs are more likely to be able to be discovered by a complex-finding algorithm. The single vertices eliminated by the haircut are unlikely to be discovered by any complex-finding algorithm, and including them lowers the edge density, clustering coefficient, and k-connectivity of the graph, as well as raising the betweenness of the adjacent vertex. The MHCS clearly highlights k-connectivity, but many other properties are also higher in the MHCS than in the original graph.\n\nIn order to assess the significance of properties in the complexes and the Y2H network as a whole, we used two different methods of generating random graphs. For the Y2H network, we generated networks with the same number of vertices and the same edge distribution by \"switching\". Switching works by choosing two random edges with different endpoints, (A, B) and (C, D), removing those edges, and replacing them with edges (A, D) and (C, B). We use the method recommended by Milo et al.25: for a network with n vertices, the process is repeated 100n times to ensure proper mixing. The end result is a random network with the same degree distribution as the original network26. This process is repeated 10 times, giving us 10 random networks for comparison.\n\nA somewhat different method was used to assess the significance of the properties of the complexes. Switching would only allow us to compare a protein complex graph with another graph of the same degree distribution, when what we really want is to compare it to other graphs from the Y2H network. Our question is \"how likely are we to see this result in the actual network where there is not a complex?\" so we seek graphs that are similar to our complexes. For each complex with at least 4 proteins, we found a \"matched\" graph that we call a pseudocomplex. A pseudocomplex P that matches a complex with n proteins is generated by taking an edge from a random triangle from the Y2H network and letting P2 = this edge and the two nodes it connects. For i > 2, we generate Pi from Pi–1 by taking a random edge in the Y2H network attached to Pi–1 and adding the vertex at the other end and all edges from this vertex to Pi–1. Repeat this process until we have the same number of vertices as the original complex and let P = Pn. We chose a random edge rather than a random neighbor so that nodes connected by multiple edges would be more likely to be chosen, making the final graph more \"complex-like\". We started with an edge from a triangle rather than a random edge for the same reason, because most (though not all) complexes contained at least one triangle. Although this bias may make pseudocomplexes more likely to contain a triangle than real complexes are, we believed it was better to be overly conservative in this respect. We considered only complexes with at least 4 proteins because fewer nodes in a connected subgraph require some measures to be unreasonably high, and this would skew our comparisons. We calculated the same measures for pseudocomplexes as we did for the complex graphs, and compared our results with the real complexes.\n\n\nResults\n\nThere were 35 studies in iPFam that involved complexes with at least 3 proteins. Some of these studies were of the same or similar complexes; we grouped studies together if they produced the exact same graph, i.e. the same proteins with the same set of interactions. This grouping gave us 13 distinct graphs. All graphs are illustrated in Figure S1 and Figure S2 along with the subgraphs they induced in the Y2H data. In some cases, it is possible that two different studies of the same complex may have produced different graphs, but we will treat all distinct graphs as separate entities. Full statistics for the complexes from iPFam are in the Supplementary material. Because we had interaction data from X-ray crystallography, we were able to analyze a reliable graph representation for these complexes.\n\nIn all except two cases, the interactions from the X-ray crystallography produced connected graphs. Most complexes were only 1-connected due to the presence of a small number of degree 1 vertices; in all cases except one, the haircut subgraphs were at least 2-connected. About half the complexes had a subgraph that was at least 3-connected. In general, the edge density could be closely correlated with the number of vertices in the complex; complexes with only 3 proteins produced cliques while those with 12 or more tended to have edge densities closer to 1/3. Clustering coefficients had a similar pattern to edge density in that the value was closely correlated with the number of vertices in the complex. Mutual clustering coefficients were more scattered, but also tended to decrease as the number of vertices increased.\n\nWhen we look at the iPFam complexes in the Y2H data, we see that 9 of the 13 have all of their proteins present, 3 have slightly more than 60 percent, and 1 has only 1 out 4 proteins present. Only in one, a complex with 3 proteins, were all of the interactions from the X-ray crystallography present in the Y2H data. With the exception of that complex, none of the complexes induced connected graphs, and they all had edge densities of less than 0.1. In all except two cases, the haircut produced an empty subgraph. Only two complexes had a subgraph that was at least 2-connected. Most graphs had clustering coefficients of 0. Average mutual clustering coefficients were higher, between 0.14 and 0.63. Comparing these results with the results obtained using the X-ray crystallography data set gives an indication of how many interactions have not been detected using a Y2H assay and how these false negatives make it difficult to detect complexes. Note that, in Figure 1, only 8 of the 32 edges in the correct graph generated by X-ray crystallography were also observed in the high-throughput graph generated by Y2H experiments.\n\nResults on k-connectivity, edge density, clustering coefficient, and mutual clustering coefficient are summarized in Figure 2, and results for normalized maximum degree and betweenness centrality are summarized in Figure 3. The left-hand graphs contain full results on the complexes, while the right-hand graphs contain comparisons with pseudocomplexes. Note that because at least 4 proteins are needed to create a pseudocomplex, the real complexes on the right-hand side are a subset of the complexes on the left-hand side. Full results are contained in the Data Files.\n\nFor each statistic, the graph on the left contains the percent of complex graphs, haircut graphs, MHCS, and all connected components that are above a given threshold. The graph on the right contains percentages of real complexes and pseudocomplexes that are above the threshold. Note that only complexes that had some interactions between their component proteins are included in these graphs.\n\nFor maximum normalized degree, the graph on the left contains the percent of complex graphs, haircut graphs, MHCS, and all connected components that are above a given threshold. The graph on the right contains percentages of real complexes and pseudocomplexes that are above the threshold. For maximum betweenness, the graphs show the percent of complexes below a threshold. Note that only complexes that had some interactions between their component proteins are included in these graphs.\n\nThe results on k-connectivity are shown at the top of Figure 2. The graph on the top left gives results on k-connectivity in the full complex graphs, the haircut graphs, the MHCS, and all connected components of complexes. From this we can observe that most complexes are at most 1-connected, but when degree 1 vertices are removed, all complexes not destroyed by this operation (39% of the total) are 2-connected. Many complexes also had a subgraph with even higher connectivity.\n\nComparisons between k-connectivity in real complexes and pseudocomplexes are shown on the top right of Figure 2. Note that this graph, unlike the other graphs comparing complexes and pseudocomplexes, gives the k-connectivity of the MHCS rather than the entire complex or pseudocomplex. This was done because most complexes and pseudocomplexes had a k-connectivity of 1. It was only looking at the MHCS that the differences between complexes and pseudocomplexes became apparent. While roughly the same number of complexes and pseudocomplexes had a 2-connected subgraph, a far higher percentage of complexes had more highly connected subgraphs. Note that pseudocomplexes were designed to have, with high probability, a triangle (a 2-connected subgraph).\n\nThe remainder of Figure 2 summarizes the results on edge density, clustering coefficient, and mutual clustering coefficient. From the raw edge density values, we can see that the edge density of most complexes is nowhere near as high as it would be if complexes were cliques or near-cliques: only about 1 in 10 complexes had an edge density above 0.7. In the comparisons with pseudocomplexes, we see that the edge density of complexes and pseudocomplexes is fairly similar, with the density of complexes being slightly higher. The difference is less dramatic, however, than it is for k-connectivity due to the high standard deviation of the pseudocomplexes: the point where the maximum difference between known complexes and pseudocomplexes can be seen (the obvious cut-off point between real complexes and pseudocomplexes), 0.5, was well within a standard deviation of the average for pseudocomplexes. The obvious cut-off point for k-connectivity, 3-connected, by contrast was more than a standard deviation away from the average of the pseudocomplexes. The number of complexes with high clustering coefficients was also quite small, but clustering coefficients had a far more dramatic contrast with pseudocomplexes, especially for lower thresholds. Again, however, there was a fairly high deviation among pseudocomplexes. Mutual clustering coefficients have higher raw values but much less of a contrast with pseudocomplexes. When the deviation of pseudocomplexes is considered, mutual clustering coefficient does not differentiate from complexes as well as k-connectivity.\n\nThere are a few further things to note about clustering coefficients and mutual clustering coefficients. Clustering coefficients were quite high in haircut graphs, but this is somewhat misleading. The haircut can remove length 2 paths from the graph but cannot remove any triangles; therefore, we would expect to increase clustering coefficient, but this increase would not necessarily help us in finding complexes. Average mutual clustering coefficient is much higher than clustering coefficient. The reason for this is that there are many more 4-cycles than triangles. While triangles are overrepresented in the Y2H network as compared to a random network of the same degree distribution produced by switching (4681 v. 1609.8, 2.9 times as many), 4-cycles are also overrepresented (98166 v. 24045.0, 4.1 times as many). The frequencies of triangles and 4-cycles relative to random networks has been calculated for a previous yeast PPI network, also with the result that both were overrepresented, with 4-cycles also overrepresented by a higher margin, though this was not stated explicitly27. This pattern does not, however, appear to hold completely true for all PPI networks; specifically, in Drosophila melanogaster, triangles appear to be more overrepresented than 4-cycles28.\n\nThis pattern also seems to hold in the complex graphs. Neither triangles nor 4-cycles were particularly prevalent in complexes relative to pseudocomplexes (which were each seeded within a triangle), but 4-cycles were more prevalent than triangles. In 50% of complexes, there were more 4-cycles as compared to matching pseudocomplexes. However, only 29% of complexes had more triangles than their matching pseudocomplexes.\n\nThe normalized results for maximum degree and comparisons with pseudocomplexes are in Figure 3. In many of the complexes we looked at, there was at least one protein of high degree that had an interaction with all or almost all of the other proteins in the complex, forming a \"star\" or a \"hub and spoke\" in the graph. This has been previously suggested by Bader and Hogue as a way to model the interactions in complexes that were found experimentally using affinity-purification8. However, there are some problems with using this idea to search for complexes in the data. The first is that we did not notice a strong correlation between proteins with high degree and proteins that appear in known complexes; roughly 30% of proteins of degree 3 or higher in our data set appeared in at least one complex, and this number remained roughly constant as we increased the degree threshold until it eventually started decreasing due to the limited number of proteins with degrees above 20. The second problem is that if we look at the protein in a complex with the most interactions with other proteins in that complex, the majority of its interactions in the Y2H data are not within the complex. Therefore, the strategy of looking for a protein of high degree and taking it and all of its neighbors as a complex seems unlikely to produce meaningful results for finding protein complexes in Y2H data.\n\nNormalized maximum betweenness is also shown in Figure 3. Note that for the panels for maximum betweenness, unlike the others, we report the number of complexes that were less than a given threshold rather than greater than the threshold. Some graphs did not have enough vertices (at least 3 in a connected component) to make a valid measure of betweenness; these were not included in the statistics. Betweenness statistics are not given for unconnected complexes because not all pairs of vertices have paths between them. Traditionally, betweenness has been used as a way to divide the PPI network into functional modules by identifying edges with high betweenness as edges between distinct modules or complexes, so it may seem odd that we are looking at betweenness within a complex. We expect betweenness values to be low, since we expect there to be few if any \"bottleneck nodes\" in the complex that many shortest paths must go through. Although the minimum betweenness was almost always 0, and average betweenness was relatively small, the maximum betweenness varied quite widely, and there were some vertices with very high normalized betweenness. Surprisingly, the maximum betweenness tended to be higher in the real complexes than in the pseudocomplexes.\n\n\nDiscussion\n\nOther binary interaction data sets, such as small-scale experimental data and literature curated interactions, were not used in this study due to the fear that they would be biased in favor of interactions in known complexes. While these interactions would be included in the data set used by an algorithm looking for unknown complexes, they should not be included in an attempt to learn the properties of complexes and what differentiates them from random.\n\nSimilarly, we chose not to use non-binary data such as affinity purification data in this study. While these data again might be used in a complex-finding algorithm, the correct way to translate the data from these non-binary experiments into the binary interactions required by graphs is not completely obvious. The two commonly used methods (clique and spoke) produce very different topological properties, and neither captures well the underlying biology. Therefore, we decided to sidestep the issue by using only binary data. Future studies may include finding a way to use these data.\n\nAs we carried out this analysis, we were always aware of the fact that our data are error-prone. We must keep in mind that the absence of an edge does not mean that there is no interaction. In order to see that we have false negatives, we need only look at the complexes with their interactions determined by X-ray crystallography and compare them to the interactions of those same proteins in the Y2H data (Figure 1 and Figure S1 and Figure S2). Presumably, if all \"real\" interactions had been detected, all of the interactions that we see in the X-ray crystallography studies would be present. False positives are a more difficult matter to detect. Again, if we compare the X-ray crystallography to the Y2H data, we see edges in the Y2H graph that weren't in the X-ray crystallography. However, we cannot simply declare these false positives. It is possible that they truly are false positives. It is also possible that while \"false\" these interactions are significant due to the fact that they appear in the same complex (e.g. we are incorrectly labeling as a neighbor what should actually be the neighbor of a neighbor). Finally, it is possible that these are true interactions that simply do not appear as part of this complex. A recent study suggests that there are many such binary interactions and that the false positive rate for Y2H data is actually much lower than previously believed29.\n\nWhile false positives may cause problems in complex-finding algorithms, our survey suggests that false positives may be less of a problem than false negatives. If we had used a cleaner data set, we would have had fewer false positives but also fewer true positives, and we would have had even more difficulty discerning complexes. Even in the data set we used, complexes often did not stand out when compared to pseudocomplexes.\n\nWhile the errors in the Y2H data are noteworthy, we do not feel that they represent a weakness in our study. To the contrary, a complex-finding algorithm would also be working in this same error-prone data. While it would be interesting to know how a complex would appear in a completely correct network, it is more useful to know how it appears in the data we have.\n\nAnother point about our data worth noting involves the pseudocomplexes used for comparison to represent \"background\" areas of the graph. Because the generating algorithm was trying to find \"complex-like\" subgraphs, some of our \"pseudocomplexes\" may in fact be unknown protein complexes. This would skew our results somewhat, but generally gives a conservative comparison; some unique features of true complexes may not be discovered, but it is less likely that noted differences between true complexes and the set of \"pseudocomplexes\" are spurious.\n\nWe found that edge density may have been overrated as a property of complexes. We found that in Y2H data, the complexes were not particularly clique-like and edge densities were nowhere near as high as most complex-finding algorithms assumed. For example, the algorithm used by King et al.30 looks for complexes with an edge density of at least 0.7 with a minimum number of proteins. If such a technique were applied to Y2H binary interaction data (the data King et al. used included multiple types of interactions, some of which were not binary), our research suggests that such a technique would find all of the proteins involved in a complex for just over a tenth of known complexes with 3 or more distinct proteins. An edge density threshold would find the MHCS of about 60% of known complexes, thus finding at least part of the complex, but this still leaves more than a third of complexes undetected. Also, on average, the edge densities in complexes were only slightly higher than the edge densities in the pseudocomplexes, which suggests that edge density may produce many false positives as well. Therefore, while edge density has a role in complex-finding algorithms, we would be skeptical of methods that purport to find complexes in Y2H data based solely on edge density.\n\nClustering coefficient has not been as popular a parameter for complex-finding algorithms as edge density, but it has long been one of the standard tools used to study the PPI network and its subgraphs. We found that clustering coefficients in real complexes were higher than those from equivalent pseudocomplexes.\n\nMutual clustering coefficient is another statistic that has not been used extensively in complex-finding algorithms, but we believe shows promise. Many complexes had high average mutual clustering coefficients as seen in Figure 2, and pseudocomplexes often have lower mutual clustering coefficients. An additional reason to believe that mutual clustering coefficient may perform well in a complex-finding algorithm is that mutual clustering coefficient considers 4-cycles as well as triangles in its calculation. As mentioned in the results section, we have found that 4-cycles are overrepresented in the Y2H network as a whole, and seem to be even more overrepresented in complexes. Both clustering coefficient and mutual clustering coefficient seem to have a correlation with complexes and would likely have a role in a new complex-finding algorithm.\n\nLooking at maximum degree, we can see that many complexes have at least one protein with interactions with a high percentage of the other proteins in the complex. At the high end, this differentiated complexes from pseudocomplexes. However, we were not able to correlate proteins of high degree with proteins present in known complexes. Also, even among high-degree proteins that were present in complexes, the majority of the neighbors of those proteins were not co-complexes. For these reasons, we are hesitant to recommend degree as an important part of a complex finding algorithm.\n\nBetweenness was one of the statistics that performed the most unexpectedly. Vertices of high betweeenness are usually believed to be vertices that exist between different biological modules. Under that assumption, we would expect all vertices in a complex to have low betweenness. However, when we looked at complexes under this assumption, we found that most complexes had at least one vertex with a higher betweenness than their pseudocomplexes counterparts. Therefore, any algorithm that partitioned the network by looking for high betweenness vertices would run the risk of dividing complexes. It is possible that betweenness could still be used in a complex finding algorithm, but likely not in the way that it has been used traditionally.\n\nThe k-connectivity of complexes, on the other hand, stood out versus the k-connectivities of the pseudocomplexes. Our results were mixed but promising. Most complexes were only 1-connected, but this was due to a small number of degree 1 vertices. When these vertices were removed by the haircut, a 2-connected subgraph usually remained, and many complexes had 3-connected or 4-connected subgraphs. The presence of 3-connected and 4-connected subgraphs is significant; because of the way we generated our pseudocomplexes, they were biased towards including a 2-connected subgraph (the triangle from which the initial edge was selected), but very few had a 3-connected subgraph. Almost none of the pseudocomplexes that were designed to mimic the connected complexes had a 4-connected subgraph.\n\nAnother feature that is noteworthy about k-connectivity is that, while some of the haircut graphs were empty, none of the others had a k-connectivity of 1. Eliminating vertices of degree 1 is not by itself enough to guarantee that a non-empty graph will be at least 2-connected, so this result is significant. It indicates that removing all degree 1 vertices from complexes also eliminates all articulation points, vertices whose removal disconnects the graph, leaving behind a graph where no one vertex can be removed to disconnect the graph.\n\nIt should also be noted that while our results on k-connectivity in the error-prone data were promising, our results in the more accurate X-ray crystallography data were even more so. In the X-ray crystallography data, all complexes had at least a 2-connected subgraph, and the majority of complexes had a 3-connected or 4-connected subgraph. This suggests that as our data become more complete and accurate, highly connected subgraphs will play an even stronger role in searching for complexes.\n\nOur analysis confirms the connection between highly connected subgraphs and protein complexes first suggested by Habibi et al.18. The fact that k-connectivity was shown to be an important indicator of protein complexes in a different type of experimental data than the one used by Habibi et al suggests that the importance of k-connectivity is real and not just an artifact of the data.\n\nIn their paper, Habibi et al.18 present an algorithm for finding complexes based on k-connectivity. We are somewhat skeptical of using vertex connectivity alone as the basis of a complex finding algorithm in Y2H data, however, because subgraphs with these connectivities are too common; it is easy to find 2- or 3-connected graphs of almost any size in the PPI network. Starting with a triangle, it is possible by adding one vertex at a time to build a 3-connected subgraph of any size up to 1689 vertices. Starting with a 4-clique, it is possible to build a 3-connected graph of any size up to 913 vertices. Nevertheless, we feel these vertex connectivity results are significant. The MHCS of graphs representing real complexes were much more highly connected than those of pseudocomplexes, despite our method of generating pseudocomplexes being (perhaps unfairly) biased towards higher k-connectivity, and less biased towards higher edge density. The presence of a highly connected MHCS was one of the statistics that most differentiated real complexes from pseudocomplexes, suggesting that k-connectivity has a role in complex-finding algorithms. The absence of articulation points and the presence of highly connected subgraphs indicates something about the structure of complexes.\n\nWe believe k-connectivity should be used in conjunction with other properties in a complex-finding algorithm. Several other properties examined in this survey, most notably clustering coefficient and mutual clustering coefficient, were also highly correlated with complexes. A complex-finding algorithm based on these data could try to build a 3- or 4-connected subgraph that also had high clustering coefficients and mutual clustering coefficients. Several existing complex-finding algorithms use multiple criteria, such as MCODE (k-core, clustering coefficient, and edge density)7, the algorithm of King et al. (clustering and edge density)30, and the Bayesian network of Qi et al. (multiple properties, including edge density, degree statistics, and clustering coefficients)31.\n\nConnectivity could also be used to evaluate candidate subgraphs produced by other complex-finding algorithms. Subgraphs found by other methods could be examined to find their most highly connected subgraph, with higher confidence scores being given to those with higher k-connectivity values for their most highly connected subgraphs.\n\nFinally, we hypothesize that the most highly connected subgraph of a complex graph may correspond to the \"core\" of a protein-complex as described by Dezso et al.32 and Gavin et al.33. If true, this would imply that k-connectivity could be used in improvements to algorithms that use the core-attachment model34,35.\n\n\nConclusion\n\nBefore designing a new algorithm to find unknown protein complexes in protein interaction data, we must understand the topological properties of known protein complexes. We conducted a principled and comprehensive survey of the topological properties of known protein complexes. We computed vertex k-connectivity, edge density, maximum normalized degree, clustering coefficient, mutual clustering coefficient, triangle (3-cycle) count, 4-cycle count, and betweenness centrality in various graphs representing known protein complexes in the high-throughput Y2H data available for new protein complex discovery. For each known protein complex, we computed these properties in the graph induced by proteins contained in the complex in the Y2H network as well as in the haircut and MHCS subgraphs of these, which are more likely to be discoverable by an automated method. To measure the significance of our results we computed the same properties as we did for the complexes on random \"complex-like\" graphs from the Y2H network.\n\nAlthough the property of edge density has been the most commonly used measure when searching for complexes in the PPI network, we found that it may not be the best graph measure for protein complex discovery. Instead, we found that k-connectivity, clustering coefficient, and mutual clustering coefficient appear to be the most effective measures for differentiating protein complexes from background pseudocomplexes in the pairwise Y2H interaction data. Importantly, our analysis suggests that k-connectivity, a graph metric which has rarely been used in the study of protein networks, would improve algorithms designed to find protein complexes in protein-protein interaction data.",
"appendix": "Author contributions\n\nSRG and DSG designed the experiments and analyzed the results. SRG obtained the data, wrote and ran the code, and drafted the manuscript, with guidance from DSG. DSG edited the manuscript. Both authors read and approved the final manuscript.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSRG and DSG were supported by NSF award DGE-0841423.\n\n\nAcknowledgment\n\nThe authors would like to thank Todd A. Gibson for helpful discussions as well as his assistance in gathering the data and various formatting and technical issues, and Daniel Houck for his help with formatting the graphs and tables. We would also like to thank Larry Hunter, Karin Verspoor and Al Goldberg for their critical reading of the manuscript.\n\n\nSupplementary material\n\nHere we give full results of all statistics on all complexes we studied.\n\nFull results of our survey of iPFam complexes are shown in Table 1. For each of these complexes, we analyzed both the high quality X-ray crystallography data from iPFam as well as the high throughput Y2H data for the proteins in the complex. There were 35 studies in iPFam that involved complexes with at least 3 proteins. Some of these studies were of the same or similar complexes; we grouped studies together if they produced the exact same graph, i.e. the same proteins with the same set of interactions. This grouping gave us 13 distinct graphs. All graphs are illustrated in Figure S1 and Figure S2, along with the subgraphs they induced in the Y2H data. In some cases, it is possible that two different studies of the same complex may have produced different graphs, but we treat all distinct graphs as separate entities.\n\nThe number of proteins (n) and interactions (m), edge density (Edge Dens.), maximum degree (Max Degree), clustering coefficient (CC), average mutual clustering coefficient (Ave MCC), average betweenness (Ave Bet.), and the vertex connectivity of the Most Highly Connected Subgraph (MHCS Connect.) for each iPFam complex. The IDs given are from the RCSB Protein Data Bank. X-ray = complex as determined by X-ray crystallography, Y2H = induced subgraph in yeast 2-hybrid data. The number in parentheses in the m Y2H column is the number of interactions from the X-ray crystallography that also occur in the Y2H network. \"N/A\" means that there were not enough vertices to calculate a given statistic.\n\nIDs are from the RCSB Protein Data Bank.\n\nIDs are from the RCSB Protein Data Bank.\n\n\nReferences\n\nAdamcsek B, Palla G, Farkas I, et al:CFinder: locating cliques and overlapping modules in biological networks. Bioinformatics. 2006; 22: 1021–1023.\n\nCui G, Chen Y, Huang DS, et al:An algorithm for finding functional modules and protein complexes in protein-protein interaction networks. J Biomed Biotechnol. 2008; 860270.\n\nSpirin V, Mirny L: Protein complexes and functional modules in molecular networks. Proc Natl Acad Sci U S A. 2003; 100: 12123–12128.\n\nPrzulj N, Wigle DA, Jurisica I: Functional topology in a network of protein interactions. Bioinformatics. 2004; 20: 340–348.\n\nBu D, Zhao Y, Cai L, et al:Topological structure analysis of the protein-protein interaction network in budding yeast. Nucleic Acids Res. 2003; 31: 2443–2450.\n\nZotenko E, Guimaraes KS, Jothi R, et al:Decomposition of overlapping protein complexes: A graph theoretical method for analyzing static and dynamic protein associations. Algorithms Mol Biol. 2006; 1: 7.\n\nBader GD, Hogue CW: An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics. 2003; 4: 2.\n\nBader GD, Hogue CW: Analyzing yeast protein-protein interaction data obtained from different sources. Nat Biotechnol. 2002; 20: 991–997.\n\nPereira-Leal JB, Enright AJ, Ouzounis CA: Detection of functional modules from protein interaction networks. Proteins. 2004; 54: 49–57.\n\nChu W, Ghahramani Z, Krause R, et al:Identifying protein complexes in high-throughput protein interaction screens using an infinite latent feature model. Pac Symp Biocomput. 2006; 11: 231–242.\n\nLi M, Chen JE, Wang JX, et al:Modifying the DPClus algorithm for identifying protein complexes based on new topological structures. BMC Bioinformatics. 2008; 9: 398.\n\nRavasz E, Somera AL, Mongru DA, et al:Hierarchical organization of modularity in metabolic networks. Science. 2002; 297: 1551–1555.\n\nGoldberg DS, Roth FP: Assessing experimentally derived interactions in a small world. Proc Natl Acad Sci U S A. 2003; 100: 4372–4376.\n\nGirvan M, Newman ME: Community structure in social and biological networks. Proc Natl Acad Sci U S A. 2002; 99: 7821–7826.\n\ndel Sol A, O'Meara P: Small-world network approach to identify key residues in protein-protein interaction. Proteins. 2005; 58: 672–682.\n\nJoy MP, Brock A, Ingber DE, et al:High-betweenness proteins in the yeast protein interaction network. J Biomed Biotechnol. 2005; 96–103.\n\nChen J, Yuan B: Detecting functional modules in the yeast protein-protein interaction network. Bioinformatics. 2006; 22: 2283–2290.\n\nHabibi M, Eslahchi C, Wong L: Protein complex prediction based on k-connected subgraphs in protein interaction network. BMC Syst Biol. 2010; 4: 129.\n\nHartuv E, Shamir R: A clustering algorithm based on graph connectivity. Inf Process Lett. 2000; 76(4–6): 175–181.\n\nFinn RD, Marshall M, Bateman A: ipfam: visualization of protein-protein interactions in pdb at domain and amino acid resolutions. Bioinfomatics. 2005; 21: 410–412.\n\nMewes HW, Frishman D, Mayer KF, et al:Mips: analysis and annotation of proteins from whole genomes in 2005. Nucleic Acids Res. 2006; 34: D169–D172.\n\nStark C, Breitkreutz BJ, Reguly T, et al:Biogrid: a general repository for interaction datasets. Nucleic Acids Res. 2006; 34: D535–D539.\n\nTarassov K, Messier V, Landry CR, et al:An in vivo map of the yeast protein interactome. Science. 2008; 320: 1465–1470.\n\nYu H, Braun P, Yildirim MA, et al:High-quality binary protein interaction map of the yeast interactome network. Science. 2008; 322: 104–110.\n\nMilo R, Kashtan N, Itzkovitz S, et al:On the uniform generation of random graphs with prescribed degree sequences.2004.\n\nRoberts J: Simple methods for simulating sociomatrices with given marginal totals. Soc Networks. 2000; 22: 273–283.\n\nPrzulj N, Corneil DG, Jurisica I: Modeling interactome: scale-free or geometric?. Bioinformatics. 2004; 20: 3508–3515.\n\nGiot L, Bader JS, Brouwer C, et al:A protein interaction map of Drosophila melanogaster. Science. 2003; 302: 1727–1736.\n\nBraun P, Tasan M, Dreze M, et al:An experimentally derived confidence score for binary protein-protein interactions. Nat Methods. 2009; 6: 91–97.\n\nKing AD, Przulj N, Jurisica I: Protein complex prediction via cost-based clustering. Bioinformatics. 2004; 20: 3013–3020.\n\nQi Y, Balem F, Faloutsos C, et al:Protein complex identification by supervised graph local clustering. Bioinformatics. 2008; 24: I250–I258.\n\nDezso Z, Oltvai ZN, Barabasi AL: Bioinformatics analysis of experimentally determined protein complexes in the yeast Saccharomyces cerevisiae. Genome Res. 2003; 13: 2450–2454.\n\nGavin AC, Aloy P, Grandi P, et al:Proteome survey reveals modularity of the yeast cell machinery. Nature. 2006; 440: 631–636.\n\nLeung HC, Xiang Q, Yiu SM, et al:Predicting Protein Complexes from PPI Data: A Core-Attachment Approach. J Comput Biol. 2009; 16: 133–144.\n\nWu M, Li X, Kwoh CK, et al:A core-attachment based method to detect protein complexes in PPI networks. BMC Bioinformatics. 2009; 10: 169."
}
|
[
{
"id": "1873",
"date": "24 Oct 2013",
"name": "Lin Gao",
"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 promising paper. I think the idea is sound, has some novelty, and can potentially improve previous results. The explanations of the figures are of particular note. In addition, the authors could try a larger benchmark complex dataset (CYC2008 category).",
"responses": [
{
"c_id": "1484",
"date": "09 Oct 2015",
"name": "Debra Goldberg",
"role": "Reader Comment",
"response": "Thank you for reading and reviewing our paper, and we are sorry that we have taken so long to respond. Thank you also for mentioning the CYC2008 set of complexes. Unfortunately, we are currently unable to redo this analysis using this set of complexes. However, we are utilizing it in our current research on complexes and complex-finding algorithms."
}
]
},
{
"id": "3973",
"date": "05 Mar 2014",
"name": "Nassim Sohaee",
"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 paper is a survey of the topological properties of known protein complexes and is mostly focused on k-connected subgraphs as a good candidate for predicting protein complexes in PPI networks. K-connected subgraphs have been previously proposed as a good alternative for predicting less dense protein complexes. I have the following comments about this paper:The idea of using k-connected subgraphs to predict protein complexes has already been investigated by other researchers. The authors should therefore clearly point out what is new about their concept and how it differs from earlier published methods. According to Sharan et al. (2007) the functional similarity of protein pairs will decrease as the distance of the path connecting them increases. Hence, a large k value results in a less functional correlation among proteins. Specifically, for k>= 3, functional similarity significantly decreases and for k<3, or k=1 or 2, the k-connected graph is dense. As this has already been investigated by others the authors need to clearly state what makes their method significant.",
"responses": [
{
"c_id": "1483",
"date": "09 Oct 2015",
"name": "Debra Goldberg",
"role": "Reader Comment",
"response": "Thank you for taking the time to read our paper, and for your comments. We have addressed some of these points below, and updated our manuscript to reflect your concerns.\"The idea of using k-connected subgraphs to predict protein complexes has already been investigated by other researchers. The authors should therefore clearly point out what is new about their concept and how it differs from earlier published methods.\"There are four major difference between our study and the Habibi study that previously examined k-connectivity. We have updated our Background section to clarify these points.First, we looked at a number of complexes using low-throughput X-ray crystallography data, something Habibi et al. did not do. Looking at complexes in the X-ray crystallography allows us to study the true topology of interactions in complexes and see the properties complexes might have in a complete and accurate interaction network and also suggests that k-connectivity may be an innate property of the interactions within a protein complex rather than an artifact of any particular type of data.Second, we use a different type of interaction data. Habibi et al. used mass spectrometry data, while we were interested in looking at k-connectivity in Y2H pairwise interaction data. We felt that the pairwise interaction data had two advantages over the mass-spectrometry data: it is not biased towards interactions in complexes, and it is truly binary, avoiding problems that come from trying to represent the non-binary mass-spectrometry data in a binary graph. The fact that the interaction data we used is of a very different type from that used by Habibi allows us to, as we mention in our Discussion, confirm that the importance of k-connectivity in indicating complexes is real and not just an artifact of one particular type of data.Third, we examine other statistics in addition to edge density and k-connectivity and analyze the performance of these statistics at determining complexes. This allows us to determine how k-connectivity might perform as a complex-finding statistic compared not only to edge density, but to other possible statistics as well. We list the full statistics we are using in the Methods, give results on them, and evaluate their performance in the Discussion.Finally, in addition to simply examining these statistics in complexes, we also looked at them in \"pseudocomplexes,\" background pieces of the PPI network designed to be \"complex-like.\" By comparing k-connectivity and other statistics in real complexes and pseudocomplexes, we were able to give further evidence these statistics may be useful in distinguishing true complexes from others.\"According to Sharan et al. (2007) the functional similarity of protein pairs will decrease as the distance of the path connecting them increases. Hence, a large k value results in a less functional correlation among proteins. Specifically, for k>= 3, functional similarity significantly decreases and for k<3, or k=1 or 2, the k-connected graph is dense. As this has already been investigated by others the authors need to clearly state what makes their method significant.\"In k-connectivity, k refers to the number of paths rather than the distance of said paths. A graph being k-connected for a high value of k does not imply that the graph has a long shortest path between any two vertices (diameter), even if the edge density of the graph is not high. Below we describe a 4-edge-connected, 13-vertex graph with edge density of 1/3 that has a shortest path between any two vertices no longer than 2, well within the range that Sharan et al. suggested could have significant functional similarity. To construct this example, number the vertices 0-12 and connect vertex n to vertices n+1, n-1, n+5, and n-5 (mod 13). We also have an example of a 4-vertex-connected graph with diameter 2 and edge density 2/7, but this is harder to describe in a paragraph."
}
]
}
] | 1
|
https://f1000research.com/articles/2-172
|
https://f1000research.com/articles/4-1031/v1
|
09 Oct 15
|
{
"type": "Method Article",
"title": "AnalogExplorer2 – Stereochemistry sensitive graphical analysis of large analog series",
"authors": [
"Ye Hu",
"Bijun Zhang",
"Martin Vogt",
"Jürgen Bajorath",
"Ye Hu",
"Bijun Zhang",
"Martin Vogt"
],
"abstract": "AnalogExplorer is a computational methodology for the extraction and organization of series of structural analogs from compound data sets and their graphical analysis. The method is suitable for the analysis of large analog series originating from lead optimization programs. Herein we report AnalogExplorer2 designed to explicitly take stereochemical information during graphical analysis into account and describe a freely available deposition of the original AnalogExplorer program, AnalogExplorer2, and exemplary compound sets to illustrate their use.",
"keywords": [
"Medicinal chemistry",
"analog series",
"computational design",
"graphical analysis",
"structure-activity relationships",
"open access software"
],
"content": "Introduction\n\nIn medicinal chemistry, analog series are typically analyzed in R-group tables. Once analog series become so large that they are difficult to represent and study in conventional R-group tables, computational tools are indispensable for their exploration. Therefore, different computational methods have been introduced for graphical analysis of analog series1–9. Many of these approaches are based on the determination of maximum common substructures (MCS) of compound series and focus on substituents of common cores, while others employ the matched molecular pair10 formalism to define analog series. Among these computational methods is AnalogExplorer9, which has been designed to systematically organize and graphically analyze analog series and associated structure-activity relationship (SAR) information. AnalogExplorer initially identifies analog series on the basis of hierarchical molecular scaffolds11 and then determines their MCS for further analysis. Accordingly, the method is not limited to the study of individual analog series but can also be applied to extract series from structurally heterogeneous compound sets. For example, AnalogExplorer is directly applicable to late-stage lead optimization sets that often contain multiple series with large numbers of analogs.\n\nHerein we introduce AnalogExplorer2, an extension of the approach, which explicitly considers stereoisomers during graphical analysis, providing a detailed account of stereochemistry and its influence on SARs. AnalogExplorer2 is publicly available. Hence, we also report an open access deposition of the original AnalogExplorer program and AnalogExplorer2 as well as exemplary data sets assembled to illustrate the workflow of graphical analysis and help users become familiar with the program12.\n\n\nMethodology\n\nAnalogExplorer systematically determines substitution sites or site combinations in analog series and divides series into subsets having varying R-groups at the same site(s). Analog series are initially identified on the basis of hierarchical molecular scaffolds11. For a series of analogs sharing the same scaffold, the maximum common substructure (MCS) is then determined, as illustrated in Figure 1, and used for R-group decomposition in order to index and identify all substitution sites and the respective R-groups for each compound in a series.\n\nShown are six structurally analogous compounds represented by their MCS (right) with five substitution sites (R1–R5). For each compound, the corresponding substituents are highlighted in blue.\n\nOn the basis of the MCS, an analog series is divided into subsets of compounds with varying R-groups at the same substitution site or site combination. The organization is compound-based such that each member of a series only occurs in one subset. Unique compound subsets provide the basis for graphical analysis, as discussed in the following. Further methodological details are provided in the original AnalogExplorer reference9.\n\nAnalogExplorer consists of three graphical components. The complete graph (Figure 2a) captures all possible substitution sites and site combinations for a series following R-group decomposition (by design it is a directed acyclic graph). Each node represents a substitution site or site combination and all compounds with varying R-groups at the site(s). The root node 0 corresponds to a (hypothetical) compound with no R-group at any site. Node 1 represents analogs that only contain R-groups at R1 and node 12 compounds with R-groups at R1 and R2 etc. Nodes are arranged in different layers. For example, layer 1 consists of all nodes with one substitution site and layer 2 of all nodes with two sites. Edges between nodes in adjacent layers indicate all possible subset relationships, i.e. an edge is drawn if the substitution site(s) represented by a node is a subset of a site combination of another node. As indicated in Figure 2a, nodes are scaled in size according to the number of analogs comprising the subset they represent and color-coded according to the mean potency of the analogs. In addition, node border thickness indicates the potency range covered by a subset. Furthermore, white (empty) nodes correspond to possible site combinations for which no analogs are currently available within a given series. In the reduced graph, all empty nodes and connecting edges are removed for clarity (Figure 2b). Thus, the reduced graph provides a convenient format for the analysis of individual series. As the third graphical component, R-group trees are provided for each substitution site and site combination, as illustrated in Figure 2c. In the R-group tree, substitution sites for a given subset are arranged in different layers, the order of which is determined by the number of unique R-groups at each site. All R-groups are displayed in the tree. Each leaf node represents an analog (colored according to its potency). Intermediate nodes represent subsets of analogs sharing the same substituents at corresponding site(s) (and are colored by mean analog potency).\n\n(a) Shown is the complete graph for a series of 25 analogs active against serotonin 7 receptor. (b) The reduced graph is displayed obtained from (a) by removing all empty nodes and edges between them. (c) The R-group tree for substitution site combination 1345 is shown. All R-groups are provided for individual tree nodes. Stereoisomers and their corresponding pKi values are given at the bottom. Abbreviation: OoM, order of magnitude.\n\nGiven its design, AnalogExplorer provides a systematic hierarchical organization of all possible substitution sites or site combinations for an analog series (complete graph) and enables the elucidation of SAR patterns within the hierarchy (reduced graph) and at further increased resolution for analog subsets (R-group trees). The approach is particularly suitable for the analysis of large analog series because subsets of such series associated with interesting SAR information can be selectively displayed and analyzed.\n\nThe explicit consideration of stereochemistry during graphical analysis at the level of R-group trees is the major methodological enhancement of AnalogExplorer2 (in addition to further increased consistency of compound mapping to MCS considering intra-molecular symmetry). In the original R-group tree structure, nodes located in the same layer and originated from the same parent node are associated with distinct R-groups. Therefore, stereoisomers having the same substituents are combined into a single leaf node. Hence if a terminal node is associated with more than one compound, stereoisomers are present. In AnalogExplorer2, stereoisomers are explicitly considered, as illustrated in Figure 2c. Each stereoisomer is represented by a single node and stereoisomers belonging to the same subset (i.e. compounds with different stereochemistry at the same site) are identified by a unique index (i.e. ‘1’ for the three stereoisomers in Figure 2c). If different subsets of stereoisomers are present in an R-group tree, incremental indices are used to identify and distinguish them (i.e. ‘1’, ‘2’ etc.).\n\nRoutines for scaffold, analog, and MCS identification, R-group decomposition, and indexing of substitution sites are implemented in Java using the OpenEye OEChem toolkit version 2.0.2 (Open Eye Scientific Software; http://www.eyesopen.com). Therefore, this toolkit is required to execute the program. All graphical components of AnalogExplorer and AnalogExplorer2 are implemented using the open source Java package JUNG version 2.0.1 (http://jung.sourceforge.net/). Potential inconsistencies with subsequent versions of OEChem or JUNG can be avoided by using the specified versions.\n\nThe executable program utilizes standard SD files as input and generates complete or reduced graphs for all or individual series, depending on the user’s preference. The initial graph layout is produced by the DAGLayout algorithm of JUNG (http://jung.sourceforge.net/) and usually interactively modified for graphical analysis. The number of compounds assigned to each node and their mean potency can be viewed by navigating the graph. R-group trees representing compound subsets are generated together with the complete or reduced graph. In each R-group tree, the substituents associated with individual nodes, compounds (leaf nodes), and corresponding potency values can also be viewed. Subsets of stereoisomers, if available, are depicted using numerical indices, as discussed. Furthermore, an output file is generated reporting compounds belonging to individual subsets.\n\n\nExemplary applications\n\nAnalogExplorer2 can be used for different types of SAR analysis, as illustrated by a few exemplary applications. Compound data were taken from ChEMBL13 version 20. Figure 3 displays the reduced graph for a series of 45 alpha-1a adrenergic receptor ligands with a total of seven substitution sites and the R-group tree for an exemplary three-site combination. The tree reveals a clear SAR pattern (with increasing potency of analogs from the bottom left to the right) and identifies six (uniquely indexed) pairs of stereoisomers among these analogs. Figure 4 provides a corresponding representation for a series of 64 matrix metalloproteinase 9 inhibitors and compares R-group trees for three substitution sites. It becomes apparent that substituents attached to R4 alone or in combination with other sites consistently yield compounds having only low potency. In Figure 5, target-specific reduced graphs are compared for a series of 32 analogs with a total of nine substitution sites and activity against two dipeptidyl peptidases (DPP4 and DPP8). The graphs generated for the same analog series display different compound potency distributions, reflecting a selectivity tendency for DPP4 over DPP8. Figure 6 shows reduced graphs for six different analog series with activity against the same kinase. The graphs reveal different structural content and a different degree of chemical exploration among these series as well as differences in the SAR information they provide. In Figure 7, two of these series are combined into a new single series by re-calculating the MCS that comprises 43 analogs with a total of eight substitution sites. The reduced graph captures the structural organization and activity information of this combined series.\n\nAt the top, the MCS for a series of 45 analogs active against alpha-1a adrenergic receptor is shown. In addition, the corresponding reduced graph (middle, left) and R-group tree for substitution site combination 247 (middle, right) are displayed. At the bottom, six pairs of stereoisomers are shown. For each compound, its pKi value is given.\n\nShown is the reduced graph for a series of 64 analogous inhibitors of matrix metalloproteinase 9. R-group trees of three substitution sites (nodes 3, 4 and 5; dashed box) are shown on the right. For each R-group tree, substituents are provided.\n\nShown are reduced graphs for analog series with inhibitory activity against dipeptidyl peptidase IV (DPP4; left) and VIII (DPP8; right). Three representative compounds associated with node 17 (dashed box) are shown at the bottom. For each compound, the potency value (pIC50) for DPP4 and DPP8 is reported in a green and red box, respectively.\n\nShown are reduced graphs for six different series of PIM2 kinase inhibitors (capital letters A–F represent series identifiers). For each series, corresponding MCS and reduced graph are shown.\n\nSeries A and E from Figure 6 are combined into a single series by determining their MCS yielding eight substitution sites (left). The reduced graph of the combined series is shown (right) that consists of 43 analogs.\n\n\nSoftware and data availability\n\nThe following tools and data sets are made publicly available without restrictions via a deposition on the ZENODO open access platform12. Three executable files of the original AnalogExplorer program are provided for different applications including the analysis of multiple analog series from a given compound set, analysis of an individual series, and selectivity analysis (according to Figure 5). With the exception of the OpenEye OEChem library, jar files of the required external libraries are also provided. In addition, all compound sets analyzed in the original publication9 are deposited. These compound sets were taken from ChEMBL version 18. Furthermore, three executable files are made available for AnalogExplorer2 (for multiple analog series, individual series, and selectivity analysis) as well as the compound sets for which graph representations are reported herein. These compounds were taken from ChEMBL version 20. A “readme” document with detailed explanations is also provided as a part of the deposition.\n\n\nConclusions\n\nThe AnalogExplorer method was designed for the systematic organization and graphical analysis of large series of analogs, which frequently originate from lead optimization efforts. Herein, an extension of the methodology has been introduced. AnalogExplorer2 explicitly accounts for all stereoisomers during graphical analysis and SAR exploration. The AnalogExplorer2 program is made freely available to the scientific community.",
"appendix": "Author contributions\n\n\n\nJB conceived the study, YH designed the stereo sensitive extension of AnalogExplorer, BZ carried out the implementation, and MV reviewed the programs. JB and YH wrote the manuscript, and 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 authors declared that no grants were involved in supporting this work. BZ is supported by the China Scholarship Council. The use of OpenEye’s OEChem Toolkit was made possible by their free academic licensing program.\n\n\nAcknowledgements\n\nMany thanks to Pat Walters for inspiring the extension of AnalogExplorer to explicitly account for stereoisomers in R-group trees.\n\n\nReferences\n\nAgrafiotis DK, Shemanarev M, Connolly PJ, et al.: SAR maps: a new SAR visualization technique for medicinal chemists. J Med Chem. 2007; 50(24): 5926–5937. PubMed Abstract | Publisher Full Text\n\nKolpak J, Connolly PJ, Lobanov VS, et al.: Enhanced SAR maps: expanding the data rendering capabilities of a popular medicinal chemistry tool. J Chem Inf Model. 2009; 49(10): 2221–2230. 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\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\nAgrafiotis DK, Wiener JJ, Skalkin A, et al.: Single R-Group Polymorphisms (SRPs) and R-cliffs: an intuitive framework for analyzing and visualizing activity cliffs in a single analog series. J Chem Inf Model. 2011; 51(5): 1122–1131. PubMed Abstract | Publisher Full Text\n\nWassermann AM, Bajorath J: Directed R-group combination graph: a methodology to uncover structure-activity relationship patterns in a series of analogues. J Med Chem. 2012; 55(3): 1215–1226. PubMed Abstract | Publisher Full Text\n\nWassermann AM, Haebel P, Weskamp N, et al.: SAR matrices: automated extraction of information-rich SAR tables from large compound data sets. J Chem Inf Model. 2012; 52(7): 1769–1776. PubMed Abstract | Publisher Full Text\n\nGupta-Ostermann D, Bajorath J: The ‘SAR matrix’ method and its extensions for applications in medicinal chemistry and chemogenomics [v2; ref status: indexed, http://f1000r.es/3rg]. F1000Res. 2014; 3: 113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang B, Hu Y, Bajorath J: AnalogExplorer: a new method for graphical analysis of analog series and associated structure-activity relationship information. J Med Chem. 2014; 57(21): 9184–9194. PubMed Abstract | Publisher Full Text\n\nKenny PW, Sadowski J: Structure Modification in Chemical Databases. In Chemoinformatics in Drug Discovery. Oprea TI, (Ed.), Wiley-VCH, Weinheim, Germany, 2005; 271–285. Publisher Full Text\n\nBemis GW, Murcko MA: The properties of known drugs. 1. Molecular frameworks. J Med Chem. 1996; 39(15): 2887–2893. PubMed Abstract | Publisher Full Text\n\nHu Y, Zhang B, Vogt M, et al.: AnalogExplorer and AnalogExplorer2. ZENODO. 2015. Data Source\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 | Free Full Text"
}
|
[
{
"id": "10815",
"date": "15 Oct 2015",
"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\nAnalysis of diverse molecular datasets is one of the most common tasks a computational chemist in pharmaceutical industry, but also in academia is facing daily. The tool presented in this manuscript – the AnalogExplorer2 supports this process and makes it easier. Enhancement to previously published AnalogExplorer presented here, namely support for handling of stereoisomers is indeed important, since in large number of complex SAR datasets molecules with the same constitution but different stereochemistry are present. And in some cases differences in bioactivity for different stereoisomers are significant.Several examples shown in the manuscript illustrate nicely the powerful visualization capabilities of this tool, including maximum common substructure visualization, substituent tree visualization and selectivity analysis. These visualizations allow to understand the SAR even for the quite complex datasets.The authors made the executable version of the tool freely available for download. This is a good news. But since the tool is based on the OEChem library only users with a valid OEChem license would be able to use it. My recommendation for the future versions of AnalogExplorer would be to make the interface to its cheminformatics processing modular, so it would be possible to plug the tool also to open source cheminformatics toolkits like RDKit or CDK and make in this way the AnalogExplorer a truly free tool for graphical analysis of complex molecular datasets.",
"responses": []
},
{
"id": "11071",
"date": "04 Nov 2015",
"name": "Carleton Sage",
"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 extension to AnalogExplorer incorporating stereochemistry, and important extension of the method. This method of analysing complicated compound data sets is sophisticated and easy to use.\n\nThe approach to communicating results is intuitive both from the perspective of the trends in the experimental data with respect to the compounds that have been made, but also, in complete graph view, provide insight into important regions of the dataset that have not yet been investigated. This tool will be very useful in the exploration of medicinal chemistry datasets in both industry and academia.\n\nI have downloaded and run the software on a windows workstation using both the provided datasets and internally generated ones – the software works as described with two minor issues cropped up. First, the software depends not only on the OEChem Toolkit, but also requires OEDepict (both licenses are available from Openeye Scientific). Second, the software depends specifically on the precise OEChem Toolkit java jar file (oejava-2014.Oct.2-Windows-x64.jar) – it will fail without the correct jar file. On balance AnalogExplorer2 is a very useful tool for exploring compound data sets. It could be made more widely useful if it were to be adapted to run on an open source chemical toolkit such as RDKit. Nonetheless, I commend the authors for making their software available to the research community.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1031
|
https://f1000research.com/articles/4-68/v1
|
13 Mar 15
|
{
"type": "Research Note",
"title": "Biomedical publications on Ebola and the 2014 outbreak",
"authors": [
"Andrea Ballabeni",
"Andrea Boggio",
"Andrea Boggio"
],
"abstract": "In this research note we examine the biomedical publication output about Ebola in 2014. We show that the volume of publications has dramatically increased in the past year. The rise reflects an impressive growth starting in the month of August, concomitant with or following the surge in infections, deaths and coverage in news and social media. Though non-research articles have been the major contributors to this growth, there has been a substantial increase in original research articles too, including many papers of basic science. The United States has been the country with the highest number of research articles, followed by Canada and the United Kingdom. We present a comprehensive set of charts and facts that, by describing the volumes and nature of publications in 2014, show how the scientific community has responded to the Ebola outbreak and how it might respond to future similar global threats and media events. This information will assist scholars and policymakers in their efforts to improve scientific research policies with the goal of maximizing both public health and knowledge advancement.",
"keywords": [
"Ebola",
"publications",
"Pubmed"
],
"content": "Introduction\n\nThe Ebola outbreak that has affected West Africa for over a year has captured the attention of audiences throughout the world. Much has been said and written about this tragic event in the popular media as well as in scientific journals1–11. The scientific community has certainly vigorously debated about the Ebola outbreak. However, no study has so far assessed the impact of the current African epidemic on scientific production. In this research note we try to fill this gap by examining the volume and nature of the biomedical publication output about Ebola in 2014. We believe this study is important as it may lead to insights as to how the global scientific and health communities react to these kinds of events. The note looks at general trends in Ebola-related publications, at the type of papers that were published and at the extent to which countries have contributed to the Ebola literature. This information will be useful to researchers and policymakers aspiring to increase the impact of scientists’ work on public health as well as on the pure advancement of knowledge.\n\n\nMethods and data\n\nAll searches were performed in PubMed (http://www.ncbi.nlm.nih.gov/pubmed/), a public search engine maintained by the United States National Library of Medicine (NLM) at the National Institutes of Health (NIH) that provides access to over 24 million citations in all fields of life sciences, mostly from the MEDLINE (Medical Literature Analysis and retrieval System Online) database. To calculate the number of publications on Ebola in 2014, we ran a search by using the ‘custom range’ function set to 2014 publications and searched for ‘ebola OR ebolavirus’ in ‘all fields’, in ‘title/abstract’ field or in ‘title’ field. We retrieved, respectively, 945, 900 and 797 citations (Figure S1) (sheet 1). We also ran the search using the ‘MeSH major topic’ filter and retrieved 945 citations (sheet 1). Upon reviewing the results, we noticed that some citations retrieved when searching only in ‘all fields’ or ‘title/abstract’ or by using the ‘MeSH’ filter were not publications discussing some specific aspect of Ebola; for this reason, we decided to only use the 797 citations retrieved with searches in the title. Throughout the paper we will therefore not specify the search field and use title as the default option. We also searched for ‘ebola’ and ‘ebolavirus’ separately. We retrieved 778 and 19 citations, respectively (Figure S2) (sheet 1). Given that both ‘ebola’ and ‘ebolavirus’ were used in publications titles, we decided henceforth to use ‘ebola OR ebolavirus’ as the default search term.\n\nTo determine recent trends on publications, we looked at the publications record on Ebola for the past 20 years (from 1995 to 2014). Using the ‘custom range’ function, we retrieved a total of 1869 citations. A year-by-year analysis shows that while the publication output did not differ substantially from 1995 to 2013, there was a staggering increase in publication output in 2014 as evidenced by the fact that 42.6% of all the papers published during the time period 1995–2014 were published in the last year alone (Figure S3) (sheet 2).\n\nWe then determined the number of publications with an abstract available. As research articles or research notes usually have an abstract but editorials, commentaries, short letters and several other types of publication usually do not, the publications with an abstract better reflect the real output in original research papers. We observed again a remarkable increase in the year 2014, which had 23.3% of all publications of the last 20 years. In particular, the publications with an abstract available in 2014 were almost 5 times the publications with an abstract available in 2013 (Figure S4) (sheet 2). Interestingly, the proportion of publications with an available abstract in 2014 was considerably lower than in all the previous years: for example, while in 2013 the proportion was 92.4%, in 2014 it was only 32.9% (Figure S5) (sheet 2). Given that an abstract, when present, is usually made available at the same time the article appears in PubMed, these results indicate that the majority of publications in 2014, differently from the previous years, were non-research papers.\n\nTo determine the article types of 2014 Ebola publications we first used the ‘article types’ filters of PubMed. ‘Comments’ were 4.3%, ‘editorials’ 11.0%, ‘interviews’ 1.1%, ‘journal articles’ 57.5%, ‘letters’ 10.4%, ‘news’ 20.5% and ‘reviews’ 2.0% (Figure S6) (sheet 3) (note that the sum of percentages is more than 100% because PubMed can assign multiple classifications to the same article). Even if these filters lead to results that may not always be corresponding to the type of items that appears in the database (not all publications are assigned to a PubMed ‘article type’), these numbers indicate the presence of many citations that were not original research articles. By searching for only abstract available citations, we instead obtained very different numbers. ‘Comments’ were 0.8%, ‘editorials’ 0.8%, ‘interviews’ 0.4%, ‘journal articles’ 96.6%, ‘letters’ 0%, ‘news’ 2.3% and ‘reviews’ 5.7% (Figure S7) (sheet 3). These numbers show, as expected, that the vast majority of publications with an abstract available did not belong to the categories of short articles like editorials but instead belonged to the ‘journal article’ category.\n\nWe then examined three other features: the language, full text availability and the citations in PubMed Commons. By using the English language filter, we found that 94.9% of the publications were written in English. By using the ‘abstract available’, the ‘full text’ and the ‘free full text’ filters we determined that 32.9% of publications had the abstract available, 94.1% had the full text available (either for a fee or at no charge) and 25.7% had the full text available at no charge. Only 0.4% of the publications had comments in PubMed Commons (Figure S8) (sheet 3). By repeating the same searches with publications with an abstract available, we observed that 96.2% of the articles were written in English, 100% (as obvious) had an abstract available, 93.5% had the full text available (either for a fee or at no charge) and 42.7% had the full text available at no charge. Again only 0.4% had comments in PubMed Commons (Figure S9) (sheet 3).\n\nWe also analyzed the language, text availability and PubMed Commons citations only for the ‘journal articles’. 94.8% of these citations were written in English, 55.2% had an abstract available, 93.2% had the full text available (either for a fee or at no charge) and 35.6% had the full text available at no charge. Only 0.7% had comments in PubMed Commons (Figure S10) (sheet 3).\n\nThese results show that most of the publications on Ebola in 2014 did not have an abstract and therefore can be safely classified as non-research papers. Moreover they show that, similarly to the case with biomedical publications in general, almost all the citations had a full text available but only in less than half of the cases the text is freely available at present. These data also show that while 96.6% of the publications with an abstract available were ‘journal articles’, only 55.2% of the ‘journal articles’ had an abstract available (Figure S11) (sheet 3). Based on this evidence we conclude that abstract availability is most likely a better criterion than the ‘journal article’ to estimate which publications are original research papers.\n\nTo analyze the particular focus of the retrieved papers, we searched for specific terms in the title/abstract field. We first searched for terms related to cell and molecular biology like ‘cell’, ‘molecule’, ‘protein’, ‘antibody’, ‘gene’ and ‘genome’. We also searched for the terms ‘drug’ and ‘vaccine’. All these terms were searched for both the singular and the plural form. The proportions of papers with these terms ranged from 2.0% (for the term ‘gene’) to 9.7% (for the term ‘vaccine’). The term ‘drug’ appeared in 4.5% of the papers (Figure S12) (sheet 4). We then repeated the analysis with terms more specifically related to the 2014 West Africa outbreak. In particular, we searched for ‘outbreak’, ‘africa’, ‘liberia’, ‘sierra leone’, ‘guinea’, ‘nigeria’, ‘preparedness’. These terms were present in 21.5%, 22.0%, 7.4%, 7.9%, 7.5%, 2.8% and 4.5% of the papers, respectively (Figure S13) (sheet 4).\n\nWe then repeated the analyses by taking in consideration only the publications with an abstract available. As expected, the proportions of papers with these terms were higher. The terms ‘drug’ and ‘vaccine’ were in the 6.5% and 20.6% of the publications, respectively (Figure S14) (sheet 4). The terms ‘outbreak’, ‘africa’, ‘liberia’, ‘sierra leone’, ‘guinea’, ‘nigeria’, ‘preparedness’ were in 45.4%, 46.2%, 19.8%, 19.8%, 21.4%, 7.6% and 9.2% of the publications with an abstract available, respectively (Figure S15) (sheet 4). As the word Africa was present in almost half of the papers with an abstract available, these results suggest, as expected, that the sharp increase in publications in 2014 was driven mostly by the West Africa outbreak. This was also confirmed by the fact that the years 2012 and 2013 had much lower proportions of papers containing the term ‘Africa’ in the title/abstract (in particular, by performing whole year analyses, we retrieved 9 papers in 2012, 4 in 2013 and 187 in 2014; with regard to publications with an abstract available, 7 papers in 2012, 4 in 2013 and 132 in 2014). Liberia, Sierra Leone and Guinea, the three countries with the most cases of infections, were present in the title or abstract with very similar proportions near to 20% whereas Nigeria, the fourth most affected country but much less affected than the first three, was present in title or abstract of less than 10% of the publications.\n\nTo obtain the numbers of publications of clinical trials, we used the ‘clinical trial’ filter of PubMed. However, the search led to zero result: no 2014 publication on Ebola was tagged as clinical trial publication. However, this filter of PubMed may not be totally accurate because some clinical trial studies may not be assigned to the ‘clinical trial’ category. Therefore, we also searched for the term ‘clinical trial’ in the title/abstract field. We retrieved 4 publications. Among these, 3 had an abstract available. As the presence of the search term in the title/abstract does not imply that the publication is in fact about a clinical trial, we examined the content of the 4 retrieved publications. We observed that only 2 publications were actually reports of an original clinical trial (Figure S16) (sheet 4).\n\nWe also analyzed the publication trends within 2014. To this purpose we calculated the volumes of publications for each month of 2014 by using the ‘custom range’ function (for January, we did not include January 1st as this would have included a few undetermined publications). In the case of papers published online before the print version, we considered the date of the online publication. As shown in Figure S17 (sheet 5), the vast majority of publications were published after the month of August, with a peak in the month of October and a plateau-like pattern during the last three months of the year. We performed the same analysis also with the publications with an abstract available. We obtained very similar results, with a peak in the months of October and November (Figure S18) (sheet 5). These results indicate that the increase in publications (both research and non-research papers) only started at the end of the summer of 2014, concomitant with the surge in infection and death cases and more intense reports on part of the mass media.\n\nWe further investigated the focus of the 2014 publication output by performing a subject-matter analysis. By looking at the title and the abstract, we determined the proportion of papers that had a relevant focus towards one or more aspects of the current 2014 outbreak. We found out that a high proportion (40.5%) of all citations had focused on some aspects of the West Africa epidemic (Figure S19) (sheet 5). Most of these publications were reports of the epidemic or articles about preparedness or the international response to the crisis. With regard to the other publications, we assigned them to one of the following categories: ‘cell/molecular biology’, ‘drugs/antibodies/vaccines’, ‘pathophysiology/epidemiology/ecology’, ‘preparedness’, ‘society/policy/ethics’ or ‘other’. Even if many papers could be assigned to more than one category, each paper was assigned only to the most applicable category. All papers focusing on drugs/antibodies/vaccines were assigned to the ‘drugs/antibodies/vaccines’ even if many of these could belong also to the ‘cell/molecular biology’ category. We observed that the category with more hits was ‘preparedness’ with 35.1% of the papers, followed by ‘pathophysiology/epidemiology/ecology’ (18.8%), ‘drugs/antibodies/vaccines’ (18.0%), ‘society/policy/ethics’ (15.0%), ‘cell/molecular biology’ (10.0%) and ‘other’ (3.2%) (Figure S19) (sheet 5). We also analyzed the distribution of these categories on a monthly basis; we observed that the categories ‘outbreak’ and ‘preparedness’ increased substantially after August, with ‘outbreak’ peaking in October and ‘preparedness’ peaking in November. The other categories also increased considerably after August. The category with the smallest growth was ‘cell/molecular biology’; this is not surprising as this is the category that is the least directly connected to the current Africa events (Figure S20) (sheet 5).\n\nIn order to corroborate this classification by subject-matter, we also used objective parameters by examining the monthly distribution of papers containing specific terms. We analyzed the monthly distribution of papers with the terms ‘outbreak’, ‘africa’ or ‘vaccine’ in the title/abstract (see Figure S12–Figure S15) (sheet 4) and found that the distribution patterns of papers for ‘outbreak’, ‘africa’ and ‘vaccine’ were similar to the patterns obtained with the subject-matter and subjective classification. In particular, the papers with the term ‘outbreak’ peaked in October and had a distribution very similar to the subject-matter category (Figure S21–Figure S23) (sheet 5). These results show that the majority of papers published in 2014 were either about the West Africa epidemic or about general preparedness topics. Nonetheless, all discipline/area categories substantially increased during the last part of the year, including papers of basic cell and molecular biology research.\n\nWe analyzed the Ebola publications by country. To this end, we used a previously used method12 that relies on the affiliation field provided by PubMed to determine the affiliation country of authors. Before 2013 this method could be used to determine the affiliation country based on the first author only. However, starting from 2014, PubMed provides for most of citations information about every author. Thus, in 2014 this method could be used to quantify the number of papers with at least one author (any author, not only the first one) with a reported affiliation in a given country12. Using this method, we calculated the Ebola publications for the 20 countries with most publications12 (the United States was the country with most total biomedical publications, with almost 20,000 citations in the year 2014, followed by China and the United Kingdom (Figure S24) (sheet 6)). The United States was the country with by far the most Ebola publications (122 citations), followed by the United Kingdom (64), France (35) and Canada (31) (Figure 25) (sheet 6). We also calculated the ‘attraction scores’ for the same 20 countries. These scores are a measure of the relative focus towards a topic/field and are calculated by dividing the topic/field-relevant papers by the total number of biomedical papers for the same country (and multiplying by 10,000)12. The ‘attractions scores’ of the 20 countries were very different, as expected by the different patterns of total and Ebola only papers (Figure S24–Figure S25) (sheet 6). The country with the highest ‘attraction score’ was the United Kingdom, followed by France, Canada and Switzerland (Figure S26) (sheet 6).\n\nWe then repeated the same analyses by taking in consideration only the papers with an abstract available. If one looks at all biomedical publications, the proportion of publications with an abstract available was very high (on average 91.3%), with very little variability (relative standard deviation was 3.1%) (Figure S27) (sheet 6). If compared to Ebola-only papers, this number is very high. In fact, only 32.9% of the Ebola papers contained an abstract (see Figure S8) (sheet 3). This shows that, in 2014, the proportions of publications types on Ebola were different from those of the average biomedical publication.\n\nWe then looked at the only publications with an abstract available (the United States was the country with most total biomedical publications, with over 180,000 citations, followed by China, the United Kingdom and Germany (Figure S28) (sheet 6). The proportion of Ebola publications attributed to the United States (79 citations) was even higher than in the case of all types of publications, followed by Canada (21) and the United Kingdom (18) (Figure S29) (sheet 6). Canada was in this case the country with the highest ‘attraction score’, followed by the United States and France (Figure S30) (sheet 6).\n\nWe finally assessed the accuracy of our method by scrutinizing the six countries with most Ebola publications (Figure S31) (sheet 7). We looked at the affiliation information of all the papers with an abstract available published by these countries to determine if any author from those countries was indeed present. As a matter of fact, 100% of the 155 analyzed papers had at least one author with at least one affiliation from the searched country; this result proved the accuracy of the method. We then determined the numbers of first and last authors with an affiliation in the searched country. With regard to the first author, we observed that in 73.6% of the papers she/he was from the searched country (China had the largest proportion (84.6%) and Canada the smallest proportion (61.9%)). With regard to the last author, we observed that in 73.9% of the papers she/he was from the searched country (the United States had the largest proportion (90.0%) and Canada had the smallest proportion (57.1%)) (Figure S32) (sheet 7) (see also USA, UK, France, Canada, China, Germany sheets).\n\nWe took a closer look at the 101 papers with an abstract available published by the United States and Canada - the two countries with most citations with an abstract available. We first examined the article types and found that the vast majority of papers with an abstract available from the United States or Canada were research papers (average 68.3%). Reviews were the second category (average 23.8%). The sum of all the other categories (like commentaries and editorials) was less than 8% of the papers with an abstract available (Figure S33) (sheet 8). These results indicate that the vast majority of papers with an abstract available were indeed research publications; also, they reveal that the quantity of reviews was higher than the one determined by the ‘review’ filter managed by PubMed.\n\nWe then examined the area/field of the same set of 101 publications. As before, we assigned papers to different categories based on the discipline/area of study focus. We observed that 29.7% were about basic ‘cell/molecular biology’, 27.7% about ‘drugs/antibodies/vaccines’, 23.8% about ‘pathophysiology/epidemiology/ecology’, 8.9% about ‘preparedness’, 6.9% about ‘society/policy/ethics’ and 3.0% about ‘other’ areas of study. We also looked at how many of these papers were specifically focused on specific aspects of the current West Africa outbreak. We observed that 13.9% of the papers dealt with some features of the 2014 epidemic. In this case, differently from before, the papers were (optionally) assigned to the ‘outbreak’ category in addition to the assignment to the discipline/area categories (Figure S34) (sheet 8). The pattern of discipline/area focus of the papers were different between the two countries, with the United States pattern more similar to the average pattern, as also anticipated by the much higher share of publications published by the United States. These results indicate that, differently from the papers without abstracts (e.g. editorials, commentaries, short letters), research papers and extensive reviews focus much more on basic cell and molecular biology or on drugs and vaccines and less on preparedness or on specific aspects of the current epidemic (see also USA and Canada sheets).\n\nWe then looked at the publication output of the three countries (Sierra Leone, Liberia and Guinea) that have been most affected by the epidemic, both in terms of cases and deaths (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-counts.html). We therefore calculated the total numbers of biomedical papers as well as the only papers on Ebola, taking also in consideration only publications with an abstract available. These countries publish very low numbers of biomedical papers: in 2014, 46 by Sierra Leone, 14 by Liberia and 45 by Guinea. All three countries had a high proportion of papers with an abstract available similarly to the 20 countries with most publications (the average was 83.6%). Despite the very small numbers of total biomedical publications, all the three countries published papers on Ebola. Sierra Leone had 4 publications with an abstract available, Liberia 4 and Guinea 2 (Figure S35) (sheet 9). As expected, the Ebola ‘attraction scores’ for these three countries were very high (sheet 9). As done before, we also controlled for the accuracy of our method for the 10 papers with an abstract available retrieved by searching the affiliation field. Nine of these papers (90% of cases) were authored by at least one person with (at least) an affiliation in one of three countries. This confirmed again the accuracy of our method as only 1 out of total 165 tested papers (so 0.6% of the tested papers) did not have any author with an affiliation truly corresponding to the searched country. We also determined the numbers of first authors and last authors that had a reported affiliation in the searched country. While there were no publications with the first or the last author with a Liberia affiliation, there were 2 publications with the first author having a Sierra Leone affiliation, 2 publications with the first author having a Guinea affiliation, 2 publications with the last author having a Sierra Leone affiliation and 2 publications with the last author having a Guinea affiliation (Figure S36) (sheet 9). Among the 9 papers with at least one author from one of these three countries, 7 focused on some aspects of the 2014 epidemic. There were also 5 original research articles but 4 of these had neither the first nor the last author from one of the three countries. The only research article with either the first or last author with an affiliation in one of the three countries was a paper from Guinea of the category ‘pathophysiology/epidemiology/ecology’ (see also Sierra Leone, Liberia and Guinea sheets).\n\n\nConclusions\n\nIn this report we have analyzed the volumes and characteristics of the publications about Ebola virus during the year 2014. These are some of the key findings:\n\n1. There were 900 citations with the terms ‘Ebola’ or ‘Ebolavirus’ in the title/abstract, including 797 citations with one of these terms in the title.\n\n2. The volume of citations was over 12-fold the volume of citations of 2013.\n\n3. The volume of citations with an abstract available was over 4-fold the volume of citations of 2013.\n\n4. The proportion of papers with an abstract available was 32.9% whereas in 2013 it was 92.4%. The proportion for all biomedical papers published by the top 20 publishing countries was 91.3%.\n\n5. 20.6% of the papers with an abstract available contained the terms ‘vaccine’ or vaccines’ in the title/abstract.\n\n6. 46.2% of the papers with an abstract available contained the term ‘Africa’ in the title/abstract.\n\n7. There were only 2 publications about an original clinical trial study.\n\n8. 85.4% of the papers were published in the last 4 months of the year.\n\n9. 81.6% of the papers with an abstract available were published in the last 4 months of the year.\n\n10. The peak of papers with the term ‘outbreak’ in the title/abstract was in October.\n\n11. The country with most publications on Ebola was the United States with 122 citations, followed by the United Kingdom with 64 citations.\n\n12. The country with most publications on Ebola with an abstract available was the United States with 80 citations, followed by Canada with 21 citations.\n\n13. Among the 80 publications with an abstract available and with at least one author affiliated to a United States institution, the first and last authors with an affiliation in this country were 67 and 72, respectively.\n\n14. Among publications with an abstract available and with either United States or Canada affiliations, 68.3% were original research papers.\n\n15. Among publications with an abstract available and with either United States or Canada affiliations, 27.7% were studies about ‘drugs/antibodies/vaccines’ and 29.7% were other types of ‘cell/molecular biology’ studies.\n\n16. There were 9 publications with an abstract available with at least one author with an affiliation in Sierra Leone, Liberia or Guinea, including 4 with either the first or the last author with an affiliation in these countries. There were also 5 original research papers with at least one author from these countries.\n\nThe data of this report show a very dramatic change in the volume and nature of Ebola publications in 2014. While non-research papers have played a major role in the staggering increase in publications, there has also been a dramatic increase of original research articles or research notes (like this same research note), including many studies on potential new drugs or vaccines or related to basic biological research. Interestingly, the increase in publications took place in the second part of 2014 although the epidemic had already started at the end of 2013. The surge in publications (research and non-research papers) was therefore concomitant with the surge in number of cases and deaths and thus certainly due to the current West Africa outbreak. Even if an analysis of the causes is beyond the scope of this note, this surge could be due to several reasons: a genuine increased interest towards a major global health threat, a perception of easiness in publishing by writing about Ebola or by simply inserting a few comments on Ebola in the title/abstract, a strategic move in anticipation of increased funding for the Ebola field and several other reasons. Anyway, the findings of this research note shows that the scientific community started publishing many more editorials, letters and comments on Ebola only during or after the massive increase in deaths and media coverage. Even if the rises in scientific papers and media coverage could be independent, it can be speculated that at least a fraction of them were in fact inter-dependent. In particular, it is plausible that many scientific publications were conceived and published as a consequence of the extensive coverage in news and social media. Future studies will shed more light on the relationship between media coverage and scientific publications during the 2014 Ebola outbreak or similar worldwide threats.\n\nThe data presented in this report and in future studies will be useful to better comprehend how the scientific community responds to this kind of global health threats with massive media coverage. This information will assist scholars, public health officials and policymakers in their efforts to improve scientific research policies with the goal of maximizing both public health and knowledge advancement.\n\n\nAdditional methodological notes\n\nPublication output was determined by using the free and public search engine PubMed. All searches were performed during February 2015. All data and searching details are included in the accompanying database containing ‘sheets 1–9’, the ‘clinical trial term’ sheet, and the ‘Canada’, ‘China’, ‘France’, ‘Germany’, ‘Guinea’, ‘Liberia’, ‘Sierra Leone’, ‘UK’ and ‘USA’ sheets.\n\n\nData availability\n\nFigshare: Dataset and selected figures of biomedical publications on Ebola in 2014. doi: 10.6084/m9.figshare.132848313",
"appendix": "Author contributions\n\n\n\nAndrea Ballabeni designed the research, collected the data, analyzed the results and wrote the manuscript. Andrea Boggio analyzed the results and wrote the manuscript. All authors 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\nSupplementary figures\n\nYear 2014.\n\nYear 2014.\n\nYear 2014.\n\nYear 2014.\n\nYear 2014.\n\nYear 2014.\n\nYear 2014\n\nYear 2014.\n\nYear 2014.\n\nYear 2014.\n\nThe number of publications about original clinical trial studies is also indicated. Year 2014.\n\nYear 2014.\n\nYear 2014.\n\nRemaining citations were assigned to one (and only one) of the indicated discipline/area categories. Year 2014.\n\nCitations were assigned to one (and only one) category, similarly to Figure S19. Year 2014.\n\nYear 2014.\n\nYear 2014.\n\n‘Attraction scores’ were calculated by dividing the number of Ebola-related citations by the total biomedical citations of the country and by multiplying by 10,000. Year 2014.\n\nYear 2014.\n\nYear 2014.\n\nYear 2014.\n\n‘Attraction scores’ were calculated by dividing the number of Ebola-related citations by the total biomedical citations of the country and by multiplying by 10,000. Year 2014.\n\nCitations (‘ebola’ or ‘ebolavirus’ in title) with abstract available are also shown. Year 2014.\n\nNumbers of citations (‘ebola’ or ‘ebolavirus’ in title) with abstract available of the six countries with most Ebola-related publications. The numbers of publications automatically retrieved or with real ‘any author’, ‘first author’ or ‘last author’ with the proper country affiliation are indicated. 100% of the citations had at least one author (‘any author’) with the proper country affiliation, thus indicating accuracy of the method. Year 2014.\n\nYear 2014.\n\nCitations were assigned only to the more relevant category, except citations related to specific aspects of the 2014 outbreak that were assigned optionally and in addition to the other categories. Year 2014.\n\nYear 2014.\n\nNumbers of citations (‘ebola’ or ‘ebolavirus’ in title) with abstract available of the three countries with most Ebola cases. The numbers of publications automatically retrieved or with real ‘any author’, ‘first author’ or ‘last author’ with the proper country affiliation are indicated. Year 2014.\n\n\nReferences\n\nBasch CH, Basch CE, Redlener I: Coverage of the ebola virus disease epidemic in three widely circulated United States newspapers: implications for preparedness and prevention. Health Promot Perspect. 2014; 4(2): 274–251. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHouseh M: Communicating Ebola through social media and electronic news media outlets: A cross-sectional study. Health Informatics J. 2015. PubMed Abstract | Publisher Full Text\n\nFung IC, Tse ZT, Cheung CN, et al.: Ebola and the social media. Lancet. 2014; 384(9961): 2207. Publisher Full Text\n\nRodriguez-Morales AJ, Castaneda-Hernandez DM, McGregor A: What makes people talk about Ebola on social media? A retrospective analysis of Twitter use. Travel Med Infect Dis. 2015; 13(1): 100–101. PubMed Abstract | Publisher Full Text\n\nLove CB, Arnesen SJ, Phillips SJ: Ebola outbreak response: the role of information resources and the national library of medicine. Disaster Med Public Health Prep. 2015; 9(1): 82–85. PubMed Abstract | Publisher Full Text\n\nSim F, Mackie P: The rising tide of Ebola. Public Health. 2014; 128(9): 769–770. PubMed Abstract | Publisher Full Text\n\nNa W, Park N, Yeom M, et al.: Ebola outbreak in Western Africa 2014: what is going on with Ebola virus?. Clin Exp Vaccine Res. 2015; 4(1): 17–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaize S: Ebola virus in West Africa: new conquered territories and new risks-or how I learned to stop worrying and (not) love Ebola virus. Curr Opin Virol. 2015; 10C: 70–76. PubMed Abstract | Publisher Full Text\n\nBeeching NJ, Fenech M, Houlihan CF: Ebola virus disease. BMJ. 2014; 349: g7348. PubMed Abstract | Publisher Full Text\n\nGatherer D: The unprecedented scale of the West African Ebola virus disease outbreak is due to environmental and sociological factors, not special attributes of the currently circulating strain of the virus. Evid Based Med. 2015; 20(1): 28. PubMed Abstract | Publisher Full Text\n\nGatherer D: The 2014 Ebola virus disease outbreak in West Africa. J Gen Virol. 2014; 95(pt 8): 1619–1624. PubMed Abstract | Publisher Full Text\n\nXu Q, Boggio A, Ballabeni A: Countries’ Biomedical Publications and Attraction Scores [v1; ref status: approved 1, http://f1000r.es/4ri]. F1000Res. 2014; 3: 292. Publisher Full Text\n\nBallabeni A, Boggio A: Dataset and selected figures of biomedical publications on Ebola in 2014. Figshare. 2014. Data Source"
}
|
[
{
"id": "7968",
"date": "17 Mar 2015",
"name": "Alfonso J. Rodriguez-Morales",
"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 interesting article.General comments:The appropriateness of the title: Ok.Whether the abstract provides an adequate summary of the article: I would include the total number of articles found in the abstract.If there is a comprehensive explanation of study design, methods and analysis, and their suitability to the investigation: Ok.Whether the conclusions are balanced and justified on the basis of the results: I suggest commenting more on the implications of the findings in regard the advance to obtain an available vaccine for Ebola.With regards to the data (if applicable), whether sufficient information has been provided for replication of the experiment, and/or if the data are in a usable format: Ok.I have only two recommendations:To further discuss why the authors only assessed the database PubMED/Medline, and why Science Citation Index and Scopus were not included. Even we can consider to include also some African database. V1 of this article appears online on March 13, 2015, however, 7 days before, on March 6, 2015, appeared online the following article:Cruz-Calderón S, Nasner-Posso KM, Alfaro-Toloza P, Paniz-Mondolfi AE, Rodríguez-Morales AJ. A bibliometric analysis of global Ebola research. Travel Medicine & Infectious Disease 2015 Epub Ahead Mar 6; available online at: http://www.sciencedirect.com/science/article/pii/S1477893915000344. doi: 10.1016/j.tmaid.2015.02.007.Then, I consider this should be cited, as a preliminary approach to this topic. Also comparing the findings between both. This analysis included PubMED/Medline, Science Citation Index and Scopus.",
"responses": [
{
"c_id": "1641",
"date": "09 Oct 2015",
"name": "Andrea Ballabeni",
"role": "Author Response",
"response": "We would like to thank Dr Alfonso Rodriguez-Morales for his review. We are glad the referee likes and approves our article. Here are our responses to his comments/suggestion. As suggested by the referee, we have now included the number of total publications in the abstract, based on an updated search performed in September 2015.With reference to the implication of the findings in regard to the advances to obtain a vaccine, we think this is a more complex topic that is beyond the purpose of this short research note. However, we certainly agree with the referee that this is an important issue that would be worth investigating/discussing in future studies and opinion pieces. With regard to the two recommendations:We think that given the comprehensiveness and breadth of the PubMed-searched databases, the results presented in this paper are a bona fide representation of the literature. In any case, even if this is beyond the scope of this short research note, we agree with the referee that it could be useful to confirm these data by using also other databases such as the Web of Science (Thomson Reuter) and the Scopus (Elsevier). To address this, we have added a few comments in the Conclusions section. Moreover, to emphasize that the data presented in this paper are based on PubMed-searches we have modified the title.Moreover, we believe that the fact that this paper is based on a search engine/databases that is free and easily accessible adds value to the results. We have added the reference suggested and authored by the referee. We think that this referenced paper, published a few days before our paper, indeed provides interesting information that supports and complements the data of our study. We have also added a short comment in the Conclusions section. Moreover, we have made a few additional changes (summarized in Amendments from Version 1 box) that hopefully will be appreciated by the referee. We would like to thank again Dr Rodriguez-Morales for his advice. Best regards,Andrea Ballabeni and Andrea Boggio"
}
]
},
{
"id": "9648",
"date": "27 Jul 2015",
"name": "Ann Kelly",
"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 sheer volume of Ebola Virus Disease (EVD) scholarship published over the past year is unprecedented in the forty-year history of the disease. This paper provides a close examination of what is reported as an ‘over 12-fold’ increase in biomedical output, scrutinizing trends in publication content, authorship, disciplinary orientations and institutional origins, as well as availability. A central conclusion of this analysis is that EVD-literature surges in late 2014, coinciding with both the peak of the outbreak in West Africa and media attention.The paper benefits from a robust methodological approach; the search criteria are well considered and rigorously applied. Some further detail about the timelines for publication might have been useful: for instance, how long from submission would a research article take to be published, and whether different timeframes exist for research as opposed to commentary pieces. This further analysis might qualify the claims regarding the simultaneity of the volume of publications and the dramatic rise in Ebola cases.That being said, the granularity of the sub-analysis—e.g. assessing the number of papers authored by researchers affiliated with institutions in West Africa—generates some intriguing insights into the shifts in scientific interest from basic Ebola science to the specific features of the current outbreak, and suggests some of the constraints of scientific collaborations in countries with weak research infrastructure. The subject analysis also offers some tantalizing leads into the prominence of certain research areas—for instance vaccines—during a time a global health crisis.My central reservation is that these connections are not explored further. The general point that media coverage and research agendas are inter-related seems hardly surprising. Indeed, scholarship in scientific communication and science and technology studies has illuminated these links as well as the politics of funding in considerable detail. With such a potentially rich dataset to hand, it is frustrating that the authors are not more ambitious in their interpretations, comparing for instance, a similar set of trends in the literature published during the Avian Flu or SARS epidemics. Has the emphasis on ‘preparedness’ shifted? In what ways has the institutional geography of authorship changed?In general, this is an intriguing article that offers a solid overview on the dynamics of scientific output in response to a global threat. My only regret is that the analytical agenda was not bolder, for it could have led to a few more surprising conclusions.",
"responses": [
{
"c_id": "1612",
"date": "09 Oct 2015",
"name": "Andrea Ballabeni",
"role": "Author Response",
"response": "We would like to thank Dr Ann Kelly for her review. We are glad for her appreciation of the paper and want to address her two concerns. The comment “how long from submission would a research article take to be published, and whether different timeframes exist for research as opposed to commentary pieces” is a very good one. Indeed we agree with the referee that these timeframes must be taken in consideration and added a short discussion in the Conclusions section to address this concerns. However, we think that an in-depth quantitative analysis of this topic is complex and not suitable for the purposes and length-constraints of this short “research note.” Future studies will certainly shed more light on these timeframes. With regard to the comment: “scholarship in scientific communication and science and technology studies has illuminated these links as well as the politics of funding in considerable detail. With such a potentially rich dataset to hand, it is frustrating that the authors are not more ambitious in their interpretations, comparing for instance, a similar set of trends in the literature published during the Avian Flu or SARS epidemics. Has the emphasis on ‘preparedness’ shifted? In what ways has the institutional geography of authorship changed?”We agree with the referee that it would be very interesting to perform these additional investigations. For this reason we have added a short discussion in the Conclusions section. However, though potentially informative, these additional analyses were beyond the purposes and the length-constraints of this short research note. Even if we certainly agree that it will be worth performing these investigations in future studies, we hope that the referee concurs that the lack of these further analyses does not diminish the quality of the data presented in this research note. In fact the scope of the “research note” was to provide a set of good data to be used in and to stimulate further and broader studies.We hope that, based on this clarification, the referee can now fully approve our article. Moreover, we have made a few additional changes (summarized in Amendments from Version 1 box) that hopefully will be appreciated by the referee. We would like to thank again Dr Kelly for her thoughtful and helpful comments. Best regards,Andrea Ballabeni and Andrea Boggio"
}
]
}
] | 1
|
https://f1000research.com/articles/4-68
|
https://f1000research.com/articles/4-1013/v1
|
07 Oct 15
|
{
"type": "Case Report",
"title": "Case Report: Pulmonary Kaposi Sarcoma in a non-HIV patient",
"authors": [
"Arber Kodra",
"Maciej Walczyszyn",
"Craig Grossman",
"Daniel Zapata",
"Tarak Rambhatla",
"Bushra Mina",
"Maciej Walczyszyn",
"Craig Grossman",
"Daniel Zapata",
"Tarak Rambhatla",
"Bushra Mina"
],
"abstract": "Kaposi Sarcoma (KS) is an angioproliferative tumor associated with human herpes virus 8 (HHV-8). Often known as one of the acquired immunodeficiency syndrome (AIDS)-defining skin diseases, pulmonary involvement in KS has only been discussed in a handful of case reports, rarely in a non-HIV patient. Herein we report the case of a 77 year-old- male who presented with a 6-week history of progressive dyspnea on exertion accompanied by productive cough of yellow sputum and intermittent hemoptysis. His past medical history was significant for Non-Hodgkin’s Follicular B-Cell Lymphoma (NHL). Patient also had biopsy-confirmed cutaneous KS. His physical exam was notable for a 2cm firm, non-tender, mobile right submandibular lymph node. Lungs were clear to auscultation. He had multiple violet non-tender skin lesions localized to the lower extremities. CT scan of the chest showed numerous nodular opacities and small pleural effusions in both lungs. A thoracenthesis was performed, showing sero-sanguineous exudative effusions. Histopathology failed to demonstrate malignant cells or lymphoma. A subsequent bronchoscopy revealed diffusely hyperemic, swollen mucosa of the lower airways with mucopurulent secretions. Bronchoalveolar lavage PCR for HHV-8 showed 5800 DNA copies/mL. It was believed that his pulmonary symptoms were likely due to disseminated KS. This case illustrates the potential for significant lung injury from KS. It also demonstrates the use of PCR for HHV-8 to diagnose KS in a bronchoalveolar lavage sample in a case when bronchoscopic biopsy was not safe. Furthermore, this case is unique in that the patient did not match the typical KS subgroups as HIV infection and other immune disorders were ruled out. Recognition of this syndrome is critical to the institution of appropriate therapy. As such, this case should be of interest to a broad readership across internal medicine including the specialties of Pulmonology and Critical Care.",
"keywords": [
"Kaposi Sarcoma",
"Lymphoma",
"Pulmonary",
"Bronchoscopy"
],
"content": "Case report\n\nThe patient was a 77 year-old Hispanic male who initially presented to the emergency room with a 6-week history of progressive dyspnea on exertion, cough productive of yellow sputum, intermittent hemoptysis, worsening fatigue and weight loss of 10 lbs. On admission, he denied fever, chills, chest pain, sick contacts, recent travel, or tuberculosis exposure and history. His past medical history was significant for Non-Hodgkin follicular B cell lymphoma (NHL) diagnosed in 1997, treated with Rituximab, Cyclophosphamide, Hydroxydaunorubicin, Oncovin and Prednisone (RCHOP) three times. Last treatment was completed 1 year prior to presentation. He also had biopsy-confirmed cutaneous Kaposi’s Sarcoma (KS), identified within the same year of presentation, treated with pegylated liposomal doxorubicin 20 mg/m2 once every 21 days for two cycles.\n\nOn admission, he had a low grade fever and was tachycardic. His physical exam was remarkable for a 2 cm firm, non-tender, mobile right submandibular lymph node. Lungs were clear to auscultation bilaterally. He was grossly anasarcic with 4 (+) pitting edema of the lower extremities. He also had multiple violet, non-tender skin lesions localized to the lower extremities, mainly around the medial aspect of his ankles and anterior thighs bilaterally.\n\nHis labs revealed a normal cardiac panel, a white blood cell (WBC) count of 8,100/mm3 with no leukocytosis. Manual differentiation of the white blood cells showed 28% neutrophils, 48% lymphocytes, 17% monocytes and 5% eosinophils. Urinalysis and urine culture were negative for infections. Blood cultures were positive for Streptococcus pneumoniae.\n\nA chest X-ray done on admission showed a right lung consolidation consistent with pneumonia thus the patient was started on empiric vancomycin 1gm every 12 hours and piperacillin-tazobactam 4.5gm every 8 hours for a duration of 8 days. A computerized axial tomographic (CT) scan of the chest, abdomen and pelvis, showed numerous nodular opacities throughout each lung which were suspected to be consistent with KS as well as small pleural effusions in both lungs (Figure 1).\n\nCT of the chest demonstrating several nodular opacities throughout both lungs. Two nodules are measured to show size. Arrows point to pleural effusions on both lungs.\n\nDespite completing a course of antibiotics for pneumonia, his symptoms did not improve. A follow-up CT scan of the chest demonstrated that the bilateral pleural effusions had worsened (Figure 2). Patient underwent a thoracentesis of the larger effusion in the right lung which showed a sero-sanguineous and exudative fluid based on chemistry analysis. Histopathology failed to demonstrate malignant cells.\n\nCT of the chest showing nodular opacities that persisted in both lung fields and worsening bilateral pleural effusions (arrows).\n\nA bronchoscopy was also performed, revealing diffusely hyperemic and edematous mucosa of the lower airways with mucopurulent secretions (Figure 3). Biopsy was not performed at this time given concern for endobronchial bleeding. Bronchoalveolar lavage (BAL) stains and cultures were negative for bacteria, fungi or acid fast bacilli. Pneumocystis jiroveci PCR and fluid cytology were also negative but PCR for HHV-8 showed 5800 DNA copies/mL. The patient had a HIV p24 rapid antigen test done which was negative.\n\nPhotographs of the bronchoscopy performed after patient’s symptoms were not improving with appropriate antibiotic therapy. (a) Diffusely hyperemic and edematous mucosa of lower airways. (b) Arrow points to airway with significant mucopurulent secretions.\n\nAfter two weeks of supportive treatment, the patient’s symptoms began to improve. He continued to have numerous indurated confluent lesions on his body and face consistent with KS. His pulmonary symptoms were thought to be due to disseminated KS. Following significant discussions with the patient and his family regarding the poor prognosis of his disease, he decided not to undergo any further antineoplastic treatments and eventually was discharged home with hospice.\n\n\nDiscussion of diagnosis\n\nKaposi Sarcoma (KS) is an angioproliferative tumor associated with human herpes virus 8 (HHV-8)1,2. It is one of the AIDS-defining skin diseases and is strongly linked to male homosexual behavior3,4. Four clinical variants of KS have been described: classic, African, iatrogenic and AIDS-related5–10. The classic variant mainly affects elderly men of Eastern European Jewish and Mediterranean origin7–9. The African, or endemic type, affects primarily men in the 4th decade of life in East and Central Africa10. Its clinical presentation is similar to the classic form but with a more aggressive variant that responds poorly to conventional treatment10. The third variant, iatrogenic KS, is related to chronic immunosuppressive drugs used in organ-transplant recipients or cancer patients5–10. This variant tends to be more aggressive, involving lymph nodes, mucosa and visceral organs. AIDS-related KS is an aggressive epidemic form that commonly affects patients with immunosuppression from AIDS3–5.\n\nPulmonary involvement in KS has only been discussed in a handful of case reports, very few of which were in non-HIV patients11–16. This is likely due to the lack of published evidence of KS in general, as well as the presence of multiple co-morbidities in most patients which may mask clear identification of pulmonary KS. In most cases, it occurs in conjunction with more extensive muco-cutaneous disease12–16. Unique manifestations that distinguish KS from other pathologic processes in the lungs have not been identified. The most common characteristics of pulmonary KS include peri-broncho-vascular and nodular opacities, thickened interlobular septa and pleural effusions17–21.\n\nSigns and symptoms such as shortness of breath, hypoxemia, and dry cough are common in pulmonary KS. Hemoptysis, fever, chest pain and respiratory failure can also occur. Additionally, enlarged mediastinal lymph nodes are frequently seen in patients with this disease16–21.\n\nThe diagnosis of pulmonary involvement in KS can often be made by a combination of clinical, radiographic and laboratory findings, in conjunction with results of a transbronchial biopsy21,22. Radiographic findings in pulmonary KS are varied and include segregated pulmonary nodules, pleural effusions, and hilar or mediastinal lymphadenopathy, all of which were also evident in our patient23–25. Several polymerase chain reaction (PCR) assays employing primers unique for HHV-8 have been described26,27. HHV-8 DNA can be identified using PCR in biopsies of KS, including AIDS-associated KS, classic KS, and endemic KS. Studies have also shown that HHV-8 DNA can be detected in the BAL of patients with pulmonary KS, as was evident in our case28–30.\n\nOur patient did not match the typical subgroups as HIV infection and other immune disorders were ruled out. Furthermore, it has been shown that KS, in non-HIV patients, clinically resembles classic KS but occurs at a younger age, is limited to the skin, and is associated with a good prognosis5. Our patient, however, demonstrated both dermatologic and pulmonary manifestations suggesting a disseminated and aggressive form of the disease.\n\n\nTreatment and management\n\nThe published literature on the treatment of KS consists mostly of retrospective series and case reports31–34. At the time of this case report, we are aware of only a few prospectively randomized trials to date that compare different treatments for KS, most of which were for AIDS-related KS35–37. This is likely due to the lack of published evidence of the disease and the presence of co-morbidities in most patients, which may limit treatment options such as in our case.\n\nCurrently, antiretroviral therapy (ART) is the first-line therapy for pulmonary KS as it is often seen in patients with HIV/AIDS31–33. The first-line treatment for KS in patients with CD4 counts greater than 350 cells/μL is still unclear and treatment has generally been palliative in nature.\n\nOnly systemic treatments, including chemotherapy and immunomodulators, have shown potential to cause regression in all sites of disease36–38. These include pegylated liposomal doxorubicin, vinblastine, alone or in combination with bleomycin, paclitaxel, oral etoposide, vinorelbine, gemcitabine and the immunomodulator recombinant interferon alfa (IFNa). Overall response rates for all of these therapies have been reported to be high and the treatments are generally well tolerated, even in the elderly population. Only one randomized trial has been conducted in which two different systemic therapies, etoposide and vinblastine, were compared in non-AIDS related KS35. That study showed no significant differences between the two treatments with regard to response rate or survival.\n\nDespite the lack of randomized trials demonstrating superiority, most clinicians consider pegylated liposomal doxorubicin the first-line therapy of choice based on a retrospective multicenter series of patients with classic KS without evidence of HIV which showed ≥50% decrease in the number of measurable lesions and the absence of new cutaneous lesions for at least eight weeks in 71% of treated patients38.\n\nOur patient received treatment with liposomal doxorubicin prior to admission resulting in improvement of his cutaneous lesions. Doxorubicin was planned to be started prior to discharge, but the patient declined further chemotherapy, electing to establish hospice care.\n\nRadiotherapy is also an accepted treatment for all forms of KS. However, due to the tendency of new lesions to develop as well as the persistence of HHV-8, despite improvement of local lesions and symptoms, there is no consensus as to when to choose radiotherapy over systemic therapy39,40.\n\n\nConclusion\n\nKS in a non-immunocompromised patient is an infrequent occurrence and pulmonary involvement makes the diagnosis even more difficult as only a handful of cases in this patient population are present in the literature. Traditional measures of treatment are aimed at curbing the underlying immunosuppression, making it difficult to treat in individuals with normal immune function. Pulmonary involvement can be ascertained by a combination of clinical, radiographic and laboratory findings, in conjunction with results of a transbronchial biopsy.",
"appendix": "Author contributions\n\n\n\nAK drafted the manuscript and selected the references. MW and DZ performed the bronchoscopy and selected the images used for the manuscript. CG helped with drafting and editing the manuscript. TR participated in reviewing the current literature on the topic of the manuscript. BM assisted MW and DZ with the bronchoscopy, helped to edit the manuscript and coordinated the submission of manuscript. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have 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\nThe authors would like to thank Mina Botros and Jesus Lanza for their critical revisions of intellectual and written content.\n\n\nReferences\n\nWahman A, Melnick SL, Rhame FS, et al.: The epidemiology of classic, African, and immunosuppressed Kaposi's sarcoma. Epidemiol Rev. 1991; 13: 178–99. PubMed Abstract\n\nChang Y, Cesarman E, Pessin MS, et al.: Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science. 1994; 266(5192): 1865–9. PubMed Abstract | Publisher Full Text\n\nDupin N, Grandadam M, Calvez V, et al.: Herpesvirus-like DNA sequences in patients with Mediterranean Kaposi's sarcoma. Lancet. 1995; 345(8952): 761–2. PubMed Abstract | Publisher Full Text\n\nMoore PS, Chang Y: Detection of herpesvirus-like DNA sequences in Kaposi's sarcoma in patients with and without HIV infection. N Engl J Med. 1995; 332(18): 1181–5. PubMed Abstract | Publisher Full Text\n\nIscovich J, Boffetta P, Franceschi S, et al.: Classic Kaposi sarcoma: epidemiology and risk factors. Cancer. 2000; 88(3): 500–17. PubMed Abstract\n\nBuonaguro FM, Tornesello ML, Beth-Giraldo E, et al.: Herpesvirus-like DNA sequences detected in endemic, classic, iatrogenic and epidemic Kaposi's sarcoma (KS) biopsies. Int J Cancer. 1996; 65(1): 25–8. PubMed Abstract | Publisher Full Text\n\nKemény L, Gyulai R, Kiss M, et al.: Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8: a new virus in human pathology. J Am Acad Dermatol. 1997; 37(1): 107–13. PubMed Abstract | Publisher Full Text\n\nGallo RC: Some aspects of the pathogenesis of HIV-1-associated Kaposi's sarcoma. J Natl Cancer Inst Monogr. 1998; 1998(23): 55–7. PubMed Abstract | Publisher Full Text\n\nMartin JN, Ganem DE, Osmond DH, et al.: Sexual transmission and the natural history of human herpesvirus 8 infection. N Engl J Med. 1998; 338(14): 948–54. PubMed Abstract | Publisher Full Text\n\nNasti G, Errante D, Santarossa S, et al.: A risk and benefit assessment of treatment for AIDS-related Kaposi's sarcoma. Drug Saf. 1999; 20(5): 403–25. PubMed Abstract | Publisher Full Text\n\nKoss CA, Jarlsberg LG, den Boon S, et al.: A Clinical Predictor Score for 30-Day Mortality among HIV-Infected Adults Hospitalized with Pneumonia in Uganda. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nChakaya JM, Bii C, Ng'ang'a L, et al.: Pneumocystis carinii pneumonia in HIV/AIDS patients at an urban district hospital in Kenya. East Afr Med J. 2003; 80(1): 30–5. PubMed Abstract | Publisher Full Text\n\nKaplan LD, Hopewell PC, Jaffe H, et al.: Kaposi's sarcoma involving the lung in patients with the acquired immunodeficiency syndrome. J Acquir Immune Defic Syndr. 1988; 1(1): 23–30. PubMed Abstract\n\nHong A, Lee CS: Kaposi's sarcoma: clinico-pathological analysis of human immunodeficiency virus (HIV) and non-HIV associated cases. Pathol Oncol Res. 2002; 8(1): 31–5. PubMed Abstract | Publisher Full Text\n\nGodoy MC, Rouse H, Brown JA, et al.: Imaging features of pulmonary Kaposi sarcoma-associated immune reconstitution syndrome. AJR Am J Roentgenol. 2007; 189(4): 956–65. PubMed Abstract | Publisher Full Text\n\nRestrepo CS, Martínez S, Lemos JA, et al.: Imaging manifestations of Kaposi sarcoma. 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PubMed Abstract | Publisher Full Text\n\nTamm M, Reichenberger F, McGandy CE, et al.: Diagnosis of pulmonary Kaposi's sarcoma by detection of human herpes virus 8 in bronchoalveolar lavage. Am J Respir Crit Care Med. 1998; 157(2): 458–63. PubMed Abstract | Publisher Full Text\n\nHoward MR, Brink NS, Whitby D, et al.: Association of Kaposi's sarcoma associated herpesvirus (KSHV) DNA in bronchoalveolar lavage fluid of HIV infected individuals with bronchoscopically diagnosed tracheobronchial Kaposi's sarcoma. Sex Transm Infect. 1998; 74(1): 27–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrenner B, Rakowsky E, Katz A, et al.: Tailoring treatment for classical Kaposi's sarcoma: comprehensive clinical guidelines. Int J Oncol. 1999; 14(6): 1097–102. PubMed Abstract | Publisher Full Text\n\nGonzález-García J, Rubio García R, Antela López A, et al.: [Pneumocystis carinii pneumonia and HIV infection: diagnosis and treatment]. Enferm Infecc Microbiol Clin. 1998; 16(Suppl 1): 36–44. PubMed Abstract\n\nGoebel FD, Jablonowski H: Therapy of special HIV-associated diseases: HCV-HIV-co-infection and AIDS-related Kaposi's sarcoma - official satellite to the 7th European Conference on Clinical Aspects and Treatment of HIV-infection, October 23, 1999 in Lisbon, Portugal. Eur J Med Res. 1999; 4(12): 507–13. PubMed Abstract\n\nMartorano LM, Cannella JD, Lloyd JR: Mucocutaneous presentation of Kaposi sarcoma in an asymptomatic human immunodeficiency virus-positive man. Cutis. 2015; 95(4): E19–22. PubMed Abstract\n\nBrambilla L, Labianca R, Boneschi V, et al.: Mediterranean Kaposi's sarcoma in the elderly. A randomized study of oral etoposide versus vinblastine. Cancer. 1994; 74(10): 2873–8. PubMed Abstract | Publisher Full Text\n\nDi Lorenzo G, Kreuter A, Di Trolio R, et al.: Activity and safety of pegylated liposomal doxorubicin as first-line therapy in the treatment of non-visceral classic Kaposi's sarcoma: a multicenter study. J Invest Dermatol. 2008; 128(6): 1578–80. PubMed Abstract | Publisher Full Text\n\nNorthfelt DW, Dezube BJ, Thommes JA, et al.: Pegylated-liposomal doxorubicin versus doxorubicin, bleomycin, and vincristine in the treatment of AIDS-related Kaposi's sarcoma: results of a randomized phase III clinical trial. J Clin Oncol. 1998; 16(7): 2445–51. PubMed Abstract\n\nDi Lorenzo G, Di Trolio R, Montesarchio V, et al.: Pegylated liposomal doxorubicin as second-line therapy in the treatment of patients with advanced classic Kaposi sarcoma: a retrospective study. Cancer. 2008; 112(5): 1147–52. PubMed Abstract | Publisher Full Text\n\nHuang KM, Hsu CH, Cheng JC, et al.: Radiotherapy of classic Kaposi's sarcoma in Taiwan, an area where classic Kaposi's sarcoma is not prevalent. Anticancer Res. 2006; 26(6C): 4659–63. PubMed Abstract\n\nCaccialanza M, Marca S, Piccinno R, et al.: Radiotherapy of classic and human immunodeficiency virus-related Kaposi's sarcoma: results in 1482 lesions. J Eur Acad Dermatol Venereol. 2008; 22(3): 297–302. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10719",
"date": "09 Nov 2015",
"name": "Michael O'Connor",
"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 very nice case report serves to remind practitioners everywhere the Kaposi's sarcoma both occurs independently of HIV disease, and can affect organ systems other than the skin. Modern diagnostic techniques and imaging, used as described in this report, allow us to document manifestations of Kaposi's that were previously undocumented.",
"responses": []
},
{
"id": "11204",
"date": "12 Nov 2015",
"name": "Chunxue Bai",
"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\nArber Kodra et al. presented a case of KS that involved the pulmonary in a HIV negative patient. This patient has past medical history of NHL and cutaneous KS. PCR for HHV-8 in BALF showed 5800 DNA copies/ ml and bronchoscopy showed diffusely hyperemic and edematous mucosa of lower airways. The authors claimed that this patient’s pulmonary symptoms were likely due to disseminated KS and the useful of PCR for HHV-8 to diagnose KS in a BALF sample in a case when bronchiscopic biopsy was not safe.KS is thought to originate from HHV8 infected lymphatic endothelial cells. HHV8 is recognized as an essential and necessary factor in the pathogenesis of KS. However, not all HHV-8 infected individuals will develop the disease. The authors examined PCR for HHV-8 in BALF sample; however, it will be more convincing if the author could provide pathological results to demonstrate the patient pulmonary symptoms were likely due to disseminated KS. In this case, the past medical history of this patient was significant for Non-Hodgkin’s Follicular B-Cell Lymphoma. NHL may also lead to nodular opacities throughout both lungs. The abnormal cells of KS form purple, red, or brown patches, plaques or tumors on the skin. It will be better if the authors could provide some pictures of cutaneous lesions. It is a little puzzled that the “lung were clear to auscultation.”, whereas CT scan showed pleural effusions on both lungs.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1013
|
https://f1000research.com/articles/3-183/v1
|
05 Aug 14
|
{
"type": "Method Article",
"title": "Important considerations for microRNA extraction methods from whole blood and peripheral blood mononuclear cells",
"authors": [
"Sadaf Atarod",
"Hannah Smith",
"Anne Dickinson",
"Xiao-Nong Wang",
"Hannah Smith",
"Anne Dickinson",
"Xiao-Nong Wang"
],
"abstract": "MicroRNAs are non-coding RNAs that negatively regulate mRNA expression and play significant roles in both health and disease. Differential microRNA expression has been used to aid diagnosis and discriminate disease stages. The accuracy and reliability of microRNA expression measurement is of utmost importance. For diagnostic investigations, microRNA expression in human peripheral blood is commonly detected using total RNA extracted using different methods. To date, no convincing data have been available showing whether microRNA expression levels are comparable when total RNA has been extracted from whole blood or peripheral blood mononuclear cells (PBMCs). The present study examined miR-146a-5p and miR-155-5p expression using total RNA extracted in parallel from whole blood and PBMCs of 14 healthy volunteers. MicroRNA expression levels were significantly different between whole blood and PBMCs. No significant difference was observed in microRNA expression between fresh and cryopreserved PBMCs (p=0.125 for both). Further observations revealed that gender differences did not influence miR-146a-5p or miR-155-5p expression regardless of using whole blood(p = 0.797 and 1.00 respectively) or PBMC (p = 0.190 and 0.898 respectively). Our results demonstrate that microRNA expression could be subjective to the methods used for total RNA extraction which highlights the importance of using uniform extraction methods.",
"keywords": [
"MicroRNAs (miRNAs) are cell- and therefore tissue-specific",
"and their expression levels impact protein translation (Sood et al.",
"2006). Nearly",
"2000 microRNA (miRNA) sequences have been identified in humans (Kozomara & Griffiths-Jones",
"2014). Numerous studies have reported specific miRNA expression levels in peripheral blood (PB) as markers of disease (Mookherjee & El-Gabalawy",
"2013",
"Patnaik et al.",
"2012). Although miRNA expression levels could be inevitably influenced by the way that total RNA is extracted",
"often studies reporting differential miRNA expression levels fail to emphasise the impact of RNA extraction methods. This could at least partially lead to significant controversies and inconsistencies in the literature related to miRNA research."
],
"content": "Introduction\n\nMicroRNAs (miRNAs) are cell- and therefore tissue-specific, and their expression levels impact protein translation (Sood et al., 2006). Nearly, 2000 microRNA (miRNA) sequences have been identified in humans (Kozomara & Griffiths-Jones, 2014). Numerous studies have reported specific miRNA expression levels in peripheral blood (PB) as markers of disease (Mookherjee & El-Gabalawy, 2013; Patnaik et al., 2012). Although miRNA expression levels could be inevitably influenced by the way that total RNA is extracted, often studies reporting differential miRNA expression levels fail to emphasise the impact of RNA extraction methods. This could at least partially lead to significant controversies and inconsistencies in the literature related to miRNA research.\n\nPAXgene Blood RNA System (PAXgene Blood RNA Tube and Paxgene Blood miRNA Kit [PAXM]) has been the gold standard for PB collection as the stabilising reagent present in the tube prevents RNA degradation and inhibits changes in gene expression due to the collection procedure (Guenther & McCluskey, 2008). However, it should be emphasised that the miRNA expression detected in whole blood is the overall outcome from total hematocytes rather than the lymphocyte fraction only. Recent studies have shown that erythrocytes also contain a high proportion of miRNAs (Hamilton, 2010). Bayatti et al. have shown that the presence of globin (globular proteins such as haemoglobin) in PB can impact RNA expression and that globin depletion can decrease total RNA quality and yield in particular when extraction is performed using the PAXgene Blood RNA kit (Bayatti et al., 2014). However, it is necessary to evaluate the miRNA expression levels in an independent cohort, as miRNAs are in general highly stable, and therefore their expression might not be affected by globin treatment. Likewise, Mookherjee et al. have only shown that miRNA expression levels correlate between whole blood and PBMCs using PAXgene Blood RNA System and mirVana miRNA kit (MM), respectively (Mookherjee & El-Gabalawy, 2013). They have not shown that the expression levels are comparable or that they agree. This has raised some confusion as the PAXgene Blood RNA system extracts from whole blood while MM from PBMCs. In addition, researchers have only tested for the relationship between one method and another rather than for agreements as recommended by Bland et al., (Bland & Altman, 1986). Performing a comprehensive statistical analysis is required when two methods are compared (Burd, 2010).\n\nIn this investigation we have measured the expression of miR-146a-5p and miR-155-5p which have extensively been investigated in whole blood and PBMCs due to their critical functions in the innate and adaptive immune system (Curtale et al., 2010; Schulte et al., 2013). We sought to compare miRNA expression levels in PB collected from 14 healthy volunteers using both extraction methods (PAXM and mirVana PARIS [MP]). Overall, we sought to address (i) whether miRNA expression detected in whole blood is comparable to that of isolated PBMCs and (ii) whether the degree of haemolysis impacts on the detected levels of miRNA expression in whole blood and PBMCs. We also investigated whether there is a difference in miR-146a-5p and miR-155-5p expression between males and females as well as fresh and cryopreserved PBMCs. The experimental design for this investigation is depicted in Figure 1.\n\nPeripheral blood was collected into various tubes as illustrated. Total RNA was extracted via three different methods and tested for purity. Reverse transcription and quantitative real-time PCR was performed for all the samples.\n\n\nMaterials and methods\n\nWhole blood was collected from 14 healthy volunteers following approval from the Newcastle and North Tyneside 2 Research Ethics Committee (STEMDIAGNOSTICS: REC-07/H0906/131) and informed consent was obtained from every volunteer for both blood collection and miRNA testing. Samples from 14 healthy volunteers (5 males and 9 females) were used to quantify miRNA expression in whole blood and PBMC.\n\nPB (2.5 ml) was collected in PAXgene Blood RNA Tubes (PreAnalytiX GmbH, Switzerland [Catalog No: 762165]) containing an RNA stabilising agent that lysed the blood cells and stabilised the intracellular RNA. The tubes were stored at -20°C until processed. PB was also collected in sodium (Na)-heparin (Sigma, UK) containing tubes for peripheral blood mononuclear cell isolation using graduated centrifugation over Lymphoprep™ (STEMCELL Technologies, Manchester, UK). The cells were then stored at -4°C before extraction. Isolated PBMCs were cryopreserved by re-suspension in freezing solution containing 350 ml of RPMI 1640 (Sigma-Aldrich, UK), 20% fetal calf serum and 10% dimethyl sulfoxide (NBS Biologicals, UK) and stored in Cryovials at -80°C.\n\nTotal RNA was extracted from isolated PBMCs using the (i) mirVana™ PARIS™ kit (MP) and (ii) mirVana™ miRNA Isolation kit (MM) (Ambion, USA [Catalog Nos: AM1556 and AM1560, respectively]) according to the manufacturer’s protocol. PAXgene Blood RNA Tubes were incubated overnight at room temperature to increase the RNA yield. Total RNA extraction from whole blood was performed using the PAXgene Blood miRNA kit (PAXM) (PreAnalytiX GmBh, Switzerland [Catalog No: 763134]), according to the manufacturer’s protocol. Aseptic techniques were followed at all stages of extraction. Total RNA quality and concentration were assessed using NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, MA). The absorbance ratios 260 nm/280 nm and 260 nm/230 nm were analysed to determine the purity of total RNA.\n\nMicroRNA specific cDNA was synthesised from 10 ng total RNA extracted from isolated PBMCs and whole blood using TaqMan MicroRNA reverse transcription (RT) kit (Applied Biosystems, Life Technologies, USA [Catalog No: 4366596]) as per the manufacturer’s protocol. The same RNA concentration was used for all the reactions. Hydrolysis probes were used for cDNA synthesis (Assay IDs: miR-155-5p-5p: 000479, miR-146a-5p: 000468, miR-451-5p: 001141, miR-23a-3p: 000399, SNORD49A [RNU49]: 001005, SNRNP27 [U6]: 001973, SNORA74A [RNU19]: 001003 and SNORD48 [RNU48]: 001006). No-enzyme control (NEC) and a negative template control (NTC) were run for every extraction and RT reaction set. The samples were then run on a thermal cycler at four different holding temperatures: 16°C for 30 minutes, 42°C for 30 minutes, 85°C for 5 minutes and finally at 4°C until storage at -4ºC.\n\nQuantitative real-time PCR (qPCR) was performed using the TaqMan method and hydrolysis probes mentioned above (Applied Biosystems by Life Technologies, CA, USA) according to the manufacturer’s protocol. Each sample was run in triplicate and every plate contained the no-template control (NTC) from the RT step and the qPCR step as well as a no-enzyme control (NEC) on a 7900HT Fast Real-Time PCR System (Life Technologues, CA, USA).\n\nThe qPCR results were analysed using SDS v2.4 software and normalised using SNORD48 as the reference control which was selected by testing a panel of four controls for stable expression within the whole blood and PBMCs. The comparative ∆∆Cq method was used to calculate fold-changes (ΔCq = Cq microRNA of interest - Cq reference control, Relative Quantification [RQ]aaaaa2-ΔΔCq and LOG transformed = LOG2RQ). Fold-change was logarithm-transformed as qPCR data are non-linear (exponential), and is transformed to decrease the heterogeneity of variance (McDonald, 2009) and also to identify the outliers present in the data (Rieu & Powers, 2009). Standard curves for three samples from both PAXM and MP were generated and a 95% confidence interval slope of the line was used to calculate the PCR efficiency (E) using the standard formula; E=10-1/Slope and % efficiency = (E-1) × 100. Mean efficiency was then calculated. Results were analysed and plotted using GraphPad PRISM v5.0 software (GraphPad Software, Inc, USA). Mann-Whitney U t-test was used to assess difference between two groups and Kruskal-Wallis one-way analysis of variance (ANOVA) for multiple groups. Spearman’s test was used to determine correlation. Bland-Altman was performed to test whether two methods agreed and if one could be interchanged with another. Significance was set at p<0.05.\n\n\nResults\n\nTotal RNA was extracted using three different extraction methods; PAXM (PAXgene Blood miRNA), MP (mirVana PARIS) and MM (miRVana miRNA) from whole blood and PBMCs. RNA purity was assessed by detecting the absorbance ratios at 260 nm/280 nm and at 260 nm/230 nm. The ratio (260 nm/280 nm) for whole blood was 1.95 – 2.35 and for isolated PBMCs was 2.00 – 2.27 (both MP and MM respectively). The ratios were ≥ 1.8 – 2.0; thus extraction was free from protein contamination which is usually absorbed at 280 nm. Absorbance ratios detected at 260 nm/230 nm showed ratios below the accepted contamination-free range of 1.5 – 2.0 (PBMCs: 0.18 – 1.83 and whole blood: 0.15 – 1.49). Therefore, the samples may have been affected by contaminants absorbed at 230 nm such as guanidine isothiocyanate present in all the three extraction kits. For RNA purity, the peak of each total RNA plot was also analysed as it could indicate contamination by phenol and/guanidine isothiocyanate. PBMCs had plot peaks at 260 nm thus confirming contaminant-free samples. However, peaks at 260 nm were absent for total RNA extracted using the PAXM (Figure 2). This further suggests that guanidine salts may have been the cause of contamination in whole blood samples extracted using PAXM.\n\n(A) PAXM: analysis of whole blood showed peaks positioned at 230 nm (n=10) and (B) MP: analysis of PBMCs showed peaks positioned at 260 nm (n=5).\n\nNEC and NTC controls were used to ensure that the RT and qPCR reactions were contaminant-free. Each control displayed no amplification (Cq > 36). This was particularly important for total RNA extracted from PBMCs, as they were non-DNase treated. Thus, any amplification in the controls may have suggested either non-specific binding of primers or presence of contamination such as genomic DNA. In addition we calculated the average efficiency of our real-time qPCR reactions (E=97.8%) which confirmed absence of reaction inhibitors. Determining qPCR efficiency is important as inhibitory compounds can affect miRNA expression and result in false positives.\n\nWe determined the most appropriate reference gene for this investigation by testing a panel of four controls that have been known for stable expression (SNORD49A [RNU49], SNRNP27 [U6], SNORA74A [RNU19] and SNORD48 [RNU48]) (Dataset a). Our results showed that SNORD48 expression was the most stable in total RNA extracted via both the PAXM (Figure 3A) and MP (Figure 3B) method. Therefore, SNORD48 was used as the reference control to normalise miR-146a-5p and miR-155-5p expression in each sample.\n\nA panel of four stably expressed miRNAs were selected and quantified to identify the most stable control for normalisation of miR-146a-5p and miR-155-5p in paired samples (n=3) (A) whole blood and (B) PBMCs.\n\nErythrocyte haemolysis has been reported to alter miRNA measurements in whole blood, plasma, serum and tissues (McDonald et al., 2011; Pritchard et al., 2012). The total RNA extracted using the three different methods was examined for degree of haemolysis by quantifying miR-451-5p and miR-23a-3p (Dataset b). Normalised ∆Cq values (miR-23a-3p – miR-451-5p) greater than seven were considered as an indicator of haemolysis. Our results showed that there was a significantly high degree of haemolysis in the total RNA extracted using the PAXM method with the ∆Cq values in the range of 9 – 11. Haemolysis was low in total RNA extracted using either of MP or MM, ∆Cq>3 (Figure 4).\n\nTotal RNA extracted using the PAXgene miRNA isolation kit. A threshold of LOG2RQ greater than 7 was indicative of haemolysis (dashed line). The degree of haemolysis was calculated by measuring the difference between miR-451-5p and miR-23a-3p expression. All data (n=5) have been log-transformed.\n\nWith inclusion of stringent quality controls, we assessed miR-146a-5p and miR-155-5p expression in total RNA extracted in parallel from whole blood using PAXM (n=14) and PBMCs using MP (n=14) (Dataset c). Our results showed that there was no correlation (Figure 5, miR-146a-5p: r=-0.352, p=0.217 and miR-155-5p: r=0.380, p=0.180) between PAXM and MP in the expression of both miR-146a-5p and miR-155-5p. In a PCR reaction, it is assumed that the target expression doubles at every reaction cycle. Bland-Altman analysis also showed that the two methods did not agree as the bias was greater than 1 which equated to more than one qPCR cycle difference between the two methods (Figure 5A and 5B). Mookherjee et al. had used MM to extract total RNA from PBMCs, then correlated miRNA expression between the PAXM and MM method (Mookherjee & El-Gabalawy, 2013). To eliminate the possibility that using MP was the reason for the non-correlation and disagreement, we tested the three different extraction methods (PAXM, MP and MM) for both miRNAs in a randomly selected cohort of five healthy volunteers (Figure 6) (Dataset d). The results demonstrated that PAXM and MP as well as PAXM and MM do not correlate nor agree. However, MP and MM methods agree with one another and can be interchanged as the bias between the two methods for both miR-146a-5p and miR-155-5p was only 0.769 (SD=0.307) and 0.892 (SD=0.802), respectively. Interestingly, normalised miRNA expression was significantly different only between PAXM and MM methods (miR-146a-5p and miR-155-5p: p<0.01). There was a higher miRNA expression in PBMCs than in whole blood for both miRNAs (Figure 7).\n\nTotal RNA was extracted (n=14) using PAXM and MP for (A) miR-146a-5p and (B) miR-155-5p expression. MicroRNA expression is within the limits of agreement but the bias is greater than one showing high disagreement between PAXM and MP. r indicates Spearman correlation. SD: Standard Deviation and bias is the mean difference. Cq values were used for this analysis. Dashed lines show the 95% lower and upper limits of agreement.\n\nThe three methods were all compared for miR-146a-5p as (A) PAXM vs MP (B) PAXM vs MM and (C) MP vs MM as well as miR-155-5p (D) PAXM vs MP (E) PAXM vs MM (F) MP vs MM. MicroRNA expression is within the limits of agreement but the bias is greater than one showing high disagreement between PAXM and MP. Bias is lower than one for MP and MM, thus the two methods agree with one another. r indicates Spearman correlation. SD: Standard Deviation and bias is the mean difference. Cq values were used for this analysis. Dashed lines show the 95% lower and upper limits of agreement.\n\n(A) miR-146a-5p and (B) miR-155-5p expression. MicroRNA expression is significantly varied across all the three different groups (p=0.002). MicroRNA expression is higher in PBMCs extracted via either MP or MM method in comparison to whole blood. **p<0.01 and ns: not significant.\n\nMany samples from patients participating in clinical studies are cryopreserved and stored as PBMCs for later use. Therefore, it is important to know the specific effects of cryopreservation on miRNA expression in PBMCs. We evaluated the impact of cryopreservation by extracting total RNA using MP kit from paired fresh and cryopreserved PBMCs (n=5) and performing RT-qPCR respectively (Dataset e). There was no correlation in miRNA expression between fresh and cryopreserved PBMCs, but the bias for both miR-146a-5p and miR-155-5p was less than 1 (Figure 8A and B). In addition, there was no statistically significant difference between fresh and cryopreserved PBMCs for both miR-146a-5p and miR-155-5p expression (p=0.125) (Figure 8C and D, respectively). Thus, expressions in fresh and cryopreserved samples do agree with one another but more samples would need to be tested as the standard deviation of bias was high for both miRNAs (miR-146a-5p: SD=2.284 and miR-155-5p: SD=1.342), which indicates a large sample variability.\n\nExpression was measured in five volunteers. Bland-Altman plots (A) miR-146a-5p and (B) miR-155-5p expression. The bias for both miRNAs is less than one. Bias is the mean difference. SD: Standard deviation. Cq values were used for this analysis. Dashed lines show the 95% lower and upper limits of agreement. Normalised miRNA expression (C) miR-146a-5p and (D) miR-155-5p. All data was log-transformed. Significance was set as p<0.05.\n\nIn order to determine whether miR-146a-5p and miR-155-5p expression differed between males (n=5) and females (n=9), further analysis was performed (Figure 9) (Dataset c). No significant difference was observed between males and females for miR-146a-5p (Whole Blood (PAXM): p=0.797, PBMC (MP): p=0.190) and miR-155-5p (PAXM): p=1.00, PBMC (MP): p=0.898) using both methods.\n\nNo significant difference in (A & B) miR-146a-5p and (C & D) miR-155-5p expression was observed between males and females when total RNA was extracted using PAXM and MP. Normalised miRNA expression was used for this analysis.\n\n\nDiscussion\n\nGlobally, miRNA expression levels are used to classify diseases and also to distinguish the diseased from the healthy population. However, non-uniform technical procedures have led to the many controversies and inconsistencies in miRNA expression results observed in various studies. In addition, failure to test the degree of haemolysis in studies using PB has led to only few reproducible experimental results. Thus, in an attempt to elucidate the importance of protocol standardization and show the impact of technical variations in miRNA expression investigations, we examined several parameters when measuring the expression of immune related miRNAs (miR-146a-5p and miR-155-5p).\n\nFirst, we performed RT-qPCR to test whether the expression of miR-146a-5p and miR-155-5p is the same in whole blood and PBMC samples using three different commercially available extraction methods (PAXM, MP and MM). Our results showed that there was no agreement between PAXM and both MP and MM for miR-146a-5p and miR-155-5p expression. PBMCs constitute only a fraction of the cells present in PB and therefore lack granulocytes, platelets and erythrocytes (Min et al., 2010). Due to the unique miRNA expression pattern in each cell-type the relative proportions of cells in blood may have an effect on the overall miRNA expression profile and the expression of their protein targets (Min et al., 2010). Some studies have shown that mature miRNA expression signature in erythrocytes is similar to that in whole blood while different when compared to PBMCs (Chen et al., 2008). We have clearly demonstrated a high degree of haemolysis in whole blood samples processed using PAXM by measuring the difference between miR-451-5p which is a marker of erythrocytes (Rasmussen et al., 2010) and miR-23a-3p that has been shown to be unaffected by haemolysis (Blondal et al., 2013). We have shown that there is a lower miR-146a-5p and miR-155-5p expression in whole blood compared to PBMCs. Several studies have shown that total RNA yield from whole blood decreases after the use of DNase step in the PAXM protocol (Asare et al., 2008; Bayatti et al., 2014; Debey et al., 2004). However, if PAXM total RNA is not DNase-treated, there may be a possibility of DNA contamination. Contrary to our results, Mookherjee et al. found a linear correlation between miR146a-5p and miR-155-5p expression in whole blood and isolated PBMCs collected from a healthy population (Mookherjee & El-Gabalawy, 2013). In their work, they did not measure the degree of haemolysis in the samples, which may partly explain the discrepancy between the two studies. Furthermore, our study compared both the strength (correlation) and level of agreement between the two methods whilst Mookherjee et al. examined only the correlation (Bland & Altman, 1986). This highlights the importance of performing the correct statistics when two methods are compared with regards to their equivalence and interchangeability (Burd, 2010).\n\nOur results suggest that isolation of total RNA from PBMCs is more reliable with higher RNA purity than isolation from whole blood via PAXgene Blood RNA Tubes. However, in clinical practice, it is easier to collect PB in PAXgene Blood RNA tubes as they have a shelf-life of two to five years without any RNA degradation. Immediate stabilisation is vital as storage of blood cells induce changes in the miRNA composition (Gaarz et al., 2010). Thus, extraction methods from whole blood must be optimised to either eliminate erythrocyte contamination or consider the expression as cumulative and design downstream experiments for miRNA protein target studies accordingly.\n\nWe evaluated whether miR-146a-5p and miR-155-5p expression was different between males and females as previous studies have shown that miRNA expression can be gender-dependent (Ji et al., 2009). We found no gender-dependent differences in the expression of miR-146a-5p and miR-155-5p.\n\nFinally, we assessed the impact of cryopreservation on the expression of both miRNAs. Gaarz et al. had shown that miRNA expression profiles of fresh and cryopreserved isolated PBMCs were not comparable (Gaarz et al., 2010). However, in our investigation we have shown that there is no significant difference in the expression of both miRNAs. This disagreement could be due to technical differences between the studies as Gaarz et al., (2010) who used TRIZOL reagent for extraction, while we used the MP kit. Bland-Altman plots showed that both fresh and cryopreserved methods agreed with each other and cryopreserved samples had lesser variability, which could be explained by the fact that any erythrocyte carried-over from the gradient centrifugation step is removed on freeze-thawing.\n\nIn conclusion, our study shows differences in miR-146a-5p and miR-155-5p expression in isolated PBMCs and whole blood. We suggest that PBMCs are not the ideal source to study and correlate miRNA protein targets where the miRNA expression had been measured in whole blood as the miRNA expression pattern in whole blood is not comparable to that in PBMCs. We also highlight the importance of having a stringent set of technical controls and performing the correct statistics to increase the reliability and reproducibility of miRNA expression studies.\n\n\nConsent\n\nAll participants to the study provided informed written consent for molecular testing and publication of the data.\n\n\nData availability\n\nF1000Research: Dataset 1. Data of miRNA extraction methods from whole blood and PBMCs, 10.5256/f1000research.4884.d33496 (Atarod et al., 2014).",
"appendix": "Author contributions\n\n\n\nS.A performed the experiments, analysed the results and wrote the manuscript. H.S. performed part of the experiments. A.M.D provided constructive comments for the discussion. XN.W interpreted the results and wrote the manuscript. All 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\nThis work was supported by the Newcastle University and the FP7 Marie Curie Initial Training Network CELLEurope (Contract No: 315963).\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 all the volunteers who donated blood for this study. 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}
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{
"id": "6354",
"date": "17 Oct 2014",
"name": "Kenneth Whitaker Witwer",
"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 design of this study is flawed, and several conclusions are not justified by the results. Since the PCR technique seems sound (and the authors are commended for including information such as PCR efficiency), there may be several subcomponents of the study that could be separated out and published as a smaller paper (e.g., the results of the gender and freezing experiments).I would encourage the authors to remove immediately the baseless claims about putative problems with the PAXgene reagents or protocol. A major (and wrong) conclusion of the paper is that the PAXgene method results in putatively excessive hemolysis, or, as the Figure 4 title incorrectly states, \"Erythrocyte contamination\". The putative difference from the other methods is described as \"significantly\" high, and although no statistical analysis is reported, one could hardly quibble with a ten-order-of-magnitude difference. However, this difference has nothing to do with hemolysis as a processing variable and in fact reflects well, not poorly, on the PAXgene method. The PAXgene tube is designed to lyse blood cells and stabilize RNA. This study is thus not an examination of methods, hemolysis or RBC contamination! It is simply a confirmation that lysed RBCs contain RBC RNA, whereas PBMCs do not contain RBC RNA. There appears to be a fundamental confusion about what constitutes hemolysis vs. red blood cell contamination. Where is the evidence that \"haemolysis has been reported to alter miRNA measurements in whole blood, plasma, serum, and tissues\"? The two cited references examine the effects of hemolysis in plasma and serum, where lysis of cells can indeed have a great effect. It is clear that the very low abundance extracellular RNA fraction of blood is influenced by an influx of cellular RNA. It is unclear how degree of hemolysis could affect detection of very high abundance cellular miRNAs in whole blood (where erythrocytes dominate anyway) or in cells like PBMC that are purified away from other components. The title does not describe the study. The study is presented incorrectly as a comparison of RNA extraction methods and of RNA expression in whole blood and PBMCs. Unfortunately, too many variables are changed at once in the study to allow any conclusions to be drawn. For example (see Figure 1): substrate type (whole blood vs PBMC); initial treatment (PAXgene tube for whole blood, heparin for PBMC); cell isolation (none for whole blood, centrifugation over a gradient for PBMC); and RNA isolation method (PAXgene for whole blood, two nearly identical Ambion kits for PBMC). The only variable that can be isolated is the use of the mirVana PARIS vs miRNA kit. Heparin, as a PCR inhibitor, may not be the best anticoagulant for use in qPCR-based studies. No special steps are described for removal of heparin.Minor pointsThere are no error bars to indicate biological or technical variability in Figure 3 or 4. The workflow of Figure 1 does not indicate that the right-hand-side pathway involved only PBMC, not whole blood. The PAXgene Blood RNA Tube and the PAXgene Blood miRNA Kit are presented as the \"gold standard\" in the field, yet the citation is a publication by the company, and it does not mention the miRNA kit. This should be replaced by a more objective assessment. More clarity is needed in the use of terms. For example, there may or may not be RNA \"expression\" differences between genders or cell types. In contrast, differences in RNA quantitation observed upon freezing, or addition of PCR inhibitors, or after purification of the same sample with different kits, are not due to \"expression\" differences. The underlying expression in the cell remains the same; the ability to characterize it changes. Citations of the literature are somewhat thin.",
"responses": [
{
"c_id": "1105",
"date": "01 Dec 2014",
"name": "Sadaf Atarod",
"role": "Author Response",
"response": "Thanks for the valuable comments. We admit full heartedly that some descriptions and statements in the text were not as clear as they should be. This has caused misunderstanding and confusion. Good efforts have been given to improve the clarity of the text and to address each comment by the reviewer. We hope the revised manuscript could convince the reviewer (and the readers) that the design of the study is sound and rational and the conclusions can be well justified by the results.As suggested, the results of gender and cryopreservation experiments have been excluded to deliver a more focused message.The title of the manuscript has also been changed to clarify the message of the \"I would encourage the authors to remove immediately the baseless claims about putative problems with the PAXgene reagents or protocol. A major (and wrong) conclusion of the paper is that the PAXgene method results in putatively excessive hemolysis, or, as the Figure 4 title incorrectly states, \"Erythrocyte contamination\". The putative difference from the other methods is described as \"significantly\" high, and although no statistical analysis is reported, one could hardly quibble with a ten-order-of-magnitude difference. However, this difference has nothing to do with hemolysis as a processing variable and in fact reflects well, not poorly, on the PAXgene method. The PAXgene tube is designed to lyse blood cells and stabilize RNA. This study is thus not an examination of methods, hemolysis or RBC contamination! It is simply a confirmation that lysed RBCs contain RBC RNA, whereas PBMCs do not contain RBC RNA.\" This article has no intention at all to claim any problems with the PAXgene protocol or to examine haemolysis or RBC contamination. The key points we intended to raise in this manuscript are 1) the use of total RNA extracted from whole blood or isolated PBMC could lead to differential results of miRNA quantification as miRNAs expressed in erythrocytes are present in total RNAs extracted from whole blood but not isolated PBMCs; 2) This inevitable fact is partly accountable for many conflicting reports in the existing literatures; 3) This is an important factor that researchers should consider when conducting miRNA research so as to extract total RNAs from most appropriate starting material (whole blood or isolated PBMC). Particularly, total RNAs extracted from the same starting material should be used for analysis of specific miRNAs and their downstream protein targets. These points have been clarified in the revised manuscript.Furthermore, Figure 4 was intended to demonstrate the fact that erythrocyte related miRNA is present and detectable when using total RNAs extracted from whole blood (not to show red blood cell contamination). The title of Figure 4 and the related contents in the text have been revised to clarify this point that it is not a contamination per se but a factor that needs to be considered when analysing, reporting results and investigating miRNA protein targets. \"There appears to be a fundamental confusion about what constitutes hemolysis vs. red blood cell contamination. Where is the evidence that \"haemolysis has been reported to alter miRNA measurements in whole blood, plasma, serum, and tissues\"? The two cited references examine the effects of hemolysis in plasma and serum, where lysis of cells can indeed have a great effect. It is clear that the very low abundance extracellular RNA fraction of blood is influenced by an influx of cellular RNA. It is unclear how degree of hemolysis could affect detection of very high abundance cellular miRNAs in whole blood (where erythrocytes dominate anyway) or in cells like PBMC that are purified away from other components.\"We believe this is a confusion caused by the inappropriate use of terminology rather than a flaw in study design. The revised manuscript has clarified these points. The discussion point of the manuscript is neither about haemolysis nor red blood cell contamination but simply about the fact that the presence of red blood cells in whole blood method may give rise to different miRNA quantification results compared to those where isolated PBMCs were used to extract total RNAs. \"The title does not describe the study. The study is presented incorrectly as a comparison of RNA extraction methods and of RNA expression in whole blood and PBMCs. Unfortunately, too many variables are changed at once in the study to allow any conclusions to be drawn. For example (see Figure 1): substrate type (whole blood vs PBMC); initial treatment (PAXgene tube for whole blood, heparin for PBMC); cell isolation (none for whole blood, centrifugation over a gradient for PBMC); and RNA isolation method (PAXgene for whole blood, two nearly identical Ambion kits for PBMC). The only variable that can be isolated is the use of the mirVana PARIS vs miRNA kit.\" This study was not a comparison of RNA extraction methods. The aim of this study was to find out whether miR-146a-5p and miR-155-5p expression detected in PBMCs mirrored that quantified in total RNA extracted from whole blood. For this reason two main extraction methods had to be used one for extraction from whole blood and the other from PBMCs. Our results clarified the misconceptions reported in the literature suggesting that the levels of miRNAs detected in whole blood and PBMCs correlate with each other. In fact miR-146a-5p and miR-155-5p are both miRNAs that are detectable in acellular sources such as serum and plasma which again reiterates that the miRNA expression quantified in whole blood would be varied from that of PBMCs. \"Heparin, as a PCR inhibitor, may not be the best anticoagulant for use in qPCR-based studies. No special steps are described for removal of heparin.\"The use of Heparin in this study was driven by the logic that Heparin is one of the most commonly used anti-coagulants for collecting PBMCs from clinical samples. We reported over 90% efficiency of the PCR reactions demonstrating the fact that the data presented in this study had not been compromised by the use of Heparin. The quality of RNA was also high as the 260/280 ratios were above 1.8 as stated in the manuscript.Minor points\"There are no error bars to indicate biological or technical variability in Figure 3 or 4. \"Error bars have been added to illustrate technical variability in Figure 3 and Figure 4, respectively. As the variability is very small the error bars are hardly visible, hence inserted the sentence “The error bars represent technical variability” in the figure legends. \"The workflow of Figure 1 does not indicate that the right-hand-side pathway involved only PBMC, not whole blood.\" The workflow of Figure 1 has been modified with an added step to indicate that the right-hand-side pathway involved only PBMCs and not whole blood. \"The PAXgene Blood RNA Tube and the PAXgene Blood miRNA Kit are presented as the \"gold standard\" in the field, yet the citation is a publication by the company, and it does not mention the miRNA kit. This should be replaced by a more objective assessment.\"The company reference has been substituted with an original research article by Viprey et al (2012) which highlights the reliability and suitability of the whole blood collection method using the PAXgene Blood RNA tubes. It is worth noting that the focus of this report is to evaluate the effect of using total RNAs collected via the PAXgene Blood RNA protocol on miRNA quantification rather than on mRNA expressions. \"More clarity is needed in the use of terms. For example, there may or may not be RNA \"expression\" differences between genders or cell types. In contrast, differences in RNA quantitation observed upon freezing, or addition of PCR inhibitors, or after purification of the same sample with different kits, are not due to \"expression\" differences. The underlying expression in the cell remains the same; the ability to characterize it changes.\"Great attention has been given to using terms with clarity and accuracy in the revised manuscript. \"Citations of the literature are somewhat thin.\"In contrast to mRNA research where large pools of literatures have investigated numerous factors that could impact on the outcome of mRNA quantification, the miRNA research is still at its infancy and very few studies have investigated the impact of various total RNA extraction methods on the outcome of miRNA quantification although many articles have been published reporting associations of differential miRNA expression with diagnostic or prognostic status of diseases. This manuscript is particularly aimed to inform the miRNA community that different sources and methods used for extracting total RNAs could have significant impact on the outcome of miRNA quantification."
}
]
}
] | 1
|
https://f1000research.com/articles/3-183
|
https://f1000research.com/articles/4-1002/v1
|
06 Oct 15
|
{
"type": "Case Report",
"title": "Case Report: Severe non-immune mediated haemolytic anaemia associated with acute hepatitis B and E co-infection in a patient with normal G6PD levels",
"authors": [
"Michael Wang",
"Florence Yap",
"Gavin Joynt",
"Florence Yap",
"Gavin Joynt"
],
"abstract": "Severe non-immune mediated haemolytic anaemia (HA) rarely occurs in acute viral hepatitis, unless patients have underlying red cell enzyme abnormalities or pre-existing liver disease. We report such a case and provide a summary of other available cases to date.Overall, young and fit patients suffering from acute viral hepatitis seem to be affected. Several viral subtypes have been associated, although we report the first hepatitis B and E co-infection case. It seems to occur when patients are recovering from hepatitis and the disease course is variable. The degree of anaemia is always severe, and is inevitably associated with increased morbidity and mortality.Several theories exist with regard to its aetiology, including the disruption of erythrocyte metabolism. Although its optimal treatment strategies remain unclear, some evidence suggests a possible role for steroid therapy.",
"keywords": [
"viral hepatitis",
"anemia",
"hemolytic",
"hemolysis",
"glucose phosphate dehydrogenase deficiency"
],
"content": "Background\n\nAlthough mild haemolysis is a well known phenomenon associated with acute viral hepatitis and occurs in approximately 4% of affected patients1, severe non-immune mediated haemolytic anaemia (HA) is rare and is usually associated with underlying red cell enzyme abnormalities2,3 or pre-existing liver diseases4,5. We report a case of severe non-immune mediated haemolytic anaemia associated with acute viral hepatitis in the absence of any underlying enzyme or hepatic abnormalities. Only 7 other such cases have been found in the current literature, and their summaries will be provided and discussed.\n\n\nCase report\n\nA 48 year old Chinese male manual worker was admitted on 4th February with acute right-upper-quadrant abdominal pain for 5 days, preceded by flu like illness. He had no past history of liver disease, anaemia or bleeding disorders. He reported recent consumption of raw seafood and unprotected sexual activities with sex workers, but denied any history of alcohol abuse or recreational drug use. He was not on any regular medications. Clinical examination revealed fever, deep jaundice and right-upper-quadrant tenderness with no signs of chronic liver disease or hepatic encephalopathy. His blood tests showed a severe hepatitis picture (Table 1) including a total bilirubin of 291 µmol/L and alanine transaminase (ALT) of 8320 IU/L. He tested positive for hepatitis B surface antigen (HBsAg), IgM antibody to hepatitis B core antigen (anti-HBc IgM) and IgM antibody to hepatitis E virus (anti-HEV IgM). His real-time polymerase chain reaction (PCR) quantification for hepatitis B virus was 4.34 log IU/ml, thus confirming the diagnosis of acute viral hepatitis B and E co-infection. His haemoglobin (Hb) level was 13.3 g/dl and dropped to 12 g/dl initially, which suggested possible mild haemolysis. His international normalised ratio (INR) was 1.5 on presentation which was also expected in severe acute hepatitis. He was empirically treated with entacevir, 0.5 mg once a day from day 1 to day 9 following admission, and vitamin K replacement, 10 mg twice a day for two days. In addition, his ceruplasmin levels were normal, and paracetamol and salicylate were not detectable.\n\nOn the following few days his ALT improved and INR normalised (Table 1). But while these parameters and his clinical status were improving, his Hb level was noted to drop to 10.8 g/dl on day 3 following admission, then to 8.9 g/dl on day 4 and finally to a nadir of 4.9 g/dl on day 5. This was accompanied by a rapid increase in total bilirubin levels, which peaked at 1132 µmol/L with an indirect bilirubin level of 461.7 µmol/L. Severe haemolysis was suspected and confirmed on further blood tests: haptoglobin of < 0.2 g/l (normal range: 0.30 to 2.00 g/l), lactate dehydrogenase of (LDH) 2493 IU/L (normal range: 106–218 IU/L), and reticulocytes of 201.1 ×109/L. His peripheral blood film showed polychromasia, which was compatible with haemolysis, with no evidence of sickle cell morphology or hereditary spherocytosis. Furthermore, he had a normal glucose-6-phosphate dehydrogenase (G6PD) level. His direct Coombs test was negative with a normal cold agglutinin titre, thus confirming non-autoimmune haemolytic anaemia. He was treated with folate, 5 mg once a day from day 5 to day 9 following admission. In addition, his bone marrow aspirate excluded any haematological malignancies and his raised WCC of 57 ×109/L was thought to be a significant leukemoid reaction in response to the haemolysis. Unfortunately, his condition was further complicated by acute renal failure (Table 1) and severe chest sepsis, requiring continuous venous venous haemofiltration (CVVH) at 3 litres ultrafiltration per hour, high dose terlipressin at 1 mg every 6 hours and noradrenaline at the range of 0 to 1.2 mcg/kg/min to target mean arterial pressure of 65 mmHg, as well as intravenous tazocin at the dose of 4.5 g three times a day. All supportive were continued until day 9 following admission. The acute severe haemolysis resolved as quickly as it occurred, after transfusing a total of 5 units of whole blood between day 5 and day 8 following admission, his Hb level stabilised (Hb 10.4 g/dl on day 9 following admission) and LFTs much improved (Table 1).\n\nUnfortunately, the patient deteriorated again on the evening of day 9. He developed increasing abdominal distension, melaena and severe metabolic acidosis (pH 6.96, BE -21). An urgent abdominal computerized tomography (CT) scan showed intramural gas and diminished mucosal enhancement of segments of large bowel with pneumoperitoneum, suggestive of perforated large bowel, likely secondary to intestinal ischaemia attributed to sepsis and vasopressors. An urgent laparotomy and subtotal colectomy was performed, and the intra-operative findings confirmed ischaemic bowel and intestinal perforation. However, the patient failed to improve and finally succumbed on day 10.\n\n\nDiscussion\n\nThe authors found seven other case reports of severe non-immune mediated haemolytic anaemia in patients with acute viral hepatitis, having normal G6PD levels and no previous liver disease (Table 2)4–8. Of the 4 cases in which the virus subtype was known, A, B and E subtypes were all involved. All the patients were young, aged between 11 and 48, with good past health. They all dropped their Hb levels significantly to below 8 g/dl, with one case reaching a nadir of 3.99 g/dl. In the majority of cases, the haemolysis presented when the patient was showing improvement from hepatitis, either clinically or evidenced by the dropping in their liver parenchymal enzyme levels. All the patients received blood transfusion and five patients received steroids, four of whom were thought to have responded. In all cases, the haemolysis was associated with a suboptimal clinical outcome, of which six patients had a prolonged hospital stay with a median duration of around 30 days. The remaining one succumbed.\n\nOur patient was co-infected with hepatitis B and E, most likely due to his sexual activity with a sex worker and consumption of raw seafood respectively, and severe haemolysis associated with this type of co-infection has not been reported in the literature before. The co-infection might have contributed to the poor outcome of this patient. Additionally, our patient’s haemolysis developed over a few days and resolved as rapidly as it developed, as the transfusion of 5 units of whole blood alone was sufficient to stabilize the Hb level (Table 1). Haemolysis with such a rapid rate of onset and resolution has not been observed in other cases.\n\nThe mechanism for non-immune mediated haemolytic anaemia in acute viral hepatitis is not clear. One theory suggests that partially oxidative metabolites that decrease erythrocyte-reduced glutathione are released in acute viral hepatitis, and this decreased level of reduced glutathione impairs the integrity of erythrocytes1,2. Some data suggest that haemolysis is caused by an extracorpuscular factor including the virus itself9–11. Other theories include endogenous hepatotoxic substances, splenomegaly and alteration of red cell osmotic fragility as potential causes of haemolysis2,4.\n\n\nConclusion\n\nSevere non-immune mediated haemolytic anaemia occurs rarely in patients with acute viral hepatitis with no G6PD deficiency or pre-existing liver disease and is associated with suboptimal outcomes. Most patients are young with good health status and are affected when they have recovered or are recovering from hepatitis. The anaemia secondary to haemolysis is so far always severe and can predispose the patients to other organ dysfunctions. Several viral subtypes are associated, although the disease mechanism is unclear, despite several proposed theories. In terms of treatment, several patients are thought to have responded to steroids in addition to transfusion, indicating its potential therapeutic role.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the next of kin of the patient.",
"appendix": "Author contributions\n\n\n\nMXW prepared the first draft of the manuscript. FHYY and GMJ conceived the case report and guided MXW in preparing the first draft of 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\nChau TN, Lai ST, Lai JY, et al.: Haemolysis complicating acute viral hepatitis in patients with normal or deficient glucose-6-phosphate dehydrogenase activity. Scand J Infect Dis. 1997; 29(6): 551–553. PubMed Abstract | Publisher Full Text\n\nKattamis CA, Tjortjatou F: The hemolytic process of viral hepatitis in children with normal or deficient glucose-6-phosphate dehydrogenase activity. J Pediatr. 1970; 77(3): 422–430. PubMed Abstract | Publisher Full Text\n\nAgarwal RK, Moudgil A, Kishore K, et al.: Acute viral hepatitis, intravascular haemolysis, severe hyperbilirubinaemia and renal failure in glucose-6-phosphate dehydrogenase deficient patients. Postgrad Med J. 1985; 61(721): 971–975. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHansbarger EA Jr, Hyun BH: Acute hemolytic anemia in viral hepatitis. Report of two cases. Va Med Mon (1918). 1963; 90: 134–139. PubMed Abstract\n\nRaffensperger EC: Acute acquired hemolytic anemia in association with acute viral hepatitis. Ann Intern Med. 1958; 48(6): 1243–1253. PubMed Abstract | Publisher Full Text\n\nVanderhoof JA, Ament ME: Severe Coombs negative hemolytic anemia in hepatitis B. West J Med. 1976; 125(3): 228–230. PubMed Abstract | Free Full Text\n\nTing PL, Chew WL, Tan BY: Viral hepatitis B induced haemolytic anaemia in a patient with normal glucose 6 phosphate dehydrogenase--a case report. Singapore Med J. 1984; 25(5): 360–361. PubMed Abstract\n\nIbe M, Rüde B, Gerken G, et al.: Coombs-negative severe hemolysis associated with hepatitis A. Z Gastroenterol. 1997; 35(7): 567–569. PubMed Abstract\n\nMartini GA, Strohmeyer G: Posthepatitis syndromes. Clin Gastroenterol. 1974; 3(2): 377–390. PubMed Abstract\n\nChan TK, Todd D: Haemolysis complicating viral hepatitis in patients with glucose-6 phosphate dehydrogenase deficiency. Br Med J. 1975; 1(5950): 131–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConrad ME, Schwartz FD, Yound AA: Infectious hepatitis; a generalized disease. A study of renal, gastrointestinal and hematologic abnormalities. Am J Med. 1964; 37(5): 789–801. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10685",
"date": "02 Nov 2015",
"name": "Rosario Notaro",
"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\nMichael Wang and co-Authors report a case of severe non-immune mediated hemolytic anemia occurred during an episode of acute E and B viral hepatitis in a patient that is not G6PD deficient.Unfortunately the manuscript does not provide strong evidence that the patient is not G6PD deficient. In fact, the Authors have measured G6PD level (the level is not shown) only during the hemolytic episode when reticulocyte count was high (201.1 ×109/L): it is well known that high reticulocyte count can mask the deficiency of G6PD. In order to prove that the patient is not G6PD deficient the Authors should show G6PD levels measured before the hemolytic episode or they should show that at DNA level the patient carried a G6PD mutation know to be associated with G6PD deficiency. In addition, a more extensive workout for inherited hemolytic anemia (pyruvate kinase deficiency, etc.) would be a good addition. Minor PointsAbstract. The abstract reports no data about the actual clinical case.Conclusion. This is a summary of the literature poorly related with the findings from the reported clinical case.Table 1: Few abbreviations are not spelt out.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1002
|
https://f1000research.com/articles/4-624/v1
|
25 Aug 15
|
{
"type": "Research Note",
"title": "The ubiquitous and ancient ER membrane protein complex (EMC): tether or not?",
"authors": [
"Jeremy G. Wideman"
],
"abstract": "The recently discovered endoplasmic reticulum (ER) membrane protein complex (EMC) has been implicated in ER-associated degradation (ERAD), lipid transport and tethering between the ER and mitochondrial outer membranes, and assembly of multipass ER-membrane proteins. The EMC has been studied in both animals and fungi but its presence outside the Opisthokont clade (animals + fungi + related protists) has not been demonstrated. Here, using homology-searching algorithms, I show that the EMC is truly an ancient and conserved protein complex, present in every major eukaryotic lineage. Very few organisms have completely lost the EMC, and most, even over 2 billion years of eukaryote evolution, have retained a majority of the complex members. I identify Sop4 and YDR056C in Saccharomyces cerevisiae as Emc7 and Emc10, respectively, subunits previously thought to be specific to animals. This study demonstrates that the EMC was present in the last eukaryote common ancestor (LECA) and is an extremely important component of eukaryotic cells even though its primary function remains elusive.",
"keywords": [
"Evolutionary cell biology",
"ER membrane protein complex (EMC)",
"Membrane contact sites (MCS)",
"ERMES",
"ER-mitochondria contact sites"
],
"content": "Introduction\n\nRecent studies suggest that the EMC (Endoplasmic Reticulum Membrane Complex) is a multifunctional, multi-subunit protein complex. In Homo sapiens, the EMC comprises ten subunits, Emc1-10, whereas in Saccharomyces cerevisiae the complex comprises only Emc1-6 (Jonikas et al., 2009). The EMC has been implicated in several cellular processes. It has been implicated in ERAD (ER-associated degradation) (Christianson et al., 2012; Jonikas et al., 2009; Richard et al., 2013) but the molecular mechanism for how EMC triggers ERAD has remained elusive. Emc6 contains a Rab5 interacting domain and has been shown to interact with Rab5A in humans during autophagosome formation (Li et al., 2013). It has also been shown that the EMC is an ER-mitochondria tether in S. cerevisiae that interacts with the outer membrane protein Tom5 of the TOM (Translocase of the Mitochondrial Outer Membrane) complex (Lahiri et al., 2014). Most recently, the EMC has been implicated in the proper assembly of multi-pass transmembrane (TM) proteins (Satoh et al., 2015). These recent findings suggest that EMC involvement in ERAD may be due to secondary effects, as cells devoid of EMC components may result in either disruption of ER-mitochondria tethering, or the misfolding of multipass membrane proteins. Thus, the primary function of the EMC is still open for debate.\n\nThe ER-mitochondria encounter structure (ERMES), also involved in ER-mitochondria tethering, is a multifunctional protein complex implicated in both lipid transfer and mitochondrial outer membrane protein assembly (AhYoung et al., 2015; Kornmann et al., 2009; Meisinger et al., 2006, Meisinger et al., 2007; Wideman et al., 2013; Wideman et al., 2010). However, ERMES as an ER-mitochondria tether is limited to a subset of eukaryote taxa (Wideman et al., 2013), suggesting that a universal ER-mitochondria tethering complex might exist. Lahiri et al. (2014) state in their title that the EMC is a conserved protein complex. However, by stating that a protein is conserved, cell biologists and biochemists often simply mean that the protein is present in S. cerevisiae (fungi) and animals. Since the clade comprising animals and fungi only accounts for one fifth of the diversity of eukaryotes (Adl et al., 2012), more work is necessary in order to support the claim made by Lahiri et al. Thus, I was prompted to investigate the taxonomic distribution of the EMC in order to (1) determine if it really is a conserved protein complex and (2) if it could possibly represent the pan-eukaryotic ER-mitochondria tether.\n\n\nMethods\n\nSequences of experimentally validated EMC components (see Table S1 for accession numbers) from H. sapiens and S. cerevisiae were used as queries in BLAST (Altschul et al., 1997) and pHMMer (Finn et al., 2011) searches into the predicted proteomes of 70 eukaryotes spanning the diversity of eukaryotes. Retrieved sequences were considered orthologous if they retrieved the original H. sapiens or S. cerevisiae EMC sequences as top hits when used as reciprocal BLAST or pHMMer queries into H. sapiens or S. cerevisiae predicted proteomes and did not retrieve any other closely related sequences (except in the case of Emc8 and Emc9, which are related). In cases in which EMC components could not be identified in this manner, transcriptomes and genomes were searched using bioinformatically validated sequences from the previous step that were retrieved from closely related species. Genomes were downloaded from public repositories and genome project websites. See Table S1 for retrieved sequences.\n\n\nResults and discussion\n\nUsing homology-searching algorithms EMC candidate proteins were identified in the vast majority of sequenced genomes representing the complete diversity of eukaryotes (Figure 1). Only Emc9 was found to be absent from most taxa and is very likely the result of a vertebrate-specific duplication of Emc8.\n\nColoured pies indicate presence of a particular subunit. Plot was generated using the Coulson plot generator (Field et al., 2013). Asterisks indicate presence of orthologue in a different member of the genus but absent in the indicated species (see Table S1). Abbreviations: Vertebrates: Hsap, Homo sapiens; Mdom, Monodelphis domesticus; Drer, Danio rerio; Xtro, Xenopus tropicalis; Ggal, Gallus gallus; Mmus, Mus musculus; Invertebrates: Cele, Caenorhabditis elegans; Dmel, Drosophila melanogaster; Bflo, Branchiostoma floridae; Nvec, Nematostella vectensis; Tadh, Trichoplax adhaerens; Unicellular Holozoa: Mbre, Monosiga brevicollis; Cowc, Capsaspora owczarzaki; Sarc, Sphaeroforma arctica; Sros, Salpingoeca rosetta; Fungi: Spom, Schizosaccharomyces pombe; Scer, Saccharomyces cerevisiae; Ncra, Neurospora crassa; Cneo, Cryptococcus neoformans; Umay, Ustilago maydis; Bden, Batrachochytrium dendrobatidis; Ncer, Nosema ceranae; Ecun, Encephalitozoon cuniculi; Pir, Piromyces sp.; Spun, Spizellomyces punctatus; Rirr, Rhizophagus irregularis; Crev, Coemansia reversa; Ccor, Conidiobolus coronatus; Cang, Catenaria anguillulae; Rall, Rozella allomyces; Apusozoa: Ttra, Thecamonas trahens; Fonticulids: Fonticula alba; Amoebozoa: Acas, Acanthamoeba castellanii; Ddis, Dictyostelium discoideum; Ehis, Entamoeba histolytica; Ppal, Polysphondylium pallidum; Excavata: Ngru, Naegleria gruberi; Gint, Giardia intesinalis; Tvag, Trichomonas vaginalis; Bsal, Bodo saltans; Lmaj, Leishmania major; Tbru, Trypanosoma brucei; Stramenopiles: Bhom, Blastocystis hominis; Alim, Aurantiochytrium limacinum; Aana, Aureococcus anophagefferens; Tpse, Thalassiosira pseudonana; Ptri, Phaeodactylum tricornutum; Psoj, Phytophthora sojae; Esil, Ectocarpus siliculosus; Ngad, Nannochloropsis gaditana; Alveolates: Ptet, Paramecium tetraurelia; Tthe, Tetrahymena thermophila; Otri, Oxytricha trifallax; Tpar, Theileria parva; Smin, Symbiodinium minutum; Tgon, Toxoplasma gondii; Cpar, Cryptosporidium parvum; Pfal, Plasmodium falciparum; Rhizaria: Bnat, Bigelowiella natans; Rfil, Reticulomyxa filosa; Archaeplastida: Crei, Chlamydomonas reinhardtii; Cmer, Cyanidioschyzon merolae; Cyp, Cyanophora paradoxa; Atha, Arabidopsis thaliana; Ppat, Physcomitrella patens; Otau, Ostreococcus tauri; Gsul, Galdieria sulphuraria; Mpus, Micromonas pusilla; Ccri, Chondrus crispus; CCTH: Ehux, Emiliania huxleyi; Gthe, Guillardia theta.\n\nA complete EMC (Emc1-8, 10) was found in at least one representative from each major lineage including animals, fungi, excavates, amoebozoa, green algae, plants, stramenopiles, alveolates, rhizaria, and haptophytes (Figure 1). The relative sequence conservation of EMC components across diverse taxa suggests that the EMC has an ancient and critical role in cellular function.\n\nAlthough previous reports suggest S. cerevisiae EMC comprises only six subunits, I identified Sop4 and YDR056C as orthologues of Emc7 and Emc10, respectively. Supporting this, Jonikas et al. (2009), the original discoverers of the EMC, show by co-immunoprecipitation analyses that Sop4 and YDR056C are interacting partners of FLAG-tagged Emc3. This experiment not only confirms my bioinformatic classification but also puts into perspective a previous study on Sop4’s role in membrane protein quality control (Luo et al., 2002). Furthermore, tracing the evolutionary history of the EMC in fungi demonstrates that Emc8 was lost only in Ascomycetes and a few basally diverging fungi whereas most fungi retain Emc8 (as well as Emc7 and 10).\n\nAlthough the EMC was identified in representative taxa from every major eukaryote supergroup, I was unable to identify even a single EMC member in the genomes of the microsporidians Nosema ceranae and Encephalitozoon cuniculi, the metamonad Giardia intestinalis, the stramenopile Blastocystis hominis, the alveolate Theileria parva, and the red alga Cyanidioschyzon merolae (Figure 1 and Figure 2). Trichomonas vaginalis, another metamonad retains only a rather divergent Emc2, that passed the test for orthology, but only weakly, suggesting that this protein is under relaxed selection, perhaps repurposed, or in the process of being lost. All other genomes from the remaining 65 species investigated contained clear representatives of EMC homologues (Figure 1).\n\nEMC 1-8 and 10 evolved prior to the divergence of the major eukaryote lineages. Green and red dashes represent gains and losses of EMC components, respectively. Coloured pies are schematic representations of which EMC components were present at different points over the course of evolution.\n\nThese disparate organisms that lack the EMC prompted the question: What cellular or biochemical features tie these diverse organisms together? The microsporidians, metamonads and B. hominis all contain reduced anaerobic mitochondria-related organelles (MROs) and also lack the EMC. However, the amoebozoan Entamoeba histolytica retains Emc1-4, 7 and 10, the apicomplexan Cryptosporidium parvum retains Emc1-4, and 8, and the fungus Piromyces sp. retains Emc1-4, 6, 7, and 10, but all three organisms also contain extremely reduced MROs. T. parva and C. merolae contain relatively normal mitochondria but completely lack the EMC. Thus, it seems that further insight into the cell biology of these organisms is required to understand why only these few species from unrelated lineages have lost the EMC. At this point, of the proposed functions of the EMC, its involvement in multipass membrane protein assembly is the best candidate for generalization to other eukaryotes. It explains the connection to ERAD as a secondary effect of misassembled multipass proteins and explains why an organism with extremely reduced mitochondria (E. histolytica) might retain the EMC. Finally, although EMC involvement as an ER-mitochondria tether is attractive, the distribution of the only known MOM-localized interactor of EMC (Tom5) has not been identified in organisms other than animals and fungi (Maćasev et al., 2004). Thus, until an ancient interaction partner is identified, the role of EMC as an ancient tether remains speculative.\n\n\nConclusions\n\nSince the vast majority of species from each major branch of eukaryotes retain the EMC it can be inferred that it was present in the last eukaryote common ancestor (LECA). Since the sequences of most of the identified EMC homologues are very similar, it can be inferred that its function has likely been retained in most eukaryote lineages. Thus, the EMC is a generalizable eukaryotic feature as is its function—whatever it might be.\n\n\nData availability\n\nAll sequence data are freely available in online databases (NCBI, JGI, or independent genome sequencing project websites).",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJGW is the recipient of the European Molecular Biology Organization (EMBO) Long-term Fellowship (ALTF 761-2014).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nI thank Joel Dacks for server access, computational time, and inspiration.\n\n\nSupplementary materials\n\nProtein sequences retrieved in this study.\n\nClick here to access the data.\n\n\nReferences\n\nAdl SM, Simpson AGB, Lane CE, et al.: The revised classification of eukaryotes. J Eukaryot Microbiol. 2012; 59(5): 429–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhYoung AP, Jiang J, Zhang J, et al.: Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly. Proc Natl Acad Sci U S A. 2015; 112(25): E3179–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAltschul SF, Madden TL, Schäffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChristianson JC, Olzmann JA, Shaler TA, et al.: Defining human ERAD networks through an integrative mapping strategy. Nat Cell Biol. 2012; 14(1): 93–105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nField HI, Coulson RMR, Field MC: An automated graphics tool for comparative genomics: the Coulson plot generator. BMC Bioinformatics. 2013; 14: 141. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinn RD, Clements J, Eddy SR: HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011; 39(Web Server issue): W29–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJonikas M, Collins S, Denic V, et al.: Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science. 2009; 323(5922): 1693–1697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKornmann B, Currie E, Collins SR, et al.: An ER-mitochondria tethering complex revealed by a synthetic biology screen. Science. 2009; 325(5939): 477–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLahiri S, Chao JT, Tavassoli S, et al.: A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria. PLoS Biol. 2014; 12(10): e1001969. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi Y, Zhao Y, Hu J, et al.: A novel ER-localized transmembrane protein, EMC6, interacts with RAB5A and regulates cell autophagy. Autophagy. 2013; 9(2): 150–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuo W, Gong X, Chang A: An ER membrane protein, Sop4, facilitates ER export of the yeast plasma membrane [H+]ATPase, Pma1. Traffic. 2002; 3(10): 730–9. PubMed Abstract | Publisher Full Text\n\nMaćasev D, Whelan J, Newbigin E, et al.: Tom22’, an 8-kDa trans-site receptor in plants and protozoans, is a conserved feature of the TOM complex that appeared early in the evolution of eukaryotes. Mol Biol Evol. 2004; 21(8): 1557–64. PubMed Abstract | Publisher Full Text\n\nMeisinger C, Pfannschmidt S, Rissler M, et al.: The morphology proteins Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria. EMBO J. 2007; 26(9): 2229–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeisinger C, Wiedemann N, Rissler M, et al.: Mitochondrial protein sorting: differentiation of beta-barrel assembly by Tom7-mediated segregation of Mdm10. J Biol Chem. 2006; 281(32): 22819–26. PubMed Abstract | Publisher Full Text\n\nRichard M, Boulin T, Robert VJP, et al.: Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex. Proc Natl Acad Sci U S A. 2013; 110(11): E1055–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSatoh T, Ohba A, Liu Z, et al.: dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors. Elife. 2015; 4: e06306. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWideman JG, Gawryluk RMR, Gray MW, et al.: The ancient and widespread nature of the ER-mitochondria encounter structure. Mol Biol Evol. 2013; 30(9): 2044–9. PubMed Abstract | Publisher Full Text\n\nWideman JG, Go NE, Klein A, et al.: Roles of the Mdm10, Tom7, Mdm12, and Mmm1 proteins in the assembly of mitochondrial outer membrane proteins in Neurospora crassa. Mol Biol Cell. 2010; 21(10): 1725–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWideman JG, Lackey SWK, Srayko MA, et al.: Analysis of mutations in Neurospora crassa ERMES components reveals specific functions related to β-barrel protein assembly and maintenance of mitochondrial morphology. PLoS One. 2013; 8(8): e71837. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "10100",
"date": "04 Sep 2015",
"name": "Sujoy Lahiri",
"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 titled “The ubiquitous and ancient ER membrane protein complex (EMC): tether or not?” authored by Jeremy G. Wideman is a comprehensive study to determine if the EMC proteins are truly conserved. Using homology searching algorithms the author has shown that except for a few branches the EMC proteins are present in the vast majority of the eukaryotes and reasoned in favor of the presence of EMC proteins in the last eukaryote common ancestor (LECA). In addition the author has also identified two genes in S. cerevisiae to be orthologues of Emc7 and Emc10 which supports the finding of Jonikas et al. where these two proteins were co-immunoprecipitated along with the other EMC proteins. In light of the increasing scientific attention on the EMC proteins over past few years and their multifaceted roles in cell physiology I find this article to be quite relevant in delivering a thorough understanding of this protein complex from the evolutionary perspective. Thus this study by Wideman will be helpful in further understanding of the biology of the EMC proteins. On its scientific merit I consider this article to be substantially important for getting published with F1000Research. However there is one major concern, which I'd like to be addressed before endorsing the acceptance of this article. The author has described Emc9 to be present only among the vertebrates. However the HomoloGene database of NCBI shows Emc9 homologs to be present in Drosophila melanogaster and Anopheles gambiae (http://www.ncbi.nlm.nih.gov/homologene/41095). I assume that the homology searching algorithm used by the author has designated the Drosophila Emc9 homolog protein NP_611731.1 as Emc8 which calls for a discussion by the author. Furthermore, this led me to explore whether Emc8 and Emc9 share any sequence homology. Using pairwise alignment of NCBI Blast (http://goo.gl/B8T0P3) between human Emc8 (NP_006058.1) and Emc9 (NP_057133.2) I found 44% sequence identity between these two proteins with 93% query coverage and an E value of 2e-57. No other Emc proteins, besides Emc8 and Emc9, share such high degree of sequence identity. This makes me curious of whether Emc8 and Emc9 could be paralogs in the vertebrates. In such case the gain of Emc9 among the vertebrates could be explained by a possible duplication of Emc8. In light of this I would request the author to elucidate possible reasons for the high degree of sequence homology between Emc8 and Emc9 and discuss the anomaly between his data as presented in this article and the HomoloGene database.",
"responses": [
{
"c_id": "1621",
"date": "05 Oct 2015",
"name": "Jeremy G Wideman",
"role": "Author Response",
"response": "Thank you for your very positive review. I have addressed your major concern by including additional phylogenetic data. Emc8 and Emc9 are now clearly shown as paralogues due to a duplication in the ancestral lineage leading to vertebrates. As such, to prevent future confusion I suggest that vertebrate Emc8 and Emc9 be renamed to Emc8a and Emc8b respectively. I hope you now find the article sufficient for approval."
}
]
},
{
"id": "10381",
"date": "24 Sep 2015",
"name": "Courtney Stairs",
"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 \"The ubiquitous and ancient ER membrane protein complex (EMC): tether or not?\" presents the distribution of EMC components across eukaryotic diversity. Using a strictly bioinformatic approach, Wideman identified homologues of the majority of the EMC in every major eukaryotic supergroup, suggesting that this complex was likely present in the last eukaryotic common ancestor. One of the most interesting findings of this study was the identification of two previously unreported EMC components (Emc7 and Emc10) in yeast. In fact, the in silico findings presented here are supported by previously published co-immunopreciptation study (Jonikas et al, 2009) that identified these two components. Surprisingly, the EMC also seems to be present in some organisms that possess highly reduced mitochondria (i.e. mitochondrion-related organelles; MROs). Although beyond the immediate scope of this study, it would be interesting to correlate the presence of various TOM components in these 'amitochondriates' with the various EMC components. Perhaps a brief comment on this in the discussion would be informative - especially since the interaction of TOM and EMC is known in yeast. In another review for this article, Sujoy Lahiri commented on the assignment of the Drosophila and Anopheles Emc8 as Emc9 on the Homologene database (NCBI). It appears as though these organisms have only one homologue of Emc8 (OR Emc9) by BLAST. It would be helpful if the author could comment on this observation - is this a mistake by Homologene? A phylogenetic analysis of these two related proteins could be helpful to determine the evolutionary origins of these proteins in animals. They also brought up concerns about the homology between these two proteins - however I think the author addresses this in the methods section where he states '...Emc8 and Emc9, which are related)...'. A system so fundamental to the cellular biology of eukaryotes is likely the result of vertical inheritance, however phylogenetic analysis of each component could help solidify this hypothesis and exclude any concerns over lateral gene transfer. Also, a single sentence describing if any of these components have distant homologues in prokaryotes (especially the recently described Lokiarchaeota) could also be informative for a non-expert audience (such as this reviewer). The data presented by Wideman (2015) is well within the scope of F1000Research and will be an invaluable resource for those studying the interactions between the ER and the mitochondria. I have no major concerns on the article and fully support its continued publication in F1000Research.",
"responses": [
{
"c_id": "1622",
"date": "05 Oct 2015",
"name": "Jeremy G Wideman",
"role": "Author Response",
"response": "Thank you for your insightful review. I have addressed the major concern from Sujoy Lahiri's review by including additional data (opisthokont Emc8/9 phylogeny).As you suspected, some of your other comments are out of the scope of the paper, but I would like to comment on them here for anyone that is interested.First, regarding your comment “it would be interesting to correlate the presence of various TOM components in these 'amitochondriates' with the various EMC components”: yes this would be interesting, however, I believe best included in a larger study on the evolution of protein import pathways. Tom5, the only known MOM interactor of EMC is found only in Opisthokonts (Macasev et al. 2004), although this has not been investigated in detail for quite some time. The protein is so short (~50aa) that it is easily missed in bioinformatic analyses; even if the protein is more widespread, it may be that it will only be identified biochemically. Additionally, most amitochondriates have extremely divergent Tom complexes (e.g. Entamoeba, microsporidians, Giardia), and it is unlikely that even if a small protein like Tom5 is present in these organisms that it will be detectable by phylogenetic analysis.Second, prokaryotes do not seem to have any close homologues (based on a preliminary BLAST into NCBI prokaryote database) but some weak homology can be detected. Further investigation is beyond the scope of this project.Third, the likelihood of HGT of EMC components is quite low in this case given the high frequency of retention of EMC across all eukaryotes. Also, for many of the proteins it is unlikely that phylogenies would resolve HGTs as many of the proteins are very short and support values would be low."
}
]
}
] | 1
|
https://f1000research.com/articles/4-624
|
https://f1000research.com/articles/4-997/v1
|
05 Oct 15
|
{
"type": "Software Tool Article",
"title": "NGSeasy: a next generation sequencing pipeline in Docker containers",
"authors": [
"Amos A Folarin",
"Richard JB Dobson",
"Stephen J Newhouse",
"Richard JB Dobson"
],
"abstract": "Motivation: Bioinformatic pipelines often use large numbers of components and deploying them incurs substantial configuration and maintenance burden that remains a significant barrier to reproducible research. Our aim is to define a new paradigm and best practices for developing, distributing and running pipelines encapsulated in Docker containers (lightweight virtualization), with a focus on next generation sequencing (NGS) workflows. This approach provides several advantages, namely: efficiency, portability, versioning and reproducibility. Using the NGSeasy pipeline, a user can quickly deploy any pipeline version in any environment (e.g. operating systems, workstations, clusters, clouds). While this might also be achieved with a virtual machine (VM); VMs lack portability, have substantial overhead (disk, CPU, RAM), and require allocated resources to be provisioned statically – Docker, to a large extent, solves these issues.Results: We demonstrate best practices for packaging and execution of a multicomponent pipeline for NGS using a set of container building blocks which are versioned, modular and reusable. We present a basic ”proof of concept” evaluation of a next generation sequencing pipeline in Docker containers, capable of producing meaningful results, that are comparable with public and ”best practice” workflows, with little to no impact on standard computing performance.Availability: Both versioned Dockerfiles and container images for each component are published on GitHub and Docker Hub, respectively. The pipeline and containers can be pulled from Docker Hub and executed on any environment capable of running the Docker platform with minimum hardware requirements for running an NGS pipeline.",
"keywords": [
"next generation sequencing",
"docker",
"container",
"variant caller",
"pipeline",
"read alignment",
"reproducible",
"bioinformatics"
],
"content": "Introduction\n\nBioinformatic pipelines are frequently composed of large numbers of loosely coupled pieces of software, each tool requiring substantial configuration, maintenance and management of dependencies. Historically to facilitate packaging and reuse of pipelines, management frameworks such as Galaxy1, Ruffus2, and Taverna3 have been developed. While these workflow management systems work well, portability and deployment complexity limit their usability.\n\nOur primary motivation for developing NGSeasy was to simplify pipeline deployment for academic and clinical labs, minimising the burden of informatic support. To achieve this, we used Docker4, an emerging container-based virtualization technology. Compared to virtual machines, Docker containers are simply a set of processes running in a multi-tenanted Linux host kernel, so are very lightweight as there is no underlying machine to emulate. These containers capture the initial investment of effort to build and configure them greatly facilitating re-use, they can be easily extended to modify or incorporate new components and shared on private or public (Docker Hub) registries.\n\nUsing NGSeasy and Docker, bioinformaticians and more importantly, researchers with fewer bioinformatic skills can very quickly deploy the pipeline to different environments e.g. development, testing and production, with the knowledge that the containers should always run consistently. Furthermore, we support multiple versions of the NGSeasy containers on Docker Hub, as each container packages its own dependencies and is versioned, the fidelity of the analysis is preserved in future execution – a requirement for reproducible research and clinical auditing5.\n\n\nMethods\n\nNGSeasy has provided us with the opportunity to start defining and thinking about best practices for building Dockerised modular pipelines. Many of these practices have been adapted in our images. Our (compbio/ngseasy-base) image forms the foundation layer on which each pipeline container application is built.\n\nAll Dockerfiles used to generate the NGSeasy images are available at https://github.com/KHP-Informatics/ngseasy.\n\nWe include what we think of as some of the best and most useful NGS \"power tools\" in compbio/ngseasy-base image (Table 1). These are all tools that allow the user to manipulate BED/SAM/BAM/VCF files in a variety of ways.\n\nOur feature rich base image, allows pipes and streamlined system calls for manipulating the output of NGS pipelines, namely, BED/SAM/BAM/VCF files. Therefore, we built these into a single development environment for NGSeasy. This image is used as the base for all of our compbio/ngseasy-* tools.\n\nA more Docker-esque approach, would be to have separate containers for each NGS tool. However, this belies the fact that many of these tools are required to interact, e.g. through pipe calls, when used as part of a streamlined pipeline.\n\nMany of the raw NGSeasy images are fairly heavy (2–4GB). As a result, we flattened all images in order to compress multiple Docker layers into one, creating an image with fewer and smaller layers, before committing and pushing to Docker Hub.\n\nWith exception of the content built into the base image, each NGSeasy pipeline component (Table 2) is encapsulated in a separate container. Using separate containers helps to minimize container size, reduce unexpected interactions between components, and maximise the re-usability of containers.\n\n\nResults and discussion\n\nA typical NGS pipeline for variant calling and discovery involves the following steps, all of which are implemented in the current version of NGSeasy (1.0-r001):\n\n1. Pre-alignment quality control\n\n2. Sequence alignment\n\n3. Raw alignment processing (e.g. local realignment around candidate indel sites and base quality score recalibration)\n\n4. Post-alignment quality control\n\n5. Variant calling\n\nNGSeasy contains all of the basic tools needed for manipulation and quality control of raw FASTQ files (Illumina focused), SAM/BAM manipulation, alignment, SAM/BAM cleaning and first pass variant discovery. The software we provide as part of NGSeasy are summarised in Table 1 and Table 2.\n\nNGSeasy follows many of the current published best practices for next generation DNA sequencing analysis, specifically, we include options to include the Genome Analysis Toolkit (GATK) recommendations for de-duplication (using Picardtools MarkDuplicates), GATK’s base quality score recalibration (BQSR) and GATK’s realignment around indels18–20.\n\nWe also include alternatives to GATK’s BQSR and indel realignment tools, specifically, BamUtil’s recab function http://genome.sph.umich.edu/wiki/BamUtil:recab), and for indel realignment, use of glia (https://github.com/ekg/glia). These options are provided for use in commercial and/or clinical laboratories who do not want to use or pay for a GATK licence.\n\nContainerised software is automatically deployed, so we have opted to provide a wide variety of tools, including multiple tools for alignment and variant calling where available.\n\nTo keep the NGSeasy pipeline small and portable, input files, indexed reference genomes and generated output should bypass the container’s root file system instead using a host mounted directory or volume (Figure 1).\n\nIn certain instances it may be necessary to inspect a running container and this can be done by injecting a new process (e.g. a shell terminal) into the container with the docker-exec command, a valuable feature for debugging or monitoring. For resource allocation, Docker uses cgroups to control memory and CPU allocation (hard or soft allocation).\n\nThe container images are only provided for software which is freely available. For software components which require registration (e.g. GATK), or are proprietary (e.g. novoalign), we provide a short Dockerfile to complete the build with the additional components which the user must acquire. We believe this is a pragmatic solution for packaging and publishing pipelines that provide the option to use components with a restricted licence. In this way we provide maximum automated deployment with the minimum burden on the end user.\n\nNGSeasy consists of a set of shell (bash) script wrappers, that orchestrate and call all parts of the Dockerised NGS pipeline - where the system calls are to docker run -i -t NGSTool instead of /bin/bash NGSTool, for example. Docker is agnostic, however, in that any workflow management software can be used to orchestrate a Docker based pipeline (eg. rufus2 or nextflow32).\n\nOur design choice was largely influenced by our desire to provide a lightweight and fairly dependency free solution, that is \"easy\" to set up and maintain. We did not want the user to be tasked with installing a large number of software dependencies before being able to run NGSeasy. In this way, NGSeasy takes advantage of the fact that any modern computer, running any operating system with Docker (or for example boot2docker https://github.com/boot2docker/boot2docker-cli) installed, will come pre-packaged with all of the basic software needed to run a NGS pipeline.\n\nNGSeasy gives the user several options to call a complete NGS pipeline, going from raw FASTQ files to aligned BAM files, variant calls (VCF) and annotations using a range of software. All options are defined in a simple configuration file that can be made, for example, using any spreadsheet application, and then saved as a tab-delimited text file. With this, the user is able to choose from a wide selection of sequence aligners, and variant callers, see Table 2.\n\nThe NGSeasy scripts enforce specific naming conventions and directory structures upon the user - allowing sensible and reproducible organisation of NGS projects and associated data on the users local machine. This also avoids all of the potential issues with typographical errors that are typical of manual input.\n\nAll NGSeasy applications are run as a non-root user within each container. This is hard coded in the NGSeasy ecosystem and provides some security for Docker containers running in shared computing environments.\n\nMany useful optimisations and recommendations were adapted from bcbio-nextgen (https://bcbio-nextgen.readthedocs.org/en/latest/ - A python toolkit providing best practice pipelines for fully automated high throughput sequencing analysis - and speedseq (https://github.com/cc2qe/speedseq) - a flexible and open source framework to rapidly identify genomic variation33.\n\nFor useful cutting edge discussion and testing of NGS pipelines, we also refer readers to the Blue Collar Bioinformatics site at http://bcb.io/.\n\nAll Dockerfiles used to generate the NGSeasy images are available at https://github.com/KHPInformatics/ngseasy along with documentation on installing and running NGSeasy. The pre-built containers are available to download from https://registry.hub.docker.com/repos/compbio.\n\nGetting and running NGSeasy is simple and outlined in the code block below.\n\nListing 1. \"Getting and running NGSeasy\"\n\n\n\nUsers should note that deploying the pipeline containers is fairly fast, dependant on network speeds, however, downloading the reference genomes and test datasets for the resources folder can take a while. For example, the install time averages at about 94 min on machines connected to relatively fast networks (i.e. > 500 Mbit/s).\n\nFor full details on obtaining, setting up and running NGSeasy, please refer to our GitHub repository documentation (https://github.com/KHPInformatics/ngseasy).\n\nSee Table 3 for our recommended system requirements. The hard disk requirements are based on our experience, and result from the fact that the pipeline/tools produce a range of intermediary and temporary files for each sample. The full NGSeasy install includes indexed genomes for hg19 and b37 for all aligners, annotation files from the GATK’s resource bundle (ftp://ftp.broadinstitute.org/bundle, 34), and all of the NGSeasy Docker images.\n\nBased on our experience, a basic NGS computing system for a small lab would consist of at least 4TB disk space, 60GB RAM and at least 32 CPU cores. Network speed is a major bottle neck when dealing with NGS sized data, and groups are encouraged to think about these issues before embarking on multi sample or population level studies - where computing requirements can very quickly escalate, and transferring NGS data between sites becomes a major rate limiting step.\n\nWe tested basic NGSeasy functionality - going from raw .fastq to .bam to .vcf - on an Illumina 100bp paired end whole exome (30x coverage) dataset available from GCAT: Genome Comparison and Analytic Testing - An analytical framework for optimizing variant discovery from personal genomes (http://www.bioplanet.com/gcat). For more details about GCAT, please refer to 35.\n\nFor this report, a basic/fast \"non-GATK\" based pipeline was tested. We skipped FASTQ quality control trimming, re-alignment around indels and BQSR. The selected pipeline first runs FastQC on the raw data, followed by read alignment using all of the selected aligners: stampy, snap, novoalign, and bowtie2. All reads were aligned to the UCSC hg19 reference genome available at http://hgdownload.cse.ucsc.edu/goldenpath/hg19/chromosomes/.\n\nThe alignment stage outputs a duplicate marked (samblaster), sorted and indexed BAM file (sambamba), annotated with the appropriate read group information (e.g. sample name, platform unit etc). The alignment stage also includes generation of basic alignment statistics using sambamba’s flagstat function, and a bed file of aligned regions using the bedtools function bamtobed - these extras steps are reflected in the average run times for NGSeasy’s alignment stage (see Table 4). Note that stampy alignment is contingent on aligning reads with bwa first, and hence, we chose not to report separate results for bwa.\n\nVariant calling was peformed using the haplotype based variant callers Platypus31 and FreeBayes30, and the resulting VCF files uploaded to the GCAT server for comparisons to the genome in a bottle (GIB) call set36. The GCAT results for the tests listed above are available at the following urls:\n\n1. All aligners + FreeBayes: http://goo.gl/G9tHRK.\n\n2. All aligners + Platypus: http://goo.gl/CB88G9.\n\nA full discussion on GIB performance statistics is beyond the scope of this paper. Briefly, for the 30x whole exome dataset, NGSeasy is achieving GIB sensitivities and specificities of 81.1–85.8% and 99.996–99.998%, respectively. There are obvious gains to be made by further pipeline optimisations, and the planned inclusion of structural variant callers and variant re-calling and filtering options.\n\nWe are presenting these results solely as a \"proof of concept\". That is, we have successfully Dockerised a full NGS pipeline, that is capable of producing meaningful results, that are comparable with public and \"best practice\" workflows.\n\nFor the testing carried out in this paper, NGSeasy was run on Rosalind, an Openstack private cloud based at Kings College London, using a virtual machine with 256GB RAM and 32 cores. We have also successfully tested NGSeasy on workstations running a wide variety of environments (OSX, Windows 7, Ubuntu 14.04).\n\nAverage representative run times for a full NGSeasy pipeline and its components are presented in Table 4.\n\nThe obvious winners for alignment, based purely on speed, are bwa and snap. The two software are comparable. The extra run time seen for snap are due to loading/reading of the indexed reference genome. Once this has been done, snap will run at speed, and is the fastest aligner these authors have seen. The reported runtime for stampy is dependent on bwa having been run first.\n\nNote, that fastQC and read quality trimming need only be applied once. After which, the pipeline is set up to test for, and skip these stages, if the have already been run - speeding up subsequent pipeline calls that use the same data. Be aware that run times will vary depending on depth, quality of data, and compute power (e.g. available RAM and CPU).\n\nBoth Platypus31 and FreeBayes30, are highly parallelisable and run at speed; Platypus being 6x faster than FreeBayes in our test, but, less sensitive than FreeBayes; the average GIB sensitivity over all aligners from Platypus versus FreeBayes was 82.40% versus 84.15%.\n\nRunning a full NGS pipeline using Docker containers had no real noticeable reduction in computing performance (run time) when compared to our original native (non-Dockered) NGS pipeline. The differences are in the milliseconds to seconds range, and largely depend on the underlying system hardware (and data quality). These observations are similar to those reported in 37.\n\nStrikingly, depending on available compute, read depth and the selected pipeline components, the observed runs times indicate that a full clinical NGS pipeline could be run, and achieve actionable results in less than 2 hours. This has major positive implications for molecular diagnostics and projects like the 100,000 Genomes Project (http://www.genomicsengland.co.uk/the-100000-genomes-project/). That is, alignment and variant calling are no longer a major bottle neck. More work is needed to speed up and improve library preparation, sequencing machine run times and solutions for variant annotation, prioritisation and clinical reporting.\n\nNGSeasy demonstrates the utility of Docker as a means to package software used in modular workflows. We envisage NGSeasy as a method for deploying drop-in analyses, in scenarios where data cannot be shared (either for size or privacy reasons) and an analysis must be carried out in-situ. In such cases, using a pipeline like NGSeasy, it is simple to develop an analysis off site, package it and deploy it on computational facilities where access to the data is provided, examples of such scenarios include the 100,000 Genomes Project and Illumina BaseSpace38 Docker ’apps’.\n\nIn addition, NGSeasy is being tested across a select group of NHS Labs (under the NHS England Open Source Initiative) for molecular diagnostic and clinical research pipelines. In particular, a version of NGSeasy has been adapted by Viapath at King’s College Hospital (publication pending; personal communication from Dr Barnaby Clark and Dr David Brawand http://www.viapath.co.uk/locations/kings-college-hospital). The advantages being, the ease of use and set up, the built in version control and the ability for audit tracking and reproducibility conferred by the use of Docker and the open source community built around GitHub.\n\nNGSeasy is under continual development. What we demonstrate here is the pre-production release and basic proof of concept evaluation of NGSeasy :a next generation sequencing pipeline in Docker containers. We want to present this to the scientific community at large, especially those working in the bioinformatics domain, and wish to encourage and invite collaboration on NGSeasy and our groups efforts to Dockerise bioinformatic pipelines.\n\nThe group is currently working on a GUI for NGSeasy and along with a modular benchmarking suite. In planned extensions, NGSeasy will provide options for consensus calling, trio/family and population based calling pipelines, human leukocyte antigen (HLA) calling, structural variant calling, cancer pipelines, more optimisations, improved logging, and the latest b38 indexed genomes.\n\nIn later versions we will publish detailed benchmarking statistics for all aligners and variant calling on whole exome, genome and clinical panels from a range of depths and platforms.\n\nDevelopment work on Docker continues at pace. The present Docker daemon, runs as root, and there remain security issues with the notion of providing access to this daemon in a shared user environment, such as a typical cluster, a solution to this exists using Linux kernel user namespaces but this is presently undergoing review.\n\n\nSoftware availability\n\n1. Container images are available from: https://registry.hub.docker.com/repos/compbio\n\n2. Latest source code, Dockerfiles, pipeline and documentation are available from: https://github.com/KHP-Informatics/ngseasy\n\n3. Link to archived source code as at time of publication: http://dx.doi.org/10.5281/zenodo.3144439\n\n4. GNU General Public License, version 2: http://www.gnu.org/licenses/oldlicenses/gpl-2.0.en.html",
"appendix": "Author contributions\n\n\n\nAF, SN: Contributed equally to this work AF: Pipeline design and Docker architecture and manuscript writing SN: Pipeline design and Docker architecture and manuscript writing RD: Manuscript writing and comments.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis paper represents independent research funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. SN, AF and RD are all funded by the National Institute For Health Research.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nGiardine B, Riemer C, Hardison RC, et al.: Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005; 15(10): 1451–1455. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodstadt L: Ruffus: a lightweight python library for computational pipelines. Bioinformatics. 2010; 26(21): 2778–2779. PubMed Abstract | Publisher Full Text\n\nWolstencroft K, Haines R, Fellows D, et al.: The Taverna workflow suite: designing and executing workflows of Web Services on the desktop, web or in the cloud. Nucleic Acids Res. 2013; 41(Web Server issue): W557–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDocker. 2014. Reference Source\n\nBoettiger C: An introduction to docker for reproducible research, with examples from the R environment. CoRR. 2014. Reference Source\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\nDanecek P, McCarthy S, Li H, et al.: bcftools — utilities for variant calling and manipulating vcfs and bcfs. The MIT/Expat License or GPL License, see the COPYING document for details. Copyright (c) Genome Research Ltd, 2015. Reference Source\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\n\nGarrison E, erik.garrison@bc.edu: vcflib: a c++ library for parsing and manipulating vcf files. Reference Source\n\nAbecasis Group. bamutil is a repository that contains several programs that perform operations on sam/bam files. all of these programs are built into a single executable, bam. Reference Source\n\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFaust GG, Hall IM: SAMBLASTER: fast duplicate marking and structural variant read extraction. Bioinformatics. 2014; 30(17): 2503–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTarasov A, Vilella AJ, Cuppen E, et al.: Sambamba: fast processing of NGS alignment formats. Bioinformatics. 2015; 31(12): 2032–4. PubMed Abstract | Publisher Full Text\n\nLi H: Seqtk is a fast and lightweight tool for processing sequences in the fasta or fastq format. Reference Source\n\nAbecasis Group. A variant tool set that discovers short variants from next generation sequencing data. Reference Source\n\nChiang C: An awk-like vcf parser. Reference Source\n\nLi H: Bwk awk modified for biological data. Reference Source\n\nVan der Auwera GA, Carneiro MO, Hartl C, et al.: Current Protocols in Bioinformatics. John Wiley & Sons, Inc., Hoboken, NJ USA. 2002; 11.\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): 1297–1303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDePristo MA, Banks E, Poplin R, et al.: A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011; 43(5): 491–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews S: Fastqc a quality control tool for high throughput sequence data. 2015. Reference Source\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPicard. 2015. Reference Source\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–1760. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangmead B, Trapnell C, Pop M, et al.: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3): R25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLunter G, Goodson M: Stampy: A statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 2011; 21(6): 936–939. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaharia M, Bolosky WJ, Curtis K, et al.: Faster and More Accurate Sequence Alignment with SNAP. 2011; 1–10. Reference Source\n\nHercus C: Novocraft. 2015. Reference Source\n\nKural D, Garrison E: Glia. 2015. Reference Source\n\nGarrison E, Marth G: Haplotype-based variant detection from short-read sequencing. 2012; 9. Reference Source\n\nRimmer A, Phan H, Mathieson I, et al.: Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Nat Genet. 2014; 46(8): 912–918. PubMed Abstract | Publisher Full Text\n\nBal HE, Steiner JG, Tanenbaum AS: Programming languages for distributed computing systems. ACM Comput Surv. 1989; 32–2. Reference Source\n\nChiang C, Layer RM, Faust GG, et al.: Speedseq: Ultra-fast personal genome analysis and interpretation. Nat Methods. 2015; 12(10): 966–968. PubMed Abstract | Publisher Full Text\n\nThe Broad Institute. The gatk resource bundle is a collection of standard files for working with human resequencing data with the gatk. 2015. Reference Source\n\nHighnam G, Wang JJ, Kusler D, et al.: An analytical framework for optimizing variant discovery from personal genomes. Nat Commun. 2015; 6: 6275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZook JM, Chapman B, Wang J, et al.: Integrating human sequence data sets provides a resource of benchmark SNP and indel genotype calls. Nat Biotechnol. 2014; 32(3): 246–51. PubMed Abstract | Publisher Full Text\n\nMatzke M, Jurkschat K, Backhaus T, et al.: PrePrints PrePrints. 2014; (1): 1–34.\n\nDickinson AG, Garcia FJ, Kain RC, et al.: Cloud computing environment for biological data. US Patent App. 13/790,596. 2013. Reference Source\n\nNewhouse SJ, Folarin A: ngseasy: ngseasy-release-0.0.1. Zenodo. 2015. Data Source"
}
|
[
{
"id": "10672",
"date": "06 Oct 2015",
"name": "Fabien Campagne",
"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 manuscript describes a software tool called NGSEasy, which consists of a set of configured docker images for writing pipelines to call genomic variations. The manuscript addresses a need because the portability and reproducibility of bioinformatics pipelines, such as the one described in this manuscript, are very real problems faced by bioinformaticians and users of these methods. As such, the manuscript is an effort to describe an approach that could help with these challenges. Despite my interest in the manuscript, I have several reservations regarding the presentation of the results that I believe should be addressed before the manuscript is accepted for publication in a peer-reviewed journal. These reservations are:The abstract indicates that the manuscript “demonstrates” best practices for packaging and executing multi-component pipelines for NGS. I am very uneasy about the term “best practices” used in a scientific context. I believe that it is very difficult to define objectively what constitutes “best practices”. Asking different experts may yield different answers, and one could argue that “best practices” are a synonym for asking an expert about his or her opinion. I believe that the manuscript would be strengthened if it described the authors’ recommendations and supported each recommendation with a clear and detailed rationale (perhaps outlining alternatives that the authors have tried and eventually rejected while developing NGSEasy, and explaining the reasons to do so). While the word “demonstrates” in the abstract suggested to me that the manuscript would describe a set of practices recommended by the authors for packaging docker containers, it appears from the Result section that no such practices are explicitly described. It is therefore possible that the authors meant to refer to a set of published practices for analysis of high-throughput sequence data. If it is the case, as suggested by the sentence at the top right of page 4, I am not sure as to what is claimed in the abstract: demonstration of published practices, or recommended practices for packaging NGS code in a docker image? The abstract and introduction define the domain of application of NGSEasy as “next-generation sequencing (NGS)”. However, the manuscript is about methods for variant calling, which is an important, but smaller scope that the full NGS data analysis. For instance, NGSEasy does not include tools for analysis of RNA-Seq. I recommend to revise the abstract and introduction to clearly indicate the scope of the software tool. A strength of the manuscript is to use the GCAT server to evaluate the pipeline, but the results are not presented in the context of the performance of other pipelines, so the readers have no easy way of knowing if the sensitivity and specificity measures presented on page 6 are competitive. For instance, on Page 6, the manuscript claims: “we have successfully Dockerized a full NGS pipleline that is capable of producing meaningful result, that are comparable with public and “best practice” workflows”. However, there is no reference for the workflows the work is compared to and no simple way to establish if the results are comparable, let alone competitive. I strongly recommend to include a comparison directly in the manuscript to help the readers objectively assess performance. The manuscript would be strengthened by providing a discussion of the limitations of the work. For instance, it is unclear what support is provided for parallelization across nodes, rather than SMP parallelization. (Multi-node parallelization is important when more than one or two samples need to be analyzed.) I am unable to locate Reference 37 using the citation information: “37. Matzke M, Jurkschat K, Backhaus T, et al.: PrePrints PrePrints. 2014; (1): 1–34. ”. This reference is used when discussing performance of docker containers and I am unable to determine if this is appropriate. A valid reference for this point is https://peerj.com/articles/1273/. The reference provided for Nextflow is wrong. The tool should be cited using the web site (http://nextflow.io) or FigShare poster, and the correct authors given credit. Minor comments:Page 3, “NGSEasy contains all the basic tools needed for manipulation and quality control..” should be toned down. Using all in a manuscript is inviting contradiction. For instance, I could point out that the NGSEasy do not contain SpeedSeq, a recently published set of tools that considerably accelerates variation calling in HTS data. Therefore, I would argue that NGSEasy does not contain all the basic tools that I would like to use. Consider revising as “NGSEasy contains a set of tools sufficient for manipulation and quality control..” Page 5. The word “all” is used again (left column, 6th paragraph). I doubt that the practice, as described, eliminates all potential issues with typo, since end-users will be writing scripts using tools in the image, and I am not sure how consistent naming conventions can fully eliminate typos when writing scripts. Page 6. last paragraph, last sentence, check the grammar (missing a “y”?).",
"responses": [
{
"c_id": "1663",
"date": "22 Oct 2015",
"name": "Stephen Newhouse",
"role": "Author Response",
"response": "Great - thanks! We are waiting for a 3rd reviewer comments before addressing all of these points."
}
]
},
{
"id": "10873",
"date": "20 Oct 2015",
"name": "Brad Chapman",
"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 NGSeasy, a containerized set of tools for variant calling on high throughput sequencing data. The implementation is open-source, maintained and well documented with instructions for getting started. The paper reports a number of useful practical results, including reproducibility through containerization, validations and timing of analyses. Below are my suggestions for helping to improve the paper and questions about the implementation details.Paper contentIn the Introduction, you should note that similar Docker-based approaches exist for making Galaxy installation easier: https://github.com/bgruening/docker-galaxy-stable You mention running at non-root in the Docker container but should also discuss that users need root privileges to install and run Docker. This currently limits usability of Docker on shared computing environments since it requires giving NGSeasy root-equivalent permissions. User namespace support is in progress and will help, but is not yet in any released versions of Docker. I agree with your security points once you have the NGSeasy approach setup, but getting there can be a challenge. You do mention this later in \"NGSeasy future developments\" and that would fit better in the initial installation section. For reporting of download/install times, please also list install times from more standard connection speeds. A majority of users will not have 500Mb/s or better download. Is it possible to download subsets of the data? It looks like it currently grabs both hg19, b37 and hs37d5, tripling the download times and space required. Digging into the code it wasn't clear how to get other mentioned genomes like hs38DH.ValidationFor the GCAT/Genome in a Bottle validations, I'd suggest reporting precision instead of specificity. Specificity is not especially useful for calling since it's dominated by true negatives. For example, the precision rates show clear differences between FreeBayes and Platypus, and also differences between novoalign and the other aligners. The specificity numbers do not reveal these. It's hard to judge the results of your validation without comparing to another best-practice pipeline like bwa + GATK HaplotypeCaller. Having these as a baseline next to your comparisons would strengthen the argument that the current implementation does a comparable job to expected best practice. It would be useful to have bwa-mem alignment results also listed in the GCAT validations. bwa-mem is a widely used aligner, separate from stampy. Do you have validation of using non-GATK tools (recab and glia) versus GATK tools in terms of the output quality? This would be useful to report. I've had good output success avoiding these step entirely but would like to see differences between avoiding the steps and using freely available alternatives.TimingsThe timing information is really useful and a great addition to the paper. I'd suggest adding some caveats to the conclusion and tables to make it clearer about the inputs, since the numbers are exome with only 30x coverage. Most standard exomes would be higher coverage and WGS is becoming increasingly standard. Some of the statements like \"alignment and variant calling are no longer a major bottle neck\" seem overextended from timings on this smaller dataset. Scaling up is not linear and things get harder for WGS projects like 100k genomes project. Has there been scaling work across non-single machine setups? Our experience is that shared network issues and managing Docker containers can dominate scaling. If the target is single multi-core machines it would be worth specifying this directly.DockerWhat is your experience with larger Docker containers and Docker Hub? Practically I've found a lot of timeout issues trying to download and manage larger images. Do you have workaround/experience with these issues? Have you successfully run workflows on non-unix systems with Boot2Docker? You list these as workstations where NGSeasy should work. We've not had good success with mounting external data into Boot2Docker instances but would have interest in re-exploring if this changed: https://github.com/chapmanb/bcbio-nextgen-vm#mac-osx-docker-supportMinorTypo: sudo make INTSALLDIR=\"/media/scratch\" all",
"responses": [
{
"c_id": "1664",
"date": "22 Oct 2015",
"name": "Stephen Newhouse",
"role": "Author Response",
"response": "Thanks for the review and comments!"
}
]
},
{
"id": "10673",
"date": "23 Oct 2015",
"name": "Michael Barton",
"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\nMy understanding of this article is that NGSeasy pipeline aims to simplify the distribution of common tools used in sequencing analysis. A still significant problem in bioinformatics is getting the third-party tools installed and working, by using Docker containers as described in this article, the authors will make this process easier. The code is available on github as described and they provide extensive documentation.MajorOne concern is the install instructions in the article. Specifically:sudo make INTSALLDIR=\"/media/scratch\" allsudo make intsallI am wary of using 'sudo' to install. I know that using tools like 'apt-get' require 'sudo' however for most bioinformatics software I prefer to install in my user directory simply to avoid any possible security problems. I took a look at the Makefile and I believe that sudo is not necessarily required to install, only the the INSTALLDIR and TARGET_BIN are owned by the user. Also there is typo here in 'intsall' The project doesn't include any NGS tools related to assembly or transcriptomics. Though not stated specifically, the tools and data described here leads me to believe this project is focused around clinical applications and human genomics. If that is the case perhaps this should be clarified in the article and the title. The Docker security issue at the end feels tagged-on. This, I think, is a pressing concern that prevents many people from using Docker on HPC machines as opposed to on-demand computing such as AWS. This is the case at the JGI where I currently work. I would suggest expanding on this point a little more to describe why it is an issue. I think a short paragraph would be useful to end the article with. This would summarise the points described above and potential impact of the work. I think Figure 1 could be expanded upon. It currently assumes a familiarity with Docker that could make it difficult to interpret without a good understanding of containers and volumes.MinorThe scripts installed from https://github.com/KHP-Informatics/ngseasy/tree/master/binThe scripts here are very large (400-800 line) bash scripts. My concern is these scripts may be difficult to maintain or debug. Writing from my own experience, when projects get larger it is worth considering if code can be refactored or made more modular to make it more maintainable. Inconsistent use of Dockerized / Dockerised between the article and the documentation I believe Docker have deprecated boot2docker and now recommend docker-toolkit. This is their recommended way of installing Docker. All data is available from the AWS EU region. This may take much longer to download outside of this region. I'm not sure if the authors can do anything about this however as using a CDN may be prohibitive. The URL https://registry.hub.docker.com/repos/compbio requires the creation of a DockerHub account to view. This is not the developers fault, however having to register is likely to result in some users not following up.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-997
|
https://f1000research.com/articles/4-147/v1
|
09 Jun 15
|
{
"type": "Research Article",
"title": "Matching Behavior as a Tradeoff Between Reward Maximization and Demands on Neural Computation",
"authors": [
"Jan Kubanek",
"Lawrence H. Snyder",
"Lawrence H. Snyder"
],
"abstract": "When faced with a choice, humans and animals commonly distribute their behavior in proportion to the frequency of payoff of each option. Such behavior is referred to as matching and has been captured by the matching law. However, matching is not a general law of economic choice. Matching in its strict sense seems to be specifically observed in tasks whose properties make matching an optimal or a near-optimal strategy. We engaged monkeys in a foraging task in which matching was not the optimal strategy. Over-matching the proportions of the mean offered reward magnitudes that would yield more reward than matching, yet, surprisingly, the animals almost exactly matched them. To gain insight into this phenomenon, we modeled the animals' decision-making using a mechanistic model. The model accounted for the animals' macroscopic and microscopic choice behavior. When the models' three parameters were not constrained to mimic the monkeys' behavior, the model over-matched the reward proportions and in doing so, harvested substantially more reward than the monkeys. This optimized model revealed a marked bottleneck in the monkeys' choice function that compares the value of the two options. The model featured a very steep value comparison function relative to that of the monkeys. The steepness of the value comparison function had a profound effect on the earned reward and on the level of matching. We implemented this value comparison function through responses of simulated biological neurons. We found that due to the presence of neural noise, steepening the value comparison requires an exponential increase in the number of value-coding neurons. Matching may be a compromise between harvesting satisfactory reward and the high demands placed by neural noise on optimal neural computation.",
"keywords": [
"matching law",
"reward magnitude",
"reinforcement learning",
"value",
"choice",
"neurons"
],
"content": "Introduction\n\nPeople and animals must make choices. It has been often reported that organisms distribute the frequency of their choices according to the relative rate of reinforcement they obtain from each choice1–4. The match between the behavioral and reinforcement distributions in a two-option task has been described by the matching law:\n\n\n\nwhere Bx and By are the rates of behavior allocated at options x and y, and Rx and Ry are the corresponding rates of reinforcement obtained from these options1,4.\n\nThis elegant relationship has provoked much discussion and research across multiple fields3,5–9. Although matching has been observed in many environments, including real-life settings10–12, there are important constraints on the conditions in which matching is observed.\n\nFirst, matching behavior in the above form is consistently observed specifically in tasks that use or can be characterized by concurrent variable interval (VI-VI) schedules of reinforcement13,14. In such tasks, after a reward is harvested by choosing an option, a reward is scheduled to become available again after a certain time interval. Once scheduled, the reward remains available at that option until it is harvested again. This critical task feature is referred to as “baiting”. Baiting may model the situations in which even low-yield sources eventually produce a reward. However, the fact that a reward remains available until the animal harvests it is unrealistic, at the very least because it entirely disregards competition. Nonetheless, in these lab-based baiting paradigms, it is a sensible strategy for the decision-maker to occasionally select even the much poorer of the two options, since due to the baiting, after a long enough interval, the animal can be sure that a reward will appear at that option6. Although such behavior is not surprising, it may seem intriguing that the proportion of choices devoted to each option in these tasks follows the matching equation. However, it has been shown that matching is an optimal or near-optimal strategy in the VI-VI baiting paradigms. In particular, in these tasks, matching follows from the maximization of reward at either the molecular (maximizing reward at each element of time)15–17 or molar (maximizing reward over the course of the experiment)13,18,19 levels. Matching does not seem to apply to reinforcement-based choice tasks in general. For instance, choice behavior under concurrent ratio-interval schedules (FR-VI) substantially deviates from matching13. Furthermore, response ratio concurrent schedules (VR-VR) usually lead to stereotypic behavior19.\n\nSecond, matching is adversely affected by the animals’ tendency to often switch from one option to the other (e.g.,1, Figure 4). This frequent switching brings the proportion of choices of the two options closer to 50:50, which results in “under-matching” of the reward proportions. Such under-matching, as well as other deviations from the matching law, can be captured using generalized forms of the matching law20–22. Nonetheless, these generalizations come at the expense of freely adjustable parameters, thus diminishing the beauty of the matching equation. To discourage this behavioral tendency, researchers often punish the animals’ frequent switching by incorporating change-over delays (COD)1,23–25. In a change-over delay paradigm, when an animal changes a choice, no reward is scheduled until a certain amount of time following the change. This effectively discourages frequent switching. When this control is in force, the animals typically exhibit the matching behavior captured by Equation 1, at least in the tasks or life situations in which reward remains baited until harvested.\n\nWe engaged monkeys in a reward-magnitude-based foraging task that featured neither a baiting schedule nor a change-over delay. In our task, animals chose an option based on the magnitude (amount) of fluid reward expected for each option. The mean magnitude ratios for the two options, 3:1, and 1.5:1, changed often and unpredictably. Intriguingly, we observed a nearly exact matching of the magnitude ratios. This is surprising because in this task, the matching behavior is not optimal—animals could harvest more reward by over-matching the magnitude ratios.\n\nTo investigate the mechanisms of this phenomenon, we described the animals’ behavior with a simple three-parameter mechanistic model rooted in reinforcement learning6,26–28. When the model was allowed to forage freely and its three parameters were optimized to maximize the reward income, the model did substantially better than the monkeys at accurately comparing the value of the two options. Our neuronal simulations suggested that the animals’ ability to compare the two values could be limited by the noise in the representation of value by populations of spiking neurons.\n\n\nMethods\n\nTwo adult male rhesus monkeys supplied by the Washington University Department of Veterinary Medicine. (macaca mulatta, monkey S: 7 kg, monkey B: 8 kg) participated in this study. Animals were housed in pairs with 12/12 hour light/dark cycles29. Monkeys were fed on Purina Monkey Chow, fruit and treats, and were provided with environmental enrichment30. We trained two male rhesus monkeys (macaca mulatta, 7 kg and 8 kg) to choose one of two targets using a saccadic eye movement or a reaching arm movement30. Tests were performed during normal working hours (9am to 5pm). The animals sat head-fixed in a custom designed monkey chair (Crist Instrument) in a completely dark room. Visual stimuli (squares of 2.3° by 2.3°) were back-projected by a CRT projector onto a custom touch panel positioned 25 cm in front of the animals’ eyes. Eye position was monitored by a scleral search coil system (CNC Engineering). All procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Washington University Institutional Animal Care and Use Committee.\n\nAnimals performed a two-alternative forced choice task. They first fixated and put their hand on a central target. After 120 ms, two white targets appeared simultaneously to the left and right of fixation. Each target was associated with a reward, described below. At the same time, the central fixation point changed color to either red or blue, instructing the monkeys that either a saccade or a reach, respectively, would be required on this trial. After a variable delay interval (0.8 s to 1.6 s), the fixation point disappeared, cueing the monkey to execute a movement to one or the other target. The animals’ behavior was very similar for choices made using saccades and reaches, and we therefore did not distinguish between the two. If they failed to make the instructed movement to within 7° of visual angle from one of the two targets within 1.5 s of fixation offset, then the animal received no reward and the start of the next trial was delayed by 2 s. Otherwise, the next trial started immediately after the reward was delivered.\n\nThe reward associated with the two targets consisted of a primary reinforcer—a drop of water, delivered by the opening of a valve for a particular length of time—combined with a secondary reinforcer—an auditory tone of the same duration. The volume of fluid delivered was proportional to the valve opening times. Our aim in designing the task was that at any one time, one target would deliver larger rewards than the other. The assignment of the richer and poorer targets to the left and right choices would change periodically, but in a way that would not be obvious to the animal or easy to determine. To accomplish this, we made many aspects of the reward delivery stochastic. At any one time, the mean reward durations for the two targets had a ratio of either 3 : 1 or 1.5 : 1. This ratio was held constant for a block of 7–17 trials (exponentially distributed with a mean of 11 trials and truncated at 17) and then changed to either 1 : 3 or 1 : 1.5. Within each block, the time that the water valve was held open in each trial was itself not held constant, but instead was drawn from a truncated exponential distribution that ranged from 20 to 400 ms. Thus, the valve open time differed from trial to trial, with an overall mean that differed for each target and changed every 7–17 trials. The effect of the exponential distribution was to make small rewards more common than large rewards, relative to the mean. This mean differed for each target and depended on the reward ratio for that block. For a reward ratio of 1.5 : 1, the mean valve open times for the richer and poorer target were centered around 140 and 70 ms, respectively. For a ratio of 3 : 1, the mean times were centered around 250 and 35 ms, respectively. To randomize reward delivery even further, the actual valve open times were multiplied by a factor ranging from 0.8 to 1.2, and this factor was changed on average every 70 trials (exponential distribution truncated to between 50 and 100 trials).\n\nThe reward magnitude of the option that the monkeys did not choose was assigned exactly in the same way as that assigned to the chosen option, that is, they were drawn stochastically from changing distributions with a particular mean. Once generated, the reward magnitudes for the unchosen option were fixed throughout the investigation.\n\nThe data are available in a .mat format at http://www.neuralgate.org/download/matchingdata or at the link below.\n\nWe modeled the monkeys’ trial-to-trial behavior using a mechanistic model. The model is grounded in reinforcement learning, a framework whose various instantiations have been applied previously to successfully explain foraging behavior6,25–28.\n\nThe model (Figure 3) first computes the value V of each option by weighing the past 3 rewards ri obtained from choosing each option:\n\n\n\nThe first two weights (w1, w2) are free parameters; the third weight is w3 = 1 – w1 – w2 such that ∑i wi = 1.\n\nThe option that was chosen is assigned a value r1 = R, where R is the reward obtained for choosing that option. The unchosen option is assigned a value r1 = ρ, where ρ is a free parameter.\n\nThe value of the two options (Vright and Vleft) are compared and a choice of the rightward option is made with probability\n\n\n\nwhere the parameter β controls the steepness of the sigmoid function (see Figure 10).\n\nThe four parameters w1, w2, ρ, and β were fitted to the monkeys’ behavior such as to maximize the log likelihood log L that the monkeys’ choices could be made by the model:\n\n\n\nwhere Pright(t) is the model’s prediction of the probability of choosing the rightward option on trial t; c(t) = 1 for the monkeys’ rightward choice on trial t and 0 for his leftward choice. The maximization was performed by the Nelder-Mead simplex direct search algorithm implemented by the function fminsearch in Matlab (The Mathworks, Inc., Natick, MA, RRID:nlx_153890). The algorithm converged in all tested conditions, and onto the same solution when run repeatedly.\n\nWe further simplified this model by approximating the three weights wi with a geometric sequence with the common ratio α (Figure 8). Given that ∑i wi = 1, we can write w1=11+α+α2, w2 = αw1 and w3 = αw2. We then fit α to minimize the mean squared error between the approximated and the actual weights.\n\nWe tested a variety of other models, none of which offered a significantly better fit. The present model is well established in the reinforcement learning literature26, has been successfully used previously6,27,28, and is a generalization of many special cases we also tested (see Results for an example).\n\nWe also tested an extended model that featured a separate set of weights for the unchosen option. This extension did not significantly improve the fit to the animals’ behavior or the ability of the freely foraging model to harvest more reward.\n\nWe further tested an extended model which in the (Vright–Vleft) term of Equation 2 featured two additional bias terms that could model the monkeys’ possible biases in choices made using saccades and reaches. These extensions had only minimal impact on the results (see Results). We therefore used the original, simpler model.\n\n\nResults\n\nMonkeys engaged in a foraging task (Figure 1) in which they selected one of two targets based on the associated reward magnitude. Specifically, one target was associated with a larger liquid reward than the other target, with mean payoff ratios of 1.5 : 1, 3 : 1, 1 : 1.5, or 1 : 3. The payoff ratio was held constant for 7–17 trials before changing to one of the opposite ratios. To further challenge the animals, the volume of juice delivered on each trial was variable, drawn from a truncated exponential distribution (see Methods for details).\n\nAnimals first fixated and put their hand on a central target. Following a short delay, two targets appeared in the periphery. The animals selected one of the targets using either an eye or hand movement, if the central cue was red or green, respectively. A choice was followed by the delivery of a liquid reward of a particular size. At any one time, one target was more valuable than the other, but individual rewards were stochastic and drawn from overlapping distributions, and which target was more valuable switched often and unpredictably. See text for details.\n\nThe monkeys chose the richer option more frequently, but not stereotypically (Figure 2A). On average, after each change of payoff ratio, the monkeys’ behavior converged in about 3 to 6 trials to a new steady state choice ratio. The fact that animals did not immediately switch over to a new steady state but required several trials to do so indicates that the animals were not aware of the transition times and integrated the reward history to converge onto the richer target. In the steady state (trial 7 following transition) the animals’ choices followed the strict matching law (Equation 1). Specifically, for a ratio of 1.5 : 1, the strict matching law dictates choosing the richer option in 60% of trials. Our two animals chose the richer option in 60.0% and 61.6% of trials, respectively. For a ratio of 3 : 1, the matching law dictates choosing the richer option on 75% of trials. The animals chose this option in 73.5% and 71.9% of trials, respectively. Only the case of 71.9% slightly deviated from its corresponding matching level of 75% (p = 0.022, t1117 = -2.29); the other three cases were indistinguishable from the corresponding matching levels (p > 0.25).\n\nThe finding that animals matched the reward proportions in this task is notable given that we did not impose specific constraints typically used to elicit matching, such as reward baiting or change-over delay punishment of frequent switching1,13,23–25.\n\nAnimals switched from one target to another often (Figure 2B), on average about once every third trial (probability to switch choice, P = 0.31). The distribution of stay durations was well approximated with an exponential (Figure 2B), which suggests (though it does not prove) that the choice the animals made on a given trial was independent of the choice the animals made on the previous trial.\n\n(A) Proportion of choices of an option as a function of each payoff ratio, aligned on a transition. The dotted black lines indicate the 3:1 and 1.5:1 proportions dictated by the matching law (Equation 1). (B) Frequency histogram of successive choices of one option. Dashed line: exponential fit.\n\nTo gain insight into the processes leading to the matching behavior, we modeled the animals’ trial-to-trial behavior using a mechanistic model. The model (see Methods for details) is grounded in reinforcement learning and its various instantiations have been applied previously to successfully explain foraging behavior in reward-based tasks6,25–28. The model (Figure 3) first computes the value V of each option. It does so by weighing the past three rewards ri obtained from choosing each option: V=∑i=13wiri. Two of the weights (w1, w2) are free parameters; the third weight is w3 = 1 – w1 – w2 such that ∑i wi = 1. An important question is what reward magnitude the animals assign to the option that was not chosen. This reward magnitude constitutes an additional free parameter, ρ. Finally, the values of the two options, Vright and Vleft, are compared and a choice of the rightward option is made with probability Pright = Ψ(Vright – Vleft), where Ψ is a simple sigmoid function (see Methods, Equation 2) whose steepness is controlled by the parameter β. This sigmoid function can implement both a sharp Vright > Vleft) comparator function when β is large, as well as a more stochastic choice when β is small.\n\nIn the model, a option is assigned the reward obtained from the according choice, of magnitude R. The unchosen option is assigned a value of ρ, a free parameter. The past three rewards obtained for each option ri are linearly weighted to obtain the value of an option, V=∑i=13wiri. The weights w1 and w2 are free parameters; w3 = 1–w1–w2. The values Vright and Vleft are then compared using a sigmoid choice function Ψ(Vright – Vleft) whose steepness is parametrized by β. This results in the model’s output: the probability of choosing the rightward option Pright in each trial. The model’s free parameters are highlighted in blue.\n\nThis framework is quite general and can represent many special cases. For instance, in a win-stay lose-shift (WSLS) model, an animal compares a just-obtained reward R against a threshold T; if R > T, the animal stays with its choice, else it shifts choice. This model is a special case of the above general framework in which free parameters w1 = 1, w2 = 0 (and so also w3 = 0), ρ = T, and β is large to achieve the sharp R > T comparator, e.g., β = 1.0.\n\nWe estimated the model’s four parameters such that the model’s predictions are close to the monkeys’ choices. The estimation was based on maximizing the likelihood of observing the monkeys’ choices given the model’s parameters (MLE; see Methods for details). The fit resulted in w1 = 0.816, w2 = 0.197 (and so w3 = -0.013), ρ = 55.1, and β = 0.023. We also tested an extended model by adding two additional parameters (one for choices made using saccades, one for choices made using reaches) at the comparator stage (see Methods for details) to account for possible biases in preferring a rightward or a leftward choice. This extended model resulted in very similar parameter fits (w1 = 0.815, w2 = 0.198 (and so w3 = -0.013), ρ = 55.3, β = 0.023). Furthermore, the biasing values (V = -4.6 and V = 8.5) were negligible compared to the large range of (Vright – Vleft) (5th percentile equal to -172.8, 95th percentile equal to 176.3). We therefore used the simpler model.\n\nThis simple model faithfully captured the animals’ behavior. When the animals’ choices were binned according to the model’s probabilistic predictions, there was a nearly linear (R2 = 0.997) relationship between the model’s predictions and the animals’ mean proportion of choices (Figure 4A). For instance, across all trials in which the model claimed that Pright = 0.4, the monkey actually chose the rightward option in close to 40% of cases. The model also explained very faithfully the animals’ matching behavior and their behavior just after the payoff ratio transition (Figure 4B). In particular, the model (dashed lines) explained R2 = 0.986±0.005 (mean±SD) of the variance in the 4 curves.\n\n(A) Proportion of choices of the rightward target (±SEM) as a function of the model’s probabilistic output, Pright. (B) Same format as in Figure 2A, with the model’s probabilistic output superimposed as dashed lines.\n\nWhen fitting the model, the model’s input (the rewards) and the outputs (choices) were held fixed; i.e., the model made the same choices as the monkeys and experienced the same rewards as the monkeys. Fixing the input and output permits us to investigate the structure of the model, i.e., to determine the mechanics of the transformation between the input and the output. However, it is also valuable to determine the model’s behavior, using the inferred parameters, when it is allowed to make choices for itself. This is important because it is conceivable that without the choice prescription, the model may show unstable behavior, such as alternating between choices or stereotypically making one choice.\n\nThis was not the case. When the model made choices by itself (i.e., on every trial the model computed a Pright and made a rightward choice with probability Pright), it still exhibited behavior similar to that of the monkeys (Figure 5). Although the model chose the richer option slightly less frequently than the monkeys (Figure 5A; 72.7% for 3:1 and 59.2% for 1.5:1), there was no significant difference between the monkeys’ and the model’s mean choice levels at the steady state for either the 3:1 or the 1.5:1 payoff ratios (trial 7 following transition, p > 0.11, t-tests). The model also exhibited trial-wise switch dynamics that were very similar to that of the monkeys (Figure 5B). In particular, the mean stay duration of the monkeys (model) was 3.2 (3.3) trials; this small difference was not significant (p = 0.19, t28984 = -1.31).\n\nThe model used the same parameter values as in Figure 4. Same format as in Figure 2, with the model’s behavior superimposed as dashed lines.\n\nA question of particular interest is why the animals exhibited matching behavior in this task. We start this inquiry by asking whether the matching behavior was optimal in this task. An ideal agent who has information about the times of the payoff transitions will converge onto the richer option in one trial and continue to choose the richer option until the time of the next transition. Choosing the richer option at steady state in 100% of trials would constitute very strong over-matching. However, our subjects were not ideal: they were not signaled when the payoff transitions occurred, and we designed the task to make it difficult for them to detect the transition times. Specifically, the transitions occurred at random, exponentially distributed intervals, such that the hazard function for transition was flat. In addition, the reward magnitude received on each trial was variable, drawn from an exponential distribution (see Methods for details).\n\nThese task attributes may make it difficult for any subject or scheme to perform the task perfectly. To obtain an estimate of how well an agent might perform the task, we released the constraints on the model’s behavior and searched for the combination of parameter values that maximized the harvested reward. This reward-maximizing (“optimized”) model converges onto w1 = 0.621, w2 = 0.310 (and so w3 = 0.069), ρ = 72.4, and β = 0.207.\n\nThis optimized model harvested substantially more reward than the monkeys (Figure 6). Choosing right and left options at random, which is equivalent to models that always choose the left or always choose the right option, will result in harvesting 105.9 ms of valve opening time per trial, which we label as random performance of 50%. The theoretical limit, achieved by an ideal agent that knows the transition times and so always selects the richer option, harvests 141.2 ms of valve open time per trial, which we label as 100%. Our moneys earned 59.4% of the reward on this scale. This was substantially more (p < 0.0001, t94306 = 13.78) than the random choice model. However, the optimized model harvests 68.6% of the reward, substantially more (p < 0.0001, t94306 = 10.99) than the monkeys. This result proves that the behavior of our monkeys was suboptimal in this task. Given the same reward environment, there is at least one physically realizable model that forages substantially better than the monkeys.\n\nThe mean reward harvested by a model that makes choices at random (defined as 50%), by the monkeys, and by the optimized model (see text for details). A theoretical maximum (100%) would be obtained by an ideal agent that has information about the payoff transitions times and always chooses the richer option. * p < 0.0001.\n\nThe behavior of this optimized model is shown in Figure 7. As expected, the model clearly over-matches the reward proportions (Figure 7A). The steady state proportions of choices of the richer option for the payoff ratios 3:1 and 1.5:1 were 85.7% and 67.2% respectively, both significantly different from the proportions dictated by the matching law (p < 0.0001). The optimized model also switches less often than the monkeys (Figure 7B), on average every 4.1 trials, compared to the 3.2 of the monkeys. The difference is significant (p < 0.0001, t26114 = -20.83).\n\nSame format as in Figure 2, for the model with parameters maximizing its reward income.\n\nTo simplify the presentation and interpretation of all that follows, we reduced the number of free parameters in the model from four to three (Figure 8). A single parameter representing an exponential kernel replaces the two weight parameters (w1 and w2). This is more biologically plausible than using multiple discrete weights. Note also that the weights of the monkeys’ data fit and the optimized model fit are well approximated by a geometric series, which is the effective result of an exponential kernel (monkeys: w1 = 0.815, w2 = 0.198, w3 = -0.013; model: w1 = 0.621, w2 = 0.310, w3 = 0.069). Taking into account the constraint ∑i wi = 1, the first weight w1 is approximated as 11+α+α2, where α is the common ratio of the sequence. Then, w2 = αw1 and w3 = αw2. We set α such as to minimize the squared error between the actual weights and the approximated weights. That common ratio was found to be α = 0.201 for the model representing the monkeys, and α = 0.424 for the optimized model. The mean square error of these fits was small, equal to 0.058 for the model of the monkeys and 0.063 for the optimized foraging model. Consequently, the geometric approximation of the weights had negligible impact on the models’ behaviors (data not shown). The common ratio α helped not only to eliminate one free parameter; it also lends itself a straightforward interpretation: The larger the α, the more weight the monkeys put on the rewards received in the more distant past. For instance, for α =1, w1=w2=w3=13. Such model would simply average the past 3 rewards. The other extreme, α = 0 (w1 = 1, w2 = w3 = 0) would only consider the last obtained reward. Henceforth, we refer to α as the model’s “memory”: The larger the α, the longer reward history is used to compute the value V.\n\nThe model is identical to the model shown in Figure 3 with the exception that the weights are approximated with a geometric sequence with the common ratio α, subject to the constraint ∑i wi = 1. This way, w1=11+α+α2, w2 = αw1 and w3 = αw2.\n\nWe next investigated the role of the individual model parameters in the reward that can be harvested in this task. We visualized the effects of each parameter while fixing the values of the other two parameters. The fixed parameter values were the values of the optimized model (α = 0.424, ρ = 72.4, β = 0.207), as this model is much closer to the optimum compared to the monkeys. The parameter α was varied between 0 and 1 in steps of 0.05; ρ between -100 and +300 in steps of 20; β from 10-4 to 102 in geometric steps of 1.78. The parameter space additionally included also the values of the monkeys and of the optimal model.\n\nIt is important to note that each two-dimensional plot of reward as a function of a parameter value only shows a slice through the reward landscape; it does not show the entire reward landscape, which for this three-parameter model is four-dimensional. Figure 9 shows the leverage of each parameters on the mean harvested reward given the fixed values of the other two parameters.\n\nEach plot shows the mean±SEM reward harvested as a function of a particular parameter value. We varied the value of a parameter while fixing the other two parameters at values of the optimized model (α = 0.424, ρ = 72.4, β = 0.207). Red: parameters of the model of the monkeys’ behavior. Blue: parameters of the optimized model.\n\nThe model’s memory, α, had only small effect on the obtained reward. In regard to this aspect of the model, there was no significant difference (p = 0.63, t94306 = -0.48) in the reward gained by the optimized model (blue) and the monkey model (red). Assuming that our model has mechanistic validity, this plot indicates that limits on memory, as captured by this parameter, are unlikely to underlie the monkeys’ suboptimal performance.\n\nThe reward assigned to the unchosen option, ρ (middle plot), had a strong leverage on the reward gained. There was a clear optimum centered around the value ρ ~ 70. The monkeys’ ρ = 55.1 fell somewhat short of the model’s ρ = 72.4. As a consequence, in regard to this parameter, the monkeys earned 2.9% less reward compared to the optimal model. Although this drop was significant (p < 0.001, t94306 = -3.68), it can explain only about one-third of the monkeys’ suboptimal performance.\n\nThe parameter defining the steepness of the sigmoid that governs the value comparison (Figure 8), β, strongly affects the reward that can be harvested (right plot). The monkey model and the optimized model differ substantially in the value of this parameter (monkeys: β = 0.023; model: β = 0.207). Compared to the optimized model which properly reached the optimum (within the convergence rules of the optimization procedure), the monkeys harvested 6.4% less reward than the model. This was a significant (p < 0.0001, t94306 = -7.59) and substantial drop in the performance.\n\nThus, the parameters ρ and β were instrumental in governing the gain in this task. Of these, the fit to the monkeys’ data suggests that their low value of β substantially impaired their performance. The effect of the relatively small value of β is plotted in Figure 10. The figure plots Pright = Ψ(Vright – Vleft), for the Ψ parameter β of the monkeys and the optimized model. The figure reveals that as a result of the relatively high β, the value comparison function of the optimized model is much steeper compared to that of the monkeys. As a result, the optimized model is better equipped to compare the two values when making a choice. In fact, the comparison function of the optimized model is so steep that it essentially acts as a perfect comparator, choosing the rightward option when Vright > Vleft and the leftward option otherwise. The monkeys were not capable of performing such a sharp value comparison. As a result, their choice appeared more stochastic in regard to the value difference.\n\nThe figure plots Pright = Ψ(Vright–Vleft), over the range of (Vright–Vleft) (5th percentile equal to -172.8, 95th percentile equal to 176.3) for the Ψ parameter β of the monkeys and the optimized model. The optimized model had β about an order of magnitude higher than the monkeys, which defines its relatively sharp decision criterion.\n\nWe next investigated why the monkeys did not achieve a steeper value comparison function given that its steepness β governs the amount of earned reward (Figure 9-right). We hypothesized that this bottleneck may be due to the noisy representation of value (and value difference) by the monkey’s decision apparatus, which is presumably implemented by value-coding neurons32,33. The neuronal representation of value (and for that matter, of any variable) is inherently noisy34. We simulated how well an ideal observer, given the spike counts of value-coding neurons, could distinguish Vright from Vleft. We will lay out an ideal case; as such, our estimate of the brain’s ability to distinguish the two values will likely be optimistic.\n\nNeurons in many regions of the brain33,35,36 increase their discharge rate (r) with increasing value (V) of the option they encode:\n\n\n\nwhere r0 is the baseline firing rate and θ is the slope of the linear relationship between firing rate and value. Thus, neurons that encode the value of the rightward option fire with rate rright = r0+θVright and neurons that encode the value of the leftward option fire with rate rleft = r0+θVleft. We set r0 = 10 sp/s. We set θ to a 50% modulation of the baseline due to value, i.e., to θ = 5 sp/s over the value range (we used V = 300 as the maximum value).\n\nNow, assume that an ideal observer, positioned as an idealized downstream decoder37, knows which neurons encode Vright and which neurons encode Vleft. The task of this ideal observer is to tell, based on the discharge rates of these neurons rright and rleft, whether Vright > Vleft. For simplicity, we first consider the case in which the ideal observer assesses the activity of only one right-value-coding and one left-value-coding neuron. To be able to obtain any information from the spiking neurons, the ideal observer must measure the number of spikes n occurring within a certain time interval T. Because our monkeys had to make relatively fast decisions, we set T = 500 ms. Within this interval, the right-value-coding neuron will produce an average of μright = rrightT spikes; the left-value-coding neuron an average μleft = rleftT spikes. These are average spike counts, however. Spikes occur stochastically; a different train of spike times will occur during each decision. We will model spike occurrence times using a homogenous Poisson process37. As a result, during each decision, the measured spike counts nright and nleft will be drawn from a Poisson (~ Gaussian for n > 10) distribution. The variance of these distributions is σ2 = μ, i.e., σright2=rrightT and σleft2=rleftT.\n\nDue to the inherent noise in the spike generation process, the spike count distributions that encode the left and right value necessarily overlap (Figure 11). As a consequence, even the ideal observer of neuronal spike counts will make erroneous judgments on whether Vright > Vleft. The probability of making a correct Vright > Vleft decision Φ can be computed by drawing a boundary between the two distributions, and evaluating the rates of misclassification as a function of all boundary values (an ROC analysis37). The area under the ROC curve then equals Φ. An alternative approach to evaluating Φ is to notice that comparison Vright > Vleft is equivalent to Vright – Vleft > 0. Thus, the ideal observer may simply evaluate whether ndiff = (nright – nleft) > 0. Assuming that the two neurons fire spikes independently of each other, it is easy to show that the mean of ndiff equals nright–nleft and its variance equals σright2+σleft2. If nright and nleft are close to normal, then their difference ndiff is, according to the central theorem, yet closer to normal. The resulting probability density function is 𝒩 (nright−nleft,σright2+σleft2)= 𝒩 (nright−nleftσright2+σleft2,1). The probability Φ that ndiff > 0 then simply amounts to the integral below the normal probability density, which evaluates to erf(nright−nleftσright2+σleft2). We are interested in the right tail (ndiff > 0), so\n\n\n\nThe plots show the distributions of spike counts n for a neuron encoding Vright and a neuron encoding Vleft. The spike counts follow a Poisson distribution. In the Poisson distribution, σ2 = μ, so the right distribution with the higher σ also has a higher α. For large enough n, the distribution approaches a Gaussian. For simplicity, the illustrated distributions are Gaussian.\n\n(Note that (nleft−nrightσright2+σleft2)×2=d', which is an often used measure of discriminability of two distributions in psychology and neuroscience.)\n\nWe presented the right-value-coding and the left-value-coding neuron with the range of values Vright and Vleft, respectively, experienced by the monkeys. Based on the spiking activity of these neurons, we plotted the probability Φ that the ideal observer could correctly choose the rightward option, i.e., Pright = Φ, as a function of Vright – Vleft (Figure 12A). The simple case of 2 independent neurons coding Vright and Vleft is shown in gray. The plot reveals that the ideal observer can only poorly determine whether Vright or Vleft is larger. There is too much noise in the spike counts.\n\nThe neuronal noise can be effectively reduced if the ideal observer can read out the activity of multiple uncorrelated neurons. In particular, if the observer averages the responses of m independently firing neurons in each (left or right) value-coding pool, then the noise variance σ2 drops by a factor of m. As a result, the distributions of the average population spike counts become thinner than those of the individual neurons shown in Figure 11. Consequently, it is easier to tell the values drawn from these thinner distributions apart. Indeed, when the observer averages spike counts over 10 independent neurons in each pool (20 all together), the observer’s value assessment improves substantially (black curve in Figure 12A).\n\n(A) Same format as in Figure 10. The figure additionally includes responses of an ideal observer whose job is to tell Vright and Vleft apart by reading out the responses of simulated spiking neurons (see text for details). The more independent neurons available to the ideal observer, the higher the ability to discriminate the two values. The gray (black) curve represent 2 (20) available neurons. (B) The number of neurons necessary to obtain a value comparison function of a particular steepness (β). The data are plotted in log-log space. In this space, the apparently linear relationship represents an exponential relationship between the two quantities. To increase β, one needs to access an exponentially higher number of independent value-coding neurons.\n\nWe plotted the minimum number of the independent value-coding neurons necessary to achieve the value comparison function of a particular value of β. The result is shown in Figure 12B. On the log-log scale plotted in the figure, there is an approximately linear relationship between the required number of neurons and the comparison function steepness β. This means that to achieve a higher β, one must employ an exponentially growing number of independent value-coding neurons. The minimum number of independent value-coding neurons to attain the β of the monkeys, in the ideal case, is 77. In contrast, the optimized model would require at least 6651 independent value-coding neurons.\n\nIt is important to stress that these numbers represent a theoretical minimum. We assumed neurons with a large (50%) modulation of their firing rates by value, assumed completely independent neurons (zero noise correlation), assumed that the ideal observer can flawlessly average the responses in the respective right and left neuronal populations, that the ideal observer has 500 ms of time to read out the spike counts during each decision, and disregarded any additional sources of noise. Therefore, the true numbers are likely to be substantially higher. Thus, this analysis suggests that increasing β to harvest more reward is very costly in terms of the number of neurons required. It is therefore likely that the neuronal noise presents a bottleneck in the animals’ attaining a steeper value comparison function.\n\nFigure 7 revealed that the optimized model strongly overmatched the proportions dictated by the matching law. We next determined how the three model parameters of the simplified model influence two characteristics of the behavioral response: the matching level and the transition rate (Figure 13). We define the matching level (ML) as the choice proportion at trial 7 following a transition. We average across all four possible transitions (i.e., 1:3 reward ratio changing to 3:1 ratio, 1:3 ratio changing to 1.5:1 ratio, etc). We then scale the data such that selecting the two targets equally (unbiased or 50% choice proportion) corresponds to ML = 0, and perfect matching (average of 60% and 75%, or 67.5%) corresponds to ML = 1, with a linear continuum between and beyond these values. We define the transition rate (TR) as the change in the proportion of choices of the richer option from trial 0 to trial 1 following transition, averaged across all four possible transitions.\n\nThe Transition Rate (TR) is defined as the change in the proportion of choices of the richer option from trial 0 to trial 1 following transition. The matching level (ML) is defined as the choice proportion at trial 7 following transition, such that ML = 0 for the 50% choice proportion and ML = 1 for the 67.5% proportion (average of 60% and 75%), with a linear continuum between and beyond these values.\n\nWe first evaluated the effects of each individual parameter on TR (Figure 14A). The analysis is similar to that of Figure 9, except that the dependent variable is TR instead of reward. We evaluate the effect of each parameter on both the optimized model (blue: α = 0.424, ρ = 72.4, β = 0.207) and on the best-fit match to the monkey performance (red: α = 0.201, ρ = 55.1, and β = 0.023). The left panel reveals that TR is a monotonic function of the model’s memory α. As expected, the shorter the model’s reward memory (i.e., the smaller the reliance on the past rewards), the faster the model transitions to a new payoff ratio. TR is also strongly dependent on ρ, showing an optimum (middle panel). This is also as expected. During steady state, the poorer option is less often chosen. Therefore the larger the reward assigned to the unchosen option, the more likely that its value will exceed that of the chosen option, causing the model to switch. This benefit applies only up to a certain point: high values of ρ lead to metronome-like switching (not shown), thus hampering TR. TR is also sensitive to the steepness of the value comparator β (right panel). For a shallow comparator (low value of β), the model fails to clearly distinguish the values of the two options and as a result transitions poorly. This is improved by using a β of higher value, with an effect that saturates at just over β = 0.01.\n\nSame format as in Figure 9 but plotting Transition Rate (A) and Matching Level (B) instead of reward as the dependent variable. We varied the value of a parameter while fixing the other two parameters at values of the monkeys (red) and of the optimized model (blue).\n\nIn a similar vein, we then investigated which parameters are important in achieving a particular ML. To do so, we repeated the previous analysis, but for ML as the dependent variable (Figure 14B). The model’s memory α has a small but noticeable effect on the ML. The longer the memory span (higher α), the higher the ML. This is as expected—reliably identifying the richer value requires a rigorous assessment of the past rewards; the weights on the past reward are maximal (w1=w2=w3→13) when α → 1. The value of the reward of the unchosen option, ρ, has strong leverage on the ML. There is an optimum at about 0 < ρ < 80, depending on the values of the other two parameters. Notably, the ρ plot reveals that the optimized model did not maximize ML. Maximizing ML may not result in maximizing reward. We revisit this question at the end of the Results section. The steepness of the value comparison function, β, also had a substantial impact on the ML. The steeper the value comparison function, the higher the ML. This is as expected: the model should include as little noise in the value comparison as possible in order to correctly identify the richer option.\n\nFinally, we investigated the possibility that animals optimized molar aspects of task performance, such as the TR and ML, instead of the parameters of the reinforcement learning model. We therefore plotted the mean harvested reward as a function of TR and ML. To obtain enough variability in these two attributes, we exhaustively tested each considered value of α, ρ, and β against each other. This resulted in 14283 different models, each associated with a TR, an ML, and a reward gain.\n\nFigure 15 shows the mean harvested reward averaged over all models that have a particular value of ML and TR. The figure reveals that the mean reward increases both with increasing ML and increasing TR. This is as expected. An ideal agent should transition to the richer option as rapidly as possible, and in the steady state should maintain as high a value of ML as possible. Furthermore, the figure reveals that at certain level, there is tradeoff between ML and TR. In particular, starting at ML ≈ 1, a further increase in ML comes at the cost of a decrease in TR.\n\nWe exhaustively varied, against each other, the values of α, ρ, and β, to arrive to a total of 14283 different models. Each was associated with a mean reward, with a Transition Rate, and with a Matching Level. The plot shows in color the mean harvested reward averaged over all models that have a particular value of Transition Rate and Matching Level. At the blank spaces, there was no model of the 14283 tested with the corresponding value of Transition Rate and Matching Level.\n\nThe model approximating the monkeys’ behavior (red cross) is positioned far from the maximum in this model-average reward landscape. There was no clear local optimum at that point, not in regard to TR, not in regard to ML, and not in regard to the particular combination of TR and ML. This suggests that the monkeys did not optimize their behavior based on TR or ML. The optimized model occupies a much more lucrative spot in this reward landscape, positioned at or near the maximum. Notably, the optimized model did not attain the highest value of ML it possibly could. Nonetheless, this allowed the model to achieve a higher TR. The plot shows that maximizing ML does not necessarily equal maximizing reward; it is important to strive for a high TR, too. However, at the high reward levels, there is a tradeoff between these two attributes of molar behavior.\n\n\nDiscussion\n\nMatching has been a widely studied and a much debated behavioral phenomenon1,3–12. In baiting tasks, in which a reward, once scheduled, is available at an option until the subject harvests it, matching is the optimal or near-optimal strategy. In particular, it has been shown that matching follows from maximization of reward at either the molecular15–17 or molar13,18,19 scales. Furthermore, at the level of mechanistic implementation, a biophysically based neural model grounded in reinforcement learning7 was also shown to reproduce matching behavior in a baiting task25.\n\nAn important question is to what extent matching applies to tasks that do not feature baiting or other control elements that render matching an optimal strategy. We engaged animals in a reward-based foraging task that featured neither baiting nor other controls to elicit matching. Surprisingly, we found that animals in our task very faithfully matched the reward proportions. This is a surprising finding because matching was not the optimal strategy in this task; we found that a model could harvest substantially more reward than the monkeys by over-matching the reward proportions. We investigated the source of the animals’ bottleneck at the mechanistic level. We found that the animals showed a relatively shallow comparison criterion that contrasts the values of the rightward and the leftward options (Figure 10). This is an important bottleneck because at least in this task, the steepness of the value comparison function has a strong effect on the earned reward (Figure 9, right). Furthermore, the steepness also has strong leverage on the level of matching (Figure 14B, right).\n\nOne possible explanation for the animals’ poor comparison of the values of the options is that the they did not properly register the amount of the delivered juice. This is unlikely, for three reasons. First, there was a nearly linear relationship between the valve open time and the amount of fluid reward delivered (data not shown). Second, the setup produced an auditory beep of the duration corresponding to the valve open time, which served as a secondary reinforcer. A trained ear can likely distinguish duration differences of less than 5%38. Third and most importantly, our pilot data showed that animals were capable of distinguishing even very small differences, namely a 105 ms from a 95 ms period of the valve opening.\n\nIf the suboptimal value comparison is not due to the registration of the reward magnitude, the bottleneck likely emerges from the internal representation of reward-related variables. There are many possible sources of noise affecting the representation of value in the brain. We considered the one that is inevitable and so at play: the noisy representation of value by spiking neurons. In a simulated representation of value by spiking neurons, we showed that the ability to discriminate two values is poor when only two neurons are considered in the discrimination (Figure 12A). That ability improves when the number of independent value-coding neurons increases (Figure 12A). Importantly, we found that the increase in the steepness of the value comparison β requires a recruitment of an exponential number of independent neurons (Figure 12B). Thus, increasing the steepness of the value comparison function is very costly in regard to neural resources.\n\nNotably, the statistical framework we employed in Figure 12 is general, not limited to the poisson noise in the spike counts. The analysis of the number of required neurons n simply rests on the fact that to reduce noise, one may average signals over m neurons; if the neurons are independent, the averaging reduces the variance in the noise by a factor of m. The simulation in Figure 12B showed that this rate of variance reduction is low with respect to an increase in the steepness of β: the relationship between m and β is exponential. Given this general statistical consideration, other forms of noise superimposed on the neuronal representations would lead to the same conclusion: To increase β, given a non-zero amount of noise in the brain, one must engage an exponentially growing number of neurons.\n\nConceivably, animals in this task could also under-match the reward proportions. However, under-matching would incur further loss (Figure 15). In this task, matching thus appears as a compromise between harvesting a sufficient amount of reward and the demands placed by noise on optimal neural computation.\n\n\nConclusions\n\nWe observed matching behavior in a task in which more reward could be harvested if animals over-matched the reward proportions. Mechanistic modeling revealed that the reward gained in this task and the level of matching strongly depend on the quality of the comparison of values of the decision options. The animals had a shallow comparison function, which dampened their reward income and their matching level. A neural simulation showed that an increase in the steepness of the comparison function is very costly (exponential explosion) in the number of the required value-coding neurons, given that there is a non-zero amount of noise in the neuronal representations. This finding identifies an important neural constraint on optimal choice.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw task data, 10.5256/f1000research.6574.d4885339",
"appendix": "Author contributions\n\n\n\nJK and LHS designed the task. JK collected the data. JK analyzed the data. JK and LHS wrote the paper. All authors have read and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests.\n\n\nGrant information\n\nThis work was supported by the grants from the NIH EY012135 and EY002687 to LHS.\n\n\nAcknowledgements\n\nWe thank Jonathan Tucker for technical assistance and Mary Kay Harmon for veterinary assistance.\n\n\nReferences\n\nHerrnstein RJ: Relative and absolute strength of response as a function of frequency of reinforcement. J Exp Anal Behav. 1961; 4(3): 267–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe Villiers P: Choice in concurrent schedules and a quantitative formulation of the law of effect. Handbook of operant behavior. 1977; 233–287.\n\nDavison M, McCarthy D: The matching law: A research review. Lawrence Erlbaum Associates, Inc. 1988. Reference Source\n\nHerrnstein RJ: The matching law Papers in psychology and economics. Harvard University Press. 2000. Reference Source\n\nTodorov JC, Hanna ES, Bittencourt de Sá MCN: Frequency versus magnitude of reinforcement: New data with a different procedure. J Exp Anal Behav. 1984; 41(2): 157–167. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLau B, Glimcher PW: Dynamic response-by-response models of matching behavior in rhesus monkeys. J Exp Anal Behav. 2005; 84(3): 555–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSoltani A, Wang XJ: A biophysically based neural model of matching law behavior: melioration by stochastic synapses. J Neurosci. 2006; 26(14): 3731–44. PubMed Abstract | Publisher Full Text\n\nLoewenstein Y, Seung HS: Operant matching is a generic outcome of synaptic plasticity based on the covariance between reward and neural activity. Proc Natl Acad Sci U S A. 2006; 103(41): 15224–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoffarnus MN, Woods JH: Quantification of drug choice with the generalized matching law in rhesus monkeys. J Exp Anal Behav. 2008; 89(2): 209–224. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVollmer TR, Bourret J: An application of the matching law to evaluate the allocation of two- and three-point shots by college basketball players. J Appl Behav Anal. 2000; 33(2): 137–150. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKraft JR, Baum WM: Group choice: the ideal free distribution of human social behavior. J Exp Anal Behav. 2001; 76(1): 21–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReed DD, Critchfield TS, Martens BK: The generalized matching law in elite sport competition: football play calling as operant choice. J Appl Behav Anal. 2006; 39(3): 281–297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStaddon JE, Motheral S: On matching and maximizing in operant choice experiments. Psychological Review. 1978; 85(5): 436–444. Publisher Full Text\n\nRachlin H: On the tautology of the matching law. J Exp Anal Behav. 1971; 15(2): 249–251. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShimp CP: Optimal behavior in free-operant experiments. Psychological Review. 1969; 76(2): 97–112. Publisher Full Text\n\nHerrnstein RJ: Derivatives of matching. Psychological Review. 1979; 86(5): 486–495. Publisher Full Text\n\nStaddon JER, Hinson JM, Kram R: Optimal choice. J Exp Anal Behav. 1981; 35(3): 397–412. Reference Source\n\nRachlin H, Green L, Kagel JH, et al.: Economic demand theory and psychological studies of choice. Psychology of Learning and Motivation. 1976; 10: 129–154.\n\nBaum WM: Optimization and the matching law as accounts of instrumental behavior. J Exp Anal Behav. 1981; 36(3): 387–403. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaum WM: On two types of deviation from the matching law: bias and undermatching. J Exp Anal Behav. 1974; 22(1): 231–242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAllen CM: On the exponent in the \"generalized\" matching equation. J Exp Anal Behav. 1981; 35(1): 125–127. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacdonall JS: Concurrent variable-ratio schedules: Implications for the generalized matching law. J Exp Anal Behav. 1988; 50(1): 55–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStubbs DA, Pliskoff SS: Concurrent responding with fixed relative rate of reinforcement. J Exp Anal Behav. 1969; 12(6): 887–895. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaum WM: Time allocation in human vigilance. J Exp Anal Behav. 1975; 23(1): 45–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSugrue LP, Corrado GS, Newsome WT: Matching behavior and the representation of value in the parietal cortex. Science. 2004; 304(5678): 1782–7. PubMed Abstract | Publisher Full Text\n\nSutton RS, Barto AG: Reinforcement learning: an introduction. IEEE transactions on neural networks / a publication of the IEEE Neural Networks Council. 1998; 9(5): 1054. Reference Source\n\nCorrado GS, Sugrue LP, Seung HS, et al.: Linear-Nonlinear-Poisson models of primate choice dynamics. J Exp Anal Behav. 2005; 84(3): 581–617.PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeo H, Barraclough DJ, Lee D: Lateral intraparietal cortex and reinforcement learning during a mixed-strategy game. J Neurosci. 2009; 29(22): 7278–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInstitute of Laboratory Animal Resources (US). Committee on Care and Use of Laboratory Animals and National Institutes of Health (US). Division of Research Resources, Guide for the care and use of laboratory animals. National Academies. 1985. Reference Source\n\nLutz CK, Novak MA: Environmental enrichment for nonhuman primates: theory and application. ILAR J. 2005; 46(2): 178–91. PubMed Abstract | Publisher Full Text\n\nKubanek J, Wang C, Snyder LH: Neuronal responses to target onset in oculomotor and somatomotor parietal circuits differ markedly in a choice task. J Neurophysiol. 2013; 110(10): 2247–2256. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPadoa-Schioppa C, Assad JA: Neurons in the orbitofrontal cortex encode economic value. Nature. 2006; 441(7090): 223–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKable JW, Glimcher PW: The neurobiology of decision: consensus and controversy. Neuron. 2009; 63(6): 733–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYarom Y, Hounsgaard J: Voltage fluctuations in neurons: signal or noise? Physiol Rev. 2011; 91(3): 917–929. PubMed Abstract | Publisher Full Text\n\nPlatt ML, Glimcher PW: Neural correlates of decision variables in parietal cortex. Nature. 1999; 400(6741): 233–8. PubMed Abstract | Publisher Full Text\n\nRoesch MR, Olson CR: Neuronal activity related to reward value and motivation in primate frontal cortex. Science. 2004; 304(5668): 307–10. PubMed Abstract | Publisher Full Text\n\nDayan P, Abbott LF: Theoretical neuroscience: Computational and Mathematical Modeling of Neural Systems. Cambridge MA, MIT Press, 2001. Reference Source\n\nCreelman CD: Human discrimination of auditory duration. J Acoust Soc Am. 1962; 34(5): 582–593. Publisher Full Text\n\nKubanek J, Snyder L: Dataset 1 in: Matching Behavior as a Tradeoff Between Reward Maximization and Demands on Neural Computation. F1000Research. 2015. Data Source"
}
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[
{
"id": "10172",
"date": "01 Sep 2015",
"name": "Bruno Averbeck",
"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 by Kubanek and Snyder presents interesting data and modeling on matching behavior. They find that animals match in a learning task where the animals should infer which of two targets will deliver the larger reward on each trial. They find matching behavior in the animals. The behavioral choice strategy of the animals is modeled using a simple value integration algorithm. The algorithm accounts well for the choices of the animals. They also show that the algorithm can significantly outperform the animals if it over-matches, i.e. if it picks the better option more often. The main difference between the improved algorithm and the animal's behavior is the decision noise or beta parameter. They then develop a second model which assumes that the noise in the animal's choice behavior is driven by limits in their population code for value. The paper is well written and the study has been carefully carried out. Overall, this is nice work. I would make one comment on the final conclusion, that the noisiness in the animal's choice behavior is driven by noise in their population code. Specifically, how can this hypothesis be differentiated from the possibility that the noise in the animal's choice behavior is a strategic choice? In other words, is the animal limited by noise in their population coding, or are they exploring for other reasons, including perhaps satisficing? Would their decision noise (the beta parameter) be the same in another task in which values have to be learned, but under different conditions?",
"responses": [
{
"c_id": "1613",
"date": "02 Oct 2015",
"name": "Jan Kubanek",
"role": "Reader Comment",
"response": "We thank this reviewer for this helpful comment. In response to this comment, we now include a new paragraph in the Discussion:\"The finding that the animals' value comparison function is relatively shallow indicates that the animals' choice behavior is relatively stochastic. The simulation of the representation of value by noisy neurons provides one possible explanation for this stochastic choice behavior. However, the stochasticity might be also due to other factors. For instance, the animals might, at least in part, use a strategy that deviates from the optimal strategy of comparing the value of the two options. A deviation from that optimal strategy might appear as an increased level of noise in the animals' choice. Another possibility is that the nervous system specifically introduces noise into certain stages of the decision machinery to promote foraging and exploration. This might be beneficial in environments with stochastic reward schedules, i.e., in which the reward obtainable for a choice is difficult to predict.\""
}
]
},
{
"id": "10361",
"date": "16 Sep 2015",
"name": "Jacqueline Gottlieb",
"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 very interesting article that thoroughly examines the “matching” behaviour in monkeys using behavioural testing and reinforcement learning models. Monkeys perform a task where they can choose between two targets associated with variable rewards. The monkeys show approximate matching of the reward ratios in their choices, even though this is not optimal in the present task. The authors carry out an exhaustive modelling effort to characterize the matching behaviour, its difference from an optimized behaviour based on RL, and the parameters that give rise to non-optimality in the choices. Based on these efforts, they conclude that a significant source of non-optimality may be in the noise of internal value representations. Overall the paper is very nicely done – it is well written and I greatly appreciate the thoroughness of the modelling efforts. I have several suggestions that may improve it: By design, the authors provided the monkeys with reward magnitudes that varied in a complicated fashion in order to prevent stereotyped behaviors. However, above and beyond this variability, the *reward ratios* fell into only 4 distinct categories. Given enough training the monkeys could, in principle, have learnt these categories and used some stereotyped strategies to switch between them. The success of the RL- model in capturing the data seems to make this possibility unlikely – but this is not conclusive and there should be some explicit analysis of this possibility. At present there is no mention of the length of training (or even, in the data provided on the website, of the *session* from which a trial came from). These are important details to include.Echoing the comment of reviewer 1, the conclusion that the source of suboptimality is in neural noise seems overdone. This is *one* possible explanation that lends itself to an elegant model, but the mapping function between behavior and neural activity is complex, and many other schemes are possible. The authors should discuss these alternative schemes.I found the Introduction a bit difficult to follow. Although individual paragraphs are well written, I was not clear where the entire narrative was going. The analysis (in the Results) focuses on non-optimal choice strategies and their possible neural bases – and the Introduction should be re-arranged to bring out this theme.",
"responses": [
{
"c_id": "1614",
"date": "02 Oct 2015",
"name": "Jan Kubanek",
"role": "Reader Comment",
"response": "1. By design, the authors provided the monkeys with reward magnitudes that varied in a complicated fashion in order to prevent stereotyped behaviors. However, above and beyond this variability, the *reward ratios* fell into only 4 distinct categories. Given enough training the monkeys could, in principle, have learnt these categories and used some stereotyped strategies to switch between them. The success of the RL- model in capturing the data seems to make this possibility unlikely – but this is not conclusive and there should be some explicit analysis of this possibility. At present there is no mention of the length of training (or even, in the data provided on the website, of the *session* from which a trial came from). These are important details to include.#Authors' response:We designed the task so that animals could not anticipate a reward ratio transition (the distribution of transition times is exponential, which has a flat hazard rate).Critically, the data show that the monkeys did not anticipate a specific reward ratio. In addition to the success of the ratio-agnostic RL model, this is conclusively demonstrated by the behavior aligned on transition (Figure 2A). If the animals anticipated a transition, there would be an increase in the proportion of choices of the richer option prior to or on transition (e.g. trial -1 or trial 0 in that figure). No such increase is observed. The figure demonstrates that once the animals reach a behavioral equilibrium, they maintain it.Moreover, if the animals anticipated a specific reward ratio, there would be no distinction in behavior between the 3:1 and 1.5:1 reward ratios (Figure 2A), or at least, the matching behavior would be profoundly degraded. Yet, the animals showed nearly exact matching of the respective ratios (Figure 2A).In response to this comment, the Methods now include the following text:\"We used an exponential distribution of reward ratio duration because an exponential distribution has a flat hazard rate, making it difficult for the animals to anticipate a transition. Indeed, animals showed no anticipation of a transition (Figure 2A).\"We now also provide the length of training and data collection in the Methods. 2. Echoing the comment of reviewer 1, the conclusion that the source of suboptimality is in neural noise seems overdone. This is *one* possible explanation that lends itself to an elegant model, but the mapping function between behavior and neural activity is complex, and many other schemes are possible. The authors should discuss these alternative schemes.#Authors' response:This is now addressed in a new paragraph in the Discussion:\"The finding that the animals' value comparison function is relatively shallow indicates that the animals' choice behavior is relatively stochastic. The simulation of the representation of value by noisy neurons provides one possible explanation for this stochastic choice behavior. However, the stochasticity might be also due to other factors. For instance, the animals might, at least in part, use a strategy that deviates from the optimal strategy of comparing the value of the two options. A deviation from that optimal strategy might appear as an increased level of noise in the animals' choice. Another possibility is that the nervous system specifically introduces noise into certain stages of the decision machinery to promote foraging and exploration. This might be beneficial in environments with stochastic reward schedules, i.e., in which the reward obtainable for a choice is difficult to predict.\"I found the Introduction a bit difficult to follow. Although individual paragraphs are well written, I was not clear where the entire narrative was going. The analysis (in the Results) focuses on non-optimal choice strategies and their possible neural bases – and the Introduction should be re-arranged to bring out this theme. #Authors' response:In response to this comment, we made the Introduction much more compact. We also entirely rewrote its last paragraph. The last paragraph now reads:\"The finding that matching behavior is observed in a task that does not impose it provides important insights into the nature of matching behavior. To shed light on the mechanism, we described the animals' behavior using a mechanistic model. The model faithfully captured the monkeys' molar and molecular behavior. We show which components of the model are important in mediating matching. We then implement the critical component by populations of spiking neurons. The mechanistic modeling revealed a bottleneck in the animals' ability to compare the values of the two options. The additional neuronal implementation suggested that this bottleneck could be due to noise in the representation of value by the neuronal populations.\""
}
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https://f1000research.com/articles/4-147
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https://f1000research.com/articles/4-188/v1
|
07 Jul 15
|
{
"type": "Case Report",
"title": "Case Report: A case report of dry tap during ventriculostomy",
"authors": [
"Sunil Munakomi",
"Binod Bhattarai",
"Binod Bhattarai"
],
"abstract": "Pneumocephalus following ventriculoperitoneal (VP) shunt insertion is an exceptionally rare occurrence. We report such an event after attempting ventricular puncture (ventriculostomy) for VP shunt insertion and then discuss the management of the same. Dry tap can lead to multiple attempts for ventriculostomy with the associated added risks of complications, as well as complicating the subsequent management. In addition, there is an increased risk of tension pneumocephalus, seizure and shunt failure due to a blockage by air bubbles. Our patient presented with features of raised intracranial pressure two months following craniotomy and evacuation of traumatic subdural hematoma. External ventricular puncture revealed egress of CSF under pressure. Upon attempting VP shunting for post-traumatic hydrocephalus, we experienced dry tap during ventricular puncture that complicated further management. We placed the proximal shunt in the presumed location of the foramen of Monro of ipsilateral frontal horn of lateral ventricle and did not remove the external ventricular drain. Post-operative CT scan revealed pneumoventriculi as the cause for the dry tap during ventricular puncture. Patient was managed with 100% oxygen. He showed gradual improvement and was later discharged. This case shows that variations in the procedure, including head down positioning, adequate cruciate dural incision prior to cortex puncture, and avoiding excessive egress of CSF can help to prevent such complications.",
"keywords": [
"Dry tap",
"Pneumocephalus",
"Management"
],
"content": "Introduction\n\nPneumocephalus is defined as the presence of air within the calvarium. It often follows trauma but is also a common sequelae of intracranial surgery1,2. Tension pneumocephalus is a life-threatening emergency that necessitates immediate surgical intervention3. It is rarely reported after cerebrospinal fluid (CSF) diversion procedures4,5. We present a rare case of tension pneumocephalus, resulting in dry tap during ventriculostomy and discuss its subsequent management.\n\n\nCase report\n\nHerein we report a case of a 35-year-old male from Nawalparasi, Nepal, who had undergone a craniotomy and evacuation of acute subdural hematoma following an automobile accident 2 months before admission to our institution. He presented with complaints of an abnormal gait, with a tendency to fall backwards and also with features of frontal lobe-related incontinence. There were no significant past medical illnesses. He was taking Sodium Valproate (300 mg oral three times daily) as seizure prophylaxis following the traumatic head injury and surgical intervention for the same 2 months previously. Fundus examination revealed the presence of papilledema. A head computerized tomography (CT) scan revealed the presence of evolving hydrocephalus. To rule out hydrocephalus ex vacuo due to volume loss and changes in CSF dynamics subsequent to the previous accident, external ventricular drainage (EVD) was placed which revealed egress of CSF under pressure. Thereafter he was scheduled for insertion of a VP shunt. During insertion of the VP shunt, there was dry tap during an attempt of ventriculostomy from the Kocher’s point. We made two further attempts to ensure the correct trajectory of the shunt end and also to reflush the shunt end to prevent blockage due to blood clots and cell debris. We placed the shunt tip in the presumed location of the foramen of Monro of the frontal horn of ipsilateral lateral ventricle. We did not remove the EVD, hoping that it would act as a safety channel for CSF bypass had we missed the correct trajectory for the VP shunt.\n\nA postoperative scan revealed the presence of tension pneumocephalus and pneumoventriculi (Figure 1 and Figure 2). The patient was managed with 100% oxygen for 3 days and was continued on antiepileptic medications at the same dose intravenously. Stringent neurological monitoring was undertaken to evaluate early neurological deterioration due to tension pneumocephalus. Pupils were routinely assessed to look for hippus (a clinical marker of epilepsy). Patient was extubated the following morning. A repeat CT scan on the 6th day post-operation showed that the proximal shunt was in the third ventricle (Figure 3) and there was complete resolution of the condition. The EVD was subsequently removed with no neurological deterioration of the patient on 7th day after operation. The patient then started to walk with support from the 8th day post-operation, and he slowly improved in gait. Patient went home walking with minimal support on the 14th day post-operation. Patient had also regained his bladder control within that time. Patient returned, walking on his own 1 month later for his follow up in the outpatient department His gait was normal with no features of retropulsion. The shunt chamber was functioning well and his bowel habits were normal. Compliance in continuation of Sodium valproate therapy (at the aforementioned dose) was also ensured.\n\n\nDiscussion\n\nPneumocephalus usually occurs after head trauma, skull base fractures, and associated CSF fistulas1. The incidence of this entity was reported to be as high as 100% following supratentorial craniotomies6. On the other hand, tension pneumocephalus is a neurosurgical emergency that requires rapid surgical intervention. Pneumocephalus as a complication of CSF diversion procedures is rare5. Diagnosis is mainly based on clinical examination and computerized tomography (CT) scan4. Two CT findings that characterise the condition – the ‘Mount Fuji’ sign and the ‘air bubble’ sign - have been described by Ishiwata et al.7.\n\nThere are two factors that are thought to be responsible for tension pneumocephalus development. The first is a decrease in intracranial pressure due to a sudden egress of CSF; the second is the presence of a craniodural defect that works as a one way valve allowing air inflow to the intracranial space and preventing outflow4. It is claimed that moderate cerebral atrophy might play a role1.\n\nThe duration of the shunt surgery must be as short as possible and CSF leakage during the connection of the shunt system must be avoided. Another factor that can lead to undesirable outcomes can be introduced during the puncturing of the cortex. Adequate cruciate incision must be given to prevent the passage of environmental air into the subdural space. Filling the subdural space on the ventriculostomy site with irrigation fluid until overflowing might help the outflow of air from the intracranial vault, reducing the risk of this rare complication. Cortical atrophy may have also had an effect on isolated air collection within the subdural space. In our case, another remote possibility for the development of pneumocephalus would be any leak in the closed drainage system of the previous EVD drain. Properly layered closure of the skin in VP shunt surgery is the most important factor for prevention of this rare complication.\n\nThe dry tap, as seen in our case, can lead to multiple attempts to attain the correct shunt trajectory, thereby increasing the risk of false trajectories and track hematomas. If there had been no EVD, then this would have led to termination of the procedure, thereby adding to the morbidity and risk of subsequent surgery. One alternative to our approach would be the use of intra-operative CT scan to ensure the diagnosis. Unfortunately this is not currently possible in developing countries like ours. We can, if available, take help of neuro-navigation tools to ensure the correct trajectory to the ventricles even in cases were in pneumocephalus occurs. This can reduce the added burden of subsequent surgeries and associated risk of anesthesia.\n\nThere is also risk of seizure and rapid neurological deterioration due to tension pneumocephalus. Once this occurs, close monitoring of the patient, rapid and accurate identification of tension pneumocephalus, and immediate surgical intervention is life-saving. Gore et al.8 have advocated the use of 100% oxygen for rapid resolution of pneumocephalus.\n\nIn conclusion, though VP shunting is one of the most common surgical procedures performed in neurosurgery, strict adherence to basic principles should be followed during the procedure so as to prevent avoidable complications such as in our case which may in times lead to sudden deterioration in the patient and also add to diagnostic and therapeutic dilemma to the concerned surgeons. One advantage in this case was the presence of an EVD as a safety bypass for CSF diversion. The limitations of our approach can be attributed to the unavailability of intra-operative CT scan and neuronavigation techniques which would have aided in early diagnosis and management in this scenario.\n\n\nConsent\n\nBoth written and verbal informed consent for publication of images and clinical data related to this case was sought and obtained from the wife of the patient.",
"appendix": "Author contributions\n\n\n\nDr Sunil reviewed the literature, designed the study and formatted the paper. Dr Binod revised and edited the final format.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThe authors declared that no funding was involved in supporting this work.\n\n\nReferences\n\nBarada W, Najjar M, Beydoun A: Early onset tension pneumocephalus following ventriculoperitoneal shunt insertion for normal pressure hydrocephalus: a case report. Clin Neurol Neurosurg. 2009; 111(3): 300–302. PubMed Abstract | Publisher Full Text\n\nKawajiri K, Matsuoka Y, Hayazaki K: Brain tumors complicated by pneumocephalus following cerebrospinal fluid shunting--two case reports. Neurol Med Chir (Tokyo). 1994; 34(1): 10–14. PubMed Abstract | Publisher Full Text\n\nMonas J, Peak DA: Spontaneous tension pneumocephalus resulting from a scalp fistula in a patient with a remotely placed ventriculoperitoneal shunt. Ann Emerg Med. 2010; 56(4): 378–381. PubMed Abstract | Publisher Full Text\n\nTuğcu B, Tanriverdi O, Günaldi O, et al.: Delayed intraventricular tension pneumocephalus due to scalp-ventricle fistula: a very rare complication of shunt surgery. Turk Neurosurg. 2009; 19(3): 276–280. PubMed Abstract\n\nUgarizza LF, Cabezudo JM, Lorenzana LM, et al.: Delayed pneumocephalus in shunted patients. Report of three cases and review of the literature. Br J Neurosurg. 2001; 15(2): 161–167. PubMed Abstract | Publisher Full Text\n\nReasoner DK, Todd MM, Scamman FL, et al.: The incidence of pneumocephalus after supratentorial craniotomy. Observations on the disappearance of intracranial air. Anesthesiology. 1994; 80(5): 1008–1012. PubMed Abstract | Publisher Full Text\n\nIshiwata Y, Fujitsu K, Sekino T, et al.: Subdural tension pneumocephalus following surgery for chronic subdural hematoma. J Neurosurg. 1988; 68(1): 58–61. PubMed Abstract | Publisher Full Text\n\nGore PA, Maan H, Chang S, et al.: Normobaric oxygen therapy strategies in the treatment of postcraniotomy pneumocephalus. J Neurosurg. 2008; 108(5): 926–929. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "9362",
"date": "11 Aug 2015",
"name": "Guo-Yi Gao",
"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\nVP shunt is the most frequent surgical technique in neurosurgery to treat hydrocephalus related with trauma, SAH, tumor and other brain diseases. Dry tap, which is a rare episode during the shunt operation, may cause confused decision and leading to complications. In this report, authors described the details of the dry tap and analysis the possible reasons behind, which is helpful for neurosurgeons to prevent this rare complication in clinical settings.",
"responses": []
},
{
"id": "10474",
"date": "24 Sep 2015",
"name": "Roman Bosnjak",
"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 analysed dry tap during ventriculostomy in the presence of EVD inserted few days before.Their explanations are reasonable. But there might be additional explanations.The EVD was inserted to rule out ex vacuo hydrocephalus. CSF egressed under pressure. Here the authors could insert VP shunt on first occasion (without previous EDV).\n\nThere is no report on patient's condition between EVD and VP insertion. If it improved and remained stable, no significant tension hydrocephalus is expected to evolve. It is my theory that the air was sucked into the ventricles and trapped subdurally at the time of VP shunt insertion when EDV was probably not stopped enough long time before VP insertion.\n\nEDV produced some minor negative pressure. During attempts of inserting ventricular catheter of VP shunt produced sucking of the air into the ipsilateral ventricle and trapping it there. Similarly, during trephination of a burr hole for insertion of VP shunt and cutting the dura, some air was sucked subdurally on that side.\n\nAs slowly evolving postraumatic hydrocephalus is mainly of malabsorptive communicative type, lumbar drainage is less invasive attempt to reveal ex vacuo hydrocephalus than EDV. EDV or LD is stopped cca. 4-6h before inserting VP catheter to reexpand the ventricles and ease targeting.",
"responses": [
{
"c_id": "1617",
"date": "24 Sep 2015",
"name": "Sunil Munakomi",
"role": "Author Response",
"response": "Respected sir,Thank you for your report.We place the EVD in our ICU in suspected cases of hydrocephalus and then plan for the shunting if there is egress of CSF under pressure and neurological improvement in the patient. This case showed neurological improvement after EVD placement. The repeat CT after the shunt did not reveal pneumocephalus. We routinely do clamp the EVD 6 hours prior to the procedure to facilitate the tap.As you have correctly mentioned ,the pneumocephalus might have resulted either during the trephination or during corticol incision prior to ventricular tap.Sorry to have missed these points in our paper."
}
]
}
] | 1
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https://f1000research.com/articles/4-188
|
https://f1000research.com/articles/4-940/v1
|
01 Oct 15
|
{
"type": "Review",
"title": "Dynamic reorganization of the actin cytoskeleton",
"authors": [
"Gaëlle Letort",
"Hajer Ennomani",
"Laurène Gressin",
"Manuel Théry",
"Laurent Blanchoin",
"Gaëlle Letort",
"Hajer Ennomani",
"Laurène Gressin",
"Manuel Théry"
],
"abstract": "Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations.",
"keywords": [
"Actin",
"Cytoskeleton",
"Polarization"
],
"content": "Introduction\n\nCellular actin assembly can generate a variety of architectures. These highly dynamic actin-based structures lie at the heart of a diverse array of cellular processes1,2. Actin filaments are found inside cells in three basic patterns: branched filament networks, parallel-, or mixed-polarity filament bundle arrays. These different types of organization can contribute to more complex structures and determine their functions3. Although most of the time many actin structures are localized to different parts of the cell, they are rarely independent and their dynamics often influence each other. In this review, we will discuss the dynamic reorganization of actin inside the cell and explore the crosstalk between different architectures.\n\n\nActin structures in the cell: formation, architecture, and functions\n\nThe cell cytoplasm provides a large reservoir of actin monomers, and this reservoir is necessary for the assembly of complex actin-based structures4. The initial step in building such a large structural array containing different types of actin filament arrangements (Figure 1) requires controlled actin assembly and the inhibition of spontaneous polymerization4. Two actin-binding proteins play a major role in regulating this process: thymosin and profilin5. Thymosins sequester actin monomers to which they bind and thus fully block filament assembly6. Profilins also bind to actin monomers but only inhibit spontaneous nucleation7. Indeed the profilin/actin complex can add on to any pre-existing free filament barbed end and therefore participate in actin elongation8.\n\nSchematic representation of the three main actin structures found in the cell: 1. Lamellipodium: dense, branched network involved in cell protrusion. 2. Filopodium: a finger-like structure located at the leading edge of the motile cell composed of aligned filaments. Filopodia sense the extracellular environment and influence the direction of cell motility. 3. Contractile structure: dynamic structure made of antiparallel and/or mixed-polarity actin filaments associated with myosin. These structures play an important role in mechanical responses, providing force generation for different cellular functions. Zoomed regions highlight the specific actin organization of the different cellular actin structures.\n\nSeveral types of proteins, classified as actin nucleators, can counteract the inhibitory effects on actin assembly by thymosin, profilin, or other monomer binding proteins9,10. These actin nucleators include the actin-related protein 2/3 (Arp2/3) complex, formins, and proteins containing WASP homology 2 (WH2) domains. However, the Arp2/3 complex11,12 is the only definitive actin nucleator, in the sense that it can overcome the limiting step in the formation of an efficient actin nucleus during assembly. Indeed, this complex contains two Arps, Arp2 and Arp3, that mimic an actin dimer11. Other actin nucleators, including formins13,14 or WH2-domain containing proteins15, appear to stabilize pre-existing dimers rather than generating or mimicking new ones16,17. Interestingly, profilin in yeast and mammalian cells can inhibit Arp2/3 complex nucleation activity, thus favoring the actin filament elongation activity of formin or Ena/VASP18,19.\n\nThe lamellipodium is a dense branched array of filaments that occurs at the leading edge of a motile cell and its formation is dictated by the activity of the Arp2/3 complex (Figure 1 and 20). This specific type of actin organization pushes forward the plasma membrane during motility1,21,22. This property relates to the lamellipodium’s optimal composition of arrays of growing actin filaments, which are oriented at ±35° with respect to the membrane. Once growing actin filaments extend beyond ~1 µm, they form parallel filament bundles and emerge as finger-like protrusions called filopodia23,24.\n\nFilopodia direct how the cell probes the extracellular matrix (ECM) (Figure 1 and 25) and control the orientation of lamellipodium26. The parallel filament bundles within the filopodium also serve as tracks for protein transport27. Filopodia are ~1–10 µm long28,29, with 10–30 actin filaments crosslinked in parallel arrays by fascin30. Structural models predict that the densely packed nature of these actin arrays is important for the filaments to resist the loads coming from the membrane, such that filament elongation (by insertion of monomeric actin at the growing tip) remains uninhibited29,31. Moreover, the filaments within the filopodium have a turnover rate of ~20 mins32 and hence are far more stable than those filaments within the lamellipodium, which have a turnover rate of ~1 min33, or even only a few seconds at the very front of the lamellipodium34.\n\nThe cell can also contain actin structures assembled from short filaments that are the sites for the action of molecular motors of the myosin family. Depending on their orientation, the short filaments can act as tracks for myosin or as contractile fibers, such as the transverse arcs or ventral stress fibers35, and the perinuclear actin cap (Figure 1 and 36). Radial and ventral stress fibers, oriented parallel to the migration axis37, are anchored at focal adhesions at one (radial) or both (ventral) ends35. Transverse arcs are formed just behind the lamellipodium35,38. Ventral stress fibers are made of filaments of >2 µm in length, whereas transverse fibers are made of shorter filaments of ~1 µm in length. These fibers contain on average 10–30 filaments by width section39. Filament polarities inside stress fibers can be random (i.e. mixed polarity), graded, or sarcomeric (i.e. anti-parallel)39,40. Contractility is triggered by myosins that mediate sliding of anti-parallel filaments along each other41. The equilibrium between contractile stress and adhesion strength can act as a modulator of cellular tension42 and of the conversion of mechanical signals (tension) into biochemical signals (focal adhesion maturation), thus regulating the communication between the cell and the ECM36,43. Indeed, the assembly of stress fibers may only occur once the cell is under mechanical stress36. Ventral fibers allow the retraction of the motile cell’s trailing edge39 and may also initiate cell motility44. By connecting the lamellipodium and the lamella, the transverse arcs, in the flattened perinuclear region45, participate in the persistence of cell motility35,46. The perinuclear cap, a structure consisting of actomyosin fibers positioned around the nucleus, regulates the shape and position of the nucleus47.\n\nThe actin structures described above are highly dynamic in terms of formation, elongation/contraction, and disassembly, and these processes can be interdependent (Figure 2). Therefore, to have a more complete understanding of cellular actin organization, it is essential to take into account the cytoskeleton dynamics inside the cell.\n\n(A) In the convergent elongation model, the transition from lamellipodium to filopodium involves the formation of parallel actin filaments from a branched network created by the Arp2/3 complex. Elongation factors, like Ena/VASP or formin, protect barbed ends from capping protein and induce the rapid polymerization of parallel bundles. (B) The transition from lamellipodium to contractile structures is triggered by the disassembly of the branched network at the rear of the lamellipodium by ADF/cofilin and myosin. Myosin induces actin filament alignment and the formation of fibers stabilized by crosslinkers, such as α-actinin. (C) The fusion of contractile structures (the transverse arcs) and non-contractile structures (the radial fibers) can lead to the formation of ventral stress fibers. In this scheme, myosins connect a transverse arc and two radial fibers and, after contraction, align the radial fibers with the transverse arc, creating a ventral stress fiber. This contractile antiparallel fiber is anchored at its two ends to focal adhesions.\n\n\nFrom one actin structure to another: dynamical transitions\n\nThe potential for filopodia to emerge from the lamellipodium near the plasma membrane (Figure 2A) raised the question of how a structure made of parallel actin bundles can arise from a densely branched actin network. Two overlapping theoretical models have attempted to explain this transition: the convergent elongation model and the nucleation model23,48,49.\n\nAccording to the convergent elongation model, filopodia are initiated by the reorganization of the branched actin network due to a fine-tuning of actin filament elongation at their growing ends25. The branched actin filaments of the lamellipodium are short due to the regulation of their growth by capping proteins50,51. An attractive hypothesis to explain the transition between short filaments in the lamellipodium and the longer filaments driving filopodium formation is that some of the barbed actin filament ends in the lamellipodium are protected from capping proteins by cellular elongation factors such as Ena/VASP proteins52,53 or formins54 and will therefore grow longer. In support of this hypothesis, Ena/VASP and formin proteins have been observed at filopodia tips30,55 and can induce filopodia formation56,57.\n\nMoreover, depletion of capping protein promotes filopodia formation at the expense of lamellipodium extension, and Ena/VASP proteins have been shown to play an important role in filopodia formation58. Indeed, Ena/VASP proteins promote the convergence of filament barbed ends and have an enhanced activity when bound to trailing barbed ends in a fascin bundle, thus allowing the trailing ends to catch up with the leading barbed ends59. Longer actin filaments can, after positional fluctuations and bending, be captured and aligned into bundles by fascin (Figure 2), depending on the angle of their association60,61. These initial thin bundles can be further reinforced by other actin filaments to form a rigid body that is necessary for filopodium growth29. Convergence of actin network filaments into filopodia-like bundles can be recapitulated by both in vitro reconstitution23,62 and Monte Carlo simulation63.\n\nThe nucleation model is supported by the observation that filopodia can form even when the lamellipodium is absent as a consequence of lack of the Arp2/3 complex or its activation64–67. In this model, formin and/or Ena/VASP promoting de novo tip nucleation form actin filaments in filopodium. Further support for this model comes from the recent observation that fibroblasts lacking Arp2/3 complex produce more prominent filopodia than wild-type cells68. However, it is not yet clear how precisely filaments are initiated in the absence of the Arp2/3 complex. In a very elegant study using fission yeast, inhibition of the Arp2/3 complex disturbed the balance of different actin structures that were, in effect, competing for actin monomers from the same reservoir and resulted in the enhanced formation of formin-dependent structures69. The abundance of filopodia when the Arp2/3 complex is knocked down might also be explained by the disruption of actin homeostasis causing an increased incidence of spontaneous assembly of actin filaments in the cytoplasm68. A proportion of these spontaneously formed actin filaments may be capped by Ena/VASP and/or formins, whose activities are enhanced by the absence of the Arp2/3 complex, to promote filopodia formation49. Together, the two models are not necessarily mutually exclusive and might be reconciled by a capture elongation model mediated by Ena/VASP or formins.\n\nTo interrogate and illustrate the dynamic transition from lamellipodium to filopodia, we performed mathematical simulations using the cytoskeleton simulation software Cytosim70. In the simulation, a lamellipodium-like branched network was grown by distributing the Arp2/3 complex-like nucleators within a broad, two-dimensional area (Figure 3A, top and 61). To create the variation of lengths among those actin filaments, formin-like (could also be Ena/VASP-like) entities were added to capture the barbed ends of growing actin filaments and accelerate filament elongation. The growing actin filaments then extended out of the lamellipodium network and merged into bundled filaments by fascin-like crosslinkers. In the simulation, a synergy between the modulation of actin filament elongation at growing barbed ends by Ena/VASP and/or formin and actin filament crosslinking into tight bundles is sufficient (Figure 3A, top) and necessary to induce filopodium formation (Figure 3A, bottom panel).\n\n(A) Emergence of filopodia-like protrusions from a lamellipodium-like network. Simulations were performed using the Cytosim software. In the top panel, the actin network grows by branched nucleation via the Arp2/3 complex, and a proportion of actin filaments grow longer due to capture of their growing barbed ends by an elongation factor (formin/VASP, green filaments). Actin filaments contact each other by chance due to thermal fluctuations and are stabilized in bundles by crosslinkers (fascin). In the bottom panel, the presence of elongation factors in the simulation is essential for the emergence of protrusions (left), while the crosslinkers are necessary to group the protrusions into one rigid bundle (right). (B) Transition between lamellipodium-like and stress fiber-like networks. Simulations were performed using the Cytosim software. In the top panel, a branched network is formed and moved towards friction points (mimicking focal adhesions nucleating dorsal fibers) associated with parallel filaments. In the contact zone, the action of crosslinkers and myosins induces the disassembly of the branched network leading to the formation of a contractile structure of anti-parallel filaments. This structure is further compacted by a slow vertical flow (~centripetal actin flow) until it co-aligns with the friction points to form one contractile fiber. In the bottom panel, in the absence of motors, the network has no tension and is thus highly curved and spread (left). The crosslinkers are essential to maintain the connectivity between the filaments and form a continuous actin structure (middle). The friction points are essential to keep the network elongated at a given length, otherwise the network collapses to one point in the middle due to the tension (right). ti and tf indicate initial time and final time of simulations (empirical).\n\nAlthough the principle of the transition from lamellipodium to filopodia may be simple, it is quite difficult to identify exactly which proteins or pathways are involved in the formation of a filopodium. There exists potential competition or redundancy between different cellular actors, illustrated by formins that constitute a large family of different isoforms71. Moreover, the interactions between filaments and the membrane could also regulate this transition. The tension produced by the membrane can determine filopodia dimensions29 and can induce filament alignment in protrusions, even in the absence of crosslinkers72.\n\nA considerable amount of information about the assembly mechanisms of contractile structures has been obtained from numerous studies using live cell imaging36,38,46,73,74. The current model for the assembly of radial fibers is based on a simple mechanism of initiation, whereby the fibers are generated by formin-mediated nucleation at focal adhesions75. Following this initiation step, the growing actin filaments are brushed into parallel bundles by the retrograde flow toward the cell center38. Radial fibers further recruit crosslinked filaments from the lamella, giving rise to an organization of filaments with graded polarity36.\n\nThe model for the formation of transverse arcs is clearly different to that of radial fibers38. Transverse arcs are assembled by the end-to-end annealing of myosin filaments and actin bundles that have come from the reorganization of the Arp2/3 complex branched network at the back of the lamellipodium (Figure 2 and 46). The reorganization of a branched network into actin bundles of mixed polarity includes several steps. First, disassembly factors such as ADF/cofilin and glia maturation factor (GMF) disconnect the network by debranching the Arp2/3 complex links76–78. Second, the released short filaments are captured by myosin and actin filament crosslinkers such as α-actinin, which are present in the lamella to trigger the formation of small bundles (Figure 2B). Third, the alignment of the filaments is enforced by the high mechanical stress produced by the interaction between focal adhesions and the ECM, and by centripetal flow at the lamellipodium/lamella interface79. Fourth, the nascent bundles are pushed away from the cell edge by the actin centripetal flow74, while condensing and forming transverse arcs, until they encounter focal adhesions and pre-formed radial fibers38,80. The radial fibers and transverse arcs will then associate with crosslinked actin bundles incorporating into the ends of radial fibers through the activity of myosin filaments.\n\nWe have also performed simulations of the transition from a branched actin network to a contractile fiber using Cytosim and the same simulation starting point using Arp2/3 complex-like nucleators as described above (Figure 3A). To mimic focal adhesions nucleating the radial fibers, two small zones of adherence (friction points) with a few parallel filaments growing toward the cell center were placed at the bottom of a growing branched network. Arp2/3 complex connections were removed to simulate the lamellipodium debranching effect mediated by ADF/cofilin or GMF, and then motors (to simulate myosins) and crosslinkers (to simulate α-actinin) were added. A slow, directed flow was added to simulate the effect of centripetal actin flow. With only these few ingredients, the transition from a branched, non-contractile network to a mixed polarity, contractile fiber emerged from numerical simulations (Figure 3B, top panel). These ingredients all seem essential, since removal of the motors, crosslinkers, or friction points all prevented the efficient formation of the contractile cable (Figure 3B, bottom panel).\n\n\nOther transitions\n\nRadial fibers can associate with transverse arcs by the incorporation of myosin II filaments and subsequently develop into ventral fibers37,38. During this process, first, two independent radial fibers connect with a pre-existing transverse arc that is pushed to the trailing edge of the motile cell by the flow. As a consequence, arc contractile forces get transmitted to radial fibers. Second, the distal parts of the transverse arc dissociate because of local stress relaxation (Figure 2C). Finally, the radial fibers fuse with what remains of the contracting transverse arc to form a ventral stress fiber that is attached to focal adhesions at both ends38.\n\nIn addition, a ventral stress fiber could be formed by the fusion of two dorsal stress fibers, without transverse arc incorporation80. This latter case has been observed in Arp2/3 complex knockdown cells38.\n\nThe mechanisms by which filopodia disassemble remain to be determined. However, stationary filopodia can be disassembled into small bundles by ADF/cofilin81. Filopodia may also develop kinks after a decrease and/or change of direction of the actin flow between the lamellipodium and lamella leading to their integration into the lamella73,74,82. In both scenarios, short actin bundles generated by filopodia disassembly would then participate in the formation of contractile structures, by feeding pre-existing contractile fibers with actin filaments.\n\n\nConclusion\n\nCellular functions depend on complex actin choreography. To orchestrate such a diversity of actin organizations, the dynamic integration of different mechanistic pathways is necessary. Some pathways are quite specific to the formation and maintenance of a particular basic actin organization, but because these actin structures may reorganize and transform, they may also indirectly participate in the emergence of other structures. The prevalence of these different basic actin organizations also varies in different cell types (e.g. lamellipodia predominate over filopodia in keratocytes and neutrophils, whereas filopodia predominate over lamellipodia in dendritic cells or neuronal growth cones). Within a given cell type, the predominance and/or existence of the different actin structures can be regulated to achieve specific functions, for example during collective migration83. Thus, focusing on the behavior of a single type of actin structure may only provide an incomplete view of its formation and maintenance in vivo. Hence, the development of more appropriate experimental systems that can reconstitute more than one actin structure at a time should improve the understanding of the complexity of cellular actin dynamics. Mathematical simulations demonstrate that only a few components and simple boundary conditions are sufficient to mediate transitions between or during the emergence of complex actin structures. These mathematical approaches may also help in elaborating more appropriate experimental systems to unveil the general laws behind dynamic cytoskeletal reorganization.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no disclosures.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe thank F. Nedelec for providing an unpublished code used to run the simulations with Cytosim. LB is supported by Agence Nationale de la Recherche (ANR) grant N° ANR-12-BSV5–0014 (Contract). HE and LG are supported by IRTELIS grant from CEA. Illustrations were made by A. Kawska at IlluScientia.com.\n\n\nSupplemental movie legends\n\nMovie S1 (related to Figure 3A)\n\nTransition of lamellipodium into filopodia: assembly of parallel bundles (filopodia) triggered by the dense branched network (lamellipodium).\n\nClick here for file.\n\nMovie S2 (related to Figure 3B)\n\nTransition of lamellipodium into contractile structures. Formation of a contractile structure generated by the combined action of 3 parameters: actin flow, molecular motors and crosslinkers.\n\nClick here for file.\n\n\nReferences\n\nPollard TD, Borisy GG: Cellular motility driven by assembly and disassembly of actin filaments. Cell. 2003; 112(4): 453–65. PubMed Abstract | Publisher Full Text\n\nBlanchoin L, Boujemaa-Paterski R, Sykes C, et al.: Actin dynamics, architecture, and mechanics in cell motility. Physiol Rev. 2014; 94(1): 235–63. PubMed Abstract | Publisher Full Text\n\nFletcher DA, Mullins RD: Cell mechanics and the cytoskeleton. Nature. 2010; 463(7280): 485–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPollard TD, Blanchoin L, Mullins RD: Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct. 2000; 29: 545–76. 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Annu Rev Cell Biol. 1987; 3: 379–421. PubMed Abstract | Publisher Full Text\n\nGoffin JM, Pittet P, Csucs G, et al.: Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers. J Cell Biol. 2006; 172(2): 259–68. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nShemesh T, Bershadsky AD, Kozlov MM: Physical model for self-organization of actin cytoskeleton and adhesion complexes at the cell front. Biophys J. 2012; 102(8): 1746–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCramer LP: Forming the cell rear first: breaking cell symmetry to trigger directed cell migration. Nat Cell Biol. 2010; 12(7): 628–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nIngram VM: A side view of moving fibroblasts. Nature. 1969; 222(5194): 641–4. PubMed Abstract | Publisher Full Text\n\nBurnette DT, Manley S, Sengupta P, et al.: A role for actin arcs in the leading-edge advance of migrating cells. Nat Cell Biol. 2011; 13(4): 371–81. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNagayama K, Yahiro Y, Matsumoto T: Stress fibers stabilize the position of intranuclear DNA through mechanical connection with the nucleus in vascular smooth muscle cells. FEBS Lett. 2011; 585(24): 3992–7. PubMed Abstract | Publisher Full Text\n\nFaix J, Rottner K: The making of filopodia. Curr Opin Cell Biol. 2006; 18(1): 18–25. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nYang C, Svitkina T: Filopodia initiation: focus on the Arp2/3 complex and formins. Cell Adh Migr. 2011; 5(5): 402–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nEdwards M, Zwolak A, Schafer DA, et al.: Capping protein regulators fine-tune actin assembly dynamics. Nat Rev Mol Cell Biol. 2014; 15(10): 677–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWear MA, Cooper JA: Capping protein: new insights into mechanism and regulation. Trends Biochem Sci. 2004; 29(8): 418–28. PubMed Abstract | Publisher Full Text\n\nKrause M, Bear JE, Loureiro JJ, et al.: The Ena/VASP enigma. J Cell Sci. 2002; 115(Pt 24): 4721–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBreitsprecher D, Kiesewetter AK, Linkner J, et al.: Molecular mechanism of Ena/VASP-mediated actin-filament elongation. EMBO J. 2011; 30(3): 456–67. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nZigmond SH, Evangelista M, Boone C, et al.: Formin leaky cap allows elongation in the presence of tight capping proteins. Curr Biol. 2003; 13(20): 1820–3. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSchirenbeck A, Bretschneider T, Arasada R, et al.: The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia. Nat Cell Biol. 2005; 7(6): 619–25. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPellegrin S, Mellor H: The Rho family GTPase Rif induces filopodia through mDia2. Curr Biol. 2005; 15(2): 129–33. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBlock J, Stradal TE, Hänisch J, et al.: Filopodia formation induced by active mDia2/Drf3. J Microsc. 2008; 231(3): 506–17. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMejillano MR, Kojima S, Applewhite DA, et al.: Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end. Cell. 2004; 118(3): 363–73. 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Publisher Full Text\n\nFaix J, Grosse R: Staying in shape with formins. Dev Cell. 2006; 10(6): 693–706. PubMed Abstract | Publisher Full Text\n\nLiu AP, Richmond DL, Maibaum L, et al.: Membrane-induced bundling of actin filaments. Nat Phys. 2008; 4: 789–93. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNemethova M, Auinger S, Small JV: Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella. J Cell Biol. 2008; 180(6): 1233–44. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAnderson TW, Vaughan AN, Cramer LP: Retrograde flow and myosin II activity within the leading cell edge deliver F-actin to the lamella to seed the formation of graded polarity actomyosin II filament bundles in migrating fibroblasts. Mol Biol Cell. 2008; 19(11): 5006–18. 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}
|
[
{
"id": "10647",
"date": "01 Oct 2015",
"name": "Klemens Rottner",
"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",
"responses": []
},
{
"id": "10648",
"date": "01 Oct 2015",
"name": "David Kovar",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-940
|
https://f1000research.com/articles/4-939/v1
|
01 Oct 15
|
{
"type": "Review",
"title": "Anti-dsDNA Antibodies are one of the many autoantibodies in systemic lupus erythematosus",
"authors": [
"Shu Man Fu",
"Chao Dai",
"Zhenhuan Zhao",
"Felicia Gaskin",
"Chao Dai",
"Zhenhuan Zhao",
"Felicia Gaskin"
],
"abstract": "Anti-dsDNA antibodies are the most studied antibodies of the lupus-related autoantibodies. The dogma is that these are the most important autoantibodies in systemic lupus erythematosus. In this review, evidence is presented to show that these antibodies (as measured by modern clinical laboratories) are not the most important autoantibodies in the diagnosis of systemic lupus erythematosus, and are of limited value in clinical correlation and in predicting disease flares. In addition, they are not likely to be the initiating autoantibodies in lupus nephritis. Thus, several pervasively held beliefs on anti-dsDNA antibodies are not valid. We suggest that anti-dsDNA antibodies should be considered as just one of the many autoantibodies associated with systemic lupus erythematosus.",
"keywords": [
"Systemic lupus erythematosus",
"anti-dsDNA antibodies",
"autoantibodies",
"lupus nephritis"
],
"content": "Introduction\n\nSystemic lupus erythematosus (SLE) is a syndrome affecting multiple organs with circulating autoantibodies of complex specificities1. Of the SLE-related autoantibodies, anti-dsDNA antibodies have received the most intense investigation. These antibodies have been cited to be specific for SLE and are the antibodies that initiate lupus glomerulonephritis. They are often thought to be of value in correlating with disease activity and predicting flares in SLE. The clinical significance of anti-dsDNA antibodies has been reviewed at the 50th anniversary for the description of anti-dsDNA antibodies in SLE2. In view of several recent publications on this topic, this review intends to provide evidence that anti-dsDNA antibodies should be treated as just one of the SLE-related autoantibodies with limited diagnostic and prognostic values in SLE.\n\n\nHistoric perspective\n\nIn order to understand the origin of the myths associated with anti-dsDNA antibodies, it is important to review briefly the history of anti-dsDNA antibodies in their association with SLE. Fifty-eight years ago, Holman and Kunkel3 reported in the July 29, 1957 issue of Science that deoxyribonuclease destroys the antigenic determinant in nucleoprotein that participates in the LE (lupus erythematosus) cell phenomenon, implicating anti-DNA antibodies in the sera of SLE patients. Subsequent to this publication, additional reports appeared to establish that circulating anti-DNA antibodies were present in patients with SLE4–7. Interest in anti-dsDNA antibodies increased after the publication of the paper by Koffler et al.8 that described the elution of anti-dsDNA antibodies from the kidneys of SLE patients with nephritis. Lupus nephritis was considered to be a prototype of immune complex nephritis in man9. Although it was emphasized that other antibody-antigens are likely to be present in lupus nephritis, it was stressed that the anti-dsDNA antibodies-dsDNA system is of paramount importance10.\n\nThe importance of anti-dsDNA antibodies in the clinical care of SLE patients has been emphasized in the past. In a remarkable paper by Tan et al.11, anti-DNA antibodies were detected in certain lupus patients prior to the onset of severe proteinuria. These antibodies were not detectable during the acute phase of lupus nephritis. Instead, circulating DNA was detected, suggesting antibody excess. Anti-DNA antibodies were measured in this study by immunodiffusion. The immunodiffusion technique measures precipitating antibodies that may have a higher binding affinity to dsDNA. Some aspects of this study were confirmed by the investigation of Hughes et al.12. Hughes et al. showed that precipitating antibodies rapidly disappeared with therapy and clinical improvement, and patients with the most severe renal disease with complement consumption and renal impairment gave the strongest precipitin lines. Since the immunodiffusion method for the detection of anti-DNA antibodies is no longer used in clinical laboratories, it should be emphasized that immunodiffusion for the analysis of anti-DNA antibodies identifies patients with anti-dsDNA antibodies of higher affinity and concentration. It is also of historical interest to note that Hughes et al.12 showed that the Farr technique is more sensitive and less specific, requiring the setting of a binding level (in their case, 20%) to make the assay specific for SLE.\n\nSince these early publications, there have been many published papers to support the importance of anti-dsDNA antibodies as a biomarker in the diagnosis, pathogenesis, and prognosis of SLE (reviewed in 2, 13, and 14).\n\n\nAnti-dsDNA antibodies are not specific or the best biomarker for SLE\n\nAnti-dsDNA antibodies have been included in the 1982 American College of Rheumatology (ACR) revised criteria for the classification of SLE and in the 1997 update of the criteria for the classification of SLE15,16. The 10th criterion includes “antibody to native DNA in abnormal titer”. Edworthy et al.17 used the Stanford Lupus Cohort (n=339) and matched controls to validate the 1982 revised ACR criteria for the classification of SLE. Because of the referral pattern, it is not surprising that anti-dsDNA antibodies were found to be the best discriminator. In a more detailed analysis of the literature, Kavanaugh et al.18 provided a more comprehensive guideline regarding the use of anti-DNA antibody tests in rheumatic disease. The ACR Ad Hoc Committee on Immunological Testing Guidelines chaired by Dr. A. F. Kavanaugh did a thorough evaluation of the literature. It came to the conclusion that the three commonly employed methods (i.e. ELISA, the Farr assay, and immunofluorescence using Crithidia luciliae as a substrate) correlated with each other when they were applied to populations of patients, but there were substantial discrepancies when these techniques were applied to individual patients. This conclusion may not be surprising, since each of these methods may detect different populations of anti-DNA antibodies due to affinities of the targeted antibodies for dsDNA and substrate differences. The Committee recommendation was that anti-DNA antibodies are useful in supporting the diagnosis of SLE in the setting of clinical presentation highly suggestive of the diagnosis. Although anti-DNA antibodies are rarely described in other rheumatologic conditions, a positive anti-DNA is not diagnostic of SLE but a negative test does not rule out the diagnosis. Regarding the correlation of anti-DNA antibodies with clinical activities, the Committee found that the correlation is modest at best. A similar conclusion was drawn regarding the correlation of anti-DNA antibodies with renal disease. It was also concluded that the presence of anti-DNA antibodies does not predict a flare of the disease. The Committee withheld its judgement regarding the usefulness of an increase in anti-dsDNA antibodies that may pre-date or may be associated with flare of disease activity because of lack of studies on this issue. These guidelines remain valid and useful more than a decade later.\n\nDespite the ample amount of publications on anti-DNA antibodies in SLE, investigations on anti-DNA antibodies as related to SLE have continued. On the technical issue regarding different assays, a recent paper by Encosson et al.19 compared IgG anti-dsDNA bead-base multiplex assay (FIDIS; Theradiag), fluoroenzyme-immunoassay (EliA; Phadia/Thermo Fisher Scientific), Crithidia luciliae immunofluorescence test (CLIFT; ImmunoConcepts) and line blot (EUROLINE; EUROIMMUN) on 187 patients with SLE, with patients with rheumatoid arthritis (RA) and progressive systemic sclerosis (pSS) as disease controls and healthy controls. It was shown that rare patients with RA and pSS were positive by the Crithidia immunofluorescence tests. The specificity of CLIFT in the authors’ laboratory was cited to be 98%. The other three assays were cited to have lower specificities. By adjusting the base line the other three tests achieved similar specificity to the CLIFT assay. In this population of SLE patients, the sensitivity of all four assays was in the low 20s. The stated conclusion was that “there is a great variability among anti-dsDNA assays and a stricter cut-off limit must be applied to acceptable SLE specificities of FIDIS, ELiA and EUROLINE”. These results were in accordance with those outlined in 18. They also suggest that the criterion for the inclusion of anti-dsDNA antibodies for the ACR classification of SLE should be modified similarly to the modification in the SLICC-12 (2012 Systemic Lupus International Collaborating Clinic Classification) criteria for SLE20. The modification is that anti-dsDNA antibody levels should be above laboratory reference range (or twice the reference range if tested by ELISA).\n\nThe variability of different anti-dsDNA assays was highlighted in an article by Compagno et al.21 published in the inaugural issue of Lupus Science & Medicine. The article described the clinical phenotype associated with various types of anti-dsDNA antibodies in patients with recent onset of rheumatic symptoms. 1073 patients were recruited from three academic centers in the three Scandinavian countries. 292 patients were found to be antinuclear antibody (ANA) positive. 292 patients were randomly selected from patients with negative ANA. These sera were assayed for anti-dsDNA antibodies by CLIFT at least three times by two commercial kits and four times by three solid phase ELISA kits. 37 patients dropped out. Of the 288 ANA-positive patients, 19.8% (n=57) carried the diagnosis of SLE. In contrast, only 2.3% (n=6) of the ANA-negative patients (n=259) were identified to have SLE. In the 288 ANA-positive sera, 39 (13.5%) sera were positive in any CLIFT and 50 (17.4%) were positive in any ELISA. Of the 259 ANA-negative sera, 20 (7.7%) were positive by any CLIFT and 49 (18.9%) were positive by any ELISA kits. There was low concordance between the CLIFT assays and ELISA assays with 25 CLIFT+ELISA-, 65 CLIFT-ELISA+, and 34 CLIFT+ELISA+. It was concluded that different anti-dsDNA antibodies are associated only modestly with nephropathy, pleuritis, alopecia, and lymphopenia.\n\nThe group of patients studied by Compangno et al.22 was followed for a median of 4.8 years. The follow-up results were astonishing. It was concluded that CLIFT is not reliable as a diagnostic tool in unselected patients with rheumatic symptoms. CLIFT had low positive predictive value for SLE, in that only one out of 36 CLIFT positive patients who were not diagnosed with SLE at entrance developed SLE. Thus, for non-SLE patients, being CLIFT positive poses little risk of developing SLE within 5 years.\n\nIn the review by Mehra and Fritzler14, it was concluded that the anti-chromatin/nucleosome antibodies may be superior to anti-dsDNA antibodies as a biomarker for SLE. In summary, this brief analysis of the literature supports the conclusion that anti-dsDNA antibodies are not specific or the best biomarker for SLE.\n\n\nA surge in anti-dsDNA antibody titer may not be a good predictor for either non-renal or renal flares in SLE\n\nThe data supporting the claim that rising titers of anti-dsDNA antibodies is a good predictor for flares in SLE have been reviewed in 2. Recently Pan et al.23 published their retrospective study on the lupus cohort at the Hospital of Special Surgery in an attempt to correlate surges in anti-dsDNA antibodies with renal and non-renal flares. It was concluded that an anti-dsDNA surge was not predictive of renal flare. Regarding non-renal flares as measured by the SELENA-SLEDAI instrument, the data showed that an anti-dsDNA surge had a sensitivity of 62%, specificity of 80%, positive predictive value of 59%, and negative predictive value of 81% for a severe SELENA-SLEDAI flare. Although the authors concluded that a surge in anti-dsDNA titer predicts a severe SELENA-SLEDAI lupus flare within 6 months, the predictive value of such a surge appears to be, at best, of modest accuracy and clinical applicability.\n\nRegarding the predictive value of a surge in anti-dsDNA antibody titer for a renal flare, this was investigated in 487 patients who had a history of lupus nephritis and an anti-dsDNA antibody titer ≥15 IU/ml at baseline (as measured by Farr assay) and who represented the treatment and placebo arms of patients being treated with a dsDNA-based bioconjugate, LJB394, in 2 clinical trial cohorts24. The results24 showed that “Changes in anti-dsDNA antibody levels were inversely correlated with changes in the C3 level (P<0.0001 in both trials). Cox proportional hazards regression models showed that changes in anti-dsDNA antibody levels correlated with the risk of renal flare. The models predicted that a point estimate of a 50% reduction in anti-dsDNA antibody levels is associated with a 52% reduction (95% confidence interval [CI] 26–68%, nominal P=0.0007) and a 53% reduction (95% CI 33–69%, nominal P<0.0001) in the risk of renal flare in the 2 trials, respectively. In the 2 trials, the incidence of renal flare was lower in patients with sustained reductions in anti-dsDNA antibodies (3.0% and 4.1%, respectively) than in patients with stable or increasing antibody levels (21.3% and 20.3%, respectively)”. The results (that only ~20% of the patients with an anti-dsDNA antibody surge had a renal flare and that 3–4% of the patients without such a surge developed a renal flare) suggest that an anti-dsDNA antibody titer surge is, at best, of modest predictive value. The changes of anti-dsDNA antibody titers cannot be used in clinical practice to treat patients prophylactically, or to assure patients without an increase in anti-dsDNA antibodies that a renal flare will not occur.\n\n\nAnti-dsDNA antibodies may not be the antibodies that initiate lupus nephritis\n\nBecause of the initial report that anti-dsDNA antibodies were eluted from kidneys in patients with lupus nephritis, it has been suggested that these antibodies may initiate lupus nephritis. In the 1971 paper that proposed SLE to be a prototype of immune complex nephritis in man9, Koffler et al. were able to demonstrate the concentration of antibodies to dsDNA in the kidney eluates from 5 out of 9 samples, suggesting that some of the cases studied may not have anti-dsDNA antibody deposits in the kidney. These authors also showed that other antigen-antibody complexes were deposited in the diseased kidneys. In a later paper10, Koffler et al. stated that “acid buffer eluates were prepared from biopsy and two kidneys from necropsy which showed no histological or clinical evidence of renal disease. Immunofluorescent study of these tissues revealed a linear deposit of γG-globulin. The eluates obtained did not contain demonstrable anti-nuclear or anti-basement membrane antibodies”. The study by Mannik et al.25 showed that antibodies of multiple specificities were eluted from the kidneys of patients who died of lupus nephritis. Many of the 25 renal eluates did not have anti-dsDNA antibodies. These findings suggest that anti-dsDNA antibodies may not be required for the pathogenesis of lupus nephritis. This hypothesis has been supported by our studies of the genetics of lupus nephritis in NZM2328 mice26,27. We found that a single locus on chromosome 4 controls the production of anti-dsDNA antibodies26. The congenic strain NZM2328.C57L/Jc4 (NZM.L/Jc4) was generated by introgressing a genetic segment of chromosome 4 from C57L/J, a non-lupus prone strain where the gene controlling anti-dsDNA antibody production is located to NZM232827. Female mice of NZM.L/Jc4 had little circulating ANA or anti-dsDNA antibodies. They developed immune complex-mediated nephritis with end-stage renal disease and early mortality in a manner similar to that of the parental strain NZM2328.\n\nRecently, Bruschi et al. have published two papers relevant to this issue28,29. They eluted antibodies from 20 renal biopsy samples from patients with lupus nephritis. They identified 12 targeted podocyte molecules. It appears that α-enolase and annexin A1 were the most commonly targeted antigens28. These patients have high titers of circulating antibodies to these two antigens and to dsDNA and C1q29. Six of the 20 renal eluates had antibodies to α-enolase without antibodies to dsDNA, and 4 of the 20 eluates had antibodies to annexin A1 without anti-dsDNA antibodies. In these patients, anti-dsDNA antibodies did not play a role in the initiation of lupus nephritis.\n\n\nConcluding remarks\n\nThis review provides evidence that anti-dsDNA antibodies have a limited value in the diagnosis of SLE. These antibodies are useful in confirming the diagnosis in the clinical settings when SLE is likely to be the diagnosis. They have limited usefulness in monitoring disease activities and in predicting flares. In contrast to the current dogma that these antibodies may initiate lupus nephritis, they are not necessary or sufficient to cause lupus nephritis. It is likely that these antibodies play an amplification role in the pathogenesis of lupus nephritis, in that they interact with Toll-like receptors (TLRs) with subsequent release of type 1 interferons that amplify the autoantibody response. They may react with DNA released from podocytes undergoing apoptosis and implanted in the glomerular basement membrane, causing further renal damage via complement fixation and/or interaction with Fc receptors. These antibodies should be considered to be one of the many autoantibodies seen in SLE patients.\n\nDespite their limited value as biomarkers, anti-dsDNA antibodies are the most studied autoantibodies, and significant information has been generated regarding autoantibody formation and B cell development from studying these antibodies13,30. Undoubtedly further studies of these antibodies will yield significant information on B cell biology. They would also be useful in studying the interaction of autoantibodies with their targeted organs31. Evidence has been accumulated to show that autoimmune diseases are the results of interactions of autoantibodies and autoreactive T cells with targeted organs. The targeted organs play an active role in this process. Autoimmunity and end organ damage are under separate genetic control32. The model proposed by us as shown in Figure 1, for the pathogenesis of autoimmune diseases in general and SLE in particular, will be useful in placing the role of autoantibodies and autoreactive T cells in proper perspective. It will also explain the puzzling clinical observations that some SLE patients are serologically active and clinically quiescent while others are clinically active and serologically quiescent33,34.\n\nThis model makes the assumption that environmental triggers act on susceptible hosts. The triggers act on both genes controlling immune responsiveness and genes for end organ damage. These are two independent yet interactive pathways. Pathway I leads to the generation of autoantibodies and autoreactive effector T cells. Pathway II provides autoantigens and/or soluble mediators that influence immune responsiveness. Pathways I and II interact at several levels as indicated by III. These interactions can lead to end organ damage. In this context, the end organ is the kidney and the autoimmune response is the production of autoantibodies to multiple autoantigens that form immune complexes to be deposited in the kidney32.\n\nIn the review article by Isenberg et al. from 20072, the authors asked, “Fifty years of anti-dsDNA antibodies: are we approaching journey’s end?”. Perhaps we can answer this question by saying that the journey should end regarding their role in the diagnosis of SLE and in their correlation with clinical activity and flares. Just as with many other autoantibodies, the events leading to the production of anti-dsDNA antibodies remain to be elucidated.\n\n\nAbbreviations\n\nACR, American College of Rheumatology; ANA, antinuclear antibody; CLIFT, Crithidia luciliae immunofluorescence test; LE, lupus erythematosus; pSS, progressive systemic sclerosis; RA, rheumatoid arthritis; SELENA, Safety of Estrogen in Lupus: National Assessment; SLE, systemic lupus erythematosus; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index; SLICC-12, 2012 Systemic Lupus International Collaborating Clinic Classification.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work is supported in part by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01-AR047988 and R01-AR049449) and a grant (TIL332615) from the Alliance for Lupus Research, New York.\n\n\nReferences\n\nTsokos GC: Systemic lupus erythematosus. N Engl J Med. 2011; 365(22): 2110–21. PubMed Abstract | Publisher Full Text\n\nIsenberg DA, Manson JJ, Ehrenstein MR, et al.: Fifty years of anti-ds DNA antibodies: are we approaching journey's end? Rheumatology (Oxford). 2007; 46(7): 1052–6. PubMed Abstract | Publisher Full Text\n\nHolman HR, Kunkel HG: Affinity between the lupus erythematosus serum factor and cell nuclei and nucleoprotein. Science. 1957; 126(3265): 162–3. PubMed Abstract | Publisher Full Text\n\nRobbins WC, Holman HR, Deicher H, et al.: Complement fixation with cell nuclei and DNA in lupus erythematosus. Proc Soc Exp Biol Med. 1957; 96(3): 575–9. PubMed Abstract | Publisher Full Text\n\nCeppelini R, Polli E, Celada F: A DNA-reacting factor in serum of a patient with lupus erythematosus diffuses. Proc Soc Exp Biol Med. 1957; 96(3): 572–74. PubMed Abstract | Publisher Full Text\n\nMiescher P, Straessler R: New serological methods for the detection of the L.E. factor. Vox Sang. 1957; 2(4): 283–87. PubMed Abstract | Publisher Full Text\n\nSeligmann M: Evidence in the serum of patients with systemic lupus erythematosus of a substance producing a precipitation reaction with DNA. CR Soc Biol (Paris). 1957; 245: 328–33.\n\nKoffler D, Schur PH, Kunkel HG: Immunological studies concerning the nephritis of systemic lupus erythematosus. J Exp Med. 1967; 126(4): 607–24. PubMed Abstract | Free Full Text\n\nKoffler D, Agnello V, Thoburn R, et al.: Systemic lupus erythematosus: prototype of immune complex nephritis in man. J Exp Med. 1971; 134(3): 169–79. PubMed Abstract | Free Full Text\n\nKoffler D, Agnello V, Carr RI, et al.: Variable patterns of immunoglobulin and complement deposition in the kidneys of patients with systemic lupus erythematosus. Am J Pathol. 1969; 56(3): 305–16. PubMed Abstract | Free Full Text\n\nTan EM, Schur PH, Carr RI, et al.: Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J Clin Invest. 1966; 45(11): 1732–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHughes GR, Cohen SA, Christian CL: Anti-DNA activity in systemic lupus erythematosus. A diagnostic and therapeutic guide. Ann Rheum Dis. 1971; 30(3): 259–64. PubMed Abstract | Free Full Text\n\nPisetsky DS: The complex role of DNA histones and HMGB1 in the pathogenesis of SLE. Autoimmunity. 2014; 47(8): 487–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMehra S, Fritzler MJ: The spectrum of anti-chromatin/nucleosome autoantibodies: independent and interdependent biomarkers of disease. J Immunol Res. 2014; 2014: 368274. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTan EM, Cohen AS, Fries JF, et al.: The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982; 25(11): 1271–7. 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\n\nEdworthy SM, Zatarain E, McShane DJ, et al.: Analysis of the 1982 ARA lupus criteria data set by recursive partitioning methodology: new insights into the relative merit of individual criteria. J Rheumatol. 1988; 15(10): 1493–8. PubMed Abstract\n\nKavanaugh AF, Solomon DH; American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines: Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody tests. Arthritis Rheum. 2002; 47(5): 546–55. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nEnocsson H, Sjöwall C, Wirestam L, et al.: Four Anti-dsDNA Antibody Assays in Relation to Systemic Lupus Erythematosus Disease Specificity and Activity. J Rheumatol. 2015; 42(5): 817–25. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPetri M, Orbai AM, Alarcón GS, et al.: Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012; 64(8): 2677–86. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCompagno M, Rekvig OP, Bengtsson AA, et al.: Clinical phenotype associations with various types of anti-dsDNA antibodies in patients with recent onset of rheumatic symptoms. Results from a multicentre observational study. Lupus Sci Med. 2014; 1(1): e000007. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCompagno M, Jacobsen S, Rekvig OP, et al.: Low diagnostic and predictive value of anti-dsDNA antibodies in unselected patients with recent onset of rheumatic symptoms: results from a long-term follow-up Scandinavian multicentre study. Scand J Rheumatol. 2013; 42(4): 311–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPan N, Amigues I, Lyman S, et al.: A surge in anti-dsDNA titer predicts a severe lupus flare within six months. Lupus. 2014; 23(3): 293–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLinnik MD, Hu JZ, Heilbrunn KR, et al.: Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus. Arthritis Rheum. 2005; 52(4): 1129–37. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMannik M, Merrill CE, Stamps LD, et al.: Multiple autoantibodies form the glomerular immune deposits in patients with systemic lupus erythematosus. J Rheumatol. 2003; 30(7): 1495–504. PubMed Abstract | Faculty Opinions Recommendation\n\nWaters ST, Fu SM, Gaskin F, et al.: NZM2328: a new mouse model of systemic lupus erythematosus with unique genetic susceptibility loci. Clin Immunol. 2001; 100(3): 372–83. PubMed Abstract | Publisher Full Text\n\nWaters ST, McDuffie M, Bagavant H, et al.: Breaking tolerance to double stranded DNA, nucleosome, and other nuclear antigens is not required for the pathogenesis of lupus glomerulonephritis. J Exp Med. 2004; 199(2): 255–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBruschi M, Sinico RA, Moroni G, et al.: Glomerular autoimmune multicomponents of human lupus nephritis in vivo: α-enolase and annexin AI. J Am Soc Nephrol. 2014; 25(11): 2483–98. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBruschi M, Galetti M, Sinico RA, et al.: Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo (2): Planted Antigens. J Am Soc Nephrol. 2015; 26(8): 1905–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCohen-Solal J, Diamond B: Lessons from an anti-DNA autoantibody. Mol Immunol. 2011; 48(11): 1328–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYung S, Chan TM: Autoantibodies and resident renal cells in the pathogenesis of lupus nephritis: getting to know the unknown. Clin Dev Immunol. 2012; 2012: 139365. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDai C, Deng Y, Quinlan A, et al.: Genetics of systemic lupus erythematosus: immune responses and end organ resistance to damage. Curr Opin Immunol. 2014; 31: 87–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGladman DD, Urowitz MB, Keystone EC: Serologically active clinically quiescent systemic lupus erythematosus: a discordance between clinical and serologic features. Am J Med. 1979; 66(2): 210–5. PubMed Abstract | Publisher Full Text\n\nGladman DD, Hirani N, Ibañez D, et al.: Clinically active serologically quiescent systemic lupus erythematosus. J Rheumatol. 2003; 30(9): 1960–2. PubMed Abstract | Faculty Opinions Recommendation"
}
|
[
{
"id": "10645",
"date": "01 Oct 2015",
"name": "George C. Tsokos",
"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",
"responses": []
},
{
"id": "10646",
"date": "01 Oct 2015",
"name": "Allan Gibofsky",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-939
|
https://f1000research.com/articles/4-938/v1
|
01 Oct 15
|
{
"type": "Review",
"title": "Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine?",
"authors": [
"Malcolm J. McConville",
"Eleanor C. Saunders",
"Joachim Kloehn",
"Michael J. Dagley",
"Eleanor C. Saunders",
"Joachim Kloehn",
"Michael J. Dagley"
],
"abstract": "A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments. How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.",
"keywords": [
"central carbon metabolism",
"virulence",
"reductive stress",
"macrophages",
"Leishmania"
],
"content": "Introduction\n\nMacrophages play key roles in the mammalian innate and adaptive immune responses1. These cells are actively recruited to sites of tissue damage and infection and are able to kill a wide range of invading bacterial, fungal, and protozoan pathogens following phagocytosis and their delivery to the lysosome compartment1. Not surprisingly, a number of medically important microbial pathogens have developed strategies to either avoid phagocytosis by macrophages or to subvert uptake into the mature lysosome compartment. The latter group either prevent maturation of the phagosomes within which they are internalized or escape into the cytosol, or both (for example, Mycobacterium tuberculosis, Salmonella spp., Trypanosoma cruzi)2,3. Other pathogens invade macrophages via phagocytosis-independent mechanisms and reside within non-hydrolytic compartments in these cells (for example, Toxoplasma gondii)4. However, a small number of pathogens are internalized into the mature phagolysosome compartment of macrophages and are capable of long-term survival and proliferation within this compartment5–7. These include the protozoan parasites belonging to the genus Leishmania which in humans cause a spectrum of diseases ranging from localized cutaneous skin lesions to disseminating mucocutaneous infections and deadly visceral infections7. Strikingly, mammalian-infective stages of Leishmania lack many of the conventional virulence determinants of other pathogens, such as a thick cell wall, or cytoprotective pigments, suggesting that they may be more dependent on physiological changes. Although some progress has been made in identifying signaling pathways and other processes that are important for Leishmania virulence in the mammalian host6,8–11, major gaps in our understanding of Leishmania amastigote survival strategies remain. Here, we summarize recent studies that suggest that intracellular survival is linked to a marked decrease in parasite growth and a rewiring of central carbon metabolism. These changes may underlie the intrinsic resistance of these parasite stages to many stresses (temperature, pH) and their tolerance of both nutrient limitation and nutrient excess (feast and famine) in this intracellular niche.\n\n\nLiving in the macrophage phagolysosome\n\nLeishmania spp. develop as flagellated promastigotes in the lumen of their sandfly vectors and are transmitted to a range of human and animal hosts when the sandfly takes a bloodmeal. After injection into the skin, promastigotes are initially internalized by neutrophils before being phagocytosed by macrophages and delivered to the mature phagolysosome compartment where they differentiate to the small, round, aflagellate amastigote stage6. The further recruitment of macrophages to the site of infection results in the formation of lesions or granuloma-like structures that are the hallmark of all Leishmania infections11,12. Macrophages are the predominant cell type within lesions and can be infected with a few to several hundred amastigotes that, depending on the species involved, reside either within individual tight-fitting vacuoles (one parasite per vacuole) or within large spacious communal vacuoles. These vacuoles have a low pH (~5.4) and contain all of the membrane and luminal markers of a mature phagolysosome, including the characteristic suite of hydrolases and the membrane NADH oxidase that generates anti-microbial oxidative burst. The Leishmania-occupied phagolysosome compartment appears to be highly dynamic, receiving a wide range of host macromolecules via fusion with vesicles from the phagocytic, endocytic, and autophagic pathways as well as the endoplasmic reticulum (Figure 1)6,13. These macromolecules are degraded by luminal hydrolases (proteases, lipases, glycosidases) to generate free sugars, lipids, and peptides/amino acids which can be taken up by amastigote plasma membrane transporters. Amastigotes can also internalize host macromolecules directly and degrade many of them within their own lysosome. Thus, the phagolysosome compartment may contain a wide array of carbon sources and essential nutrients, in contrast to other compartments in the endo-lysosomal network14. Consistent with this notion, Leishmania are auxotrophic for many essential nutrients, including purines, vitamins, heme, and a range of amino acids, which must be scavenged from the lysosome. Similarly, a number of Leishmania mutants have been generated with defects in pathways for de novo synthesis of other metabolites (glycine, amino sugars) or nutrient salvage pathways (nucleotide/nucleoside/purine base) that retain virulence in animal models, suggesting considerable redundancy in nutrient uptake/de novo biosynthetic pathways15–18. Indeed, we have previously proposed that the complex auxotrophic requirements of these parasites may underlie their tropism for this intracellular niche6 (Figure 1). Interestingly, the Gram-negative bacterium Coxiella burnetii, one of the few other microbial pathogens to survive long term within in the macrophage phagolysosome, exhibits a similar broad range of nutrient auxotrophies19. Therefore, the macrophage phagolysosome may represent a relatively permissive intracellular niche with regard to nutrient availability, if microbes can establish suitable strategies for inhibiting or evading the activation of highly effective host cell microbiocidal processes.\n\nThis compartment is predicted to contain a range of carbon sources (sugars, amino acids, and fatty acids) and essential nutrients (major auxotrophic requirements listed in insert) that are delivered to the phagolysosome via different endocytic pathways, autophagy, lysosomal membrane transporters, and fusion with the endoplasmic reticulum (ER). Macromolecules delivered to this compartment are degraded by a barrage of luminal hydrolases or internalized by amastigotes and degraded within their own hydrolytically active lysosomes, or both. Arg, arginine; EE, early endosome; Glc, glucose; Glc6P, glucose 6-phosphate; GlcA, glucuronic acid; GlcN, glucosamine; His, histidine; Ile, isoleucine; LE, late endosome; Leu, leucine; Lys, lysine; Man, mannose; Phe, phenylalanine; Rib, ribose; TAG, triacylglycerol; Trp, tryptophan; Tyr, tyrosine; Val, valine; Xyl, xylose.\n\n\nLeishmania amastigotes enter a quiescent state and exhibit a stringent metabolic response\n\nUp until recently, information on the growth rate and metabolic state of Leishmania amastigotes in inflammatory lesions and granulomas was limited. A number of studies have tracked changes in Leishmania parasite load in both susceptible and resistant murine models by monitoring changes in parasite numbers or by following transgenic parasites lines expressing luciferase or fluorescent reporter proteins20–23. These studies suggest that Leishmania amastigotes undergo progressive and continuous replication in susceptible mice strains (such as BALB/c), leading to systemic infection and death. In contrast, while parasite numbers increase in resistant mice strains (such as C57BL/6) during early stages of infection, numbers subsequently plateau and eventually are reduced to a low level as a protective host immune response develops. Thus, net changes in Leishmania parasite burden are determined by both parasite growth rate and the rate of parasite clearance or dissemination to other tissues (or both), which will vary with the immune status of the host. Recently, two distinct approaches have been developed to more precisely determine both the growth rate and metabolic state of Leishmania amastigotes in vivo. In the first approach, transgenic L. major lines were generated expressing a photo-convertible fluorescent protein and used to monitor both amastigote dissemination in inflammatory lesions and overall protein turnover as a proxy of their growth and metabolic state24. This study showed that there was very little migration of L. major amastigote-infected macrophages into or out of these lesions and that intracellular amastigotes exhibited surprisingly low rates of protein turnover and, by inference, replication. Interestingly, the slow rate of parasite replication in these tissues appeared to reflect, at least partially, the production of sub-lethal concentrations of nitric oxide by lesion macrophages24.\n\nIn the second approach, the growth rate of L. mexicana amastigotes in inflammatory lesions in susceptible BALB/c mice was measured by labeling infected mice with heavy water (2H2O)25. 2H2O labeling results in the incorporation of deuterium into a wide range of metabolic precursors in both host tissues and resident parasite populations, and the subsequent incorporation of these building blocks into macromolecules can be used to determine the turnover of key cellular components (DNA, RNA, proteins, and lipids). With this novel approach, L. mexicana amastigotes were found to divide at a very slow, but constant, rate (t1/2 ~12 days on the basis of DNA turnover) throughout lesion development25. The growth rate of lesion parasites was substantially slower than in cultured macrophages, supporting the notion that parasite growth in lesions is constrained, by either autonomous or host-microbicidal responses. Furthermore, the empirically determined amastigote growth rates closely matched those calculated from overall parasite burden (total parasites and parasites per macrophage), suggesting that parasite killing in BALB/c lesions is rare and that infected lesion macrophages are very long-lived. The 2H2O labeling approach was further extended to measure global rates of RNA and protein turnover in lesion amastigotes25. Both processes were found to be repressed to a greater extent than in non-dividing insect (promastigote) stages, suggesting that lesion amastigotes enter into a semi-quiescent state in which major energy-consuming processes are specifically repressed.\n\nMetabolite profiling and 13C-stable isotope labeling of isolated lesion amastigotes have suggested that entry into this metabolically quiescent state is associated with major rewiring of key fluxes in central carbon metabolism26,27. In particular, lesion amastigotes have dramatically reduced rates of glucose and amino acid uptake and use these carbon sources much more efficiently than rapidly replicating or non-dividing promastigotes27 (Figure 2). Specifically, both dividing and non-dividing promastigote stages take up more glucose than is needed to maintain or increase biomass and exhibit high levels of overflow metabolism (secretion of partially oxidized glucose end-products, such as acetate, succinate, and alanine). In contrast, amastigotes exhibit much reduced rates of glucose uptake but negligible rates of overflow metabolism (glucose-sparing) (Figure 2). This switch to a more economical metabolism in amastigotes has been termed the stringent response and is associated with reduced uptake of other potential carbon sources, such as amino acids27. This response appears to be hard-wired into the amastigote differentiation process as a similar downregulation of glucose and amino acid uptake also occurs in in vitro differentiated amastigotes regardless of the availability of glucose or other carbon sources in the medium.\n\nThe differentiation of Leishmania promastigotes (insect stage) to amastigotes (macrophage host) is associated with major changes in central carbon metabolism. Promastigotes exhibit high rates of glucose and (non-essential) amino acid uptake that are co-catabolized via the major pathways of central metabolism. Promastigotes also take up fatty acids, but these are primarily incorporated into membrane lipids and not used as carbon sources (downward arrow). Amastigotes also preferentially use glucose as a carbon source. However, they exhibit much lower (~10-fold) rates of sugar and amino acid uptake and overflow metabolism (note that amastigotes continue to take up essential amino acids but primarily use these for protein synthesis). Amastigotes also actively catabolize fatty acids in the tricarboxylic acid (TCA) cycle, as a result of reduced glucose uptake. The downregulation of hexose/amino acid uptake in amastigotes (stringent response) is hardwired to differentiation, as it occurs in vitro irrespective of nutrient levels and is coupled to a reduced growth rate27.\n\nHow this stage-specific switch in metabolism is regulated remains largely undefined. Leishmania are unusual in lacking conventional gene-specific transcriptional regulation (and transcription factors) and constitutively transcribe gene-rich regions of their genome as long polycistronic mRNAs that are subsequently processed to generate individual mRNA28. As a result, the levels of most protein-encoding mRNAs remain constant in both dividing and non-dividing developmental stages29. Similarly, most metabolic enzymes are constitutively expressed and any stage-specific differences in protein levels, where present, are modest (generally less than a twofold) or variable (or both) across different Leishmania species30,31. Post-translational mechanisms are therefore likely to play an important role in the induction of the amastigote stringent response. There is accumulating evidence that several key nutrient transporters involved in glucose and amino acid uptake are downregulated in amastigotes. In the case of glucose transporters, downregulation can be mediated by ubiquitination of the cytoplasmic tail and internalization and degradation of the transporter in the parasite lysosome32. Ubiquitination or sumoylation has also been shown to regulate key pathways, such as fatty acid β-oxidation33. The upstream signals and processes that trigger these changes are poorly defined. However, amastigote differentiation is associated with marked changes in the phosphorylation state of many proteins, including those involved in stress responses34, and several protein kinases8,9 and phosphatases8–10,35 have been shown to be essential for virulence, suggesting that different signaling cascades may be required for the activation of the stringent response.\n\n\nWhat is the function of metabolic quiescence?\n\nThe finding that Leishmania amastigotes enter a slow growth/metabolically quiescent state was unexpected given the available evidence suggesting that the phagolysosome compartment contains a variety of potential carbon sources. One explanation for this apparent paradox is that the phagolysosome, while containing high levels of some carbon sources, may be growth-limiting with regard to the availability of other (micro)nutrients. Consistent with this notion, intracellular amastigote growth in ex vivo infected macrophages and in vivo is promoted by increasing the availability of select amino acids, such as arginine36–41. In the latter case, it remains unclear whether promotion of amastigote growth is due to increased availability of arginine, an essential amino acid, or conversion of arginine to growth-promoting polyamines by the host cell arginase. Moreover, active salvage of arginine by intracellular parasites may deplete arginine pools in the macrophage and affect the capacity of these host cells to generate nitric oxide via inducible nitric oxide synthase41, further complicating the interpretation of these supplementation experiments. Similarly, there is strong evidence that phagolysosomal levels of micronutrients, such as iron and heme, can regulate intracellular parasite growth42–46. Host cell transporters in the macrophage phagolysosomal membrane pump iron and heme out of the phagolysosome lumen to the cytosol and thus are important determinants of amastigote growth42,43,47–49. In response, Leishmania amastigotes upregulate expression of a surface ferric reductase (that converts Fe3+ to Fe2+) and a ferrous (Fe2+) iron transporter, LIT1, allowing efficient salvage of iron, an essential cofactor in many parasite enzymes, including the parasite iron superoxide dismutase (FeSOD) and iron-sulphur containing enzymes involved in the mitochondrial tricarboxylic acid (TCA) cycle. Interestingly, L. amazonensis mutants that lack the LIT1 transporter are unable to retain viability when promastigote stages reach stationary phase or to effectively differentiate to amastigotes43. Differentiation was found to be dependent on FeSOD-mediated conversion of superoxide to hydrogen peroxide, which appears to stimulate amastigote differentiation. Thus, iron restriction within the phagolysosome may have a dual effect of preventing induction of the stringent response as well as limiting operation of energy-generating pathways in the mitochondria. Together, these studies suggest that selected nutrient restriction occurs in the phagolysosome and that Leishmania adapt to this niche by upregulating the expression of specific nutrient sensing and salvage pathways as well as downregulating global energy requirements (stringent response).\n\nThe Leishmania amastigote stringent response is induced in response to elevated temperature and reduced pH in culture, suggesting that these physiological changes may protect parasites from these specific environmental stresses or that it is part of a programmed stress response to multiple stresses (that can be triggered by these key signals in vivo) or both. In support of the latter proposal, the stringent response is enhanced in lesion amastigotes compared with cultured (axenic) amastigotes. As mentioned above, amastigote growth in developing lesions may be restricted by sublethal concentrations of reactive nitrogen species (RNS), which can inactivate many enzymes in the mitochondrial TCA cycle and respiration chain containing iron-sulphur clusters50. A switch to increased dependency on glycolysis and an overall reduction in basal energetic requirements would reduce amastigote vulnerability to macrophage-derived RNS. Interestingly, a number of other bacterial pathogens that invade macrophages also appear to be dependent on sugars as their major carbon source51, and decreased bacterial respiration is associated with resistance to a range of external stresses, including microbicidal NO and drug treatments52.\n\nThe stringent response may also protect amastigotes from nutrient excess. The concept that nutrient excess can lead to cellular stress is now well established in diseases such as obesity, metabolic syndrome, and diabetes53,54 but less commonly considered in microbes, particularly those in intracellular niches55. Metabolic stress induced by nutrient overload (that is, excess glucose) can occur as a result of multiple mechanisms, of which the most prevalent are increased production of mitochondrial NADH (that is, increased NADH/NAD+ ratio) and concomitant elevated production of endogenous reactive oxygen species (ROS) as a result of leakage of electrons from the mitochondrial respiratory chain53. Leishmania are potentially highly vulnerable to reductive stress, as they lack the capacity to transcriptionally downregulate TCA cycle enzymes involved in NADH generation and, owing to the compartmentalization of glycolytic enzymes into modified peroxisomes, termed glycosomes, also appear to have lost classic allosteric regulatory mechanisms that result in feedback inhibition of glycolysis6 (Figure 3). The absence of allosteric feedback mechanisms in upper glycolysis means that glycolytic fluxes are largely regulated by glucose uptake rates. Leishmania promastigotes can exploit high concentrations of glucose and avoid excessive flux into the TCA cycle (with concomitant NADH production) by secreting partially oxidized intermediates, such as alanine, acetate, and succinate into the medium26 (Figure 2). A similar strategy is used by other microorganisms, such as Saccharomyces cerevisiae, during periods of rapid growth on fermentable carbon sources56. However, the profligate use of carbon sources and secretion of partially oxidized intermediates is likely to be deleterious for intracellular parasite stages and could also impact on host cell physiology. The global downregulation of amastigote nutrient transporters after activation of the stringent response32 may constitute an important strategy for minimizing nutrient uptake and reductive stress within the restrictive environment of the phagolysosome32.\n\nLeishmania amastigotes appear to depend primarily on the uptake and catabolism of sugars scavenged from the macrophage phagolysosome. Hexose phosphates are catabolized in the glycolytic and pentose phosphate pathway (PPP) and converted to intracellular and surface glycoconjugates (GPI, N-glycans, mannogen). Key enzymes involved in glycolysis are partially or exclusively sequestered within glycosomes (modified peroxisomes), and ATP and NAD+ within this organelle are regenerated by fermentation of phosphoenolpyruvate to succinate (succinate fermentation pathway, or SFP) or pyruvate66. The end-products of glycosomal catabolism are further catabolized in the mitochondrion, together with acetyl-CoA generated by fatty acid β-oxidation, to produce anabolic precursors, such as glutamate. Most of the glutamate (and other non-essential amino acids) in amastigotes is synthesized de novo rather than taken up from macrophages. Excess NADH production in the mitochondrion might lead to increased endogenous reactive oxygen species (ROS) production via the respiratory chain. The gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBP), is also required for amastigote survival in vivo. This enzyme is sequestered in glycosomes with phosphofructokinase (PFK) and might allow amastigotes to transiently use other carbon sources or regulate glycolytic fluxes by cycling FBP back to fructose 6-phosphate (futile cycling), or both. αKG, α-ketoglutarate; AcCoA, acetyl-CoA; Fru6P, fructose-6-phosphate; Glc6P, glucose-6-phosphate; GlcNAc6P, N-acetylglucosamine-6-phosphate; Glu, glutamate; Man6P, mannose-6-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Rib5P, ribose-5-phosphate; Triose-P, triose phosphates.\n\n\nRewiring of carbon metabolism may also be used to deal with nutrient excess?\n\nActivation of the Leishmania stringent response in amastigotes is linked to additional changes in carbon metabolism that could also contribute to parasite survival within macrophages. Detailed 13C-tracer studies27 have shown that lesion amastigotes, in common with promastigote, appear to preferentially use sugars, although rates of uptake are much lower than in promastigotes. Whereas most compartments within the endolysosomal system of macrophages are thought to contain low luminal concentrations of sugars, the (phago)lysosome compartment may be an exception. Macrophages constitutively internalize a wide range of complex glycoproteins, proteoglycans, and glycosaminoglycans that are degraded by lysosomal glycosidases to generate free sugars or oligosaccharides. Leishmania hexose transporters57 and enzymes involved in the catabolism of host-derived amino sugars are essential for Leishmania virulence58,59. Furthermore, intracellular growth of Leishmania amastigotes in cultured macrophages can be stimulated by the addition of glycosaminoglycans such as hyaluronan, highlighting the importance of amino sugar catabolism for Leishmania survival and virulence58,59.\n\nAmastigotes also co-utilize fatty acids as a significant carbon source (Figure 3). This contrasts with promastigotes that preferentially co-utilize non-essential amino acids, aspartate, alanine, and glutamate with glucose26,27. The increased β-oxidation of fatty acids in amastigotes appears to be a direct consequence of reduced glucose uptake by this stage27, and the resultant acetyl-CoA produced by fatty acid oxidation is used primarily to top up the TCA cycle (anapleurosis) providing intermediates for the biosynthesis of amino acids, such as glutamate, glutamine, and aspartate (catapleurosis) (Figure 3). Pharmacological inhibition of enzymes involved in the synthesis of non-essential amino acids via the TCA cycle results in complete inhibition of amastigote growth and survival27. These amino acids are required for nucleotide, thiol and amino-sugar biosynthesis, and the dependence on de novo synthesis is consistent with the finding that amino acid uptake by amastigotes is limited57,59,60. Similarly, genetic disruption of fatty acid β-oxidation or proteins involved in the mitochondrial respiratory chain also results in a loss of virulence33,61. Together, these studies suggest that amastigotes are highly dependent on sugar and fatty acids scavenged from the lumen of the phagolysosome.\n\nParadoxically, Leishmania amastigote mutants lacking the key gluconeogenic enzyme, fructose 1,6-bisphosphatase (FBPase), are also poorly virulent in mice62. FBPase catalyzes the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate and is expressed in the same glycosome compartment as the glycolytic enzyme, phosphofructokinase (PFK), that catalyzes the reverse reaction62 (Figure 3). The functional significance of the constitutive expression of these two enzymes in the same organelle remains unclear. Sugar levels in the phagolysosome could fluctuate in response to changes in membrane transport and the delivery of cargo to this compartment, leading to periods of sugar starvation and transient dependency on gluconeogenesis for the synthesis of essential glycoconjugates, DNA/RNA synthesis, and production of reducing equivalents via the pentose phosphate pathway63. In this context, co-expression of both FBPase and PFK could allow Leishmania amastigotes to rapidly respond to changes in carbon source availability. However, lesion-derived amastigotes exhibit very low rates of amino acid uptake and intracellular stages appear to be dependent on glucose catabolism even when infected macrophages are supplied with excess amino acids27. Furthermore, Leishmania lack a glyoxylate cycle and therefore are unable to switch to using fatty acids (a likely plentiful carbon source in this compartment) as a sole gluconeogenic carbon source. It is possible that FBPase may have acquired non-enzymatic functions, other than its role in gluconeogenesis, that account for the dependency of intracellular stages on this enzyme. FBPase has recently been shown to regulate glycolysis in mammalian cells via at least two mechanisms, one of which involves transcriptional regulation of signaling proteins and is not dependent on its enzymatic activity64. Alternatively, FBPase may be required for parasite growth under growth conditions in which glucose uptake and glycolysis are still active (Figure 3). This has recently been shown to be the case in Toxoplasma gondii, another intracellular parasite that resides in a distinct vacuolar compartment and is also primarily dependent on glucose catabolism for growth65. As with L. major, genetic disruption of T. gondii FBPase resulted in strong attenuation of intracellular growth in host cells and loss of virulence in animal models. Loss of virulence of the T. gondii ∆FBPase mutant was associated with increased glycolytic flux at the expense of glucose flux into other essential metabolic pathways. Thus, under normal growth conditions, T. gondii FBPase may function in a futile (ATP-consuming) metabolic cycle with the PFK and potentially restrict excessive flux through glycolysis and ensure balanced growth. Whether metabolic cycling between FBPase and PFK occurs in the Leishmania amastigote’s glycosome and the extent to which it regulates glycolytic fluxes remains to be determined.\n\n\nConclusions\n\nLeishmania parasites are unusual in their capacity to proliferate long-term within the mature phagolysosome compartment of host macrophages. It is likely that the complex nutritional requirements of Leishmania and the need to have access to a broad range of metabolites underlie Leishmania’s tropism for this hostile intracellular niche. However, successful colonization of this niche must have been linked to the parallel evolution of strategies for combating a range of host cell microbicidal processes (ROS, RNS, hydrolases) that are normally effective at eradicating pathogens that are delivered to this compartment. Intriguingly, Leishmania amastigotes lack many of the virulence factors found in promastigotes or other microbial pathogens (cell walls, surface coats, protective pigments, and so on), suggesting that the extraordinary resilience of these pathogens is dependent on more fundamental physiological changes that confer cytoprotection against a variety of stresses. Very recent studies, using new fluorescent protein reporters and stable isotope (2H,13C) labeling approaches for measuring amastigote physiology and metabolism in vivo, suggest that amastigotes enter into a semi-quiescent growth state in vivo. This state is distinct from that observed in non-dividing promastigotes and appears to be programmed by differentiation signals independent of external nutrient levels. It is proposed that induction of the stringent metabolic response may (i) prevent depletion of essential limiting (micro)nutrients in the phagolysosome compartment, (ii) reduce the bioenergetic needs of amastigotes and hence their dependence on high-energy-yielding pathways (such as oxidative phosphorylation) that are highly susceptible to inhibition by RNS/ROS, and (iii) minimize endogenous reductive stress induced by excessive utilization of abundant carbon sources in the phagolysosome and overflow metabolism. Thus, the stringent metabolic response may protect amastigotes from both feast and famine within this compartment. Further studies are needed to understand how amastigote metabolism is regulated in the absence of significant gene-specific transcriptional regulation, while the identification of key steps in carbon metabolism that are essential for amastigote virulence opens up new opportunities for the development of novel anti-microbial strategies.\n\n\nAbbreviations\n\n2H2O, heavy water; FBPase, fructose-1,6-bisphosphatase; FeSOD, iron superoxide dismutase; PFK, phosphofructosekinase; RNS, reactive nitrogen species; ROS, reactive oxygen species; TCA, tricarboxylic acid.",
"appendix": "Author contributions\n\n\n\nAll authors were involved in the preparation of this manuscript, have agreed to the final content, and read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nWork from the McConville laboratory was supported by NHMRC grant APP1059545.\n\n\nAcknowledgments\n\nWe thank members of the McConville lab for thoughtful discussions. MJM is a National Health and Medical Research Council (NHMRC) Principal Research Fellow.\n\n\nReferences\n\nMuraille E, Leo O, Moser M: TH1/TH2 paradigm extended: macrophage polarization as an unappreciated pathogen-driven escape mechanism? Front Immunol. 2014; 5: 603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRussell DG: Who puts the tubercle in tuberculosis? Nat Rev Microbiol. 2007; 5(1): 39–47. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuleme HM, Reguera RM, Berard A, et al.: Infection with arginase-deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis. J Immunol. 2009; 183(12): 8068–8076. PubMed Abstract | Publisher Full Text | Free Full Text\n\nda Silva MFL, Zampieri RA, Muxel SM, et al.: Leishmania amazonensis arginase compartmentalization in the glycosome is important for parasite infectivity. PLoS One. 2012; 7(3): e34022. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWhite JK, Mastroeni P, Popoff JF, et al.: Slc11a1-mediated resistance to Salmonella enterica serovar Typhimurium and Leishmania donovani infections does not require functional inducible nitric oxide synthase or phagocyte oxidase activity. J Leukoc Biol. 2015; 77(3): 311–320. PubMed Abstract | Publisher Full Text\n\nMittra B, Cortez M, Haydock A, et al.: Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels. J Exp Med. 2013; 210(2): 401–416. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHuynh C, Andrews NW: Iron acquisition within host cells and the pathogenicity of Leishmania. Cell Microbiol. 2008; 10(2): 293–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiguel DC, Flannery AR, Mittra B, et al.: Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence. Infect Immun. 2013; 81(10): 3620–3626. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRenberg RL, Yuan X, Samuel TK, et al.: The heme transport capacity of LHR1 determines the extent of virulence in Leishmania amazonensis. PLoS Negl Trop Dis. 2015; 9(5): e0003804. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWhite C, Yuan X, Schmidt PJ, et al.: HRG1 is essential for heme transport from the phagolysosome of macrophages during erythrophagocytosis. Cell Metab. 2013; 17(2): 261–270. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuynh C, Sacks DL, Andrews NW: A Leishmania amazonensis ZIP family iron transporter is essential for parasite replication within macrophage phagolysosomes. J Exp Med. 2006; 203(10): 2363–2375. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlannery AR, Huynh C, Mittra B, et al.: LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms. J Biol Chem. 2011; 286(26): 23266–23279. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAuger C, Lemire J, Cecchini D, et al.: The metabolic reprogramming evoked by nitrosative stress triggers the anaerobic utilization of citrate in Pseudomonas fluorescens. PLoS One. 2011; 6(12): e28469. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nEisele NA, Ruby T, Jacobson A, et al.: Salmonella require the fatty acid regulator PPARδ for the establishment of a metabolic environment essential for long-term persistence. Cell Host Microbe. 2013; 14(2): 171–182. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLobritz MA, Belenky P, Porter CBM, et al.: Antibiotic efficacy is linked to bacterial cellular respiration. Proc Natl Acad Sci U S A. 2015; 112(27): 8173–8180. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTeodoro JS, Rolo AP, Palmeira CM: The NAD ratio redox paradox: why does too much reductive power cause oxidative stress? Toxicol Mech Methods. 2013; 23(5): 297–302. PubMed Abstract | Publisher Full Text\n\nWellen KE, Thompson CB: Cellular metabolic stress: considering how cells respond to nutrient excess. Mol Cell. 2010; 40(2): 323–332. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh A, Crossman DK, Mai D, et al.: Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response. PLoS Pathog. 2009; 5(8): e1000545. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaman S, Lippman SI, Zhao X, et al.: How Saccharomyces responds to nutrients. Annu Rev Genet. 2008; 42: 27–81. PubMed Abstract | Publisher Full Text\n\nBurchmore RJS, Rodriguez-Contreras D, McBride K, et al.: Genetic characterization of glucose transporter function in Leishmania mexicana. Proc Natl Acad Sci U S A. 2003; 100(7): 3901–3906. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNaderer T, Heng J, Saunders EC, et al.: Intracellular survival of Leishmania major depends on uptake and degradation of extracellular matrix glycosaminoglycans by macrophages. PLoS Pathog. 2015; 11(9): e1005136. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaderer T, Heng J, McConville MJ: Evidence that intracellular stages of Leishmania major utilize amino sugars as a major carbon source. PLoS Pathog. 2010; 6(12): e1001245. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRodriguez-Contreras D, Feng X, Keeney KM, et al.: Phenotypic characterization of a glucose transporter null mutant in Leishmania mexicana. Mol Biochem Parasitol. 2007; 153(1): 9–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDey R, Meneses C, Salotra P, et al.: Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence. Mol Microbiol. 2010; 77(2): 399–414. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nNaderer T, Ellis MA, Sernee MF, et al.: Virulence of Leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1,6-bisphosphatase. Proc Natl Acad Sci U S A. 2006; 103(14): 5502–5507. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBerg M, Vanaerschot M, Jankevics A, et al.: Metabolic adaptations of Leishmania donovani in relation to differentiation, drug resistance, and drug pressure. Mol Microbiol. 2013; 90(2): 428–442. PubMed Abstract | Publisher Full Text\n\nLi B, Qiu B, Lee DSM, et al.: Fructose-1,6-bisphosphatase opposes renal carcinoma progression. Nature. 2014; 513(7517): 251–255. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBlume M, Nitzsche R, Sternberg U, et al.: A Toxoplasma gondii gluconeogenic enzyme contributes to robust central carbon metabolism and is essential for replication and virulence. Cell Host Microbe. 2015; 18(2): 210–220. PubMed Abstract | Publisher Full Text\n\nRodriguez-Contreras D, Hamilton N: Gluconeogenesis in Leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase. J Biol Chem. 2014; 289(47): 32989–33000. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "10642",
"date": "01 Oct 2015",
"name": "Syamal 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",
"responses": []
},
{
"id": "10643",
"date": "01 Oct 2015",
"name": "Marilyn Parsons",
"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",
"responses": []
},
{
"id": "10644",
"date": "01 Oct 2015",
"name": "Norma W. Andrews",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-938
|
https://f1000research.com/articles/3-205/v1
|
28 Aug 14
|
{
"type": "Method Article",
"title": "Detecting miRNA Mentions and Relations in Biomedical Literature",
"authors": [
"Shweta Bagewadi",
"Tamara Bobić",
"Martin Hofmann-Apitius",
"Juliane Fluck",
"Roman Klinger",
"Tamara Bobić",
"Martin Hofmann-Apitius",
"Juliane Fluck",
"Roman Klinger"
],
"abstract": "Introduction: MicroRNAs (miRNAs) have demonstrated their potential as post-transcriptional gene expression regulators, participating in a wide spectrum of regulatory events such as apoptosis, differentiation, and stress response. Apart from the role of miRNAs in normal physiology, their dysregulation is implicated in a vast array of diseases. Dissection of miRNA-related associations are valuable for contemplating their mechanism in diseases, leading to the discovery of novel miRNAs for disease prognosis, diagnosis, and therapy.Motivation: Apart from databases and prediction tools, miRNA-related information is largely available as unstructured text. Manual retrieval of theseassociations can be labor-intensive due to steadily growing number of publications. Despite the fact that several databases host miRNA-associations derived from text, lower sensitivity has motivated the need for an improvised framework. Additionally, the lack of a standard corpus for miRNA-relations has caused difficulty in evaluating the available systems.We propose methods to automatically extract mentions of miRNAs, species, genes/proteins, disease, and relations from scientific literature. Our generated corpora, along with dictionaries, and miRNA regular expression are freely available for academic purposes. To our knowledge, these resources are the most comprehensive developed so far.Results: The identification of specific miRNA mentions reaches a recall of 0.94 and precision of 0.93. Extraction of miRNA-disease and miRNA-gene relations lead to an F1 score of up to 0.76. A comparison of the information extracted by our approach to the databases miR2Disease and miRSel for the extraction of Alzheimer's disease related relations shows the capability of our proposed methods in identifying correct relations with improved sensitivity. The published resources and described methods can help the researchers for maximal retrieval of miRNA-relations and generation of miRNA-regulatory networks.Availability: The training and test corpora, annotation guidelines, developed dictionaries, and supplementary files are available at http://www.scai.fraunhofer.de/mirna-corpora.html",
"keywords": [
"Functionally important non-coding RNAs (ncRNAs) are now better understood with the progress of high-throughput technologies. Discovery of the major class of ncRNAs",
"microRNAs (miRNAs1) has further facilitated the molecular aspects of biomedical research."
],
"content": "Introduction\n\nFunctionally important non-coding RNAs (ncRNAs) are now better understood with the progress of high-throughput technologies. Discovery of the major class of ncRNAs, microRNAs (miRNAs1) has further facilitated the molecular aspects of biomedical research.\n\nMicroRNAs are a large group of small endogenous single-stranded non-coding RNAs (17–22nt long) found in eukaryotic cells. They post-transcriptionally regulate gene expression of specific mRNAs by degradation, translational inhibition, or destabilization of the targets (transcripts of protein-coding genes)2. Esquela-Kerscher et al. have reported on miRNAs involvement in almost every regulation aspect of biological processes such as apoptosis, and stress response3. Wubin et al. demonstrated that miR-29a regulatory circuitry plays an important role in epididymal development and its functions4. Additionally, tissue-specificity of miRNAs has been shown to provide a better clue of their fundamental roles in normal physiology5.\n\nDysregulation of miRNAs and their ability to regulate repertoires of genes (as well as co-ordinate multiple biological pathways) has been linked to several diseases6,7. One example is chronic lymphocytic leukemia where in about 68% of the cases miRNA genes (miR15 and miR16) are missing or down-regulated8. Thus, uncovering the relations between miRNAs and diseases as well as genes/proteins is crucial for our understanding of miRNA regulatory mechanisms for diagnosis and therapy9,10.\n\nSeveral databases, prediction algorithms and tools are available, providing insight into miRNA-disease and miRNA-mRNA associations. Although the detailed target recognition mechanism is still elusive, several algorithms attempt to predict miRNA targets. However, a limited precision of 0.50 and recall of 0.12 has been reported when evaluated against proteomics supported miRNA targets11. Despite the fact that these resources provide insight into miRNA-associated relationships, the majority of relations are scattered as unstructured text in scientific publications12. Figure 1 shows the growth of publications in MEDLINE and in addition depicts the normalized growth of publications that reference the keyword “microRNA”.\n\nThe dotted line points out the relative increase of miRNA-related publications per year in comparison to the growth of MEDLINE (as of 31 December, 2013).\n\nSome databases such as miR2Disease and PhenomiR store manually extracted relations from literature. The miR2Disease database13 contains information about miRNA-disease relationships with 3273 entries (as of the last update on March 14, 2011). PhenomiR14 is a database on miRNA-related phenotypes extracted from published experiments. It consists of 675 unique miRNAs, 145 diseases, and 98 bioprocesses from 365 articles (Version 2.0, last updated on February 2011). TarBase11 hosts more than 6500 experimentally validated miRNA targets extracted from literature.\n\nHowever, manual retrieval of relevant articles and extraction of relation mentions from them is labor-intensive. A solution is to use text-mining techniques. Moreover, the vast majority of the research in this direction is mainly focused around extraction of protein-protein interactions15. On the contrary, miRNA relation extraction is still naive. The shift of focus towards identification of miRNA-relations is slowly establishing with the rise in systems approaches to investigate complex diseases. The manually curated database miRTarbase16 incorporates such text-mining techniques to retrieve miRNA-related articles. Recently, the miRCancer database has been constructed using a rule-based approach to extract miRNA-cancer associations from text17. As of June 14, 2014, this database contains 2271 associations between 38562 miRNAs and 161 human cancers from 1478 articles.\n\nText-mining technologies are established for a variety of applications. For instance, the BioCreative competition18,19 and BioNLP Shared Task20–22 series have been conducted to benchmark text mining techniques for gene mention identification, protein-protein relation extraction and event extraction, among others.\n\nTo our knowledge, only limited work has been carried out in the area of miRNA-related text-mining. Murray et al. considered miRNA-gene associations from PubMed database using semantic search techniques23. For their analysis, experimentally derived datasets were examined, combined with network analysis and ontological enrichment. Regular expressions were used to detect miRNA mentions. The authors optimized the approach to reach 100% accuracy and recall for detecting miRNAs mentions as in miRBase. Relations were identified based on a manually curated rule set. The authors extracted 1165 associations between 270 miRNAs and 581 genes from the whole MEDLINE.\n\nThe freely available miRSel12 database integrates automatically extracted miRNA-target relationships from PubMed abstracts. A set of regular expressions is used for miRNA recognition that matches all miRBase synonyms and generic occurrences. The authors reach a recall of 0.96 and precision of 1.0 on 50 manually annotated abstracts for miRNA mention identification. Further, the relations between miRNA and genes were extracted at sentence level employing a rule-based approach. They evaluated on 89 sentences from 50 abstracts resulting in a recall of 0.90 and precision of 0.65. Currently, it hosts 3690 miRNA-gene interactions11.\n\nIn contrast to the previous text-mining approaches focusing purely on miRNA gene relations, we extend the information extraction approach additionally to retrieve miRNA-disease relations. Furthermore, we evaluate our approach using a larger corpus to achieve robustness. We differentiate between actual miRNA mentions (refered to as Specific miRNAs) and co-referencing miRNAs (Non-Specific miRNAs). We evaluated three different relation extraction approaches, namely co-occurrence, tri-occurrence and machine learning based methods.\n\nTo support further research, our corpora are made publicly available in an established XML format as proposed by Pyysalo et al.24, as well as the regular expressions used for miRNAs named entity recognition. In addition, our dictionary for trigger term detection and general miRNA mention identification are made available. To our knowledge, the annotated corpora as well as the information extraction resources are the most comprehensive developed so far.\n\n\nMethods\n\nMentions of miRNAs consisting of keywords (case-insensitive and not containing any suffixed numerical identifier) such as “Micro-RNAs” or “miRs” are annotated as Non-Specific miRNA. Names of particular miRNAs such as miRNA-101, suffixed with numerical identifiers are labeled as Specific miRNA. Numerical identifiers (separated by delimiters such as “,”, “/”, and “and”) occurring as part of specific miRNA mentions are annotated as a single entity. Box 1 depicts the annotation of specific miRNA mentions (including an example for part mentions). In addition, Disease, Gene/Protein, Species, and Relation Trigger are annotated. The detailed annotation guideline for annotating specific miRNA mentions is available as a supplementary file.\n\nHere “-181b”, and “-181c” are the part mentions annotated as a single entity along with “miR-181a” in box. A non-specific miRNA mention is shown in italics.\n\nInteresting results were obtained from miR-181a, -181b, and -181c. These set of brain-enriched miRNAs are down-regulated in glioblastoma. However, miR-222, and miR-128 are strongly up-regulated.\n\nMentions of disease names, disease abbreviations, signs, deficiencies, physiological dysfunction, disease symptoms, disorders, abnormalities, or organ damages are annotated as Disease. Possessive terms such as “Diabetic patients” are not marked. Mentions referring to proteins/genes which are either single word (e.g. “trypsin”), multi-word, gene symbols (e.g. “SMN”), or complex names (including of hyphens, slashes, Greek letters, Roman or Arabic numerals) are annotated as Gene/Protein. Only those organisms that are having published miRNA sequences and annotations represented in miRBase database are labeled as Species. Any verb, noun, verb phrase, or noun phrase associating miRNA mention to either labeled disease or gene/protein term is annotated as Relation Trigger.\n\nWe restrict the relationship extraction to sentence level and four different interacting entity pairs: Specific miRNA-Disease (SpMiR-D), Specific miRNA-Gene/Protein (SpMiR-GP), Non-Specific miRNA-Disease (NonSpMiR-D), and Non-Specific miRNA-Gene/Protein (NonSpMiR-GP). Relevant triples, an interacting pair co-occurring with a Relation Trigger are defined to form a relation and can belong to one of the four above mentioned Relation classes.\n\nThe annotation has been performed using Knowtator25 integrated within the Protégé framework26.\n\nWe develop a new corpus based on MEDLINE, annotated with miRNA mentions and relations. Out of 27001 abstracts retrieved using the keyword “miRNA”, 201 are randomly selected as training and 100 as test corpus. Two annotators have been involved in the annotation. The first annotator annotated the training corpus iteratively to develop guidelines and built the consensus annotation. The second annotator followed these guidelines and annotated the same corpus. Table 1 provides the inter-annotator agreement (measured as F1 and Cohen’s κ) for the test corpus. An example annotation is shown in Box 1.\n\nTable 2 shows the number of annotated concepts in the training and test corpora for each entity class and the count for manually extracted relations (triplets), categorized for different interacting entity pairs. Table 3 provides the overall statistics of the published corpora (additional information about the corpus is given in the README supplementary file).\n\nCounts of manually annotated entities in the training and the test corpora as well as annotated sentences describing relations.\n\nFor identification of specific miRNA mentions in text (cf. Table 4), we developed regular expression patterns using manual annotations of miRNA mentions as the basis. Similarly, a dictionary has been generated for general miRNA recognition. The regular expression patterns are represented in the format as defined by Oualline et al.27. In this work, several aliases are defined (cf. Table 5) to be used in the final regular expression patterns for specific miRNA identification, given in Table 4. Detected entities are resolved to a unique miRNA name and disambiguated (e. g. hsa-microRNA-21 to hsa-mir-21 and microRNA 101 to mir-101). Unique miRNA terms are mapped to human miRBase database identifiers through the mirMaid Restful web service. For those names where we do not retrieve any database identifiers, we fall back to another organism mention found in the abstract (if any), using the NCBI taxonomy dictionary (see below), otherwise we retain the unique name.\n\nAliases used to form the final regular expression, see Table 5, are highlighted in bold.\n\nAliases used in regular expression patterns for miRNAs identification (highlighted in bold).\n\nWe detect Species with a dictionary-based approach. The built dictionary consists of all the concepts from the NCBI taxonomy corresponding to only those organisms mentioned in miRBase.\n\nSimilarly, for identification of Disease and Gene/Protein mentions in text we adapted a dictionary-based approach. To detect Disease, we apply three dictionaries: MeSH, MedDRA28 and Allie. For Gene/Protein, a dictionary29 based on SwissProt, EntrezGene, and HGNC is included. Gene synonyms which could be potentially tagged as miRNAs are removed to overcome redundancy. For example, genes encoding microRNA, hsa-mir-21 are named as miR-21, miRNA21 and hsa-mir-21, the gene symbol of MIR16 membrane interacting protein of RGS16 is MIR16, which can represent a miRNA mention.\n\nThe relation trigger dictionary comprises of all interaction terms from the training corpus, together with additional spelling variants (manually added to the list, also made freely available).\n\nFor all named entity recognition performed, the dictionary-based system ProMiner29 is used.\n\nWe consider three approaches for addressing automatic extraction of interacting entity pairs from free text, described in the following.\n\nThe co-occurrence approach serves as a baseline. Assuming all interactions to be present in isolated sentences, this approach is complete but may be limited in precision. Reducing the number of false positives can be achieved by filtering with the dictionary of relation triggers. The rationale behind this filter is that the interaction is more likely to be described if such a term is present (we refer to that as tri-occurrence).\n\nTo increase the precision, we use a machine learning-based approach formulating the relation detection as a binary classification problem: each instance (consisting of a pair of entities) is classified either as not-containing a relation or belonging to one of the four-relation classes. Our system uses lexical and dependency parsing features. We evaluate linear support vector machines (SVM)30 as implemented in the LibSVM library, as well as LibLINEAR, a specialized implementation for processing large data sets31, and naive Bayes classifiers32. For more details, we refer to Bobić et al.33.\n\nLexical features capture characteristics of tokens around the inspected pair of entities. The sentence text can roughly be divided into three parts: text between the entities, text before the entities, and text after the entities. Stemming34 and entity blinding is performed to improve generalization. Features are bag-of-words and bi, tri, and quadri-gram based. This feature setting follows Yu et al. and Yang et al.35,36. The presence of relation triggers is also taken into account, using the previously described manually generated list. Next to lexical features, deep parsing provides an insight into the entire grammatical structure of the sentence37. We analyzed the vertices v (tokens from the sentence) in the dependency tree from a lexical (text of the token) and syntactical (POS tag) perspective. Edges e in the tree correspond to the information about the grammatical relations between the vertices. Extracting relevant information from the dependency parse tree is usually done following the shortest dependency path hypothesis38. Lexical and syntactical e-walks and v-walks on the shortest path are created by alternating sequence of vertices and edges, with the length of 3. We capture the information about the common ancestor vertex, in addition to checking whether the ancestor node represents a verb form (e.g. POS tag could be VB, VBZ, VBD, etc.). Finally, the length of the shortest path (number of edges) between the entities is considered as a numerical feature.\n\n\nResults and discussion\n\nIn the following, we present results for named entity recognition and relation extraction. This section concludes with two use-case analyses.\n\nAmong the 201 abstracts present in the training corpus, 82% contained general miRNA mentions, in comparison to specific miRNAs with 45%. In Table 6, results for miRNA entity recognition are reported. Non-specific miRNA recognition is close to perfect. Specific miRNA mention recognition has an F1 measure of 0.94.\n\nHere only complete match results are presented. The performance of named entity recognition is evaluated using recall (R), precision (P) and F1 score.\n\nFor disease mention recognition, combined dictionaries, based on three established resources, resulted in 0.79 and 0.69 F1 score for the training and test corpus respectively. Boundary matches result for the same reported 0.88 of F1, providing the possibility for detection of similar text strings for better recall. Genes/proteins dictionary showed a performance of 0.84 and 0.85 of F1 in training and test corpus respectively.\n\nMost abstracts in the test corpus are associated to human (71), followed by mouse (16), and rat (8). Pig has 2 abstracts, zebrafish, HIV-1, HSV-1, and Caenorhabditis elegans 1 each. The evaluation of the relation trigger dictionary (cf. Table 6) suggests that it covers a substantial part of the vocabulary with recall of 0.86 for the training and 0.79 for the test corpus.\n\nOnly 11.5% of miRNA-related associations occur outside the sentence level, thus, our work focused on relations at sentence level. Sentences in which co-occurring entity pairs do not participate in any relation are tagged as false. A comparison of the different relation extraction approaches is shown in Figure 2. The co-occurrence based approach leads to 100% recall for relation extraction. The recall is not diminished using the tri-occurrence approach while the precision increases between 4pp (percentage points) and 17pp when compared to the co-occurrence based approach, reducing false positives (cf. Figure 2). However, the precision reaches less than 60%. Using the machine-learning based classification, precision is increased up to 76% for specific miRNA-gene relations. The F1 measure is not substantially different but a trade-off between precision and recall can be observed. This is true for all three methods (LibLINEAR, SVM, and Naive Bayes). Most relation extraction approaches are dependent on the performance of named entity recognition. The impact of error propagation coming from an automated entity recognizers is evaluated by applying the tri-occurrence method on the automatically annotated training and test corpus, here termed as “NERTri”. Compared to the results on the gold standard entity annotation a drop of 13 pp for NonSpMiR-D, 7pp for NonSpMiR-GP, 22pp for SpMiR-D, and 30pp for SpMiR-GP in F1 is observed for the test corpus.\n\nOn the x-axis, different entity pair relations are represented as SpMiR-D for Specific miRNA-Disease, SpMiR-GP for Specific miRNA-Gene/Protein, NonSpMiR-D for Non-Specific miRNA-Disease, and NonSpMiR-GP for Non-Specific miRNA-Gene/Protein.\n\n\nUse case analysis\n\nFor the impact analysis of the proposed approach, we compare the extracted information with two databases, namely miR2Disease and miRSel. We focus on relations and articles concerning Alzheimer’s disease.\n\nAlzheimer’s disease (AD) is ranked sixth for causing deaths in major developed countries39. It affects not only individuals but also incurs a high cost to the society. Recently, miRNAs have shown close associations with AD pathophysiology40. Increasing the need to identify new therapeutic targets for AD, after major set backs due to failed drugs, motivates the need to look in this direction. In silico methods, such as the one proposed in this work, can aid in building miRNA-regulatory networks specific to AD, for further analysis such as identifying the mechanisms, sub-networks, and key targets.\n\nThe database miR2Disease is queried to return all miRNA-disease relations occurring in Alzheimer’s disease. We compare that dataset with all miRNA-disease relations from MEDLINE applying our tri-occurrence approach, retrieving 41 abstracts with 159 relations. Obtained triplets have been manually curated to remove 51 false positives. The results are summarized in Table 7. The miR2Disease database returns 28 evidences from 9 articles. Among these, only 14 evidences are present in abstracts. Moreover, 16 evidences are extracted from one full text document41. Only two evidences are identified at abstract level among these 16 evidences. Overall, 26 miRNAs identified by miR2Disease are in relation with Alzheimer’s disease. Therefore, our text-based extraction proposes approximately three times more relations than the database provides.\n\nMiRNA-Alzheimer’s disease relation retrieved from MEDLINE and in miR2Disease database.\n\nThe analysis of 17 false negative relations which are in the database but not found by our approach shows that most of the relations could be found only in full text and that the automatic system misses four miRNA-Alzheimer’s disease relations from abstracts. Manual inspection reveals that in three out of these missing four evidences the disease name is not mentioned in the sentence (relation occurred at co-reference level).\n\nWe apply our relation detection NERTri to 100 abstracts from PubMed retrieved using the query “alzheimer disease” [MeSH Terms] OR (“alzheimer disease”[All Fields] OR “alzheimer”[All Fields]) AND (“micrornas”[MeSH Terms] OR “micrornas”[All Fields] OR “microrna”[All Fields]) AND (“2001/01/01”[PDAT]:“2013/7/4”[PDAT]). Manual inspection leads to 184 miRNA-gene relations (Table 8) in 39 abstracts.\n\nComparison of miRNA-gene relations retrieval for Alzheimer’s disease in MEDLINE.\n\nThe found relations are compared with the content of miRSel. Among the 37 abstracts from the PubMed query, miRSel contained only 12 abstracts with 56 miRNA-gene relations (cf. Table 8). False negatives in our approach when compared with miRSel could not be directly identified as the database is not downloadable and searchable for disease specific relations. However, low intersection between miRSel and NERTri can be observed.\n\nIn summary, our approach provides AD related gene-microRNA relations from PubMed which have not been available in the database before.\n\nOverall, the results are promising when compared with the miR2Disease and miRSel databases and indicate that we can extend the databases to a large extent with new relations. Such an approach makes it much easier to keep databases up to date. Nevertheless full text processing would most certainly increase the recall of automatic processing.\n\n\nConclusion\n\nIn this work, we proposed approaches for identification of named entities of classes diseases and genes/proteins and relations of those entities with miRNAs, from biomedical literature. Distinguishing between two types of miRNA mentions has enabled us to achieve better recall and precision in document retrieval and relations identification. Three different relation extraction approaches are compared, showing that the tri-occurrence based approach should be the first reliable choice among all others. The tri-occurrence based approach is comparable to a machine learning-based method but considerably faster. In comparison to two well established databases, we have shown that additional useful information can be extracted from MEDLINE using our proposed methods.\n\nTo the best of our knowledge, this is the first work where manually annotated corpora containing information about miRNAs and miRNA-relations is published. Moreover, the corpora and methods provided represent useful basis and tools for extracting the information about miRNAs-associations from literature. This work serves as an important benchmark for current and future approaches in automatic identification of miRNA relations. It provides the basis for building a knowledge-based approach to model regulatory networks for identification of deregulated miRNAs and genes/proteins.\n\n\nData availability\n\nCorpora availability: http://www.scai.fraunhofer.de/mirnacorpora.html\n\nArchived corpora at time of publication: F1000Research: Dataset 1. Manually annotated miRNA-disease and miRNA-gene interaction corpora, 10.5256/f1000research.4591.d3463942",
"appendix": "Author contributions\n\n\n\nSB, RK, JF, and MHA conceived and designed the overall research strategy. SB carried out all the development work and performed the analysis. She is the major contributor of manuscript preparation and principal annotator. TB developed the machine learning-based workflow for relation extraction, transformed corpora into the standard format, and contributed to manuscript writing. JF supported in use-case analysis and paper writing. MHA is the scientific supervisor for this work. RK contributed to critical discussions, analysed the results and a major contributor in correcting and writing manuscript. All authors read and approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nShweta Bagewadi was supported by University of Bonn. Tamara Bobić was partially funded by the Bonn-Aachen International Center for Information Technology (B-IT) Research School during her contribution to this work at Fraunhofer SCAI.\n\n\nAcknowledgements\n\nWe would like to thank Heinz-Theo Mevissen for all the support during implementation of the dictionaries and regular expressions in ProMiner. We acknowledge Anandhi Iyappan for her contribution as the second annotator. We are also grateful to Harsha Gurulingappa for all his support and fruitful discussions during this work. We would like to thank Ashutosh Malhotra for proof reading the manuscript.\n\n\nReferences\n\nLee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993; 75(5): 843–54. PubMed Abstract | Publisher Full Text\n\nBartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004; 116(2): 281–297. 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In Proceedings of the 2006 Conference of the North American Chapter of the Association for Computational Linguistics on Human Language Technology: companion volume: demonstrations. New York, Association for Computational Linguistics. 2006; 273–275. Publisher Full Text\n\nGennari JH, Musen MA, Fergerson RW, et al.: The evolution of Protégé: an environment for knowledge-based systems development. Int J Hum Comput Stud. 2003; 58(1): 89–123. Publisher Full Text\n\nOualline S: Vi iMproved. New Riders Publishing, Thousand Oaks, CA, USA, 2001. Reference Source\n\nBrown EG, Wood L, Wood S: The medical dictionary for regulatory activities (MedDRA). Drug Saf. 1999; 20(2): 109–17. PubMed Abstract | Publisher Full Text\n\nFluck J, Mevissen HT, Oster M, et al.: ProMiner: Recognition of Human Gene and Protein Names using regularly updated Dictionaries. In Proceedings of the Second BioCreative Challenge Evaluation Workshop, Madrid, Spain. 2007; 149–151. 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Publisher Full Text\n\nYu H, Qian L, Zhou G, et al.: Extracting protein-protein interaction from biomedical text using additional shallow parsing information. In Biomedical Engineering and Informatics, 2009. BMEI ’09. 2nd International Conference on, 2009; 1–5. Publisher Full Text\n\nYang Z, Lin H, Li Y: BioPPISVMExtractor: a protein-protein interaction extractor for biomedical literature using svm and rich feature sets. J Biomed Inform. 2010; 43(1): 88–96. PubMed Abstract | Publisher Full Text\n\nDe Marneffe MC, Manning CD: Stanford typed dependencies manual. 2010. Reference Source\n\nBunescu RC, Mooney RJ: A shortest path dependency kernel for relation extraction. In Proceedings of the conference on Human Language Technology and Empirical Methods in Natural Language Processing, Association for Computational Linguistics. HLT ’05, Stroudsburg, PA, USA, 2005; 724–731. Publisher Full Text\n\nThies W, Bleiler L, Alzheimer’s Association: 2011 Alzheimer’s disease facts and figures. Alzheimers Dement. 2011; 7(2): 208–244. PubMed Abstract | Publisher Full Text\n\nCheng L, Quek C, Sun X, et al.: Deep-sequencing of microRNA associated with Alzheimer’s disease in biological fluids: From biomarker discovery to diagnostic practice. Frontiers in Genetics. 2013; 4(150).\n\nHébert SS, Horré K, Nicolaï L, et al.: Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/beta-secretase expression. Proc Nat Acad Sci U S A. 2008; 105(17): 6415–6420. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBagewadi S, Bobi T, Hofmann-Apitius M, et al.: Dataset 1. manually annotated miRNA-disease and miRNA-gene interaction corpora. F1000Research. Data Source"
}
|
[
{
"id": "5973",
"date": "29 Aug 2014",
"name": "Sofie Van Landeghem",
"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 manuscript presents a manually annotated corpus of miRNA entities, genes/proteins and diseases, as well as the relations between them. The authors have used this dataset to develop NER and relation detection tools, which perform quite well in terms of precision, recall and F-score. The resulting miRNA relations detected automatically in Medline can be useful to extend existing databases with reliable literature information. All datasets are made freely available.This research domain is highly relevant and it is great seeing text mining efforts focus specifically on miRNAs and their relation with diseases. The creation of a manually annotated training set certainly helps to advance this field. Two unfortunate design choices are the limitation to abstracts, even though so much data is readily available in PMC OA, and the missing support for cross-sentence relations, but these issues would definitely make for interesting future work.Major questions/remarksHow exactly are candidate instances, relations and relation triggers defined? During manual annotation, can their be relations annotated without relation triggers or does this never occur? And what do relation triggers look like if they're not part of a relation? (cf. Table 2). Finally, how would the ML method perform if you would give it only tri-occurrence-based candidate instances?I was hoping to see more implementation/evaluation details, specifically concerning the differences between LibSVM, LibLINEAR and Naive Bayes. It is not even stated which of these methods performed best.How exactly are detected entities resolved to unique miRNA names? Because no details are given, can we assume that this step is not as complicated/ambiguous as gene name normalization for instance? Can database identifiers be retrieved for non-human cases? Do the evaluation results pertain to the textual symbols, or is the normalization to unique IDs also taken into account?Could the authors clarify how they have ensured that data from the test set was not used in any way to develop the regular expressions for NER? I find it striking that the F-scores to detect miRNA are higher on the test set, or is it simply the case that miRNA entities are expressed in a homogeneous fashion throughout literature?The evaluation set presented in Table 7 seems rather limited. Would it not be feasible to compare the newly presented methods on bigger datasets, such as those discussed in related work (miRCancer DB, Murray et al), or expand the scope of the evaluation beyond Alzheimer's disease? Additionally, why are only 100 abstracts retrieved for Alzheimer? Is this because the evaluation is done (partly) manually?Minor questionsWhy are species mentions restricted to those occurring in miRBase, and why are only human-specific prefixes defined in the regular expression?Is the gene name dictionary built for human genes only, for the miRBase species only, or all?How useful are the non-specific miRNA mentions? I could see their value in trying to resolve co-reference relations across sentences, but this does not seem to be the aim in this study. In this sense, I find this statement puzzling: \"Distinguishing between two types of miRNA mentions has enabled us to achieve better recall and precision in document retrieval and relations identification\".Was Table 1 constructed using exact string matching? It would be interesting to see both numbers for stringent criteria as well as those for allowing partial mis-matches (e.g. slightly different entity span).How can there be 39 articles with relations if the query only returned 37? (Table 8 + surrounding text)I was expecting the four last rows of Table 2 to add up to the same number as the \"positive entity pairs\" number in Table 3?How were the 41 abstracts in the second section of \"use case analysis\" selected? Was there not more information to be found in Medline?Minor writing commentsI don't see the need to \"normalize\" the number of miRNA publications, multiple Y-axis tend to complicate data plots. Personally, I would use the same (logarithmic) scaleI would make a more obvious distinction between the manual curation efforts and the development of the NER and relation detection tools, for instance by placing the first 3 sections of Methods in a different \"Data curation\" section.Second to last sentence of the \"Motivation\" paragraph in the abstract: \"regular expression\" should be plural\"Relation extraction paragraph\": \"an automated entity recognizers\"\"classes diseases\" in Conclusion paragraphI had trouble reading/understanding this sentence: \"Boundary matches result for the same reported 0.88 of F1\"The claim that 11.5% of miRNA relations are across sentences should be justified by a citation. Further, I personally think this is a significant portion, and wouldn't use the phrase \"only 11.5%\".",
"responses": [
{
"c_id": "1080",
"date": "13 Nov 2014",
"name": "Roman Klinger",
"role": "Author Response",
"response": "We thank the referee for the detailed comments. We will address them and then submit a new version together with detailed comments."
},
{
"c_id": "1111",
"date": "23 Dec 2014",
"name": "Shweta Bagewadi",
"role": "Author Response",
"response": "COMMENT: \"This research domain is highly relevant and it is great seeing text mining efforts focus specifically on miRNAs and their relation with diseases. The creation of a manually annotated training set certainly helps to advance this field. Two unfortunate design choices are the limitation to abstracts, even though so much data is readily available in PMC OA, and the missing support for cross-sentence relations, but these issues would definitely make for interesting future work.\"RESPONSE: As rightly pointed out by the reviewers, we agree with the limitation of our design choice. We also agree that it makes an interesting future work. However, as stated by Shah et al.(Shah, Perez-Iratxeta, Bork, & Andrade, 2003) abstracts provides the best proportion of keywords for extracting biological information. We have included the below text in Corpus selection, annotation and properties sub-section of Methods explaining the choice:Shah et al.27 showed that abstracts provide a comprehensive description of key results obtained from a study, whereas full text is a better source for biological relevant data. Thus, we choose to build the corpus for abstracts only. Additional text has been included in Conclusion and future work section:The proposed methods encourage future work of implementing the same for full-text articles to elucidate many more relations from Biomedical literature. Non-specific miRNA mentions identification could prove highly beneficial for co-reference resolution in full-text articles, in addition to abstracts.We are currently working on a more robust miRNA-relation identification workflow implemented in UIMA framework and JAVA for full text. We will include the co-reference resolution approach once the complete workflow has been validated. However, this manuscript is our first attempt to detect miRNA relations and to use them in a bigger integrative modeling approach, NeuroRDF, we recently developed, please refer to Iyappan et al. (Iyappan, Bagewadi, Page, Hofmann-Apitius, & Senger, 2014).Major questions/remarksCOMMENT: \"How exactly are candidate instances, relations and relation triggers defined?\"RESPONSE: Description for candidate instances, and relation trigger is provided in Named entities annotation sub-section in Methods. The describing text is provided below:“Mentions of miRNAs consisting of keywords (case-insensitive and not containing any suffixed numerical identifier) such as “Micro-RNAs” or “miRs” are annotated as N on-S pecific miRNA. Names of particular miRNAs such as miRNA-101, suffixed with numerical identifiers are labeled as S pecific miRNA. Numerical identifiers (separated by delimiters such as “,”, “/”, and “and”) occurring as part of specific miRNA mentions are annotated as a single entity. Mentions of disease names, disease abbreviations, signs, deficiencies, physiological dysfunction, disease symptoms, disorders, abnormalities, or organ damages are annotated as Disease. Possessive terms such as “ Diabetic patients” are not marked. Mentions referring to proteins/genes which are either single word ( e.g. “trypsin”), multi-word, gene symbols ( e.g. “SMN”), or complex names (including of hyphens, slashes, Greek letters, Roman or Arabic numerals) are annotated as Gene/Protein. Only those organisms that are having published miRNA sequences and annotations represented in miRBase database are labeled as Species. Any verb, noun, verb phrase, or noun phrase associating miRNA mention to either labeled disease or gene/protein term is annotated as Relation Trigger.” For description of relations, please look into Relations annotation sub-section of Methods. The text available in manuscript is provided below:“We restrict the relationship extraction to sentence level and four different interacting entity pairs: S pecific miRNA-D isease (SpMiR-D), S pecific miRNA-G ene/P rotein (SpMiR-GP), N on-S pecific miRNA-D isease (NonSpMiR-D), and N on-S pecific miRNA-G ene/P rotein (NonSpMiR-GP). Relevant triples, an interacting pair co-occurring with a R elation T rigger are defined to form a relation and can belong to one of the four above mentioned Relation classes.”COMMENT: \"During manual annotation, can their be relations annotated without relation triggers or does this never occur? And what do relation triggers look like if they're not part of a relation? (cf. Table 2).\"RESPONSE: The relations are defined as a tri-occurrence, where two entities (in our case miRNA-genes/proteins or miRNA-disease) co-occur along with a relation trigger term in a single sentence. During manual annotations we considered only those sentences where two entities co-occur with a relation trigger as relation. If there occurred a sentence where miRNA and gene/proteins entity appeared but without a relation trigger, we did not tag them as relations. Thus, relations without trigger term never occur.Here is an example of relation trigger (target), which does not participate in any relation (from PubMED ID: 21346322):“As single miRNAs are often predicted to target up to hundreds of individual transcripts, miRNAs are able to broadly affect the overall protein expression state of the cell.”In the above sentence there are two Non-specific miRNA entities along with one Relation trigger term. Since we do not have the second entity mention, such as genes/proteins or diseases, we do not tag this sentence as a relation instance. However, the above-mentioned named entities are tagged to their respective classes.Considering the reviewer’s comment we have modified the text in Relations annotation sub-section of Methods as follows:Relevant triples, an interacting pair (from one of the above-mentioned) co-occurring with a RELATION TRIGGER in a sentence is defined to form a relation and can belong to one of the four above-mentioned Relation classes. On the contrary, if an interacting pair does not co-occur with any RELATION TRIGGER then we do not tag such pair as a relation.COMMENT: \"Finally, how would the ML method perform if you would give it only tri-occurrence-based candidate instances?\"RESPONSE: We are not fully sure what is meant with this comment. Does the reviewer mean to only provide positive instances for training? In that case, a one-class SVM could be used, but we do not see a reason to believe that this could provide improved performance over a two-class SVM. If the reviewer means to only use features based on the tri-occurrence, this would provide another baseline for the machine learning approach.COMMENT: \"I was hoping to see more implementation/evaluation details, specifically concerning the differences between LibSVM, LibLINEAR and Naive Bayes. It is not even stated which of these methods performed best.\"RESPONSE: We would like to thank the reviewer for interest in more detailed results. We have now included the result of the other machine learning methods in Figure 2. Additionally, text discussing these results have been included in Relation extraction sub-section in Results and Discussion as below:Using the machine-learning based classification, precision is increased up to 76% for specific miRNA-gene relations for both LibLINEAR and LibSVM methods, although Naïve Bayes is not far behind. Similarly, these two methods performed nearly the same for specific miRNAs-disease relations, the F 1 measure is not substantially different but a trade-off between precision and recall can be observed. An increase in F 1 measure is observed for non-specific miRNA relations when Naïve Bayes method is applied, out performing other strategies. Nevertheless, preference of the method highly depends on the compromise one chooses, whether better recall or precision. Overall, better recall and acceptable precision can be achieved with tri-occurrence method. COMMENT: \"How exactly are detected entities resolved to unique miRNA names? Because no details are given, can we assume that this step is not as complicated/ambiguous as gene name normalization for instance? Can database identifiers be retrieved for non-human cases? Do the evaluation results pertain to the textual symbols, or is the normalization to unique IDs also taken into account?\"RESPONSE: The detected miRNA entities have been resolved using the mirMaid REST service (described in “Named Entity Recognition Section”). Normalization of miRNA names is not as complicated as gene names since the authors follow naming scheme that can be captured with good regular expressions. However, the major challenge what we see is resolving the miRNA names to the right species. We have used species dictionary to support non-human miRNA normalization. The evaluation results are done using the normalized names as given in miRBase. In addition, we can make the miRBase IDs also available. Considering the reviewer’s comment we have included text which explains the normalization step in detail, given below:Detected entities are resolved to a unique miRNA name and disambiguated to adhere to standard naming conventions. Each identified miRNA entity has been resolved to its base form ( e. g. hsa-microRNA-21 to hsa-mir-21 and microRNA 101 to mir-101). Manual inspection of the test corpus for species distribution revealed that 71% of the documents belonged to human, followed by mouse (15%), rat (8%). Pig has 2 abstracts, zebrafish, HIV-1, HSV-1, and Caenorhabditis elegans 1 each (cf. Supplementary Figure A for the distribution). Thus, we assumed that most of the abstracts belonged to human and resolved the identified miRNA entities to human identifier in miRBase. Unique miRNA terms are mapped to human miRBase database identifiers through the mirMaid Restful web service. For those names where we do not retrieve any database identifiers, we fall back to another organism mention found in the abstract (if any), using the NCBI taxonomy dictionary (see below) (cf. Supplementary Figure B), otherwise we retain the unique normalized name (cf. Box 2).MIR0000007:MIMAT0015092@MIRBASE|MI0000002@MIRBASE|cel-lin-4|lin-4MIR0000008: miR-171|microRNA 171MIR0000005:MIMAT0000416@MIRBASE|has-miR-1|miRNA-1Box 2: Represents the un-normalized and normalized entities that are mapped to miRBase identiers. Here MIR0000007, MIR0000008, and MIR0000005 are internal identifiers used by ProMiner. COMMENT: \"Could the authors clarify how they have ensured that data from the test set was not used in any way to develop the regular expressions for NER? I find it striking that the F-scores to detect miRNA are higher on the test set, or is it simply the case that miRNA entities are expressed in a homogeneous fashion throughout literature?\"RESPONSE: We first randomly retrieved 300 abstracts from the PubMed using the keyword “miRNA”. From these we manually selected 301 abstracts that contain gene/proteins or disease terms without looking in detail for any relation term. We randomly split these in train (201) and test (100) set (described in Corpus selection, annotation and properties section) before the annotation step. Thus, we are confident that we have not used test set for development of regular expression. The miRNA naming convention has been in effect quite early after discovery of miRNAs. This has led to uniform naming usage relative to genes/proteins. In our experience, from manual annotation, we can conclude that most authors follow one of the patterns, among a set of naming schemes, to mention miRNAs in publications. Our regular expression pattern has been developed to be robust enough to capture all these patterns. Since, the test corpus is smaller than the training set (we built our regular expression on training set) we expect the performance to be better when a set of common naming schemes is followed for mentioning miRNAs.COMMENT: \"The presented in Table 7 seems rather limited. Would it not be feasible to compare the newly presented methods on bigger datasets, such as those discussed in related work (miRCancer DB, Murray et al), or expand the scope of the evaluation beyond Alzheimer's disease?\"RESPONSE: We agree with the reviewers that the evaluation set in Table 7 is limited, but currently this is the only database where manually miRNA-disease relations are available. Also, another reason could be that the database has not been updated since April 2008.We appreciate the suggestion of the reviewer for extending the validation to other datasets, but our department is heavily working within the field of Neurodegeneration, we would like to limit ourselves to diseases that fall into this domain. COMMENT: Additionally, why are only 100 abstracts retrieved for Alzheimer? Is this because the evaluation is done (partly) manually?RESPONSE: Using the keyword search (described in “Extraction of miRNA-gene relations for Alzheimer’s diseases from full MEDLINE” section) we retrieved 124 abstracts. Yes, since we manually selected relevant abstracts among these, we retained only 100 abstracts.Minor questionsCOMMENT: Why are species mentions restricted to those occurring in miRBase, and why are only human-specific prefixes defined in the regular expression?RESPONSE: miRBase is the primary database that publishes miRNA sequences and annotations. miRBase registry assigns unique names to all miRNAs for publication (just like HUGO for gene names). This database has published a list of species for which miRNA sequences have been identified, which means only for these organisms miRNAs have been discovered as of today. Thus, we restrict ourselves to miRBase-listed organisms.We currently developed the proposed workflow to primarily capture human miRNA relations since we have curated diseases and gene/proteins dictionary only for humans. However, even if the miRNA mentions are given for other organism such as “cel-lin-4” our method captures “lin-4” which is later resolved to other organisms during the normalization process. We used human-specific prefixes in our regular expression for simplicity and instant capture of human related miRNAs. Also, we included other unique prefixes “lin-4” and “let-7” since these were the first miRNAs identified in nematodes even before the naming convention was in place. However this can be easily extended to other organisms using the three-letter code provided by miRBase. COMMENT: \"Is the gene name dictionary built for human genes only, for the miRBase species only, or all?\"RESPONSE: For the current manuscript preparation we have used gene/proteins dictionary that has been built for human only. However, we plan to extend this to other miRBase species in future.COMMENT: \"How useful are the non-specific miRNA mentions? I could see their value in trying to resolve co-reference relations across sentences, but this does not seem to be the aim in this study. In this sense, I find this statement puzzling: \"Distinguishing between two types of miRNA mentions has enabled us to achieve better recall and precision in document retrieval and relations identification\".\"RESPONSE: The reviewer has aptly pointed out the interesting future work we planned to implement. Considering the reviewer’s comment, we have improved the description in the pointed text to follows:In this work, we proposed approaches for identification of relations between miRNAs and other named entities such as diseases, and genes/proteins from biomedical literature. In addition, details of named entity recognition for all the above entity classes have been described. We distinguished two types of miRNA mentions, namely Specific (with numerical identifiers) and Non-Specific (without numerical identifiers). Non-specific miRNAs entity recognition has enabled us to achieve better recall and precision in document retrieval.The proposed methods encourage future work of implementing the same for full-text articles to elucidate many more relations from Biomedical literature. Non-specific miRNA mentions identification could prove highly beneficial for co-reference resolution in full-text articles, in addition to abstracts. Extending the current approach to other model organisms such as mouse, and rat can help in revealing important relations for translational research. Inclusion of additional named entities such as drugs, pathways, etc. could lead to an interesting approach for detection of putative therapeutic or diagnostic drug targets through a gene-regulatory network generated from identified relations. COMMENT: \"Was Table 1 constructed using exact string matching? It would be interesting to see both numbers for stringent criteria as well as those for allowing partial mis-matches (e.g. slightly different entity span).\"RESPONSE: Yes, Table 1 represents the exact string match results. As requested by the reviewer, we have now included results for partial mis-matches (called as partial match) in Table 1. Additionally, we have included discussion text to the pointed section as follows:Table 1 provides the inter-annotator agreement (measured as F 1, for both exact and boundary match, and Cohen’s κ) for the test corpus. Exact string match occurs only when both the annotators annotate identical strings, whereas in partial match fraction of the string has been annotated by either of the annotators.It is evident (cf. Table 1) that in almost all cases partial match performs better than exact string match, indicating variations in span of mentioned entities. COMMENT: \"How can there be 39 articles with relations if the query only returned 37? (Table 8 + surrounding text)\"RESPONSE: We thank the reviewer for pointing out the error. The typo error has been corrected in the revised manuscript. The correct number of articles has been re-checked for 37 articles.COMMENT: \"I was expecting the four last rows of Table 2 to add up to the same number as the \"positive entity pairs\" number in Table 3?\"RESPONSE: We thank the reviewer for pointing out the mistake in the statistics. We have now re-checked the statistics using the published corpus and have updated the manuscript with correct statistics along with the README file in our website (http://www.scai.fraunhofer.de/mirna-corpora.html).COMMENT: \"How were the 41 abstracts in the second section of \"use case analysis\" selected? Was there not more information to be found in Medline?\"RESPONSE: We applied NERTri approach (tri-occurrence based approach applied on the entities identified by ProMiner system) to retrieve 41 abstracts, which have miRNA-Alzheimer’s disease relations at sentence level. We did not identify any more relations than the provided (as of 4th July, 2013). However, we assume that there could be some false negatives occurring due to the error propagation from automated entity recognizers (cf. Relation extraction sub-section in Results for details). Also, relations that occur at document level could have been missed out since our current focus is more on sentence level relations.From another point of view, for last 25 years Alzheimer’s disease research has mainly focused on amyloid-beta deposits that lead to neuronal death and tangle formation. However, the amyloid hypothesis has not been successful in late-phase drug trials (Delay & Hébert, 2011; Golde, Schneider, & Koo, 2011). The focus of miRNAs research on Alzheimer’s is relatively new, there are in total 187 articles in PubMed in comparison to 1713 articles on miRNA research in Breast cancer (as of 25th Nov 2014). Thus, we can conclude that the number of articles related to miRNA-Alzheimer’s disease research is rather limited and relatively new.We have modified the pointed out text in the manuscript to the following for clarity:For comparison, we retrieved miRNA-disease relations from MEDLINE using NERTri approach, resulting in 41 abstracts containing 159 relations. Obtained triplets have been manually curated to remove 51 false positives. False negatives have not been accounted, which may result in loss of information (cf. Relation extraction section). Comparison between the relations obtained from miR2Disease and NERTri are summarized in Table 7. Minor writing commentsCOMMENT: \"I don't see the need to \"normalize\" the number of miRNA publications, multiple Y-axis tend to complicate data plots. Personally, I would use the same (logarithmic) scale\"RESPONSE: The growth of miRNA publications has been normalized using the number of articles published in PubMed for the given year. The decision to normalize values has been taken due to low number of articles for miRNA, e.g. 37 articles related to miRNA were published in comparison to 561169 articles in the whole MEDLINE for the year 2002. Thus, we would prefer to keep the scale as it is. We addressed that comment and are now using only one axis in the depiction.COMMENT: \"I would make a more obvious distinction between the manual curation efforts and the development of the NER and relation detection tools, for instance by placing the first 3 sections of Methods in a different \"Data curation\" section.\"RESPONSE: We thank the reviewer for her suggestion. We have now moved the first three sections of Methods under “Data curation and corpus selection” subsection. Hope this is inline with what the reviewer had in mind during the suggestion.COMMENT: \"Second to last sentence of the \"Motivation\" paragraph in the abstract: \"regular expression\" should be plural\"RESPONSE: As rightly pointed out by the reviewer, we have corrected the above text in the manuscript.COMMENT: \"Relation extraction paragraph\": \"an automated entity recognizers\"RESPONSE: We thank the reviewer for the suggestion. However, “relation extraction” has an entirely different meaning when compared to \"an automated entity recognizers\". We assume that the reviewer meant to change the “Named entity recognition” paragraph title. We appreciate the suggestion and have now changed the “Named entity recognition” paragraph title to “Automated named entity recognition”. We hope this is in agreement with the reviewer.COMMENT: \"\"classes diseases\" in Conclusion paragraph\"RESPONSE: As rightly pointed out by the reviewer, we have corrected the above text in the manuscript.COMMENT: \"I had trouble reading/understanding this sentence: \"Boundary matches result for the same reported 0.88 of F1\"\"RESPONSE: The “boundary matches” refers to “partial matches”. For clarity we have now modified the text and included reference to the provided supplementary file as below:Partial matches result for the same reported 0.88 of F 1, providing the possibility for detection of similar text strings for better recall (cf. Supplementary Table B). COMMENT: \"The claim that 11.5% of miRNA relations are across sentences should be justified by a citation. Further, I personally think this is a significant portion, and wouldn't use the phrase \"only 11.5%\".\"RESPONSE: We have now included text describing the approach through which we obtained the statistic, shown below:We queried MEDLINE for “miRNA and Epilepsy” documents, among which 16 documents containing miRNA-related relations were manually selected. To avoid any biased approach we choose Epilepsy disease domain. Manual inspection of these articles revealed 11.5% of miRNA-related associations occur outside the sentence level. Thus, our work focused on relations at sentence level. REFERENCES:Delay, C., & Hébert, S. S. (2011). MicroRNAs and Alzheimer’s Disease Mouse Models: Current Insights and Future Research Avenues. International journal of Alzheimer’s disease, 2011, 894938. doi:10.4061/2011/894938Golde, T. E., Schneider, L. S., & Koo, E. H. (2011). Anti-Aβ therapeutics in Alzheimer’s disease: The Need for a Paradigm Shift. Neuron, 69(2), 203–213. doi:10.1016/j.neuron.2011.01.002.Anti-AIyappan, A., Bagewadi, S., Page, M., Hofmann-Apitius, M., & Senger, P. (2014). NeuroRDF : Semantic Data Integration Strategies for Modeling Neurodegenerative Diseases. Proceedings of the 6th International Symposium on Semantic Mining in Biomedicine (SMBM2014) (pp. 11–18). Aveiro, Portugal.Shah, P. K., Perez-Iratxeta, C., Bork, P., & Andrade, M. A. (2003). Information extraction from full text scientific articles: where are the keywords? BMC bioinformatics, 4, 20. doi:10.1186/1471-2105-4-20"
}
]
},
{
"id": "5975",
"date": "17 Oct 2014",
"name": "Filip Ginter",
"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 paper presents an annotated corpus of miRNA, gene/protein, disease, and species mentions, together with their miRNA-specific relations. Further, the authors implement a simple dictionary-based method for their extraction from text. This is a little studied, but highly relevant text mining target. Numerically, the results look rather promising. The paper is relatively easy to follow, but could be expanded somewhat to be more self-contained. More about that later.The main problem I have when reading the paper is that it does not give me a good intuitive insight into how difficult this problem actually is. This relates to several individual passages in the text that left me wondering whether I understood correctly:As for miRNA detection: On page 4, second paragraph, the authors mention that a prior study achieved 100% accuracy on miRNA detection task. Not being told more details, this either means the task is trivial, or that experiment is flawed. As for relation detection: On page 7, first paragraph, the authors mention that moving from occurrence to tri-occurrence \"does not diminish recall\" which in that context is 100%. The way I understand this is that each and every positive sentence in the test data does have a relation trigger which is present also present in the training data. Is that really possible? That would seem to disagree with Table 6. How can you add a trigger filter but keep 100% recall?I think the reader would benefit from more discussion on the variance of miRNA names and trigger expressions, to gain an intuitive grasp of the difficulty of the task. Which leads me to the regular expressions described in tables 4 and 5. Specifically, I do not understand the alias Let and Lin. Are these individual miRNAs? Why would you want to define a re-useable alias for individual miRNAs? Alternatively, I am misunderstanding something here, in which case I would appreciate clarification: what are these alias symbols, and how do I know they generalize? The impression I get from the regular expressions is that miRNA naming is highly regular and very simple. Is that the case really?In the results and discussion section, I am perplexed by the 0.79 vs 0.69 F-score train/test difference for disease mentions. Do you have an insight as to why specifically disease mentions would have such a major difference when the other entities do not? This is especially puzzling since Table 2 shows disease as the largest class, i.e. it should exhibit least noise in the results.Some other not so major points:I don't know what is meant by \"improvised framework\" in the abstract.I am not sure what is the status of relations that do not have a trigger. Or are there such?Where does the number 11.5% of cross-sentence relations come from? Counting in the corpus? (just checking)The previous point about 11.5% of cross-sentence relations makes me then wonder how can the co-occurrence approach reach 100% recall? Have these cross-sentence relations been simply deleted from the data?Towards the end of the methods section, the paper describes the use of \"deep parsing\". Through the citation I'm guessing this relates to Stanford Dependencies. I think the paper really should give some detail about how this parsing was done.",
"responses": [
{
"c_id": "1079",
"date": "13 Nov 2014",
"name": "Roman Klinger",
"role": "Author Response",
"response": "We thank the referee for the detailed comments. We will address them and then submit a new version together with detailed comments."
},
{
"c_id": "1112",
"date": "23 Dec 2014",
"name": "Shweta Bagewadi",
"role": "Author Response",
"response": "COMMENT: \"The main problem I have when reading the paper is that it does not give me a good intuitive insight into how difficult this problem actually is. This relates to several individual passages in the text that left me wondering whether I understood correctly:\"RESPONSE: Considering reviewer’s concern, we have now included the below text in Related Work sub-section in Introduction:Since the miRNA naming convention has been formalized very early in comparison to other biological entities such as genes and proteins, applying text-mining approaches is relatively simple17. Thus, most of the previously applied text mining approaches for miRNA detection has been based on keywords. miRCancer uses keywords to obtain abstracts from PubMed, further miRNA entities have been identified using regular expressions based on prefix and suffix variations. Similarly, miRWalk database uses keyword search approach to download abstracts and applies curated dictionary (compiled from six databases) for miRNA identification of human, rat, and mouse species24. TarBase, miR2Disease, miRTarBase, and several others have followed related search strategies. However, several authors still tend to use naming variations for acronyms, abbreviations, nested representations, etc. for listing miRNAs. Additionally, in contrast to the previous text-mining approaches focusing purely on miRNA gene relations, we extend the information extraction approach additionally to retrieve miRNA-disease relations.COMMENT: \"As for miRNA detection: On page 4, second paragraph, the authors mention that a prior study achieved 100% accuracy on miRNA detection task. Not being told more details, this either means the task is trivial, or that experiment is flawed.\"RESPONSE: As described by Murray et al., they developed a regular expression for miRNA identification using the stems identified by term frequency analysis (“miR”, “mirn”, “mirna”, and “microRNA”) and later optimized it to attain 100% accuracy and recall against miRBase. However, the procedure is not fully clear to us. Below we provide the exact text as mentioned in Murray et al.“Identifying miRNAs Using the stems identified by term-frequency analysis (‘‘miR’’, ‘‘mirn’’, ‘‘mirna’’ and ‘‘microRNA’’), we developed regular expression patterns to identify novel miRNA terms. Regular expressions were optimized to achieve 100% accuracy and recall against miRBase.9–11 Regular expressions were designed to identify novel miRNas with, and without, species identifiers (e.g. hsa-miR-1 and mir-1). Regular expressions were used to identify novel miRNAs by mining the entire National Library of Medicine’s PubMed abstract collection. All identified miRNas were curated into preferred terms to encompass synonymic variants; false positive hits were identified and filtered out during this process.” The authors’s do not however validate their regular expression against a larger corpus.Identification of miRNA mentions could be trivial if the variants are captured in a single regular expression. This issue we have addressed in our manuscript. The more challenging task is normalizing the miRNA mentions to correct database identifier.We have modified the pointed out text to the following:The authors claim to have optimized the approach to reach 100% accuracy and recall for detecting miRNAs mentions as in miRBase.COMMENT: \"As for relation detection: On page 7, first paragraph, the authors mention that moving from occurrence to tri-occurrence \"does not diminish recall\" which in that context is 100%. The way I understand this is that each and every positive sentence in the test data does have a relation trigger which is present also present in the training data. Is that really possible? That would seem to disagree with Table 6. How can you add a trigger filter but keep 100% recall?\"RESPONSE: We describe a relation as a tri-occurrence (non-specific miRNAs- relation trigger -gene/proteins or disease). As rightly pointed out by the reviewer, the relation is true only when a relation trigger is present. We agree with the reviewer that this is not completely correct and the sentence could be mis-leading. The tri-occurrence approach cannot be 100% as there could be some entities that could be missed out during the automated named entity recognition. On the other hand, we assume that the entity recognition reaches 100% and all the relations (entity pair with a relation trigger) are retained in tri-occurrence. Thus, the assumption that there is no diminish in recall. We have corrected the corresponding text in out revised manuscript for clarity, as given below:The recall is not diminished using the tri-occurrence approach as the true entity pairs remain constant, while the precision increases between 4pp (percentage points) and 17pp when compared to the co-occurrence based approach, reducing false positives (cf. Figure 2).In our work, we assume that all the entities have been identified giving a recall of 100% for both co-occurrence and tri-occurrence based approaches.In Relation to the extraction sub-section of Results and Discussion we have already tried to address this issue in the following text:“Most relation extraction approaches are dependent on the performance of named entity recognition. The impact of error propagation coming from automated entity recognizers is evaluated by applying the tri-occurrence method on the automatically annotated training and test corpus, here termed as “NERTri”. Compared to the results on the gold standard entity annotation a drop of 13 pp for NonSpMiR-D, 7pp for NonSpMiR-GP, 22pp for SpMiR-D, and 30pp for SpMiR-GP in F 1 is observed for the test corpus.”COMMENT: \"I think the reader would benefit from more discussion on the variance of miRNA names and trigger expressions, to gain an intuitive grasp of the difficulty of the task. Which leads me to the regular expressions described in tables 4 and 5. Specifically, I do not understand the alias Let and Lin. Are these individual miRNAs? Why would you want to define a re-useable alias for individual miRNAs? Alternatively, I am misunderstanding something here, in which case I would appreciate clarification: what are these alias symbols, and how do I know they generalize? The impression I get from the regular expressions is that miRNA naming is highly regular and very simple. Is that the case really?\"RESPONSE: Considering the reviewer’s comment, we have now modified the description in Automated entity recognizers sub-section in methods to:Detected entities are resolved to a unique miRNA name and disambiguated to adhere to standard naming conventions as authors use several morphological variants to report the same miRNA term. For example, miR-107 can be represented as miRNA-107, MicroRNA-107, MicroRNA 107, has-mir-107, mir-107/108, micro RNA 107 and 108, micro RNA (miR) 107 and so on. Thus, the identified miRNA entity has been resolved to its base f form ( e. g. hsa-microRNA-21 to hsa-mir-21 and microRNA 101 to mir-101) following the miRBase naming convention.The relation trigger dictionary comprises of all interaction terms from the training corpus. After reviewing the training corpus for relation trigger terms, we retrieved not one but many variants of the same relation trigger occurring in alternative verb-phrase groups. For example, “change in expression” can be represented in one of the following verb-phrases: Change MicroRNA-21 Expression, Expression of caveolin-1 was changed, Change in high levels of high-mobility group A2 expression, change of the let-7e and miR-23a/b expression, expression of miR-199b-5p in the non-metastatic cases was significantly changed, etc. To allow flexibility for capturing relation trigger along with its variants spanning over different phrase length, we first manually represented all the relations in its root form, such as “regulate expression” to “regulate” (cf. Relation_Dictionary.txt file in supplementary). The base form has been extended manually to different spelling variants, e.g. regulate to regulatory, regulation, etc., the detailed listing of variants is provided in Supplementary file Word_variations.txt. Not all combination of the root forms is logical; target and up-regulation terms cannot be combined to form a relation trigger. Thus, we additionally defined a set of relation combinations that are allowed (see Permutation_terms.txt in supplementary for all combinations).Lin and Let were the first known miRNAs, identified in nematode. Naming convention for miRNAs were later developed after their identification leading to recognition of microRNAs as a class of small regulatory molecules. The original names of “lin” and “let” are still used as is. Yes, Let and Lin are individual miRNA types/families. The regular expression can get very complicated when one wants to re-use. Thus, we tried to simplify it and split it into several small regular expressions to be used in bigger complex one. We have defined the aliases following a simple representation; the user can redefine the aliases if needed. Since the regular expression follows a standard representation (Oualline, 2001) we assume that it should be flexible to be adapted to different representations for implementations in other frameworks or programming languages. We partially agree with the reviewers that the miRNA naming is regular. However, the naming can have many variants in several combinations of our developed regular expressions capturing the variant descriptions in publications. We have tried our best to capture as many variants as possible. Also, we have improved the regular expression aliases for better understanding. COMMENT: \"In the results and discussion section, I am perplexed by the 0.79 vs 0.69 F-score train/test difference for disease mentions. Do you have an insight as to why specifically disease mentions would have such a major difference when the other entities do not? This is especially puzzling since Table 2 shows disease as the largest class, i.e. it should exhibit least noise in the results.\"RESPONSE: Disease mentions vary in the way they are represented. A disease entity can occur as multi-word with case variation and synonym combination, such as “Chronic Lymphocytic Leukemia” could be also represented as “Chronic Leukemia (lymphocytic)”. Resolving the acronyms for diseases can be tricky as well, for example “AD” could be resolved to “Alzheimer’s Disease” or “Atopic Dermatitis”. This leads to ambiguity during tagging of the disease entities. Nested disease names are common where abbreviations are represented within the disease name itself, e.g. “Alzheimer’s (AD) Disease”. Thus, there could be large difference in the way disease names are tagged in training and test corpus by our NERTri approach in comparison to manual annotation. The performance varied between these two corpora when considering the exact match. However, we report a performance of 0.88 of F 1 for both train and test corpus, showing that partial matches perform better in disease entity identification.Considering reviewer’s concern we have included a sentence in Performance evaluation of named entity recognition sub-section in results and Discussion:The low score for disease identification could be due to the variation in disease mentions, such as multi-word, synonym combination, nested names, etc.Some other not so major points: COMMENT: I don't know what is meant by \"improvised framework\" in the abstract.RESPONSE: We have improved the text in Abstract for clarity as shown below:Additionally, most of the published miRNA entity recognition methods are keyword based, further subjected to manual inspection for retrieval of relations. Despite the fact that several databases host miRNA-associations derived from text, lower sensitivity and lack published details for miRNA entity recognition and associated relations identification has motivated the need for developing comprehensive methods that are freely available for the scientific community. COMMENT: \"I am not sure what is the status of relations that do not have a trigger. Or are there such?\"RESPONSE: The relations are defined as a tri-occurrence, where two entities (in our case miRNA-genes/proteins or miRNA-disease) co-occur along with a relation trigger term in a single sentence. During manual annotations we considered only those sentences where two entities co-occur with a relation trigger as relation. If there occurred a sentence where miRNA and gene/proteins entity appeared but without a relation trigger, we did not tag them as relations. Thus, relations without trigger term never occur.Here is an example of relation trigger (target), which does not participate in any relation (from PubMED ID: 21346322):“As single miRNAs are often predicted to target up to hundreds of individual transcripts, miRNAs are able to broadly affect the overall protein expression state of the cell.” In the above sentence there are two Non-specific miRNA entities along with one Relation trigger term. Since we do not have the second entity mention, such as genes/proteins or diseases, we do not tag this sentence as a relation instance. However, the above-mentioned named entities are tagged to their respective classes.Considering the reviewer’s comment we have modified the text in Relations annotation sub-section of Methods as follows:Relevant triples, an interacting pair (from one of the above-mentioned) co-occurring with a RELATION TRIGGER in a sentence is defined to form a relation and can belong to one of the four above-mentioned Relation classes. On the contrary, if an interacting pair does not co-occur with any RELATION TRIGGER then we do not tag such pair as a relation. COMMENT: Where does the number 11.5% of cross-sentence relations come from? Counting in the corpus? (just checking)RESPONSE: We have now included text describing the approach through which we obtained the statistic, shown below:We queried MEDLINE for “miRNA and Epilepsy” documents, among which 16 documents containing miRNA-related relations were manually selected. Manual inspection of these articles revealed 11.5% of miRNA-related associations occur outside the sentence level. Thus, our work focused on relations at sentence level.COMMENT: The previous point about 11.5% of cross-sentence relations makes me then wonder how can the co-occurrence approach reach 100% recall? Have these cross-sentence relations been simply deleted from the data?RESPONSE: As pointed out by the reviewer, we have modified the pointed text as follows:If all the entities are correctly identified then co-occurrence based approach leads to 100% recall for relation extraction. The recall is not diminished using the tri-occurrence approach, as the true entity pairs remain constant, approach while the precision increases between 4pp (percentage points) and 17pp when compared to the co-occurrence based approach, reducing false positives (cf. Figure 2). However, overall the precision reaches less than 60%. In our work, we assume that all the entities have been identified giving a recall of 100% for both co-occurrence and tri-occurrence based approaches.In our work, we assume that all the entities have been identified giving a recall of 100% for both co-occurrence and tri-occurrence based approaches. The cross-sentence has not been deleted from data and is freely available for researcher’s to use the corpus and build a co-reference approach for the same.COMMENT: \"Towards the end of the methods section, the paper describes the use of \"deep parsing\". Through the citation I'm guessing this relates to Stanford Dependencies. I think the paper really should give some detail about how this parsing was done.\"RESPONSE: Yes, the “deep parsing” in the manuscript refers to Stanford Dependencies. The following pointed text has been modified in the manuscript:Next to lexical features, dependency parsing (created using Stanford parser) provides an insight into the entire grammatical structure of the sentence 37 and was performed using the Stanford CoreNLP library (http://nlp.stanford.edu/software/corenlp.shtml). Dependency parsing follows the shortest dependency path hypothesis38."
}
]
}
] | 1
|
https://f1000research.com/articles/3-205
|
https://f1000research.com/articles/4-930/v1
|
30 Sep 15
|
{
"type": "Review",
"title": "Cytotoxic granule secretion by lymphocytes and its link to immune homeostasis",
"authors": [
"Geneviève de Saint Basile",
"Fernando E. Sepulveda",
"Sophia Maschalidi",
"Alain Fischer",
"Fernando E. Sepulveda",
"Sophia Maschalidi",
"Alain Fischer"
],
"abstract": "The granule-dependent cytotoxic activity of T and natural killer lymphocytes has progressively emerged as an important effector pathway not only for host defence but also for immune regulation. The analysis of an early-onset, severe, primary immune dysregulatory syndrome known as hemophagocytic lymphohistiocytosis (HLH) has been decisive in highlighting this latter role and identifying key effectors on the basis of gene mutation analyses and mediators in the maturation and secretion of cytotoxic granules. Studies of cytotoxicity-deficient murine counterparts have helped to define primary HLH as a syndrome in which uncontrolled T-cell activation in response to lymphocytic choriomeningitis virus infection results in excessive macrophage activation and inflammation-associated cytopenia. Recent recognition of late-onset HLH, which occurs in a variety of settings, in association with hypomorphic, monoallelic mutations in genes encoding components of the granule-dependent cytotoxic pathway or even in the absence of such mutations has broadened our view about the mechanisms that underlie the perturbation of immune homeostasis. These findings have led to the development of a model in which disease occurs when a threshold is reached through the accumulation of genetic and environmental risk factors. Nevertheless, validation of this model will require further investigations.",
"keywords": [
"haemophagocytic lymphohistiocytosis",
"cytotoxic",
"natural killer lymphocytes"
],
"content": "Granule-dependent cytotoxic activity is a key regulator of immune homeostasis\n\nThe role of cytotoxic lymphocytes in defending the organism against virally infected cells and tumor cells has long been recognized. Through the polarized secretion of granules containing cytotoxic proteins, cytotoxic lymphocytes can rapidly kill their cognate target cells1. However, only recently have studies of inherited deficiencies of lymphocyte cytotoxic activity in humans highlighted the importance of lymphocyte cytotoxicity in the resolution of inflammation2.\n\nHemophagocytic lymphohistiocytosis (HLH) syndrome is a life-threatening immune dysregulation condition characterized by an excessive inflammatory response and hypercytokinemia. It is generally triggered by an infective agent, such as the members of the human herpes virus family. HLH manifests as the massive expansion and activation of polyclonal CD8+ T cells; this probably results from the failure of cytotoxic T lymphocyte (CTL) and natural killer (NK) cells to clear antigen-presenting cells (APCs) and therefore terminate an immune response3,4. Uncontrolled T-cell activation leads to macrophage activation, a pro-inflammatory cytokine storm, cytopenia, coagulopathy, multi-organ cellular infiltration, and organ dysfunction5–7. The link between cytotoxicity and lymphocyte homeostasis was first demonstrated 15 years ago, following the identification of perforin deficiency in a subgroup of patients with an inherited form of HLH (familial HLH, or FHL)2. Undoubtedly, this step was decisive in the characterization of the other causes of inherited HLH and in the identification of key effectors that mediate the exocytic machinery in cytotoxic lymphocytes8,9. Naturally occurring or engineered mice with a similar cytotoxicity defect have proven to be very useful tools for further understanding the underlying pathophysiological mechanism4,10,11. The observation that a relatively mild cytotoxic defect can be associated with defective immune surveillance or atypical HLH onset or both has now raised the question of the underlying individual’s risk factors that are associated with a subtle cytotoxic defect to drive disease onset.\n\n\nStudies of inherited defects of cytotoxicity have revealed critical effectors of cytotoxic granule exocytosis\n\nThe sequence of events by which T/NK cytotoxic lymphocytes kill targets is now fairly well characterized. When cytotoxic lymphocytes recognize their cognate target cells, they form a transient cellular conjugate and an immunological synapse (IS) at the area of cell-cell contact1,12. Within the very first minutes of target cell interaction, the actin network, which was previously positioned across the entire contact area, is progressively depleted from the center of the synapse, as recently highlighted by the use of rapid, super-resolution imaging methods13–15. The microtubule-organizing centre (MTOC) rapidly moves toward the target cell, while the cytotoxic granules (containing the cytotoxic proteins perforin and granzymes) migrate along microtubules and cluster around the MTOC8,16. At the IS, the centrosome can touch the membrane and then deliver polarized cytotoxic granules17. The granules fuse with the presynaptic membrane and secrete their contents into the synaptic cleft. This accurate, polarized secretion of lytic reagents ensures that cytotoxic cells destroy only the bound target cell and not bystander cells. Within the synaptic cleft, perforin oligomerizes, creates pores in the target cell membrane, and thus enables the pro-apoptotic granzymes to access the target cytosol18–20. The mechanism of granzyme uptake has long been subject to debate. By using time-lapse microscopy techniques that pinpoint the moment at which perforin permeabilizes the target cell plasma membrane within the IS, researchers observed that the time course of target cell apoptosis after pore formation is very rapid (that is, within 10 mins)21,22. In contrast to what has been proposed in other studies23–25, this suggests that granzymes cross the plasma membrane and are not taken up in endosomes.\n\nIn addition to perforin deficiency2, which accounts for about one third of the FHL cases, several inherited forms of HLH are characterized by failure to deliver cytotoxic granule contents. The identification of the underlying molecular causes has contributed to our understanding of the key steps in the secretion of cytotoxic granules at the IS8. Biallelic mutations in UNC13D (encoding Munc13-4, accounting for about one third of FHL cases), STX11 (encoding syntaxin 11, about 5% of FHL cases), and STXBP2 (encoding syntaxin-binding protein 2, also known as Munc18-2, about 20% of FHL cases) led to the occurrence of HLH in FHL types 3, 4, and 5, respectively26–29. In about 10% of FHL cases, the molecular defect remains uncharacterized. Biallelic mutations in RAB27A (encoding the small GTPase Rab27a) and LYST (encoding lysosomal trafficking regulator) account for the development of HLH in Griscelli syndrome30 and Chédiak-Higashi syndrome31, respectively. Remarkably, each of these molecules mediates a discrete, non-redundant step in cytotoxic granule exocytosis at the IS. Rab27a and Munc13-4 are respectively required for the granule docking and priming steps at the plasma membrane, whereas syntaxin 11 interacts with Munc18-2 to enable granules to fuse with the plasma membrane8. Although the role of LYST is less well understood, it may regulate a late granule maturation step32. The effector molecules’ partners and interconnections have been progressively characterized to reveal the overall picture of granule exocytosis. Notably, interaction between Rab27a and Munc13-4 was shown to be mandatory for tethering the cytotoxic granules at the IS in order to complete the exocytic process33. Munc13-4 interacts with several syntaxin isoforms, among them syntaxin 1134. Furthermore, Rab27a binds to three different members of the synaptotagmin-like (SLP1-3) family expressed in cytotoxic cells that have partially overlapping functions in granule transport and docking35–37.\n\nIt has been proposed that direct contact between polarized centrosomes and the plasma membrane drives cytotoxic granule delivery at the IS17,38. However, there is evidence to suggest that alternative mechanisms are involved, such as the observation that very rapid, effective cytotoxic granule secretion can precede MTOC polarization in some CTL-target cell conjugates39. In the latter study, inhibition of MTOC polarization did not prevent cytotoxic granule release. Furthermore, the Slp3/Rab27a complex expressed in cytotoxic cells was shown to interact with a kinesin motor and to mediate the terminal transport of polarized cytotoxic granules toward the IS35. In view of the diversity of in vivo settings in which cytotoxic cells are triggered, one can legitimately hypothesize that granule delivery may occur via several different routes. Indeed, recent research has shown that cytotoxic cells are heterogeneous and change their killing performance over time and as a function of antigenic stimulation40,41. Timescale studies of single NK cells or CTLs have revealed a progressive increase in the rapidity and efficiency of killing during serial killing, which also varies according to the avidity of antigen recognition40,41. It remains not well understood how what has been shown in vitro applies in vivo, such as the nature of target cells and the strength of triggering signal.\n\n\nThe use of animal models to characterize the pathophysiology of hemophagocytic lymphohistiocytosis\n\nAnimal models of primary HLH in which cytotoxicity-deficient mice are challenged with a virus have proven to be invaluable for understanding the pathogenesis of HLH under defined conditions. It has been demonstrated that after lymphocytic choriomeningitis virus (LCMV) infection of perforin-deficient mice, hyperactive CTLs and high levels of interferon-gamma (IFN-γ) are the driving forces behind the development of fatal HLH4. LCMV’s potent induction of HLH might be due, at least in part, to its ability to infect APCs and thus strongly stimulate a T-cell response without the need for antigen cross-presentation. Likewise, Epstein-Barr virus (EBV), a major trigger of HLH in humans, can directly infect B cells, which also have an antigen presentation function and trigger prolonged antigenic stimulation when not eliminated by cytotoxic lymphocytes. In addition to viral priming, antigen persistence and prolonged presentation were shown to be critical in the development of primary HLH in murine models4,10. This appears to contrast with the onset of primary HLH in newborn infants or even fetuses with cytotoxicity defects, since a pathogen trigger cannot be identified in many cases42,43. Although as-yet-unknown microorganisms may act as the trigger, this observation suggests that, in contrast to the situation in mice, the granule-dependent cytotoxic pathway in humans also has a role in T-cell homeostasis in the absence of an external stimulus (as is also the case for the Fas/FasL pathway). It has been shown that the elimination of a rare, antigen-presenting dendritic cell (DC) population by CD8+ T cells in a negative feedback loop is a critical determinant of the magnitude of T-cell responses44,45. Thus, the elimination of specific APC populations probably determines the activation status and survival of hyper-reactive T cells and acts as a rheostat by limiting T-cell responses. Whether the granule-dependent cytotoxic pathway is also participating to check self-reactive T/B cells is therefore a possibility that needs to be further investigated.\n\nIn mice, the degree of cytotoxicity impairment appears to be the best predictor of the development and severity of HLH, as shown by studies of the time course of HLH onset in various HLH-prone strains with defects in the granule-dependent cytotoxic pathway10,46. The same is true in humans10,46 (Figure 1A). In genetically determined murine models of HLH, the cytotoxicity of both T cells and NK cells is impaired. However, in contrast to CD8+ T-cell depletion, NK cell depletion in perforin-deficient mice did not prevent the development of manifestations of HLH4. CTLs were thus considered to be the main players in the development of HLH. However, recent work has revealed that T cells and NK cells have a non-redundant cytotoxic function in HLH: CTLs mediate LCMV viral clearance, whereas NK cells limit hyperactivation of CTLs11. This finding further suggests that the perforin-dependent cytotoxic activity of NK cells has a key role in the maintenance of immune homeostasis and the prevention of immunopathology47,48. However, the underlying mechanism through either direct or indirect T/NK cell interactions remains to be characterized. Furthermore, one cannot fully exclude the participation of other potentially cytotoxic cells such as invariant NK T (iNKT) cells and CD4+ T-cell subsets, including specific regulatory T (Treg) populations, in this setting. It is also worth noting that in syntaxin 11-deficient mice, which display a milder cytotoxic defect and less severe HLH than perforin-deficient mice, blockade of inhibitory receptors of T-cell exhaustion (such as PD1/PDL1) dramatically increases the severity of HLH and results in fatal disease49. This finding indicates that T-cell exhaustion is another important modulator of HLH severity.\n\n(A) A gradient of hemophagocytic lymphohistiocytosis (HLH) severity correlates with the defect in cytotoxic activity of lymphocytes that results from various genetic defects in humans and mice. Null mutations are considered in this image. (B) Evolving view of the risk factors inducing HLH development. Mild to extreme immune stimuli, in combination with severe (null) mutations, hypomorphic mutations, monoallelic mutation in several or one of the genes involved in HLH, appear to determine an individual’s risk for developing HLH. HLH risk lies above the red line in the hatched area.\n\nThe in vivo failure of cytotoxic cells to eliminate target cells leads to a fatal cytokine storm, a hallmark of HLH. Previous research has shown that the threshold of T-cell activation determines whether a lytic synapse (which is induced at low antigen concentrations and which enables cytotoxic activity) or a stimulatory synapse (induced at high antigen concentrations and which enables both cytotoxic activity and IFN-γ production) is formed50. Remarkably, it was recently shown that cytotoxicity-deficient lymphocytes form longer contacts with their cognate target, thus resulting in many successive rounds of Ca2+ flux into cytotoxic cells and triggering of pro-inflammatory cytokine secretions51. Thus, the cytokine storm as observed in HLH likely depends on both quantity and quality of contacts formed between cytotoxic cells and APCs.\n\nPhagocytosis of blood cells by macrophages (known as hemophagocytosis) is another hallmark of primary HLH, although it can be observed in a variety of infectious or inflammatory disorders52. A study of perforin-deficient mice has revealed that IFN-γ specifically triggers this process, which can be reproduced in wild-type mice by inducing the sustained elevation of IFN-γ. Direct, IFN-γ-dependent activation of macrophages prompts the development of severe, consumptive anemia and other types of cytopenia, probably through direct changes in the macrophages’ endocytic uptake53. These results indicate that hemophagocytosis is actually an adapted response to sustained or severe inflammation. Further details on the role of macrophages and other inflammatory cells in the pathophysiology of HLH have been provided in recent reviews5,6,54.\n\nAlthough cytotoxic lymphocytes exert a key role in the development of primary HLH, other immune cells and signaling pathways may also contribute. It has been shown that MyD88, which mediates Toll-like receptor (TLR) and interleukin-1 (IL-1) signaling, is required for HLH development in Unc13d-deficient mice, suggesting that innate immune cells contribute to the development of HLH55. Moreover, high levels of IL-4 or repeated TLR9 stimulation in wild-type mice can induce the development of an HLH-like syndrome56,57. Hence, proteins from the cytotoxic exocytic pathway may have additional functions in other immune cell types (such as inflammatory cells), the absence of which modulates the pathogenesis of HLH. More generally, any regulatory molecule involved in an inflammatory pathway might contribute to the development of the manifestations of HLH.\n\n\nWhat is the minimum level of cytotoxic activity required to preserve immune homeostasis?\n\nGenetically determined forms of HLH can occur even when a cytotoxicity defect is only partial or apparently absent. This is the case in X-linked lymphoproliferative syndrome (XLP). Patients with XLP are extremely vulnerable to EBV infection and most go on to develop HLH58. There are two genetic forms: XLP-1 and XLP-2. Firstly, XLP-1 results from a deficiency in the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)59–61. SAP-deficient CTLs and NK cells are selectively impaired in their cytotoxic response to infected B cells; the response requires interaction between SLAM family receptors and subsequent SAP-dependent signaling in T lymphocytes but not in other cell types62,63. Secondly, XLP-2 results from a deficiency in the X-linked inhibitor of apoptosis protein (XIAP) (also known as BIRC4)64. However, XIAP-deficient CTLs and NK cells exhibit apparently normal in vitro cytotoxic responses (regardless of the SLAM-receptor dependency). The cytotoxic activity of iNKT cells is known to be activated by EBV-infected B cells65. Indeed, the exacerbated apoptosis of XIAP-deficient iNKT cells, induced by EBV infection, might be involved in the development of HLH in XLP-2. Alternatively, the mechanisms underlying EBV-driven HLH in XLP-2 may differ completely from those observed in XLP-1 and other inherited forms of HLH. In a setting of XIAP deficiency, the accumulation of apoptotic cells and the persistence of EBV-infected cells might trigger abnormal inflammation and contribute to the development of HLH. This hypothesis is supported by the observation that XIAP deficiency in mice results in excessive DC death and inflammasome activation66.\n\nIt is difficult to assess the minimal level of cytotoxic activity required for the maintenance of immune homeostasis. Hypomorphic mutations in HLH genes that preserve residual cytotoxicity significantly delay the onset of HLH but predispose patients to hematological cancers67–69. Adult patients with HLH have been found to carry a monoallelic mutation in one or more FHL genes70–73. These findings suggest that the accumulation of heterozygous mutations that partially impair the granule-dependent cytotoxic pathway may have an additional functional impact. This hypothesis could be tested by studying inter-crossed animal models of HLH with monoallelic mutations. The concept whereby a monoallelic mutation in cytotoxicity-related genes can lead to immune disturbance also requires far more investigation, particularly in much larger cohorts of patients with induced HLH versus healthy controls. When the cytotoxicity defect is mild, the relative weight of additional genetic and environmental factors in HLH triggering is probably greater. It is tempting to speculate that (i) “extreme” stimuli may be sufficient to induce sporadic HLH development in any individual and (ii) the overall risk is augmented by the accumulation of genetic variants promoting excessive or poorly regulated immune response (Figure 1B). Along these lines, mutations in genes controlling inflammatory processes may also contribute to HLH. Recently, de novo activating mutations in the nucleotide-binding domain of inflammasome component NLRC4, associated with high levels of inflammatory cytokines in general and of IL-18 in particular, were found to be linked to recurrent HLH74–76. Since impaired cytotoxicity was not detected in that setting, this finding highlights the role of additional molecules in the pathophysiological process leading to HLH. By coupling next-generation sequencing to animal model studies, it should now be possible to determine whether HLH can be a polygenic condition in adults.\n\n\nConcluding remarks\n\nOver the last few decades, characterization of the molecular bases of primary HLH has highlighted the critical role of CTL activity in the control of immune homeostasis and has identified key effectors of cytotoxic granule exocytosis and their specific functions along the cytotoxic pathway. Broader knowledge of the scope of HLH occurrence has prompted the hypothesis whereby HLH is a “threshold” disease. A combination of both genetic factors and environmental factors (infections, self-antigens, and so on) is needed for the development of HLH in a context of residual cytotoxicity. Some cases of HLH do not appear to be directly related to a cytotoxicity defect, indicating that other genes, notably involved in macrophage-related inflammation, regulating the same disease pathway also have a role. Characterizing the synergistic connections between the various risk factors for HLH will be a key challenge in the coming years.\n\n\nAbbreviations\n\nAPC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBV, Epstein-Barr virus; FHL, familial hemophagocytic lymphohistiocytosis; HLH, hemophagocytic lymphohistiocytosis; IFN-γ, interferon-gamma; IL, interleukin; iNKT, invariant natural killer T; IS, immunological synapse; LCMV, lymphocytic choriomeningitis virus; MTOC, microtubule-organizing centre; NK, natural killer; SAP, signaling lymphocyte activation molecule-associated protein; SLAM, signaling lymphocyte activation molecule; TLR, Toll-like receptor; XIAP, X-linked inhibitor of apoptosis protein; XLP, X-linked lymphoproliferative syndrome.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have 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\nStinchcombe JC, Bossi G, Booth S, et al.: The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity. 2001; 15(5): 751–61. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nStepp SE, Dufourcq-Lagelouse R, Le Deist F, et al.: Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science. 1999; 286(5446): 1957–9. PubMed Abstract | Publisher Full Text\n\nKägi D, Seiler P, Pavlovic J, et al.: The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses. Eur J Immunol. 1995; 25(12): 3256–62. 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}
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[
{
"id": "10627",
"date": "30 Sep 2015",
"name": "Loïc Dupré",
"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",
"responses": []
},
{
"id": "10629",
"date": "30 Sep 2015",
"name": "Ilia Voskoboinik",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-930
|
https://f1000research.com/articles/4-928/v1
|
30 Sep 15
|
{
"type": "Review",
"title": "Hot topics in biodiversity and climate change research",
"authors": [
"Barry W. Brook",
"Damien A. Fordham",
"Damien A. Fordham"
],
"abstract": "With scientific and societal interest in biodiversity impacts of climate change growing enormously over the last decade, we analysed directions and biases in the recent most highly cited data papers in this field of research (from 2012 to 2014). The majority of this work relied on leveraging large databases of already collected historical information (but not paleo- or genetic data), and coupled these to new methodologies for making forward projections of shifts in species’ geographical ranges, with a focus on temperate and montane plants. A consistent finding was that the pace of climate-driven habitat change, along with increased frequency of extreme events, is outpacing the capacity of species or ecological communities to respond and adapt.",
"keywords": [
"biodiversity",
"climate change",
"global change",
"conservation"
],
"content": "Introduction\n\nIt is now halfway through the second decade of the 21st century, and climate change impact has emerged as a “hot topic” in biodiversity research. In the early decades of the discipline of conservation biology (1970s and 1980s), effort was focused on studying and mitigating the four principal drivers of extinction risk since the turn of the 16th century, colourfully framed by Diamond1 as the “evil quartet”: habitat destruction, overhunting (or overexploitation of resources), introduced species, and chains of extinctions (including trophic cascades and co-extinctions). Recent work has also emphasised the importance of synergies among drivers of endangerment2. But the momentum to understand how other aspects of global change (such as a disrupted climate system and pollution) add to, and reinforce, these threats has built since the Intergovernmental Panel on Climate Change reports3 of 2001 and 2007 and the Millennium Ecosystem Assessment4 in 2005.\n\nScientific studies on the effects of climate change on biodiversity have proliferated in recent decades. A Web of Science (webofscience.com) query on the term “biodiversity AND (climate change)”, covering the 14 complete years of the 21st century, shows the peer-reviewed literature matching this search term has grown from just 87 papers in 2001 to 1,377 in 2014. Figure 1 illustrates that recent scientific interest in climate change-related aspects of biodiversity research has outpaced—in relative terms—the baseline trend of interest in other areas of biodiversity research (i.e., matching the query “biodiversity NOT (climate change)”), with climate-related research rising from 5.5% of biodiversity papers in 2001 to 16.8% in 2014.\n\nNumber of refereed papers listed in the Web of Science database that were published between 2001 and 2014 on the specific topic “biodiversity AND (climate change)” (blue line, secondary y-axis) compared to the more general search term “biodiversity NOT (climate change)”.\n\nInterest in this field of research seems to have been driven by a number of concerns. First, there is an increasing societal and scientific consensus on the need to measure, predict (and, ultimately, mitigate) the impact of anthropogenic climate change5, linked to the rise of industrial fossil-fuel combustion and land-use change6. Biodiversity loss and ecosystem transformations, in particular, have been highlighted as possibly being amongst the most sensitive of Earth’s systems to global change7,8. Second, there is increasing attention given to quantifying the reinforcing (or occasionally stabilising) feedbacks between climate change and other impacts of human development, such as agricultural activities and land clearing, invasive species, exploitation of natural resources, and biotic interactions2,9. Third, there has been a trend towards increased accessibility of climate change data and predictions at finer spatio-temporal resolutions, making it more feasible to do biodiversity climate research10,11.\n\nWhat are the major directions being taken by the field of climate change and biodiversity research in recent years? Are there particular focal topics, or methods, that have drawn most attention? Here we summarise major trends in the recent highly cited literature of this field.\n\n\nFiltering and categorising the publications\n\nTo select papers, we used the Web of Science indexing service maintained by Thomson Reuters, using the term “biodiversity AND (climate change)” to search within article titles, abstracts, and keywords. This revealed 3,691 matching papers spanning the 3-year period 2012 to 2014. Of these, 116 were categorised by Essential Science Indicators (esi.incites.thomsonreuters.com) as being “Highly Cited Papers” (definition: “As of November/December 2014, this highly cited paper received enough citations to place it in the top 1% of [its] academic field based on a highly cited threshold for the field and publication year”), with five also being classed as “Hot Papers” (definition: “Published in the past two years and received enough citations in November/December 2014 to place it in the top 0.1% of papers in [its] academic field”). The two academic fields most commonly associated with these selected papers were “Plant & Animal Science” and “Environment/Ecology”.\n\nNext we ranked each highly cited paper by year, according to its total accumulated citations through to April 1 2015, and then selected the top ten papers from each year (2012, 2013 and 2014) for detailed assessment. We wished to focus on data-oriented research papers, so only those labelled “Article” (Document Type) were considered, with “Review”, “Editorial”, or other non-research papers being excluded from our final list. Systematic reviews that included a formal meta-analysis were, however, included. We then further vetted each potential paper based on a detailed examination of its content, and rejected those articles for which the topics of biodiversity or climate change constituted only a minor component, or where these were only mentioned in passing (despite appearing in the abstract or key words).\n\nThe final list of 30 qualifying highly cited papers is shown in Table 1, ordered by year and first author. The full bibliographic details are given, along with a short description of the key message of the research (a subjective summary, based on our interpretation of the paper). Each paper was categorised by methodological type, the aspect of climate change that was the principal focus, the spatial and biodiversity scale of the study units, the realm, biome and taxa under study, the main ecological focus, and the research type and application (the first row of Table 1 lists possible choices that might be allocated within a given categorisation). Note that our choice of categories for the selected papers was unavoidably idiosyncratic, in this case being dictated largely by the most common topics that appeared in the reviewed papers. Other emphases, such as non-temperature-related drivers of global change, evolutionary responses, and so on, might have been more suitable for other bodies of literature. We also did not attempt to undertake any rigorous quantification of effect sizes in reported responses of biodiversity to climate change; such an approach would have required a systematic review and meta-analysis, which was beyond the scope of this overview of highly cited papers.\n\nSummary of the ten most highly cited research papers based on the search term: “biodiversity AND (climate change)”, for each of 20129,13,14,23,26,32,34,36,40,45, 201315–17,21,27,30,31,33,37,39 and 201418–20,22,24,25,28,29,35,38, as determined in the ISI Web of Science database. Filters: Reviews, commentaries, and opinion pieces were excluded, as were papers for which climate change was not among the focal topics of the research. The first row of the Table is a key that shows the possible categorisations that were open to selection (more than one description might be selected for a given paper); n is the number of times a category term was allocated.\n\n\nAnalysis of trends, biases and gaps\n\nBased on the categorisation frequencies in Table 1 (counts are given in the n columns adjacent to each category), the “archetypal” highly cited paper in biodiversity and climate change research relies on a database of previously collated information, makes an assessment based on future forecasts of shifts in geographical distributions, is regional in scope, emphasises applied-management outcomes, and uses terrestrial plant species in temperate zones as the study unit.\n\nMany papers also introduced new methodological developments, studied montane communities, took a theoretical-fundamental perspective, and considered physiological, population dynamics, and migration-dispersal aspects of ecological change. Plants were by far the dominant taxonomic group under investigation. By contrast, relatively few of the highly cited paper studies used experimental manipulations or network analysis; lake, river, island and marine systems were rarely treated; nor did they focus on behavioural or biotic interactions. Crucially, none of the highly cited papers relied on paleoclimate reconstructions or genetic information, despite the potential value of such data for model validation and contextualisation12. Such data are crucial in providing evidence for species responses to past environmental changes, specifying possible limits of adaptation (rate and extent) and fundamental niches, and testing theories of biogeography and macroecology.\n\nAt the time of writing, 5 of the 30 highly cited papers listed in Table 1 (16%) also received article recommendations from Faculty of 1000 experts (f1000.com/prime/recommendations)9,13–16 with none of the most recent (2014) highly cited papers having yet received an F1000 Prime endorsement.\n\n\nKey findings of the highly cited paper collection for 2012–2014\n\nA broad conclusion of the highly cited papers for 2012–2014 (drawn from the “main message” summaries described in Table 1) is that the pace of climate change-forced habitat change, coupled with the increased frequency of extreme events15,17 and synergisms that arise with other threat drivers9,18 and physical barriers19, is typically outpacing or constraining the capacity of species, communities, and ecosystems to respond and adapt20,21. The combination of these factors leads to accumulated physiological stresses13,15,22, might have already induced an “extinction debt” in many apparently viable resident populations14,23–25, and is leading to changing community compositions as thermophilic species displace their more climate-sensitive competitors13,26. In addition to atmospheric problems caused by anthropogenic greenhouse-gas emissions, there is mounting interest in the resilience of marine organisms to ocean acidification27,28 and altered nutrient flows16.\n\nAlthough models used to underpin the forecasts of climate-driven changes to biotic populations and communities have seen major advances in recent years, as a whole the field still draws from a limited suite of methods, such as ecological niche models, matrix population projections and simple measures of change in metrics of ecological diversity7,12,29. However, new work is pushing the field in innovative directions, including a focus on advancements in dynamic habitat-vegetation models30–32, improved frameworks for projecting shifts in species distributions29,33,34 and how this might be influenced by competition or predation35,36, and analyses that seek to identify ecological traits that can better predict the relative vulnerability of different taxa to climate change37,38.\n\nIn terms of application of the research to conservation and policy, some offer local or region-specific advice on ecosystem management and its integration with other human activities (e.g., agriculture, fisheries) under a changing climate18,24,35,39. However, the majority of the highly cited papers used some form of forecasting to predict the consequences of different climate-mitigation scenarios (or business-as-usual) on biodiversity responses and extinctions20–22,33,40, so as to illustrate the potentially dire consequences of inaction.\n\n\nFuture directions\n\nThe current emphasis on leveraging large databases for evidence of species responses to observed (recent) climate change is likely to wane as existing datasets are scrutinised repeatedly. This suggests to us that future research will be forced to move increasingly towards the logistically more challenging experimental manipulations (laboratory, mesocosm, and field-based). The likelihood of this shift in emphasis is reinforced by the recent trend towards mechanistic models in preference to correlative approaches41. Such approaches arguably offer the greatest potential to yield highly novel insights, especially for predicting and managing the outcomes of future climate-ecosystem interactions that have no contemporary or historical analogue. Along with this work would come an increasing need for systematic reviews and associated meta-analysis, to summarise these individual studies quantitatively and use the body of experiments to test hypotheses.\n\nTechnological advances will also drive this field forward. This includes the development of open-source software and function libraries that facilitate and standardise routine tasks like validation and sensitivity analysis of projection or statistical models42,43, as well as improved access to data layers from large spatio-temporal datasets like ensemble climate forecasts10 and palaeoclimatic hindcasts44. An increasing emphasis on cloud-based storage and use of off-site high-performance parallel computing infrastructure will make it realistic for researchers to undertake computationally intensive tasks31 from their desktop.\n\nThese approaches are beginning to emerge, and a few papers on these topics already appear in the highly cited paper list (Table 1). This includes the innovative exposure of coral populations to varying carbon dioxide concentrations, and the meta-analyses of tundra plant response to experimental warming45 and marine organisms to ocean chemistry27. Such work must also be underpinned by improved models of the underlying mechanisms and dynamic processes, ideally using multi-species frameworks that make use of ensemble forecasting methods for improved incorporation of scenario and climate model uncertainty10. Such an approach can account better for biotic interactions41 via individual-based and physiologically explicit “bottom-up” models of adaptive responses31. Lastly, there must be a greater emphasis on using genetic information to integrate eco-evolutionary processes into biodiversity models46, and on improving methods for making the best use of retrospective knowledge from palaeoecological data12.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no disclosures or conflicts of interest.\n\n\nGrant information\n\nThis work was supported by Australian Research Council Discovery Grant DP120101019 (Brook) and Future Fellowship FT140101192 (Fordham).\n\n\nReferences\n\nDiamond JM: “Normal” extinctions of isolated populations. In Extinctions, MH Nitecki (Ed.), Chicago University Press, 1984; 191–246. Reference Source\n\nBrook BW, Sodhi NS, Bradshaw CJ: Synergies among extinction drivers under global change. Trends Ecol Evol. 2008; 23(8): 453–460. PubMed Abstract | Publisher Full Text\n\nIPCC. Intergovernmental Panel on Climate Change: Fourth (AR4) and Fifth (AR5) Assessment Reports: Working Group II Report on Impacts, Adaptation, and Vulnerability. 2001, 2007. Reference Source\n\nMEA. Millennium Ecosystem Assessment: Ecosystems and Human Well-being: Scenarios. Island Press, 2005. Reference Source\n\nUrban MC: Accelerating extinction risk from climate change. Science. 2015; 348(6234): 571–573. Publisher Full Text | Faculty Opinions Recommendation\n\nBrook BW, Rowley N, Flannery TF: Kyoto: doing our best is no longer enough. Nature. 2007; 450(7169): 478–478. PubMed Abstract | Publisher Full Text\n\nDawson TP, Jackson ST, House JI, et al.: Beyond predictions: biodiversity conservation in a changing climate. Science. 2011; 332(6025): 53–58. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPereira HM, Navarro LM, Martins IS: Global biodiversity change: The bad the good, and the unknown. Annu Rev Environ Resour. 2012; 37: 25–50. Publisher Full Text | Faculty Opinions Recommendation\n\nMantyka-Pringle CS, Martin TG, Rhodes JR: Interactions between climate and habitat loss effects on biodiversity: a systematic review and meta-analysis. Glob Chang Biol. 2012; 18(4): 1239–1252. Publisher Full Text\n\nFordham DA, Wigley TML, Brook BW: Strengthening forecasts of climate change impacts with multi-model ensemble averaged projections using MAGICC/SCENGEN 5.3. Ecography. 2012; 35(1): 4–8. Publisher Full Text\n\nHijmans RJ, Cameron SE, Parra JL, et al.: Very high resolution interpolated climate surfaces for global land areas. Int J Climatol. 2005; 25(15): 1965–1978. Publisher Full Text | Faculty Opinions Recommendation\n\nFordham DA, Brook BW, Moritz C, et al.: Better forecasts of range dynamics using genetic data. Trends Ecol Evol. 2014; 29(8): 436–443. PubMed Abstract | Publisher Full Text\n\nSunday JM, Bates AE, Dulvy NK: Thermal tolerance and the global redistribution of animals. Nat Clim Chang. 2012; 2: 686–690. Publisher Full Text\n\nZhu K, Woodall CW, Clark JS: Failure to migrate: lack of tree range expansion in response to climate change. Glob Chang Biol. 2012; 18(3): 1042–1052. Publisher Full Text\n\nAnderegg WR, Plavcova L, Anderegg LD, et al.: Drought's legacy: multiyear hydraulic deterioration underlies widespread aspen forest die-off and portends increased future risk. Glob Chang Biol. 2013; 19(4): 1188–1196. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBoetius A, Albrecht S, Bakker K, et al.: Export of algal biomass from the melting arctic sea ice. Science. 2013; 339(6126): 1430–1432. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSmale DA, Wernberg T: Extreme climatic event drives range contraction of a habitat-forming species. Proc Biol Sci. 2013; 280(1754): 20122829. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBates AE, Barrett NS, Stuart-Smith RD, et al.: Resilience and signatures of tropicalisation in protected reef fish communities. Nat Clim Chang. 2014; 4(1): 62–67. Publisher Full Text | Faculty Opinions Recommendation\n\nJantz P, Goetz S, Laporte N: Carbon stock corridors to mitigate climate change and promote biodiversity in the tropics. Nat Clim Chang. 2014; 4: 138–142. Publisher Full Text\n\nBurrows MT, Schoeman DS, Richardson AJ, et al.: Geographical limits to species-range shifts are suggested by climate velocity. Nature. 2014; 507(7493): 492–495. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWarren R, VanDerWal J, Price J, et al.: Quantifying the benefit of early climate change mitigation in avoiding biodiversity loss. Nat Clim Chang. 2013; 3(7): 678–682. Publisher Full Text | Faculty Opinions Recommendation\n\nScheffers BR, Edwards DP, Diesmos A, et al.: Microhabitats reduce animal's exposure to climate extremes. Glob Chang Biol. 2014; 20(2): 495–503. PubMed Abstract | Publisher Full Text\n\nDullinger S, Gattringer A, Thuiller W, et al.: Extinction debt of high-mountain plants under twenty-first-century climate change. Nat Clim Chang. 2012; 2: 619–622. Publisher Full Text\n\nShoo LP, O'Mara J, Perhans K, et al.: Moving beyond the conceptual: specificity in regional climate change adaptation actions for biodiversity in South East Queensland, Australia. Reg Environ Change. 2014; 14(2): 435–447. Publisher Full Text\n\nZhu K, Woodall CW, Ghosh S, et al.: Dual impacts of climate change: forest migration and turnover through life history. Glob Chang Biol. 2014; 20(1): 251–64. PubMed Abstract | Publisher Full Text\n\nGottfried M, Pauli H, Futschik A, et al.: Continent-wide response of mountain vegetation to climate change. Nat Clim Chang. 2012; 2(2): 111–115. Publisher Full Text | Faculty Opinions Recommendation\n\nHarvey BP, Gwynn-Jones D, Moore PJ: Meta-analysis reveals complex marine biological responses to the interactive effects of ocean acidification and warming. Ecol Evol. 2013; 3(4): 1016–1030. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHennige SJ, Wicks LC, Kamenos NA, et al.: Short-term metabolic and growth responses of the cold-water coral Lophelia pertusa to ocean acidification. Deep Sea Res Part II To Stud Oceanogr. 2014; 99: 27–35. Publisher Full Text | Faculty Opinions Recommendation\n\nRadosavljevic A, Anderson RP: Making better MAXENT models of species distributions: complexity, overfitting and evaluation. J Biogeogr. 2014; 41(4): 629–643. Publisher Full Text | Faculty Opinions Recommendation\n\nFranklin J, Davis FW, Ikegami M, et al.: Modeling plant species distributions under future climates: how fine scale do climate projections need to be? Glob Chang Biol. 2013; 19(2): 473–483. PubMed Abstract | Publisher Full Text\n\nScheiter S, Langan L, Higgins SI: Next-generation dynamic global vegetation models: learning from community ecology. New Phytol. 2013; 198(3): 957–969. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHickler T, Vohland K, Feehan J, et al.: Projecting the future distribution of European potential natural vegetation zones with a generalised, tree species-based dynamic vegetation model. Global Ecol Biogeogr. 2012; 21(1): 50–63. Publisher Full Text | Faculty Opinions Recommendation\n\nHazen EL, Jorgensen S, Rykaczewski RR, et al.: Predicted habitat shifts of Pacific top predators in a changing climate. Nat Clim Chang. 2013; 3(3): 234–238. Publisher Full Text | Faculty Opinions Recommendation\n\nFordham DA, Akçakaya HR, Araujo MB, et al.: Plant extinction risk under climate change: are forecast range shifts alone a good indicator of species vulnerability to global warming? Glob Chang Biol. 2012; 18(4): 1357–1371. Publisher Full Text\n\nSchmitz OJ, Barton BT: Climate change effects on behavioral and physiological ecology of predator-prey interactions: Implications for conservation biological control. Biol Control. 2014; 75: 87–96. Publisher Full Text\n\nUrban MC, Tewksbury JJ, Sheldon KS: On a collision course: competition and dispersal differences create no-analogue communities and cause extinctions during climate change. Proc Biol Sci. 2012; 279(1735): 2072–2080. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFoden WB, Butchart SH, Stuart SN, et al.: Identifying the world's most climate change vulnerable species: a systematic trait-based assessment of all birds, amphibians and corals. PLoS One. 2013; 8(6): e65427. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPearson RG, Stanton JC, Shoemaker KT, et al.: Life history and spatial traits predict extinction risk due to climate change. Nat Clim Chang. 2014; 4: 217–221. Publisher Full Text\n\nHannah L, Roehrdanz PR, Ikegami M, et al.: Climate change, wine, and conservation. Proc Natl Acad Sci U S A. 2013; 110(17): 6907–6912. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchloss CA, Nuñez TA, Lawler JJ: Dispersal will limit ability of mammals to track climate change in the Western Hemisphere. Proc Natl Acad Sci U S A. 2012; 109(22): 8606–8611. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFordham DA, Akçakaya HR, Brook BW, et al.: Adapted conservation measures are required to save the Iberian lynx in a changing climate. Nat Clim Chang. 2013; 3: 899–903. Publisher Full Text\n\nWatts MJ, Fordham DA, Akçakaya HR, et al.: Tracking shifting range margins using geographical centroids of metapopulations weighted by population density. Ecol Modell. 2013; 269: 61–69. Publisher Full Text\n\nLurgi M, Brook BW, Saltré F, et al.: Modelling range dynamics under global change: which framework and why? Methods Ecol Evol. 2015; 6(3): 247–256. Publisher Full Text\n\nLiu Z, Otto-Bliesner BL, He F, et al.: Transient simulation of last deglaciation with a new mechanism for Bolling-Allerod warming. Science. 2009; 325(5938): 310–314. PubMed Abstract | Publisher Full Text\n\nElmendorf SC, Henry GH, Hollister RD, et al.: Global assessment of experimental climate warming on tundra vegetation: heterogeneity over space and time. Ecol Lett. 2012; 15(2): 164–175. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nThuiller W, Münkemüller T, Lavergne S, et al.: A road map for integrating eco-evolutionary processes into biodiversity models. Ecol Lett. 2013; 16(Suppl 1): 94–105. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "10634",
"date": "01 Oct 2015",
"name": "Jonathan Rhodes",
"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",
"responses": []
},
{
"id": "10635",
"date": "01 Oct 2015",
"name": "Bernhard Schmid",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-928
|
https://f1000research.com/articles/4-927/v1
|
30 Sep 15
|
{
"type": "Research Article",
"title": "Colonoscopy in patients with inflammatory bowel disease: self-reported experience, understanding, anxieties and tolerance of the procedure",
"authors": [
"Samantha Morgan",
"Christopher Alexakis",
"Lucy Medcalf",
"Vivek Chhaya",
"Penny Neild",
"Andrew Poullis",
"Richard Pollok",
"Christopher Alexakis",
"Lucy Medcalf",
"Vivek Chhaya",
"Penny Neild",
"Andrew Poullis",
"Richard Pollok"
],
"abstract": "Objective: To address Inflammatory Bowel Disease (IBD) patients’ attitudes, understanding and tolerance of colonoscopy and assess whether there are specific factors that influence these parameters.Design: structured questionnaire-based survey. Tolerance of various aspects of colonoscopy procedure graded on a scale 1-5, 5 representing most intolerance/burden (worries/concerns about the procedure/risks, bowel preparation, disruption to life, procedural discomfort and travel concerns).Setting: London teaching hospital - St Georges HospitalPatients: Consecutive patients with established IBD attending the specialist IBD clinicResults: 98 patients responded (46% male). Mean age was 43.2 years. 33 had Ulcerative Colitis (UC), 50 had Crohn’s Disease (CD), and 11 were unsure of diagnosis. Mean number of colonoscopies was 3.7. Females were more worried about the procedure than males (3.0 vs 2.1, p<0.05), were less tolerant of bowel preparation (3.5 vs 2.3, p<0.05), experienced more disruption to their lives (2.9 vs 1.9, p<0.05) and were more troubled by travel concerns (2.0 vs 1.4, p<0.05). Patients with the disease for ≥ 5 years experienced significantly more discomfort than patients with the disease for a shorter duration (3.2 vs 2.7 p<0.05). Patients aged ≥55 years are significantly less worried about the procedure (2.7 vs 2.0, p = <0.05) and tolerate the bowel preparation better (3.1 vs 2.4, p = <0.05). The majority of the patients felt colonoscopy was ‘bearable’ (53%) with only 13% describing it as ‘very unpleasant’. 55% would have the procedure ‘as frequently as required’ if their physician felt it appropriate.Conclusions: Our research highlights a significant difference in the perception of colonoscopy by gender and age. Overall our findings reveal a preparedness to undergo colonoscopy as required despite an increasing requirement for this test. The differences highlighted should prompt endoscopy units to accommodate and make allowances for these different perceived tolerance in routine clinical activity.",
"keywords": [
"Inflammatory bowel disease",
"Colonoscopy",
"tolerance"
],
"content": "Introduction\n\nInflammatory Bowel Disease (IBD), made up primarily of Ulcerative Colitis (UC) and Crohn’s Disease (CD), is a chronic relapsing and remitting intestinal condition that affects an estimated 240,000 patients in the UK1. Medical treatment of IBD may include combinations of steroids, 5-aminosalicylic acids (5-ASAs), immunomodulators and biologic therapies. Failure of medical therapy may lead to surgery. Optimising treatment is dependent on various surrogate markers of inflammation, radiological imaging and direct endoscopic assessment of mucosal inflammation2. More recently, the importance of mucosal healing, as determined at colonoscopy, has been underscored as a key indicator of therapeutic success2,3. Mucosal healing is associated with improved health-related quality of life4, results in a longer time to relapse in CD5, and ultimately in fewer hospital admissions6.\n\nNon-invasive surrogate markers of inflammation such as faecal calprotectin may offer an alternative in the assessment of disease activity in IBD but are yet to be fully evaluated as an alternative to direct visualisation of mucosal healing7. Colonoscopy therefore currently remains the gold standard test for assessing mucosal healing. This potentially means an increasing burden of endoscopic procedures from the current level for IBD patients in the future8. However, colonoscopy is invasive, time consuming and costly, and may be viewed as an unpleasant or intolerable procedure by patients. With this in mind, it is important to assess patients’ perception and tolerance of colonoscopy, particularly since it may be performed increasingly more often to assess mucosal healing. We therefore aimed to assess the perceptions of a cohort of IBD patients consecutively recruited through a specialist IBD clinic at a large regional hospital with respect to their understanding, experience and attitudes towards colonoscopy.\n\n\nMethods\n\nOur study was performed at St George’s Hospital (SGH), which is a large 800 bed regional teaching hospital in central London with a catchment area of about 1.3 million patients. The department of gastroenterology runs weekly IBD specialist clinics. The endoscopy unit performs over 5000 procedures per year and is both a regional bowel cancer screening centre and national colonoscopy training centre. Colonoscopies were either performed by a senior clinician or by a trainee under their direct supervision. Consecutive patients with an established diagnosis of IBD attending the weekly specialist IBD clinic were invited to take part in this study by completing a short questionnaire. Data was collected over a 4 month period between September and December 2013. Data was collected anonymously. There was no specific exclusion criteria and the inclusion criteria was a confirmed diagnosis of IBD with a history of at least one colonoscopy. Consent was presumed by patients’ willingness to complete the survey. They were informed verbally by the clinic staff, that the data they provided would be used in a study to evaluate patients’ perceptions, understanding tolerance and experience of colonoscopy. Ethical approval was sought from the local Health Research Authority, who felt after consideration of the methodology, that specific ethical approval was not required for this study. The questionnaire comprised of nine questions and was divided into categories described below. A copy of the questionnaire in full is available as Supplementary materials.\n\n\nDemographics, disease characteristics and understanding of the procedure\n\nPatients were asked to self-report their age and sex and provide details of their IBD subtype and how long they had had their diagnosis as they understood it. They were asked to estimate how many colonoscopies they had undergone since diagnosis. Patients were also asked to demonstrate their understanding of the indications for colonoscopy. They were provided with eight potential indications, four of which are accepted indications and four which were “sham” indications. Choices available - 1) to assess how severe the disease is, 2) to see if you have constipation (sham), 3) to see if your treatment is working, 4) to see if there is another cause for your symptoms, 5) to get samples of the bowel wall (biopsies), 6) to see if you are digesting food properly (sham), 7) to get blood sample (sham), and 8) to check for intestinal worms (sham).\n\n\nExperience of procedure\n\nThe patients were asked to address the burden of various aspects of the procedure using a numerical grading score 1 to 5, where 1 represents least burden and 5 most burden. Parameters evaluated included: worries/concerns about the procedure/risks, bowel preparation, disruption to life, procedural discomfort and travel to and from hospital. They were then given the opportunity to describe the entire colonoscopy experience using one of the following qualitative statements: not unpleasant, neither unpleasant nor pleasant, bearable, unpleasant, and very unpleasant.\n\nFinally, the patients were asked to comment on how often they felt they could tolerate the procedure (once a year, once every 2 years, once every 3 years, once every 5 years, or as often as their doctor felt it appropriate). They were also given a space to add further comments about any aspect of their colonoscopy experience.\n\nData was collected, stored and analysed on StatView™ 5.0.1 statistics program (Abacus Corporation, Baltimore, Maryland, USA). Where appropriate, comparison of continuous data was performed using the student’s t-test.\n\n\nResults\n\n94/295 patients completed the questionnaire (32% response rate). 46% were male. Mean age was 43.2 years (male 47.1 years; female 39.9 years). Table 1 presents combined demographic, disease and tolerance data on the entire cohort of responders. More than 60% of responders to the questionnaire had their disease for longer than 5 years. The most burdensome aspect of the procedure reported was bowel preparation and procedural discomfort.\n\nAll statistics corrected to 1 decimal place\n\nTable 2 shows the intergroup comparison data. Of note, females found all aspects of colonoscopy more burdensome than their male counterparts, with all parameters reaching statistical significance bar procedural discomfort. Of interest, older patients (>55 years) reported less concerns about the procedure and associated risk, and also were less burdened by bowel preparation. Finally, patients with a longer disease duration reported higher burden of procedural discomfort.\n\nAll statistics shown to 1 DP. UC - ulcerative colitis CD - Crohn's disease *p<0.05\n\nPatients demonstrated good knowledge in their understanding of the indications for colonoscopy, with 69.2% answering all four indications correctly. Figure 1 illustrates patient responses to the individual indications for colonoscopy. Figure 2 indicates the qualitative summary statements by IBD patients as to general tolerability of the procedure. The majority of patients thought colonoscopy was bearable (53%), with only a small minority (13%) describing it as very unpleasant. Figure 3 indicates the frequency that respondents would be prepared to tolerate colonoscopy in future. The majority of patients (55%) would have the procedure ‘as frequently as required if their physician felt it appropriate’. A very small minority (7%) responded that they would only prepared to have colonoscopy every 5 years.\n\n\nDiscussion\n\nMucosal healing has evolved as a key endpoint in the assessment of therapeutic response to medical therapy in IBD patients. Requests for colonoscopy are therefore likely to continue to increase in this patient group making it particularly important to gain insight into the patients’ perceptions of this procedure. A good patient experience in the endoscopy unit is critical in facilitating long term medical management and continued engagement in services in this cohort. We have found that both women and young patients have a heightened concern about this procedure although actual reported discomfort following the procedure did not differ in these sub-groups from the rest of the cohort. Patients with an IBD disease duration of more than 5 years expressed significantly more procedural discomfort than patients with a shorter duration.\n\nOur research highlights a significant difference in the perception of colonoscopy between men and women. Females had a significantly worse perception of colonoscopy in four key areas: concerns about the procedure and associated risks; tolerance of bowel preparation; disruption to life and travel concerns to and from hospital, but not procedural discomfort itself. The results illustrate that women have higher pre-procedural anxiety than men. There are limited studies differentiating between gender and pre-procedure anxiety in patients with IBD, but our findings are supported by other work examining procedural anxiety in a non-IBD population undergoing colonoscopy9,10.\n\nIn our cohort, females showed a tendency towards increased procedural discomfort compared with males, approaching statistical significance. The majority of previous work on this subject suggests that females experience more discomfort during colonoscopy11–13, although this is not a universal finding14. It has been suggested that performing colonoscopy on females may be more difficult and more uncomfortable because of previous gynaecological surgery or differing colonic and pelvic anatomy12, and of note in this respect the procedure usually takes longer in women15.\n\nBowel preparation in colonoscopy is another important area of patient concern, with one study suggesting it is the most unpleasant part of the whole process16. This was mirrored in our group of patients with bowel preparation perceived as the most burdensome aspect of the whole episode (joint top with procedural discomfort). In our study, women were significantly less satisfied with bowel preparation compared to men, a finding supported by others17. The negative symptoms associated with bowel preparation may magnify pre-procedural anxiety in this group of patients18.\n\nOur results showed significant age-related differences in two measured outcomes. Patients aged 55 years or more were less worried about the procedure and risks. Secondly, this group reported that they found bowel preparation less burdensome than their younger counter parts. In keeping with our findings, a study showed older people expressed less discomfort associated with bowel preparation than younger patients and were overall, more satisfied17.\n\nHowever, we did not observe differences in procedural discomfort between the two age groups. The literature in this respect is conflicting. A Finnish retrospective study reported that older patients tolerate the procedure better than younger patients9. Conversely, Kim et al. reported no significant age-related differences19. Other studies have indicated older patients may tolerate the procedure less12. These inconsistencies between studies may reflect differences in the indication for and the underlying pathologies of the respective patient groups studied. Additionally, ‘older age’ is categorized differently between studies.\n\nOur study showed that patients who had IBD for more than 5 years expressed significantly more discomfort than patients having the disease for less than 5 years. This finding may relate to underlying disease factors, such as inflammation, stricturing or reduced intestinal compliance resulting in increased technical difficulty and reduced patient tolerance. One large study reported that higher doses of sedation are required amongst IBD patients with active disease20. An alternative explanation is that endoscopists now administer lower doses than historically in response to tighter monitoring and auditing of sedation practices. In a single centre study from the UK of sedation practices over a 10 year period sedation rates for outpatient diagnostic upper endoscopy reduced by 54%21. Certainly, in light of the 2004 NECPOD report ‘Scoping our Practice’22, sedation in all patient groups, particularly the elderly, has come under close scrutiny, and inevitably has influenced recent practice in endoscopic sedation. Kale et al. suggested endoscopy sedation should be individualised to the specific need of the IBD patient, particularly those that have active disease20 and this is clearly pertinent to the vulnerable subgroups we have identified, namely women and young patients.\n\nThe majority of our patients were prepared to undergo colonoscopy as frequently as their physician felt it appropriate. Similarly the majority also found colonoscopy ‘bearable’. These findings support the evidence that colonoscopy is generally associated with high levels of patient satisfaction and willingness to return23. This is a particularly important factor in patients with IBD, given that they are likely to undergo repeated procedures, especially when they enter into colorectal cancer surveillance programs. Our data suggests a high level of understanding of the indications of the procedure, which may further explain why most are prepared to undergo colonoscopy as frequently as needed.\n\nThis study has some limitations, particularly since responses were obtained retrospectively which opens it up to recall bias. If the most recent colonoscopy was a negative experience, irrespective of previous positive experiences, this may have impacted adversely. In one study, a fifth of patients reported that colonoscopy was more uncomfortable than they had expected24 and a previous experience of pain during colonoscopy was found to influence the perceived experience of pain during a subsequent procedure9. We sought to evaluate the patients’ qualitative perception of colonoscopy but a more objective and validated verification of discomfort during colonoscopy (or immediately following) might have led to different conclusions. We did not assess the patients’ requirement for sedation and analgesia, which may also have later influenced patients’ perception given the recognised retrograde-amnesic effect of benzodiazepines but we feel our study design best reflects the patient experience in routine clinical practise. Finally, our questionnaire did not include questions relating to ‘procedural embarrassment’, which has been significantly linked to non-compliance in endoscopic screening programs25.\n\nProviding patients with a positive experience is clearly important in maintaining continued engagement with medical services. Our findings indicate steps need to be taken to address the particular concerns of women and young patients with IBD in advance of their procedure. How might we ameliorate perceived pre-procedural patient concerns better? Consultation prior to colonoscopy with a clinician is associated with increased patient satisfaction26. This should form part of the consent process that is ideally performed before the day of procedure and after the clinic consult, as is the practice for the national bowel cancer screening programme. However, this may not always be logistically feasible. Music during the procedure has been shown to improve procedural experience and reduce anxiety amongst women and increases wellbeing amongst men, as well as significantly reducing discomfort27,28. Similarly, continuity of endoscopist for each procedure also lowers anxiety in females29, although this approach may be difficult to adopt in NHS endoscopy units that usually have pooled waiting lists and endoscopic trainees. Reviewing previous sedation requirements and recorded pain scores should be routine endoscopic practice to individualise the sedation plan. The analgesic adjunct of inhaled Nitrous oxide, a quick acting potent analgesic and anxiolytic, may also be helpful reducing excessive intravenous sedation30. By taking all these factors into account the global patient experience can be improved.\n\nIn conclusion, our research highlights a significant difference in the perception of colonoscopy dependent on gender, age of the patient and disease duration. These results should prompt endoscopy units to improve the experience of colonoscopy particularly amongst these subgroups of patients with IBD. In the future surrogate markers such as faecal calprotectin may reduce the burden of colonoscopy. In the meantime, further research is required to develop simple clinical tools to better identify and improve the experience of vulnerable patients that would otherwise tolerate the colonoscopic experience poorly.\n\n\nData availability\n\nF1000Research: Dataset 1. Table showing raw data collection from questionnaires, 10.5256/f1000research.6889.d10284131",
"appendix": "Author contributions\n\n\n\nSM: Article write up and data collection; CA: Article write up and data collection; LM: Data collection; VC: Data collection; PN: Data collection; AP: Data collection; RP: Article write up and data collection.\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\nSupplementary material\n\nQuestionnaire: 'Patients with IBD: survey of attitudes to colonoscopy'.\n\nClick here to access the data.\n\n\nReferences\n\nInformation about IBD - Crohn’s and Colitis UK. Reference Source\n\nBenitez JM, Meuwis MA, Reenaers C, et al.: Role of endoscopy, cross-sectional imaging and biomarkers in Crohn’s disease monitoring. Gut. 2013; 62(12): 1806–16. PubMed Abstract | Publisher Full Text\n\nPeyrin-Biroulet L, Ferrante M, Magro F, et al.: Results from the 2nd Scientific Workshop of the ECCO. I: Impact of mucosal healing on the course of inflammatory bowel disease. J Crohns Colitis. 2011; 5(5): 477–83. PubMed Abstract | Publisher Full Text\n\nCasellas F, Barreiro de Acosta M, Iglesias M, et al.: Mucosal healing restores normal health and quality of life in patients with inflammatory bowel disease. Eur J Gastroenterol Hepatol. 2012; 24(7): 762–9. PubMed Abstract | Publisher Full Text\n\nD’Haens G, Noman M, Baert F, et al.: Endoscopic healing after infliximab treatment for Crohn’s disease provides a longer time to relapse. Gastroenterology. 2002; 122: A100. (abstract).\n\nRutgeerts P, Diamond RH, Bala M, et al.: Scheduled maintenance treatment with infliximab is superior to episodic treatment for the healing of mucosal ulceration associated with Crohn’s disease. Gastrointest Endosc. 2006; 63(3): 433–42. PubMed Abstract | Publisher Full Text\n\nD’Haens G, Ferrante M, Vermeire S, et al.: Fecal calprotectin is a surrogate marker for endoscopic lesions in inflammatory bowel disease. Inflamm Bowel Dis. 2012; 18(12): 2218–24. PubMed Abstract | Publisher Full Text\n\nAhmad A, Cowling T, Laverty A, et al.: Changing trends in IBD hospital admissions and management in England, 2001–02 to 2010–11. United European Gastroenterol J. 2014; 2(1 Suppl): A132–A605. Reference Source\n\nYlinen ER, Vehviläinen-Julkunen K, Pietilä AM: Effects of patients’ anxiety, previous pain experience and non-drug interventions on the pain experience during colonoscopy. J Clin Nurs. 2009; 18(13): 1937–44. PubMed Abstract | Publisher Full Text\n\nBytzer P, Lindeberg B: Impact of an information video before colonoscopy on patient satisfaction and anxiety - a randomized trial. Endoscopy. 2007; 39(8): 710–14. PubMed Abstract | Publisher Full Text\n\nRistikankare M, Hartikainen J, Heikkinen M, et al.: The effects of gender and age on the colonoscopic examination. J Clin Gastroenterol. 2001; 32(1): 69–75. PubMed Abstract | Publisher Full Text\n\nLai W, Fung M, Vatish J, et al.: How gender and age affect tolerance of colonoscopy. Gut. 2013; 62(Suppl 2): A45–A46. (abstract). Publisher Full Text\n\nMaslekar S, Hughes M, Gardiner A, et al.: Patient satisfaction with lower gastrointestinal endoscopy: doctors, nurse and nonmedical endoscopists. Colorectal Dis. 2010; 12(10): 1033–8. PubMed Abstract | Publisher Full Text\n\nEckardt VF, Kanzier G, Willems D, et al.: Colonoscopy without premedication versus barium enema: a comparison of patient discomfort. Gastrointest Endosc. 1996; 44(2): 177–80. PubMed Abstract | Publisher Full Text\n\nBernstein C, Thorn M, Monsees K, et al.: A prospective study of factors that determine cecal intubation time at colonoscopy. Gastrointest Endosc. 2005; 61(1): 72–5. PubMed Abstract | Publisher Full Text\n\nMorgan J, Roufeil L, Kaushik S, et al.: Influence of coping style and precolonoscopy information on pain and anxiety of colonoscopy. Gastrointest Endosc. 1998; 48(2): 119–27. PubMed Abstract | Publisher Full Text\n\nLin OS, Schembre DB, Ayub K, et al.: Patient satisfaction scores for endoscopic procedures: impact of a survey-collection method. Gastrointest Endosc. 2007; 65(6): 775–81. PubMed Abstract | Publisher Full Text\n\nBessissow T, Van Keerberghen CA, Van Oudenhove L, et al.: Anxiety is associated with impaired tolerance of colonoscopy preparation in inflammatory bowel disease and controls. J Crohns Colitis. 2013; 7(11): e580–7. PubMed Abstract | Publisher Full Text\n\nKim WH, Young JC, Jeong YP, et al.: Factors affecting insertion time and patient discomfort during colonoscopy. Gastrointest Endosc. 2000; 52(5): 600–05. PubMed Abstract | Publisher Full Text\n\nKale V, Dunne C, Ahmed M, et al.: Patients with intestinal inflammation require more sedation during colonoscopy. Gut. 2013; 62: A9–A10. (abstract). Publisher Full Text\n\nMulcahy HE, Hennessy E, Connor P, et al.: Changing patterns of sedation use for routine out-patient diagnostic gastroscopy between 1989 and 1998. Aliment Pharmacol Ther. 2001; 15(2): 217–20. PubMed Abstract | Publisher Full Text\n\nNCEPOD - Scoping our practice. 2004. Reference Source\n\nChartier L, Arthurs E, Sewitch MJ: Patient satisfaction with colonoscopy: a literature review and pilot study. Can J Gastroenterol. 2009; 23(3): 203–09. PubMed Abstract | Free Full Text\n\nde Jonge V, Sint Nicolaas J, Lalor EA, et al.: A prospective audit of patient experiences in colonoscopy using the Global Rating Scale: a cohort of 1,187 patients. Can J Gastroenterol. 2010; 24(10): 607–13. PubMed Abstract | Free Full Text\n\nBleiker EM, Menko FH, Taal BG, et al.: Screening behaviour of individuals at high risk for colorectal cancer. Gastroenterology. 2005; 128(2): 280–87. PubMed Abstract | Publisher Full Text\n\nVignally P, Gentile S, Grimaud F, et al.: Pertinence of a pre-colonoscopy consultation for routine information delivery. Gastroenterol Clin Biol. 2007; 31(12): 1055–61. PubMed Abstract | Publisher Full Text\n\nBjörkman I, Karlsson F, Lundberg A, et al.: Gender differences when using sedative music during colonoscopy. Gastroenterol Nurs. 2013; 36(1): 14–20. PubMed Abstract | Publisher Full Text\n\nCosta A, Montalbano LM, Orlando A, et al.: Music for colonoscopy: A single-blind randomized controlled trial. Dig Liver Dis. 2010; 42(12): 871–76. PubMed Abstract | Publisher Full Text\n\nFidler H, Hartnett A, Cheng Man K, et al.: Sex and familiarity of colonoscopists: patient preferences. Endoscopy. 2000; 32(6): 481–82. PubMed Abstract | Publisher Full Text\n\nEmmanouil DE, Quock RM: Advances in understanding the actions of nitrous oxide. Anesth Prog. 2007; 54(1): 9–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgan S, Alexakis C, Medcalf L, et al.: Dataset 1 in: Colonoscopy in patients with inflammatory bowel disease: self-reported experience, understanding, anxieties and tolerance of the procedure. F1000Research. 2015. Data Source"
}
|
[
{
"id": "11540",
"date": "27 Jan 2016",
"name": "Vassiliki Tsikitis",
"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 article “Colonoscopy in patients with inflammatory bowel disease: self-reported experience, understanding, anxieties and tolerance of the procedure,” the authors comment that the perception of colonoscopy significantly differs by gender and age of this unique patient population, where colonoscopy is required in frequent intervals. Females had a significantly worse perception of colonoscopy and a higher pre-procedural anxiety overall. Patients over the age of 55 were less worried about the procedure, and patients with prolonged duration of IBD expressed more discomfort with it. The survey is only based on a single hospital-institution and included 98 patients, of whom 46% were male. Although there are significant limitations in the study, it brings up an important point about how we physicians prepare our patient for an upcoming procedure.Overall good article, but the population examined was too small to make any significant findings.",
"responses": []
},
{
"id": "12490",
"date": "21 Mar 2016",
"name": "Agata Mulak",
"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 single-center study assessing self-reported experience, understanding, anxiety and tolerance of colonoscopy in 94 patients with inflammatory bowel disease (IBD). The study addresses important issues and has potential practical implications as understanding patients’ experience of the procedure is crucial for service improvement, particularly in IBD patients undergoing repetitive colonoscopies in the course of their diseases. The manuscript is written in a comprehensive way. However, there are some methodological shortcomings of the study affecting the reliability of the conclusions.Major comments:In the Methods section it should be described what kind of sedation (if any) is used in the Center and what is the proportion of colonoscopies involving sedation or anesthesia. This is an important factor affecting patients’ experience. Was the bowel preparation procedure the same in all examined patients? The comparison between two age groups: younger patients (<55 years) vs older subjects (≥55 years) is not quite representative. Firstly, due to the different number of patients in each group (76 vs 18 subjects). Secondly, female patients were significantly younger than male patients (39.9 vs 47.1 years, respectively; p<0.05). Therefore sex could be a confounding factor in the comparison between these two groups. Were there any differences in patients’ experience between subjects with ulcerative colitis and Crohn’s disease? It has been previously reported that female patients’ preference for the same sex endoscopists may significantly affect the level of pre-procedural anxiety and overall experience in female patients. It was not addressed in the questionnaire. Additional comments:Please verify the number of patients given in the abstract, because according to the text and Table 1 it should be 94 instead of 98. A written informed consent is usually required in this kind of study. It is said that colonoscopies were performed either by a senior clinician or by a trainee (under supervision). In fact, the endoscopists’ skill and experience could importantly determine patients’ discomfort during colonoscopy.",
"responses": []
},
{
"id": "16816",
"date": "10 Oct 2016",
"name": "Alyssa Parian",
"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 retrospective review of 94 IBD patients at a single center evaluating the IBD patient experience of colonoscopy. The importance of colonoscopy and mucosal healing requires more frequent colonoscopic exams and determining how to make the experience as pleasant as possible can improve patient satisfaction and patient compliance with recommended colonoscopy intervals. However, there are several missing components which are essential for determining how we can improve the patient's experience with colonoscopy. Overall, these findings don't provide any tangible items for the institution change in order to improve patient experience and more data should be included in this study which should be easily gleaned from the chart.\nThe type of bowel prep used plays a role in patients satisfaction and tolerance of the bowel preparation. Smaller volume bowel preps have shown to have increased patient satisfaction and patient compliance. It should be reported if there is a standard bowel prep at the institution and evaluate which bowel prep was best tolerated amongst patients.\n\nThe type of anesthesia also contributes to the overall patient experience and tolerance of the procedure. Is it typical in this center to use conscious sedation with narcotics/benzodiazepams or propofol sedation? Were both used and a comparison can be made as to which was better tolerated?\n\nThe disease activity could also influence the patients perception of the procedure. Was disease activity assessed and reported as this can confound the results.\n\nThe small size of the population studied limits full evaluation of additional factors that may contribute to IBD patient experience of colonoscopy.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-927
|
https://f1000research.com/articles/4-926/v1
|
30 Sep 15
|
{
"type": "Research Article",
"title": "Predictors of psychiatric rehospitalization among elderly patients",
"authors": [
"Chun Yin Terry Wong"
],
"abstract": "The population of Hong Kong and the proportion of elderly people have been increasing rapidly. The aim of this retrospective cohort study is to determine predictive factors for psychiatric rehospitalization within 2 years among elderly patients who were discharged from psychiatric wards, in attempt to reduce their rehospitalization rate and to reintegrate them into the community. Patients aged 65 and over, who were discharged from psychiatric wards of Pamela Youde Nethersole Eastern Hospital from 1 March 2010 to 29 February 2012, were identified. Rehospitalization within 2 years after discharge was the primary outcome measure, and the time to rehospitalization was measured as the secondary outcome. Patients were subgrouped into readmitted and non-readmitted groups. Logistic regression and Cox regression analyses were applied to the potential predictive factors with odds ratios and hazard ratios obtained, respectively, for the significant findings. Kaplan-Meier survival curves were plotted for graphical representation of the study results in survival analysis. 368 individuals satisfying the study criteria were identified. The same four factors were shown to be significantly associated with rehospitalization in both multiple logistic regression and Cox regression survival analysis. Referral to other psychiatric disciplines upon discharge (p< 0.001, OR=0.325, HR=0.405) was associated with a lower rehospitalization risk and correlated to a longer time to rehospitalization. History of suicidal behaviors (p< 0.001, OR=4.906, HR=3.161), history of violent behaviors (p< 0.001, OR=5.443, HR=3.935) and greater number of previous psychiatric admissions (p< 0.001, OR=1.250, HR=1.121) were associated with a higher rehospitalization risk and predicted earlier rehospitalization. The rehospitalization rate of elderly patients was 5.2% at 1 month, 9.5% at 3 months, 15.0% at 6 months, 17.1% at 1 year, 18.8% at 1.5 year and 20.9% at 2 years.",
"keywords": [
"risk factors",
"psychiatric",
"readmission",
"rehospitalization",
"elderly",
"geriatric"
],
"content": "Background\n\nThe population in Hong Kong and the proportion of elderly people are increasing rapidly. Pamela Youde Nethersole Eastern Hospital is a regional hospital under the Hospital Authority serving the eastern district of Hong Kong Island. The characteristics of elderly patients were different from that of general adults, for example, more of them suffered from cognitive disorders, they were more often hospitalized due to medical comorbidities, and they might require placement such as old age homes after discharge. Despite many psychiatric readmission studies were already available in the literature, surprisingly very few of them were targeted to elderly patients only.\n\nRehospitalization had been regarded as a useful indicator to measure quality of care provided by hospitals worldwide1,2. Since there are only a small number of elderly psychiatric rehospitalization studies available, studies involving elderly patents and patients of all ages (including the elderly) were reviewed. Socio-demographic factors that were identified as significant in previous studies included: age3–10, gender5,8,11–14, ethnicity15, marital status14,16–20, education level13, and type of residence5,6,8,10,15,17,22,23. Clinical factors significantly associated with rehospitalization included: length of inpatient stay3,5,9,10,16,19,24,25, primary psychiatric diagnosis4,5,8,9,11,14,18,19,21,22,24,26, presence of psychiatric comorbidities4,17,27, presence of medical comorbidity21,27, cognitive impairment3,25, referral for aftercare services6,7,29,30, history of suicidal behaviors12, history of violence11,25,28,31 and number of previous psychiatric admissions7,8,15,20,25,32,33. Many of the articles adopted a retrospective design and involved psychiatric patients of all ages, while very few were specifically targeted elderly patients20,22,27,33,34, and no such data was available for elderly psychiatric patients in Hong Kong. Therefore this study was conducted to identify risk factors in attempt to reduce rehospitalization, so mental health services could be utilized more effectively in view of the increasing needs from the aging population.\n\n\nMethods\n\nThe aim of this retrospective cohort study is to determine predictors for psychiatric rehospitalization over 2 years among elderly patients after they were discharged from psychiatric wards.\n\nPatients aged 65 and over, who were discharged from the psychiatric wards of Pamela Youde Nethersole Eastern Hospital from 1 March 2010 to 29 February 2012, formed the study population. For patients having repeated discharge episodes during the study period, only the first discharge episode was included as the index episode for that patient to avoid duplication of data from the same individuals.\n\nPatients who died during inpatient stay at the index episode, or had not received mental health care in any psychiatric clinics under the Hospital Authority after discharge, including those who were followed up by private psychiatrists or overseas psychiatric care systems, were excluded due to a lack of information to analyze their outcome.\n\nA list of patients satisfying the study criteria was generated from the Clinical Data Analysis and Reporting System (CDARS) of the Hospital Authority’s Medical Records Office. The socio-demographic and clinical factors of each patient were determined by reviewing their medical records in the Hospital Authority’s Clinical Management System (CMS) and case notes as charted by their respective case doctors. The CMS contained information on patients who had received health services under the Hospital Authority including their demographics, inpatient discharge summaries, outpatient consultation notes, physical or psychiatric diagnoses and medications that were prescribed. Data was collected electronically and entered in Microsoft Office Excel version 2010.\n\nRehospitalization was defined as the primary outcome.\n\nSocio-demographic factors included age (upon discharge), gender, ethnicity, marital status, education level and type of residence (upon discharge). Clinical factors included priority follow-up (PFU) status (see Appendix for more details), length of inpatient stay, primary psychiatric diagnosis (as determined from the medical coding with ICD-9-CM in CMS by respective case doctors), presence of psychiatric comorbidities (as reflected by more than one psychiatric diagnosis recorded in CMS in the index episode), number of chronic physical illnesses (which required regular outpatient follow ups by other specialties), MMSE scores, referral to other psychiatric disciplines upon discharge (including community psychiatric nurses, clinical psychologists, social workers or day hospital), history of suicidal behaviors (including suicide attempts and self-harm behaviors in lifetime), history of violent behaviors (in lifetime) and number of previous psychiatric admissions.\n\n\nData analysis\n\nThe null hypothesis was that none of the identified factors are associated with rehospitalization.\n\nAfter descriptive statistical studies, univariate tests were conducted on all independent variables to identify possible significant factors for subsequent analysis. For categorical factors, a Chi-squared test was performed, and Fisher’s exact test was conducted for factors with an expected cell count less than 5. All the continuous factors in this study were not normally distributed as determined by the Shapiro-Wilk test and Mann-Whitney U test was conducted. Significant and marginally significant factors with a p value of <0.100 identified in the univariate analysis were included in the subsequent multiple logistic regression analysis to identify significant factors (with a p value of <0.050) predicting rehospitalization in 2 years after discharge.\n\nSurvival analysis was further conducted to identify factors predicting earlier rehospitalization. The rehospitalization rates at different time points within the 2 years were calculated. Univariate analysis with simple Cox regression was performed to all variables. Those with a p value of <0.100 were included in the subsequent multiple Cox regression analysis, to determine variables that significantly (with a p value of <0.050) correlated with time to rehospitalization. Kaplan-Meier survival curves were also plotted.\n\nThe protocol of this study was approved by the Hospital Authority’s Research Ethics Committee of Hong Kong East Cluster (Reference number: HKEC-2014-049). Data were analyzed through IBM Statistical Product and Service Solutions (SPSS) software version 22.\n\n\nResults\n\nFigure 1 presents the formation of the study population and separation into the subgroups. Of all 454 discharge episodes from psychiatric wards of Pamela Youde Nethersole Eastern Hospital with patients aged 65 and over from 1 March 2010 to 29 February 2012, 368 individuals formed the study population after screening for the exclusion criteria. 77 individuals were readmitted while 291 individuals were not during the 2 year follow up period. The cumulative rehospitalization rate at 2 years was 20.9%.\n\nConcerning the socio-demographic characteristics of the study population, the median age was 76 and male to female ratio was 1 to 1.61. 98.1% of individuals were Chinese. 47% of the study population was married, 39.7% was widowed, 7.9% was divorced or separated and 5.4% was single. For education level, 41.6% was less than primary, 31% was primary, 20.6% was secondary and only 6.8% was tertiary or above. 52.7% of the study population lived at home with family or friends, 33.7% was living in placement, 9% lived alone at home and 4.6% lived with maid at home. Regarding the clinical characteristics of the study population, the median length of inpatient stay for the study population was 27 days. The median for number of chronic physical illnesses was 2 and number of previous admissions was 0. 96.2% of individuals had non-PFU status. For the primary psychiatric diagnosis, 37.2% had cognitive disorders, 32.9% had depressive disorders, 23.1% had psychotic disorders, 4.1% had bipolar disorders and the remaining 2.7% suffered from other mental illnesses. 20.1% of the study population suffered from psychiatric comorbidities. Regarding MMSE scores, 32.3% of the study population scored 11–20, 18.5% scored 21–26, 13.6% scored less than 10, 9% scored over 26, and 26.6% did not have scores documented. 69.8% of individuals had referral to other psychiatric disciplines upon discharge. Lastly, 24.2% had history of suicidal behaviors and 15.8% had history of violence in their lifetime.\n\nUnivariate analysis for socio-demographic factors and clinical factors of the study population are shown in Table 1 and Table 2 respectively. Results of subsequent multiple logistic regression are shown in Table 3. Four significant factors were identified with their odds ratios calculated. Referral to other psychiatric disciplines upon discharge (p< 0.001, OR=0.325) was associated with a lower risk of rehospitalization, while the other three factors including history of suicidal behaviors (p< 0.001, OR=4.906), history of violence (p< 0.001, OR=5.443) and number of previous admissions (p< 0.001, OR=1.250) were associated with higher risk of rehospitalization. The overall accuracy of this predictor model was 83.4%.\n\ncChi-squared test, fFisher’s exact test, mMann Whitney U test, IQR interquartile range\n\ncChi-squared test, fFisher’s exact test, mMann Whitney U test\n\nPFU priority follow-up, MMSE Mini-mental State Examination, IQR interquartile range\n\nSignificant p<0.050, OR odds ratio, CI confidence interval\n\nOdds ratios were adjusted by gender and ethnicity\n\nSurvival analysis with Cox regression was then conducted to examine factors that predicted earlier rehospitalization. For individuals who did not readmit but were deceased before completion of the 2 year follow up period, the date of death was taken as the date of censoring. Taking different time points in the Kaplan-Meier survival curve as plotted in Figure 2, the cumulative readmission rate of the study population was 5.2% at 1 month, 9.5% at 3 months, 15.0% at 6 months, 17.1% at 1 year, 18.8% at 1.5 year and lastly 20.9% at 2 years upon completion of the follow up period. Most of the rehospitalization occurred within the first 6 months after discharge where the slope of the curve was steepest.\n\nUnivariate analysis by simple Cox regression was performed and the results are shown in Table 4. The significant results of subsequent multiple Cox regression survival analyses are presented in Table 5. The same four significant factors were identified in the multiple Cox regression analysis as that in the previous logistic regression analysis. Hazard ratios were calculated and Kaplan-Meier survival curves were plotted. Referral to other psychiatric disciplines upon discharge (p< 0.001, HR=0.405) correlated to a longer time to rehospitalization (Figure 3), while history of suicidal behaviors (p< 0.001, HR=3.161), history of violence (p< 0.001, HR=3.935) and number of previous psychiatric admissions (p< 0.001, HR=1.121) were predictive factors for earlier rehospitalization (Figure 4, Figure 5, Figure 6 respectively).\n\nSignificant p<0.050, HR hazard ratio, CI confidence interval\n\nHazard ratios were adjusted by age, gender and ethnicity\n\n\nDiscussion\n\nThis study involved Chinese and non-Chinese elderly psychiatric patients in Hong Kong, and by comparison between the two groups, ethnicity was not identified as a significant factor. However, it should be noted that too few cases were non-Chinese and so the statistical comparison between them might not be meaningful. In comparison to other studies involving psychogeriatric patients only, most studies did not find type of residence to be a significant factor in psychiatric readmission, except 1 study22 which reported higher readmission risk in patients with little or no supervision in living arrangements following discharge. However, this study did not find any significant differences in outcome regarding rehospitalization between patients who lived alone, with others, or in placement.\n\nThe same four clinical factors were shown to be significantly associated with rehospitalization with a p value of <0.001 in both multiple logistic regression and Cox regression survival analysis, including referral to other psychiatric disciplines upon discharge, history of suicidal behaviors, history of violence and number of previous psychiatric admissions. With adequate aftercare services6,7,29,30 by referral to other disciplines for support upon discharge, patients were more likely to comply with treatment plans and have their problems managed in outpatient settings, which reduced the need for psychiatric rehospitalization. Moreover, a study12 had reported that patients with previous suicidal attempts were more often readmitted, and similar findings had been shown in studies that patients having violence behaviors previously were at higher risk of readmission11,25,28,31. Psychiatric rehospitalization was often required when patients displayed risks of harming self or others. Furthermore, studies had extensively reported that patients with greater number of previous admissions were predisposed to rehospitalization7,8,15,20,25,32,33. These patients might be more often rehospitalized due to suboptimal control on symptoms relating to their mental health problems and inadequate support in the community.\n\nConcerning factors that were more specific to the elderly, 1 reviewed article27 reported that elderly patients without medical comorbidity were more likely rehospitalized, however, number of chronic illnesses was not identified as a significant risk factor. Elderly patients were more likely to be hospitalized due to poor physical conditions. Sometimes psychiatric symptoms had already been treated in general wards, but readmission to general wards were not accounted in this study. Besides, having a cognitive disorder or a lower MMSE score would not affect the risk of rehospitalization. However, it was noted that over one-fourth (26.6%) of the study population did not have their MMSE scores documented, and therefore the results might differ if the MMSE scores from all study individuals were available.\n\nFirstly, this was a retrospective study and the correctness of data depended heavily on the information in CMS. Some variables could have been underestimated or changed during the follow up period. Secondly, the study population involved the psychiatric unit in a regional hospital in Hong Kong only. The findings might not be generalized to other parts of the world.\n\nData on patients’ characteristics were retrieved through medical records rather than self-reporting from patients and this could minimize recall bias. Besides, this study involved elderly patients only and the findings were more specific towards psychogeriatric patients.\n\nThese findings were important to the daily practice of psychiatrists and especially the psychogeriatricians, since most of the previous rehospitalization studies in the literature were not specifically targeted to elderly patients. There was very little information available regarding risk factors that predicted psychiatric rehospitalization of elderly patients in Hong Kong prior to this study. In view of the aging population and increasing need for psychiatric services in Hong Kong, this study could help in identifying elderly patients with a high rehospitalization risk for more intensive treatments and better discharge planning based on their risk factors, including history of suicidal behaviors, history of violent behaviors and greater number of previous psychiatric admissions. Factors including cognitive disorders, lower MMSE scores, comorbid chronic physical illnesses, marital status, type of residence, age, gender, ethnicity, PFU status, education level, length of inpatient stay, and presence of psychiatric comorbidities did not affect the risk of rehospitalization in the elderly psychiatric patients. On the other hand, referral to other psychiatric disciplines upon discharge was highly encouraged to prevent rehospitalization.\n\nA larger prospective study should be carried out to determine risk factors for psychiatric rehospitalization in elderly patients. Future research could also investigate daily functioning and quality of life of elderly patients for a better understanding on their condition after discharge from psychiatric hospitals.\n\n\nConclusion\n\nThis retrospective cohort study has provided important information regarding psychiatric rehospitalization of elderly patients. Rehospitalization was mainly affected by clinical characteristics and occurred mostly within the first 6 months after discharge. Among the significant factors for rehospitalization, history of suicidal behaviors, history of violent behaviors and greater number of previous psychiatric admissions were associated with a higher rehospitalization risk. Referral to other psychiatric disciplines upon discharge predicted a better outcome and was highly recommended for elderly patients.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data of elderly patients discharged from psychiatric wards within the study period, 10.5256/f1000research.7135.d10339936",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe author wishes to thank Dr Dunn Lai Wah, Dr Lai Xin and Dr Tsui Kwan Yee for their advice. The work was part of the dissertation for completing HKCPsych Part III Examination.\n\n\nAppendix\n\nSince 1982, mental patients in Hong Kong were categorized into non-PFU (ordinary), PFU (target) and PFU (subtarget) groups depending on their risk level. Patients in PFU (subtarget) group were considered to have the highest violence risk.\n\n\nReferences\n\nFischer C, Lingsma HF, Marang-van de Mheen PJ, et al.: Is the readmission rate a valid quality indicator? A review of the evidence. PLoS One. 2014; 9(11): e112282. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilne R, Clarke A: Can readmission rates be used as an outcome indicator? BMJ. 1990; 301(6761): 1139–1140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaron K, Hays JR: Characteristics of readmitted psychiatric inpatients. Psychol Rep. 2003; 93(1): 235–238. PubMed Abstract | Publisher Full Text\n\nChang CM, Lee Y, Lee Y, et al.: Predictors of readmission to a medical-psychiatric unit among patients with minor mental disorders. Chang Gung Med J. 2001; 24(1): 34–43. PubMed Abstract\n\nLin CH, Chen WL, Lin CM, et al.: Predictors of psychiatric readmissions in the short- and long-term: a population-based study in Taiwan. Clinics (Sao Paulo). 2010; 65(5): 481–489. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOiesvold T, Saarento O, Sytema S, et al.: Predictors for readmission risk of new patients: the Nordic Comparative Study on Sectorized Psychiatry. Acta Psychiatr Scand. 2000; 101(5): 367–373. PubMed Abstract | Publisher Full Text\n\nSilva NC, Bassani DG, Palazzo LS: A case-control study of factors associated with multiple psychiatric readmissions. Psychiatr Serv. 2009; 60(6): 786–791. PubMed Abstract | Publisher Full Text\n\nThornicroft G, Gooch C, Dayson D: The TAPS project. 17: Readmission to hospital for long term psychiatric patients after discharge to the community. BMJ. 1992; 305(6860): 996–998. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYussuf AD, Kuranga SA, Balogun OR, et al.: Predictors of psychiatric readmissions to the psychiatric unit of a tertiary health facility in a Nigerian city - a 5-year study. Afr J Psychiatry (Johannesbg). 2008; 11(3): 187–190. PubMed Abstract | Publisher Full Text\n\nZilber N, Hornik-Lurie T, Lerner Y: Predictors of early psychiatric rehospitalization: a national case register study. Isr J Psychiatry Relat Sci. 2011; 48(1): 49–53. PubMed Abstract\n\nDayson D, Gooch C, Thornicroft G: The TAPS project. 16: Difficult to place, long term psychiatric patients: risk factors for failure to resettle long stay patients in community facilities. BMJ. 1992; 305(6860): 993–995. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsang YKA, Wong SL: Re-hospitalization of Psychiatric Residents of a Long-stay Care Home. Nurs Health. 2014; 2(6): 122–125. Reference Source\n\nWieselgren IM, Lindstrom LH: A prospective 1-5 year outcome study in first-admitted and readmitted schizophrenic patients; relationship to heredity, premorbid adjustment, duration of disease and education level at index admission and neuroleptic treatment. Acta Psychiatr Scand. 1996; 93(1): 9–19. PubMed Abstract | Publisher Full Text\n\nWoo BK, Golshan S, Allen EC, et al.: Factors associated with frequent admissions to an acute geriatric psychiatric inpatient unit. J Geriatr Psychiatry Neurol. 2006; 19(4): 226–230. PubMed Abstract | Publisher Full Text\n\nYamada MM, Korman M, Hughes CW: Predicting rehospitalization of persons with severe mental illness. J Rehabil. 2000; 66(2): 32–39. Reference Source\n\nFeigon S, Hays JR: Prediction of readmission of psychiatric inpatients. Psychol Rep. 2003; 93(3 Pt 1): 816–818. PubMed Abstract | Publisher Full Text\n\nFrick U, Frick H, Langguth B, et al.: The revolving door phenomenon revisited: time to readmission in 17’145 [corrected] patients with 37’697 hospitalisations at a German psychiatric hospital. PLoS One. 2013; 8(10): e75612. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarekatain M, Maracy MR, Hassannejad R, et al.: Factors associated with readmission of patients at a university hospital psychiatric ward in Iran. Psychiatry J. 2013; 2013: 685625. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJaramillo-Gonzalez LE, Sanchez-Pedraza R, Herazo MI: The frequency of rehospitalization and associated factors in Colombian psychiatric patients: a cohort study. BMC Psychiatry. 2014; 14: 161. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorrow-Howell NL, Proctor EK, Blinne WR, et al.: Post-acute dispositions of older adults hospitalized for depression. Aging Ment Health. 2006; 10(4): 352–361. PubMed Abstract | Publisher Full Text\n\nIrmiter C, McCarthy JF, Barry KL, et al.: Reinstitutionalization following psychiatric discharge among VA patients with serious mental illness: a national longitudinal study. Psychiatr Q. 2007; 78(4): 279–286. PubMed Abstract | Publisher Full Text\n\nMercer GT, Molinari V, Kunik ME, et al.: Rehospitalization of older psychiatric inpatients: an investigation of predictors. Gerontologist. 1999; 39(5): 591–598. PubMed Abstract | Publisher Full Text\n\nWalker R, Minor-Schork D, Bloch R, et al.: High risk factors for rehospitalization within six months. Psychiatr Q. 1996; 67(3): 235–243. PubMed Abstract | Publisher Full Text\n\nManu P, Khan S, Radhakrishnan R, et al.: Body mass index identified as an independent predictor of psychiatric readmission. J Clin Psychiatry. 2014; 75(6): e573–577. PubMed Abstract | Publisher Full Text\n\nTulloch AD, David AS, Thornicroft G: Exploring the predictors of early readmission to psychiatric hospital. Epidemiol Psychiatr Sci. 2015; 1–13. PubMed Abstract | Publisher Full Text\n\nBobo WV, Hoge CW, Messina MA, et al.: Characteristics of repeat users of an inpatient psychiatry service at a large military tertiary care hospital. Mil Med. 2004; 169(8): 648–653. PubMed Abstract | Publisher Full Text\n\nPrince JD, Akincigil A, Kalay E, et al.: Psychiatric rehospitalization among elderly persons in the United States. Psychiatr Serv. 2008; 59(9): 1038–1045. PubMed Abstract | Publisher Full Text\n\nZhang J, Harvey C, Andrew C: Factors associated with length of stay and the risk of readmission in an acute psychiatric inpatient facility: a retrospective study. Aust N Z J Psychiatry. 2011; 45(7): 578–585. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcGrew JH, Bond GR, Dietzen L, et al.: A multisite study of client outcomes in assertive community treatment. Psychiatr Serv. 1995; 46(7): 696–701. PubMed Abstract | Publisher Full Text\n\nCallaly T, Hyland M, Trauer T, et al.: Readmission to an acute psychiatric unit within 28 days of discharge: identifying those at risk. Aust Health Rev. 2010; 34(3): 282–285. PubMed Abstract | Publisher Full Text\n\nKent S, Yellowlees P: Psychiatric and social reasons for frequent rehospitalization. Hosp Community Psychiatry. 1994; 45(4): 347–350. PubMed Abstract | Publisher Full Text\n\nZhou Y, Rosenheck RA, Mohamed S, et al.: Retrospective assessment of factors associated with readmission in a large psychiatric hospital in Guangzhou, China. Shanghai Arch Psychiatry. 2014; 26(3): 138–148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMonnelly EP: Instability before discharge and previous psychiatric admissions as predictors of early readmission. Psychiatr Serv. 1997; 48(12): 1584–1586. PubMed Abstract | Publisher Full Text\n\nStoudemire A, Hill CD, Dalton ST, et al.: Rehospitalization rates in older depressed adults after antidepressant and electroconvulsive therapy treatment. J Am Geriatr Soc. 1994; 42(12): 1282–1285. PubMed Abstract | Publisher Full Text\n\nTombaugh TN, McIntyre NJ: The mini-mental state examination: a comprehensive review. J Am Geriatr Soc. 1992; 40(9): 922–935. PubMed Abstract | Publisher Full Text\n\nWong CYT: Dataset 1 in: Predictors of psychiatric rehospitalization among elderly patients. F1000Research. 2015. Data Source"
}
|
[
{
"id": "10954",
"date": "17 Dec 2015",
"name": "Xiao Shifu",
"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\nWe are very interested in this article, for it might be helpful in some clinical practice. However, we need the authors to answer some questions:\n\n1. How do you select the possible factors that might be related with re-admission? Why should these preclude the factors of psychiatric drugs;\n\n2. We suggest that the measurement data should be represented by mean and its standard deviation.",
"responses": [
{
"c_id": "1734",
"date": "18 Dec 2015",
"name": "Chun Yin Terry Wong",
"role": "Author Response",
"response": "Thanks a lot for your comments. In response to your questions:The possible factors included in the study were the most common predictors of psychiatric rehospitalizations that were identified from the previous studies in the literature. This study investigated if these factors could also affect the risk of rehospitalization in the Chinese elderly psychiatric patients. Since the use of different types of psychiatric drugs had not been shown to be associated with rehospitalization risk in the studies reviewed, this factor was not included as an independent variable. All the continuous variables in the study were not normally distributed. Therefore median and IQR were chosen to represent these data since they would be less affected by the extreme values."
}
]
},
{
"id": "12079",
"date": "25 Jan 2016",
"name": "W.Sue T Griffin",
"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, Wong and his colleagues examined several factors that might predict re-hospitalization rates among elderly patients to a psychiatric unit. The study is a retrospective analysis of data from patients mostly at the Pamela Youde Nethersole Eastern Hospital in Hong Kong – possible predictive factors obtained “in part” from previous studies in the same domain were sometimes included. The reported main factors related to re-admission included a history of multiple admissions to hospital, and suicidal and or violent behaviors. It isn’t surprising to note that patients with a history of suicidal or violent behaviors were at greater risk for re-admission as they require “acute” intervention (s). This is important as most re-admissions occurred in the first 6 months after discharge, raising concerns for the need for close outpatient follow up to ensure patient adherence to treatment plans. Importantly, and perhaps related to this, referral of patients to appropriate psychiatric disciplines was associated with fewer re-admissions.It was surprising that no significant differences were noted in the study, regarding outcomes between patients who lived alone or with others. This, as well as future studies to include patients with chronic psychiatric diseases may allow discoveries regarding relationships between outcomes as they may be affected by inclusion of psychiatric symptoms in patient histories when a patient is admitted to hospital in non-psychiatric wards.In summary, information reported in this study adds to our limited knowledge regarding the principle reasons for re-admission of elderly patients to hospital and, thus, may lead to improvements in patient care. Moreover, based on outcomes reported in this study, hospitals may benefit by relief from the usual challenges of inpatient bed availability and reimbursement from third party payers. More generally, health care costs might be reduced and family and other caregiver burdens lessened.",
"responses": [
{
"c_id": "1779",
"date": "26 Jan 2016",
"name": "Chun Yin Terry Wong",
"role": "Author Response",
"response": "Thank you so much for your review and your comments."
}
]
},
{
"id": "11435",
"date": "10 May 2016",
"name": "Jeffrey Braithwaite",
"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 was a useful paper and an informative read. The title is clear, although it might be indicated where the study was located (Hong Kong). I personally prefer structured abstracts.The methods and data analysis are well described. Sensible limitations, including that the study was restricted to one psychiatric unit, are provided. The author makes the valid point that few studies of this type are about elderly patients, and this study fills a gap in our knowledge, although it is confirmatory of much that we already know in the broader domain of psychiatric rehospitalisaton.",
"responses": [
{
"c_id": "1975",
"date": "12 May 2016",
"name": "Chun Yin Terry Wong",
"role": "Author Response",
"response": "Thank you very much for your review and comments."
}
]
}
] | 1
|
https://f1000research.com/articles/4-926
|
https://f1000research.com/articles/4-924/v1
|
30 Sep 15
|
{
"type": "Opinion Article",
"title": "Circulating nucleic acids: a new class of physiological mobile genetic elements",
"authors": [
"Indraneel Mittra"
],
"abstract": "Mobile genetic elements play a major role in shaping biotic genomes and bringing about evolutionary transformations. Herein, a new class of mobile genetic elements is proposed in the form of circulating nucleic acids (CNAs) derived from the billions of cells that die in the body every day due to normal physiology and that act intra-corporeally. A recent study shows that CNAs can freely enter into healthy cells, integrate into their genomes by a unique mechanism and cause damage to their DNA. Being ubiquitous and continuously arising, CNA-induced DNA damage may be the underlying cause of ageing, ageing-related disabilities and the ultimate demise of the organism. Thus, DNA seems to act in the paradoxical roles of both preserver and destroyer of life. This new class of mobile genetic element may be relevant not only to multi-cellular organisms with established circulatory systems, but also to other multi-cellular organisms in which intra-corporeal mobility of nucleic acids may be mediated via the medium of extra-cellular fluid.",
"keywords": [
"Mobile genetic elements",
"horizontal gene transfer",
"circulating nucleic acids",
"circulating DNA",
"circulating chromatin",
"DNA damage",
"ageing"
],
"content": "Background\n\nBarbara McClintock published her classic paper on mobile genetic elements (MGEs) in 19501, but it took the scientific community several decades to appreciate the enormity of her discovery. Today, it is recognized that MGEs occur widely in nature in prokaryotes, archaea and eukaryotes and play a major role in shaping their genomes and bringing about evolutionary transformations and adaptation2–5. Their bizarre behavior of moving from one part of the genome to another distinguishes them from the functioning of conventional genetic elements.\n\nMGEs belong to two classes viz, intra-genomic and inter-genomic. Intra-genomic MGEs are transposable elements (TEs) or transposons which constitute nearly 50% of the human genome but are variable among species and comprise 1%–5% of prokaryotic genomes6. Inter-genomic transposable elements, on the other hand, underlie horizontal or lateral gene transfer (HGT) whereby segments of DNA are transferred from one organism to another7–9. Although HGT is known to occur extensively in bacteria and are responsible for development of antibiotic resistance10, increasing evidence of HGT between other organisms is coming to light. For example, HGT between prokaryote and eukaryote, eukaryote and eukaryote, eukaryote and prokaryote has been reported7,11. Although the initial claims of presence of bacterial DNA in human genome were dismissed as erroneous12,13, recent evidence has confirmed the presence of bacteria DNA sequences in about one-third of healthy humans and in greater numbers in cancer cells14. A recent analysis of public databases of transcriptome sequences of multiple organisms discovered that human beings have picked up at least 145 genes from other species during the course of evolution11. Thus, HGT results in what is called a ‘web of life’ rather than a steadily bifurcating evolutionary tree8.\n\n\nCirculating nucleic acids as a new class of mobile genetic elements\n\nBased on a recent finding15, a new class of mobile genetic elements is proposed viz, circulating nucleic acids, which are produced as a result of normal physiology and operate intra-corporeally or within the body of an organism. Circulating nucleic acids (CNAs) in the form of fragmented DNA and chromatin (DNAfs and Cfs) are known to circulate in blood and are derived from the hundreds of billions of cells that die through apoptosis in the adult human body on a daily basis16,17. These fragments have a size range of between 100bp–1000bp, have a half-life of 10–15 minutes and are ultimately removed by the liver18,19. The presence of Cfs (nucleosomes) in blood can be detected by a sandwich ELISA assay15, but whether naked DNA circulates as such remains an open question since the possibility cannot be excluded that DNAfs isolated from plasma/serum are in fact products of the DNA purification process.\n\nResults of a recent study summarized below have revealed that CNAs can act as mobile genetic elements15. DNAfs and Cfs isolated from blood of healthy volunteers and cancer patients are actively taken up by cells in culture whereupon they rapidly accumulate in their nuclei and associate themselves with their chromosomes. The intracellular DNAfs and Cfs trigger a DNA-damage-repair-response (DDR) with up-regulation of multiple pathways of DNA damage and repair that facilitate their integration into host cell genome. Presence of human DNA in recipient mouse cell chromosomes could be detected by FISH while whole-genome sequencing uncovered tens of thousands of human reads in mouse cells. The integration of DNAfs and Cfs is stable and presence of extraneous DNA was demonstrable in single-cell clones developed from treated cells which had undergone numerous cell divisions. Genomic integration of DNAfs and Cfs results in phosphorylation of H2AX indicative of dsDNA breaks and up-regulation of apoptotic pathways in a proportion of cells. When injected intravenously into mice, DNAfs and Cfs integrate into cells of a variety of organs in the body, activate H2AX and the apoptotic marker active Caspase-3.\n\nWhether genomic integration of CNAs occurs preferentially in a site-specific manner or is random is not known; but in either case, integration of CNAs would give rise to somatic mutations in the host genome. Since integration of CNAs occurred in all organs of the body examined15, it may not be far-fetched to imagine that CNA-integration also occurs in germ cells. Genomic integration of CNAs would lead to DNA rearrangements, translocations and deletions20 – changes that are hallmarks of ageing, and large DNA rearrangements and cell to cell variations in gene expression are typical of ageing cells21,22.\n\n\nCNAs integrate into host-cell genomes by a unique mechanism\n\nAccording to the model depicted in the Figure, CNAs integrate into the genome by a unique mechanism in which activation of DDR plays a central role15. When DNAfs and Cfs enter into a cell, the latter mistakenly perceives the intracellular DNAfs and Cfs with dsDNA breaks in their two ends as damaged “self” DNA and activates DDR even before DNA damage has actually occurred. The activated DDR joins up multiple disparate DNAfs and Cfs into long concatemers by non-homologous-end-joining as a part of the repair process. It is the integration into the host cell genomes of the concatemers by homologous or non-homologous recombination that brings about damage to DNA. Thus, paradoxically, the activation of DDR brings about damage to DNA rather than preserving DNA integrity. This model of DNA damage and repair in which DDR precedes DNA damage is the reverse of the classical model based on damage induced by ionizing and UV-radiations and chemicals wherein DDR is activated after DNA damage. It is possible that this model of DNA damage and repair that facilitates CNAs integration may apply to horizontally transferred DNA in other organisms in nature.\n\nNHEJ = non-homologous end-joining; HR = homologous recombination; NHR = non-homologous recombination. Reproduced with permission from Mittra et al., J Biosci. 2015. 40: 91–111.\n\n\nImplications\n\nAlthough xenobiotics and DNA damaging agents constantly damage human DNA, these are usually transient and do not inflict permanent damage. CNAs, on the other hand, are ubiquitous, physiological and continuously arising, inflicting repeated damage and mutations to the somatic DNA. This naturally suggests that the somatic genome is not stable but remains in a state of turbulence characterized by DNA damage, mutations and rearrangements leading to DNA mosaicism and cell-to-cell variation in genomic structure and function. Indeed, cell-to-cell variations are being increasingly uncovered in the human body and are related to ageing21,23–25. The above events and the accompanying genomic instability may give rise to cancerous transformations which is compatible with the steep rise in the incidence of cancer with increasing age26. CNAs may also play an etiological role in several other disease conditions which are characterized by elevated levels of DNAfs and Cfs. These include auto-immune disorders27, and a host of acute and chronic human pathologies, namely, sepsis28, trauma29, burns30, organ transplantation31, diabetes32, myocardial infarction33, stroke34 and renal failure35.\n\n\nConclusions\n\nCNAs are a new class of physiological, continuously arising intra-corporeal mutagenic agents that might be responsible for ageing, age-related disabilities and ultimately the demise of the organism. Thus, DNA seems to act in paradoxical roles of both preserver and destroyer of life. This new class of intra-corporeal mobile genetic elements may be relevant not only to multi-cellular organisms which have a developed circulatory system, but also to other multi-cellular organisms in which intra-corporeal mobility of CNAs may be mediated via the medium of extra-cellular fluid.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Department of Atomic Energy, Government of India, through its grant CTCTMC to Tata Memorial Centre awarded to IM.\n\n\nAcknowledgement\n\nI thank Dr. Amit Dutt for helpful suggestions.\n\n\nReferences\n\nMcClintock B: The origin and behavior of mutable loci in maize. Proc Natl Acad Sci USA. 1950; 36(6): 344–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrost LS, Leplae R, Summers AO, et al.: Mobile genetic elements: the agents of open source evolution. Nat Rev Microbiol. 2005; 3(9): 722–732. PubMed Abstract | Publisher Full Text\n\nDeininger PL, Moran JV, Batzer MA, et al.: Mobile elements and mammalian genome evolution. Curr Opin Genet Dev. 2003; 13(6): 651–658. PubMed Abstract | Publisher Full Text\n\nKazazian HH Jr: Mobile elements: drivers of genome evolution. Science. 2004; 303(5664): 1626–1632. PubMed Abstract | Publisher Full Text\n\nBrookfield JF: Evolutionary genetics: Mobile DNAs as sources of adaptive change? Curr Biol. 2004; 14(9): R344–345. PubMed Abstract | Publisher Full Text\n\nCurcio MJ, Derbyshire KM: The outs and ins of transposition: from mu to kangaroo. Nat Rev Mol Cell Biol. 2003; 4(11): 865–877. PubMed Abstract | Publisher Full Text\n\nKeeling PJ, Palmer JD: Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet. 2008; 9(8): 605–618. PubMed Abstract | Publisher Full Text\n\nSoucy SM, Huang J, Gogarten JP: Horizontal gene transfer: building the web of life. Nat Rev Genet. 2015; 16(8): 472–482. PubMed Abstract | Publisher Full Text\n\nLevin HL, Moran JV: Dynamic interactions between transposable elements and their hosts. Nat Rev Genet. 2011; 12(9): 615–627. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPolz MF, Alm EJ, Hanage W: Horizontal gene transfer and the evolution of bacterial and archaeal population structure. Trends Genet. 2013; 29(3): 170–175. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCrisp A, Boschetti C, Perry M, et al.: Expression of multiple horizontally acquired genes is a hallmark of both vertebrate and invertebrate genomes. Genome Biol. 2015; 16: 50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInternational Human Genome Sequencing Consortium, Adekoya E, Ait-Zahra M, et al.: Initial sequencing and analysis of the human genome. Nature. 2001; 409(6822): 860–921. PubMed Abstract | Publisher Full Text\n\nStanhope MJ, Lupas A, Italia MJ, et al.: Phylogenetic analyses do not support horizontal gene transfers from bacteria to vertebrates. Nature. 2001; 411(6840): 940–944. PubMed Abstract | Publisher Full Text\n\nRiley DR, Sieber KB, Robinson KM, et al.: Bacteria-human somatic cell lateral gene transfer is enriched in cancer samples. PLoS Comput Biol. 2013; 9(6): e1003107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMittra I, Khare NK, Raghuram GV, et al.: Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes. J Biosci. 2015; 40(1): 91–111. PubMed Abstract | Publisher Full Text\n\nFliedner TM, Graessle D, Paulsen C, et al.: Structure and function of bone marrow hemopoiesis: mechanisms of response to ionizing radiation exposure. Cancer Biother Radiopharm. 2002; 17(4): 405–426. PubMed Abstract | Publisher Full Text\n\nvan Nieuwenhuijze AE, van Lopik T, Smeenk RJ, et al.: Time between onset of apoptosis and release of nucleosomes from apoptotic cells: putative implications for systemic lupus erythematosus. Ann Rheum Dis. 2003; 62(1): 10–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElshimali YI, Khaddour H, Sarkissyan M, et al.: The clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Int J Mol Sci. 2013; 14(9): 18925–18958. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGauthier VJ, Tyler LN, Mannik M: Blood clearance kinetics and liver uptake of mononucleosomes in mice. J Immunol. 1996; 156(3): 1151–1156. PubMed Abstract\n\nNevers P, Saedler H: Transposable genetic elements as agents of gene instability and chromosomal rearrangements. Nature. 1977; 268(5616): 109–115. PubMed Abstract | Publisher Full Text\n\nVijg J, Dollé ME: Large genome rearrangements as a primary cause of aging. Mech Ageing Dev. 2002; 123(8): 907–915. PubMed Abstract | Publisher Full Text\n\nBahar R, Hartmann CH, Rodriguez KA, et al.: Increased cell-to-cell variation in gene expression in ageing mouse heart. Nature. 2006; 441(7096): 1011–1014. PubMed Abstract | Publisher Full Text\n\nJacobs KB, Yeager M, Zhou W, et al.: Detectable clonal mosaicism and its relationship to aging and cancer. Nat Genet. 2012; 44(6): 651–658. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaurie CC, Laurie CA, Rice K, et al.: Detectable clonal mosaicism from birth to old age and its relationship to cancer. Nat Genet. 2012; 44(6): 642–650. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcConnell MJ, Lindberg MR, Brennand KJ, et al.: Mosaic copy number variation in human neurons. Science. 2013; 342(6158): 632–637. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCancer Research UK. All Cancers (C00-C97 Excl. C44) Average Number of New Cases per Year and Age-Specific Incidence Rates, UK, 2009–2011. Reference Source\n\nPisetsky DS, Ullal AJ: The blood nucleome in the pathogenesis of SLE. Autoimmun Rev. 2010; 10(1): 35–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRhodes A, Wort SJ, Thomas H, et al.: Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients. Crit Care. 2006; 10(2): R60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLam NY, Rainer TH, Chan LY, et al.: Time course of early and late changes in plasma DNA in trauma patients. Clin Chem. 2003; 49(8): 1286–1291. PubMed Abstract | Publisher Full Text\n\nChiu TW, Young R, Chan LY, et al.: Plasma cell-free DNA as an indicator of severity of injury in burn patients. Clin Chem Lab Med. 2006; 44(1): 13–17. PubMed Abstract | Publisher Full Text\n\nLui YY, Woo KS, Wang AY, et al.: Origin of plasma cell-free DNA after solid organ transplantation. Clin Chem. 2003; 49(3): 495–496. PubMed Abstract | Publisher Full Text\n\nButt AN, Shalchi Z, Hamaoui K, et al.: Circulating nucleic acids and diabetic complications. Ann NY Acad Sci. 2006; 1075: 258–270. PubMed Abstract | Publisher Full Text\n\nChang CP, Chia RH, Wu TL, et al.: Elevated cell-free serum DNA detected in patients with myocardial infarction. Clin Chim Acta. 2003; 327(1–2): 95–101. PubMed Abstract | Publisher Full Text\n\nTsai NW, Lin TK, Chen SD, et al.: The value of serial plasma nuclear and mitochondrial DNA levels in patients with acute ischemic stroke. Clin Chim Acta. 2011; 412(5–6): 476–479. PubMed Abstract | Publisher Full Text\n\nMittra I, Nair NK, Mishra PK: Nucleic acids in circulation: Are they harmful to the host? J Biosci. 2012; 37(2): 301–312. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10595",
"date": "13 Oct 2015",
"name": "Silvia Gravina",
"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 Opinion Article entitled “Circulating nucleic acids: a new class of physiological mobile genetic elements”, Indraneel Mittra investigated the topic of circulating nucleic acids as a new class of continuously arising DNA damaging agents. The author discusses a recent work (Mittra et al., 2015) which describes how free nucleic acids not only can enter cell nuclei freely, but they integrate in the host genome and activate proteins of DDR and apoptotic pathways. In particular, in the Opinion Article, the author hypothesizes that cell free DNA continuously arising induced damage can be the cause of aging and age related diseases.The manuscript is certainly well written and attractive, and discusses a very novel yet important topic in the aging field. My only concern is that in two parts of the manuscript the author jumps to conclusions too quickly, and I would suggest rephrasing certain statements. More specifically: pg2, mid right; “Whether genomic integration of CNAs occurs preferentially in a site-specific manner or is random is not known; but in either case, integration of CNAs would give rise to somatic mutations in the host genome.”This has to be demonstrated yet. We don’t really know if CNA integration is a direct cause of somatic mutations in the host genome. I would suggest making this statement milder (i.e.” Whether genomic integration of CNAs occurs preferentially in a site-specific manner or is random is not known. It is reasonable to speculate that integration of CNAs may give rise to somatic mutations in the host genome.” or “a fascinating hypothesis yet to be tested is that that integration of CNAs may give rise to somatic mutations in the host genome”.) Same for pg2 bottom part; “CNAs, on the other hand, are ubiquitous, physiological and continuously arising, inflicting repeated damage and mutations to the somatic DNA”. I would suggest here-as well- a milder statement (i.e., CNAs, on the other hand, are ubiquitous, physiological and continuously arising, inflicting repeated damage and potentially causing mutations to the somatic DNA”).",
"responses": []
},
{
"id": "11047",
"date": "11 Nov 2015",
"name": "Manchanahalli Rangaswamy Satyanarayana Rao",
"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 details a new paradigm in cancer genetics based on the recent original article published by the same group. The idea of the origin of the cancer phenotype based on the circulating nucleic acid/ chromatin fragments is really thought provoking and will generate a lot of interest in the cancer researchers. The manuscript is well written summarizing the salient features of their recently published article.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-924
|
https://f1000research.com/articles/4-922/v1
|
29 Sep 15
|
{
"type": "Correspondence",
"title": "Does CXCR3 chemokine receptor expression by CD8+ T cells affect their moving towards or only their binding to virus-infected monocytes?",
"authors": [
"Johannes M. Dijkstra"
],
"abstract": "This correspondence concerns a recent publication in Immunity by Hickman et al.1 who analyzed the effect of Cxcr3 knockout on migration of CD8+ T cells towards and within vaccinia virus-infected mouse ears. They found that Cxcr3 knockout had no effect on CD8+ T cell migration into the infected ears, a relatively mild effect on virus clearance, and an effect on the contact of CD8+ T cells with virus-infected cells. Curiously, despite having these basically sound and interesting data, Hickman et al. exaggerated the effect on virus clearance (“dramatically impaired virus clearance”) and focused their conclusions on assumed differences in migration towards infected cells (“CXCR3 chemokine receptor enables local CD8+ T cell migration”) rather than on better proven differences in binding to infected cells. I believe that from the data presented by Hickman et al. on the effect of Cxcr3 knockout a migration effect independent from the binding effect cannot be concluded beyond discussion. The fact that CXCR3 is a chemokine receptor, and that most researchers consequently expect a chemokine-gradient-dependent migration effect of the Cxcr3 knockout mutation, increases the risk of misleading readers when approached through the Hickman et al. narrative. The here-initiated discussion of their article may help to avoid such a misleading.",
"keywords": [
"CXCR3",
"T cell",
"CD8",
"virus",
"monocyte",
"migration",
"binding",
"intravital multiphoton microscopy"
],
"content": "\n\nHickman et al.1 established a vaccinia virus (VV) mouse ear-infection model, and investigated the importance of CXCR3 expression by CD8+ T cells for their ability to locate and bind to infected cells, and to help eradicate the virus. The receptor CXCR3 can bind the chemokines CXCL9 and CXCL10, of which genes in this mouse ear model were found to be abundantly expressed by virus-infected tissue1. Using a number of techniques, Hickman et al. assayed the migration of CD8+ T cells into the infected ears, including into dense areas of infected monocytes, and to the draining lymph nodes. Among the techniques used were intravital multiphoton microscopy (MPM) experiments, which allowed them beautifully to follow the migration of individual CD8+ T cells. Recipient mice included wildtype (wt; albino C57BL/6), CXCR3-/- and CD8-/- genotypes, while CD8+ lymphocytes used for grafting were isolated from CFP OT-I and CXCR3-/- DsRed OT-I transgenic mice. The recombinant vaccinia viruses used were VV-NP-eGFP and VV-NP-S-eGFP, of which only the latter contains the CD8+ T cell determinant peptide sequence SIINFEKL which can be recognized in the MHC context of this system by the T cell receptor transgenically expressed in OT-I mice. Hickman et al. investigated a number but not all possible combinations of these mice, cells and viruses, and their different effects on virus replication and CD8+ T cell migration.\n\nFindings by Hickman et al. were that (1) viral clearance by Cxcr3-/- CD8+ T cells was somewhat impaired at intermediate times of infection, (2) migration of Cxcr3+/+ and Cxcr3-/- CD8+ T cells into infected ears and also to draining lymph nodes was similarly efficient, (3) both Cxcr+/+ and Cxcr3-/- CD8+ T cells could be found in dense areas of VV-infected monocytes, although the relative abundance was about 1.4-fold higher for Cxcr3+/+ CD8+ T cells (see their Figure 5D), (4) Cxcr3-/- CD8+ T cells were more motile than Cxcr3+/+ CD8+ T cells in dense areas of infected monocytes (see their Figure 5F), (5) Cxcr3+/+ CD8+ T cells probably could bind better to virus-infected cells than found for Cxcr3-/- CD8+ T cells (see their Figures 5H and 5I), and (6) could also kill these cells more efficiently (see their Figure 6).\n\nTogether with the high-quality pictures and videos, the above listed data, most of which appear solid, contained enough substance for publication. For reasons that are not supported by the data, however, in their narrative Hickman et al. exaggerated the effect of the Cxcr3 knockout on viral clearance, and speculated as a major model of explanation that Cxcr3 knockout reduced penetration of CD8+ cells into infected cell areas. Here I expand on the two main issues, I and II, followed by a direct listing of the eight questions/issues as I placed them on the Immunity site (the first two therein overlap with the below items I and II). I moved the discussion to F1000Research to improve the visibility of what I hope should be a valuable public discussion.\n\n(I) Upon comparison of the development of VV-infection in ears of wt and Cxcr3-/- mice, Hickman et al. obtained results that they presented in their Figure 3F and summarized as “Likewise, we saw no overt changes in morbidity after epicutaneous VV infection; however, Cxcr3 deficiency interfered with viral clearance at intermediate times of infection (days 5–7)”. This statement is correct in stressing the modesty of the effect, but neglects that at day 5 post infection (p.i.) the virus titer is more than four-fold higher in wt than in Cxcr3-/- mice. This fact should have been either noted and discussed or the data re-evaluated in case of an error. This issue would probably not have remained unnoticed if the main narrative had properly stressed that the Cxcr3-/- knockout effect on virus clearance was rather modest and restricted to days 5–7 p.i. Instead, however, the Summary section of the article says “Cxcr3-/- mice exhibited dramatically impaired CD8+-T-cell-dependent virus clearance”, and this interpretation may have prevented co-authors, editors and reviewers from taking a thorough look at the day 5 p.i. result.\n\n(II) Whether the Figure 3F issue will seriously affect our understanding of CXCR3 function is questionable, but that might well be the case with another issue. By proper quantification, Hickman et al. show that the Cxcr3-/- CD8+ T cells are more motile and less abundant than Cxcr3+/+ CD8+ T cells in dense areas of infected monocytes (see their Figures 5D and 5F), which may be explained by differences in binding to the virus-infected cells (see their Figures 5H and 5I). However, without support by proper evidence and at least without proper quantification, the authors portray as their major model that the Cxcr3-/- CD8+ T cells have relative difficulties to enter dense areas of infected monocytes. The evidence should come from data presented in Figure 5C and movie S6, but the data is finally drawn together in a rather subjective and non-quantitative interpretation by the authors “Overall, however, whereas high numbers of wt T cells entered fields of virus-infected cells, Cxcr3-/- T cells hesitated at the perimeter of heavily infected areas of the tissue (Figure 5C; Movie S6).” Their Figure 5D shows that the percentages of Cxcr3+/+ CD8+ T and Cxcr3-/- CD8+ T cells are on average around 44% and 32%, respectively, and I wonder, even if the relatively small differences in presence levels could in part be explained by differences in penetration efficiencies, how these penetration efficiencies could reliably be judged without proper quantification. The proper assay for quantifying the penetration efficiencies would probably be the MPM technique shown in Figure 5E, but if I interpret the Figure 5E results correctly, the Cxcr3-/- CD8+ T cells have no apparent deficit in penetrating the virus-infected region, they only more readily leave. The lower presence of the Cxcr3-/- CD8+ T cells compared to the Cxcr3+/+ CD8+ T cells might be entirely explained by the lesser binding to the virus-infected cells, causing faster migration within this region and easier leaving of the region. It seems to me that, because CXCR3 is a chemokine receptor, Hickman et al. forcefully concentrate on a model different from binding. In my opinion, they should have focused on trying to find reasons for the differences in binding efficiencies, rather than assume without proper evidence that Cxcr3-/- CD8+ T cells have difficulties infiltrating regions of infected cells. Hickman et al. expressed their belief in the reduced penetration model in their title “CXCR3 chemokine receptor enables local CD8(+) T cell migration for the destruction of virus-infected cells”, in their description of the Figure 5D result “Although wt and Cxcr3-/- T cells were equally distributed outside of infected areas of the dermis, a higher percentage of wt T cells than of Cxcr3-/- T cells penetrated virus-infected areas (Figure 5D)”, and at several other sites in the article.\n\nIn summary, Hickman et al. produced good and interesting data, but they harmed their own article by an exaggerated and misleading narrative. I would like to invite Hickman et al. to address the above concerns, as well as a few other issues as they were already placed on the Immunity site:\n\n1. The major conclusion of the authors regarding the importance of CXCR3 for clearance of vaccinia virus from infected mouse ears appears to be summarized in their statement: “Likewise, we saw no overt changes in morbidity after epicutaneous VV infection; however, Cxcr3 deficiency interfered with viral clearance at intermediate times of infection (days 5–7)”. I wonder how this conclusion agrees with the day 5 result depicted in Figure 3F, which says that the virus-titer in wt mice is about four-fold higher. This observation is unlikely to be within chance variation, given the small SEM values and the fact that “All experiments were repeated at least three times with n = 2–5 mice/group”. Probably the p<0.0001 value depicted in the figure refers to this measurement.\n\n2. The authors conclude that Cxcr3-/- CD8+ cells show reduced penetration of dense areas of infected monocytes compared to wt CD8+ cells. However, the only parameter that appears to be measured reliably is the number of cells present, and if I interpret Figure 5E correctly, the Cxcr3-/- cells are not impaired in their ability to penetrate the infected area but only leave more easily (and then often come back again). From Figure 5C and movie S6, I can’t follow the interpretation by the authors that “Overall, however, whereas high numbers of wt T cells entered fields of virus-infected cells, Cxcr3-/- T cells hesitated at the perimeter of heavily infected areas of the tissue (Figure 5C; Movie S6).”\n\n3. The authors conclude that “Cxcr3-/- CD8+ T Cells activate and home normally”. Whereas most of their experimental data were obtained at day 5 p.i., for the “normal homing” conclusion in regard to draining lymph nodes they looked at day 2 p.i. (Figure 4). As far as I understand, for “homing” some degree of specificity should be shown. For the presence of transferred cells in infected ears the authors provide evidence of active homing in Figure S1, because, as they show, the transferred cells do not traffic to uninfected ears. I can see little evidence that the transferred cells detectable in the draining lymph node already at day 2 after infection are there because of active homing? Also in regard to their Figure 1B findings, day 2 p.i. may not be an appropriate time point to examine specific homing.\n\n4. The authors write “Together, these data show that CXCR3 is not required for SIINFEKL-expressing VV-induced activation of OT-I CD8+ T cells in vivo”, which most readers will interpret as that the SIINFEKL peptide was relevant. This is potentially misleading as the authors do appear not to have analyzed the effect of the SIINFEKL-epitope on the activation of Cxcr3-/- OT-1 CD8+ cells.\n\n5. It might be easier to interpret the findings if data measuring the outcome of the infection in CD8-/- hosts in the context of the different cell transfers were included. The report seems to concentrate on measurements at day 5 after infection. Did, for example, the mice survive?\n\n6. This is a study about clearance of virus from the mouse ear. Figure S3B shows that about 50% of the infected cells are CD45-negative, and consist mostly of keratinocytes as the authors explain. The virus infections of monocytes and keratinocytes should probably be understood as communicating barrels. Then why is it that for most experiments the authors chose not to show any results for the CD45-negative cells, although they systematically show virus titers and numbers of infected monocytes per ear? I feel this is an exaggeration of the principle to avoid unnecessary complexity in scientific stories.\n\n7. In Figure 4B the authors show that two days after infection the endogenous CD8+ cells in the draining lymph nodes do not express detectable IFNγ levels. But, apparently, at this day the inflammation had hardly started (see Figure 2C). Nevertheless, in other experiments measuring at day 5 p.i., at which the inflammation is pronounced, the authors seem to use the Figure 4B result to define all IFNγ+ CD45+ cells as transferred CD8+ T cells (see Figures 6B-D and the main text discussion of these results). With regard to IFNγ expression by endogenous cells, it would be interesting to have further justification why the data of a non-inflamed situation (day 2 p.i.) were extrapolated to an inflamed situation (day 5 p.i.).\n\n8. As a small point, it is unclear what is shown by the green color in Figure 4D, where green stains CD69 as well as virus infected cells.",
"appendix": "Competing interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThe author declared that no funding was involved in supporting this work.\n\n\nReferences\n\nHickman HD, Reynoso GV, Ngudiankama BF, et al.: CXCR3 chemokine receptor enables local CD8+ T cell migration for the destruction of virus-infected cells. Immunity. 2015; 42(3): 524–37. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11197",
"date": "13 Nov 2015",
"name": "Joanna R Groom",
"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\nWithout changing the main findings of the article “CXCR3 Chemokine Receptor Enables Local CD8+ T cell Migration for the Destruction of Virus-Infected Cells” (Hickman et al. Immunity 2015), the author wishes to clarify some discussion points. Firstly, the author claims the Hickman et al., “exaggerated the effect of CXCR3 knockout on viral clearance”. The data being referred by the author is Figure 3E,F, which demonstrates an increase in infected inflammatory monocytes/ear at days 5-7 in CXCR3-/- OT-I transferred animals and ear viral titres respectively. Indeed there is a disconnected in the Day 5 data, where the CXCR3-/- OT-I transferred animals have a lower titer, but higher infected monocytes compared to WT animals. This data should be re-evaluated by Hickman et al., for this discrepancy. However, the author fails to recognise or mention any data provided in Figure 6 E-J, which revisits the reduced viral titre in CXCR3-/- OT-I transferred animals. This data provides significant evidence of reduced clearance of infected monocytes and viral titer. All this data, appears to be generated on Day 5 of infection, thus conclusions of Hickman et al. are valid. The author should comment on the data presented in Figure 6 prior to acceptance of this critique. The second, and major area of clarification refers to the discussion of whether CXCR3-/- is required for entry into areas of high infection or the binding of infected cells. The images and supplementary movies, show the clear accumulation of CXCR3-/- OT-I cells at the perimeter of highly infected tissues, which has led Hickman et al. to highlight the role for CXCR3-dependent migration into these areas. One issue complicating the interpretation of this data is the phenotype in the CXCR3-/- animals that have large, high density regions of infected cells. These areas are missing in WT or co-transferred (both genotypes) OT-I animals, due to the clearance of viral infected monocytes. Therefore, the accumulation of OT-I cells at the periphery of these heavily infected areas of tissue can not be equally assessed, and the data presented using co-transferred genotypes is somewhat less compelling. Experiments that transfer WT OT-I cells into animals following establishment of infection could help answer this question, but as the data stands, there is not a clear indication that the accumulation of CXCR3-/- OT-I cells is due to the infection or CXCR3-dependent migration. Hickman et al., observe that although reduced, CXCR3-/- OT-I cells do areas of high infection and have compelling data (Figure5 E-I) indicating that once there, these cells are more mobile, and have reduced contacts with infected issue. These experiments indicate a mechanism for CXCR3 beyond entering the site of high infection. Hickman et al., conclude that “these data show that relative to WT cells CXCR3-/- CD8+ T cells exhibit decreased presence in virus-infected regions and shorter contacts with infected cells”, however the author is not satisfied with the discussion of the major phenotype within infected tissue – the reduced binding of CXCR3-/- OT-I cells to infected monocytes. This critique highlights the need for following studies that primarily focus on the mechanism of CXCR3 within infected viral tissues; migration and penetration into high viral areas verses the binding of infected cells – or both. The study from Hickman et al., describes data where both mechanisms are likely to play a role and they present this data openly. Hickman et al., have published an exhaustive study investigating the role for CXCR3 in the clearance of skin Vaccinia Virus infection by CD8+ cells. Overall this author provides an evaluation of the Hickman et al., paper that may enrich discussions on the mechanism of CXCR3, but does not alter the results or interpretation of the manuscript.",
"responses": [
{
"c_id": "1697",
"date": "16 Nov 2015",
"name": "Johannes M. Dijkstra",
"role": "Author Response",
"response": "Dear Dr. Groom,Thank you for your review, it is highly appreciated.I am glad that you agree with me that Hickman et al. should re-evaluate their Fig. 3F. After I will have received all reviewer reports, I will rewrite my correspondence and incorporate your request to discuss the Fig. 3F issue in regard to other day 5 findings presented by Hickman and co-workers. The simplest explanation would be that an error occurred during the construction of Fig. 3F, but it is up to Hickman and co-workers to elaborate on that. In regard to viral clearance the Hickman et al. study is about a time-window effect (days 5-7), whereas they found that the times of total clearance appeared to be similar in the different backgrounds. Whether a restricted time-window effect can be considered \"dramatically impaired viral clearance\" may be a matter of taste, but because of their writing style it took me a while to realize that their study was only about a time-window effect. I can only assume that co-authors and reviewers had the same problem, because as soon as one does focus on days 5-7, one immediately sees the discrepancy of the Fig. 3F day 5 result. I will not change that part of my criticism, because I think that it is fair.As for the discussion on whether CXCR3 is important for entering the areas of infected monocytes or for binding to the infected cells. You appear to agree with me that both mechanisms could be (partially) responsible, and that the evidence should have come from a direct comparison between CXCR3-/- OT-I cells and WT OT-I cells. In regard to the reduced penetration model you state \"the data presented using co-transferred genotypes is somewhat less compelling\" whereas for the reduced binding model you state that the authors \"have compelling data (Figure5 E-I) indicating that once there, these cells (note by me: CXCR3-/- OT-I cells) are more mobile, and have reduced contacts with infected issue\". So we largely agree on that matter, although I may feel more strongly than you that for the reduced penetration model Hickman et al. did not present any convincing evidence. The compelling part of their reduced contact model derives from the fact that they properly quantified the times of contacts and speeds of migration through the infected cell areas, and the \"less compelling\" part from their reduced penetration model derives from the fact that they did not quantify it. Nevertheless, Hickman et al. chose to highlight the penetration model as the major conclusion in their title, which says that CXCR3 enables migration. By all means I think that this is wrong from logic point of view, as they should have only highlighted the model for which they provided evidence and not the model for which they did not provide solid evidence. I appreciate that you add some nuance to the debate, but I probably will not change my text on this matter. I fully agree with your statement \"This critique highlights the need for following studies that primarily focus on the mechanism of CXCR3 within infected viral tissues; migration and penetration into high viral areas verses the binding of infected cells – or both.\" The important part of discussions like the current one is to help sharpen the questions for future research, and I thank you again for your contribution.Sincerely,Johannes Dijkstra"
}
]
}
] | 1
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https://f1000research.com/articles/4-922
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https://f1000research.com/articles/4-916/v1
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28 Sep 15
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{
"type": "Review",
"title": "Chemoprevention of cancer: current evidence and future prospects",
"authors": [
"Vassiliki Benetou",
"Areti Lagiou",
"Pagona Lagiou",
"Areti Lagiou",
"Pagona Lagiou"
],
"abstract": "Cancer chemoprevention refers to the use of agents for the inhibition, delay, or reversal of carcinogenesis before invasion. In the present review, agents examined in the context of cancer chemoprevention are classified in four major categories—hormonal, medications, diet-related agents, and vaccines—and the main representatives of each category are presented. Although there are serious constraints in the documentation of effectiveness of chemopreventive agents, mainly stemming from the long latency of the condition they are addressing and the frequent lack of intermediate biomarkers, there is little disagreement about the role of aspirin, whereas a diet rich in vegetables and fruits appears to convey more protection than individual micronutrients. Among categories of cancer chemopreventive agents, hormonal ones and vaccines might hold more promise for the future. Also, the identification of individuals who would benefit most from chemopreventive interventions on the basis of their genetic profiles could open new prospects for cancer chemoprevention.",
"keywords": [
"Cancer",
"chemoprevention",
"aspirin",
"nutrients",
"supplements",
"antiestrogens",
"antiandrogens",
"SERMs",
"aromatase inhibitors",
"vaccines",
"HBV",
"HPV"
],
"content": "Introduction\n\nCancer is a leading cause of death worldwide, ranking second in economically developed countries1,2. With several forms of cancer being poorly controlled through treatments, which themselves have serious side effects, and with the unavoidable limitations of cancer screening programs, chemoprevention and its potential have generated much hope and interest during the last decades. Cancer chemoprevention is the inhibition or reversal of carcinogenesis (before invasion) by intervention with pharmacologically active agents3. The concept was introduced by Sporn and colleagues in the mid-1970s4.\n\nMore than a decade ago, one of us (PL) contributed to an effort to summarize available options for chemoprevention and its potential5. In the present article, we examine the current evidence on chemopreventive agents and whether there has been any progress in terms of discovery or use of these agents in the context of cancer prevention.\n\n\nCancer chemopreventive agents\n\nCancer chemopreventive agents can be classified in four major categories: hormonal, medications, diet-related agents, and vaccines.\n\n\nA. Hormonal chemopreventive agents\n\nAll hormonal chemopreventive agents are relevant to steroid-related cancers. They can be classified in two subcategories: antiestrogens and antiandrogens.\n\ni. Selective estrogen receptor modulators. Selective estrogen receptor modulators (SERMs) form a diverse group of compounds that exhibit a varying level of tissue-specific estrogen receptor (ER) activity that can be antagonistic but also agonistic depending on the target tissue6,7. More specifically, SERMs exert an antagonistic activity on breast tissue and an agonist activity on skeletal system. Some SERMs have been reported to demonstrate an ER agonistic effect on the vagina and an antagonistic effect on the endometrium, the latter when combined with estrogen.\n\nDuring the last decade, strong evidence on the effectiveness of SERMs for breast cancer prevention has accumulated8. A meta-analysis combining data from nine clinical trials and comparing use of SERMs (tamoxifen, raloxifene, arzoxifene, or lasofoxifene) with placebo reported a significant decrease in breast cancer incidence with treatment compounds, both during treatment and for at least 5 years after completion9. The reduction in risk was confined to ER-positive invasive breast cancer (for all SERMs used) and ductal carcinoma in situ (for all, except raloxifene). Strengthening the evidence from the abovementioned meta-analysis, a recently updated analysis from the International Breast Cancer Intervention Study I (IBIS-I) provided evidence for a long-term protective effect of tamoxifen after its cessation, for at least 20 years after use10.\n\nRisks associated with tamoxifen are increased occurrence of thromboembolic events, endometrial cancer, and all-cause mortality8,9,11. Raloxifene has been associated mainly with an increase in thromboembolic events. The risk-to-benefit ratio of treatment with tamoxifen or raloxifene depends on age, race, breast cancer risk, and history of hysterectomy. Over the course of a 5-year period, postmenopausal women with an intact uterus have been reported to have a better risk-to-benefit ratio for raloxifene compared with tamoxifen, whereas for postmenopausal women without a uterus the risk-to-benefit ratio was similar for the two compounds8,12. Notwithstanding the reported results on cancer incidence, overall breast cancer mortality has not been shown to decrease in chemoprevention trials with SERMs conducted so far, and consequently some are questioning their role in reducing the overall burden of breast cancer13.\n\nii. Aromatase inhibitors. Aromatase inhibitors (AIs) inhibit the enzyme aromatase, which catalyzes the aromatization procedure that converts androgens into estrogens. Recent data suggest that anastrozole and exemestane are both associated with reduced breast cancer incidence among women at increased risk for the disease8,14–16. They are well tolerated, although some researchers point out that careful monitoring of adverse effects related to joint pain and menopausal symptoms should be implemented in large clinical trials17,18. AIs can be used as an alternative chemoprevention agent for high-risk postmenopausal women who desire chemoprevention and have contraindications for SERM use14,19.\n\nRegarding recommendations for the use of antiestrogens in breast cancer prevention, the American Society of Clinical Oncology practice guidelines indicate the administration of tamoxifen for 5 years in women 35 years and older at increased risk of breast cancer in order to reduce the risk of ER-positive breast cancer. In postmenopausal women, 5-year regimens with raloxifene or exemestane should also be discussed as options19. In the UK, the National Institute for Health and Care Excellence also provides detailed guidance on the use of tamoxifen or raloxifene for pre- or postmenopausal women who are at risk of familial breast cancer but who are not at increased risk of thromboembolic disease or endometrial cancer20.\n\nDespite recommendations, the use of SERMs as primary prevention drugs for breast cancer in clinical practice is considered limited19–21. Several reasons have been invoked, including fear of adverse effects, the lack of reasonably accurate and feasible methods for assessing individual risk, the lack of a marker to monitor cancer risk reduction, insufficient public and professional information, and medication costs8.\n\nBoth testosterone and dihydrotestosterone (DHT) are essential for normal growth and functioning of the prostate. The role of antiandrogens in prostate cancer prevention relies on the hypothesis that androgens may be implicated in the etiology of prostate cancer and that suppressing DHT synthesis may inhibit carcinogenesis22. 5-alpha-reductase is the enzyme that converts testosterone to the more active intracellular androgen DHT; the antiandrogens 5-alpha-reductase inhibitors (5-ARIs) block the process by inhibiting this enzyme.\n\nTwo 5-ARIs, finasteride and dutasteride, have been tested as chemopreventive agents for prostate cancer. Two large randomized placebo-controlled trials, the Prostate Cancer Prevention Trial with finasteride and the Reduction by Dutasteride of Prostate Cancer Events (REDUCE), have reported a decreased incidence of low-grade prostate cancer; in both, however, an absolute increase in high-grade prostate cancer has also been observed23–25.\n\nA meta-analysis of randomized clinical trials reported that 5-ARIs reduce the risk of prostate cancer among men who are screened regularly by using prostate-specific antigen (PSA) level26. The beneficial effects, however, were confined to men with PSA levels of less than 4.0 ng/mL. Evidence was insufficient with respect to the optimal age to initiate treatment or duration of chemoprevention. Uncertainty was also expressed with respect to the impact of 5-ARIs on tumors with the greatest lethal potential, including those with Gleason scores of 8 to 10. In 2010, the US Food and Drug Administration (FDA) evaluated the results of trials and supported the conclusion drawn by the Oncologic Drugs Advisory Committee that finasteride and dutasteride do not have a favorable risk-to-benefit profile in order to be proposed for chemoprevention of prostate cancer among healthy men25.\n\n\nB. Medications\n\nIn the past, only aspirin and other anti-inflammatory drugs would have been classified under this category of chemopreventive agents. More recently, however, interest has emerged about a potential cancer chemopreventive role of statins and metformin.\n\nInflammation is linked to carcinogenesis and hence it is reasonable to assume that agents with anti-inflammatory effects, like the non-steroidal anti-inflammatory drugs (NSAIDs), could have cancer chemopreventive properties. The main representative of NSAIDs is aspirin, but other compounds like indomethacin and piroxicam are included in this class of medications. Various hypotheses have been invoked to explain the chemopreventive properties of NSAIDs5,27. Most prominent among them is the hypothesis about cyclooxygenase (COX) inhibition. COX-1 and -2 are enzymes necessary for the synthesis of inflammatory prostaglandins from arachidonic acid, and NSAIDs inhibit these enzymes. COX-2 is believed to be overexpressed in the early stages of colon carcinogenesis. Selective COX-2 inhibitors have also been developed.\n\nA large body of evidence, from both randomized trials and observational epidemiological studies, has strengthened the hypothesis that regular prophylactic aspirin use reduces incidence of and mortality from colorectal cancer in the general population. A favorable effect of aspirin has also been reported with respect to recurrence of adenomatous polyps as well as polyp load in individuals with hereditary colon cancer27–31. Although data are less extensive, studies have shown reductions in incidence of and mortality from esophageal, stomach, and other gastrointestinal cancers as well as inverse, though small in magnitude, associations with breast, prostate, and lung cancers27.\n\nIssues that remain to be clarified are the optimal dose and duration of use and appropriate ages for use in average-risk individuals. Reduced incidence and mortality have been seen for all daily doses of above 75 mg, but there is no clear indication of a greater reduction with increasing dose32. In a recent systematic review, the authors concluded that prophylactic aspirin use for at least 5 years at daily doses ranging from 75 to 325 mg, starting between ages 50 and 65, has a favorable risk-to-benefit profile for cancer prevention in the average-risk general population in the developed world for both sexes. Larger benefits were observed for 10-year use, whereas longer use still seems beneficial33.\n\nNevertheless, benefits need to be balanced against harms. The side effects of aspirin and NSAIDs, attributed to inhibition of COX-1 activity in platelets, include gastrointestinal track bleeding and intracranial or extracranial hemorrhage, but serious incidents are not common at ages of less than 70 years27,34. Overall, it seems important for evidence-based recommendations regarding the use of aspirin in chemoprevention to be integrated with those for cardiovascular disease prevention.\n\nStatins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are used as cholesterol-lowering drugs but have also drawn attention as potential cancer chemopreventive agents35–37. Reduction of mevalonate synthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase has been invoked as a possible mechanism for a statin-induced suppression of tumor growth, induction of apoptosis, and inhibition of angiogenesis38,39.\n\nIn a meta-analysis of 40 studies, a modest decrease in colorectal cancer risk with statin use was observed, which, however, was statistically significant among observational studies but not among randomized controlled trials40. Reports for reduction of risk with respect to other cancer sites, such as prostate and gastric cancer as well as esophageal cancer (especially adenocarcinoma among patients with Barrett’s esophagus), have also appeared in the literature37,41,42.\n\nOverall, however, there is currently no conclusive evidence for a cancer chemopreventive effect of statins37,43. Of note, statin use among cancer patients before diagnosis has been associated with reduced total and site-specific mortality44.\n\nMetformin is a commonly prescribed drug for type 2 diabetes and belongs in the biguanide class. The crucial role of energy metabolism in cell growth and proliferation implies that antidiabetic or metabolism-altering drugs may hold preventive and therapeutic value, and, in this context, mechanisms for a potential cancer preventive effect of metformin have been proposed45.\n\nEpidemiologic studies indicate that diabetics treated with metformin have a decreased cancer risk compared with those on other antidiabetic medications46,47. Although evidence of a cancer chemopreventive effect of metformin among diabetics is accumulating, the question remains as to whether metformin can exert similar beneficial effects in non-diabetics45,48,49.\n\n\nC. Diet-related agents\n\nSeveral micronutrients have attracted the attention of the scientific community as potential cancer-preventive agents. Among them, diet-derived antioxidants have been studied intensively on account of the protection they convey against oxidative stress. Current evidence on chemopreventive effects of antioxidants and other micronutrients is summarized below.\n\nCarotenoids are fat-soluble red/orange pigments with antioxidant properties and comprise more than 600 compounds. Of the approximately 50 found in human diets, only about half can be absorbed. Carotenoids are found in vegetables and include xanthophylls (e.g., lutein) and carotenes (e.g., beta-carotene and lycopene). Beta-carotene and other carotenoids can be converted to retinol and therefore are referred to by some as “pro-vitamin A”50.\n\nBeta-carotene is one of the most studied carotenoids. Observational epidemiologic studies have shown a beneficial effect of beta-carotene dietary intake on cancer prevention, but large clinical trials conducted during the 1990s did not confirm these findings; on the contrary, they demonstrated a detrimental effect. Thus, in the Alpha-Tocopherol Beta-Carotene (ATBC) Cancer Prevention Study, beta-carotene supplementation was associated with an increase in lung cancer risk as well as in risk of other cancers, notably prostate and stomach51. Similarly, in the Beta-Carotene and Retinol Efficacy Trial (CARET), beta-carotene and retinol supplementation were found to increase lung cancer risk52. Overall, the evidence about a protective effect of foods rich in carotenoids against cancers of the mouth, lung, pharynx, and larynx as well as about a protective effect of foods rich in beta-carotene against esophageal cancer is evaluated as probable; there is, however, strong evidence that beta-carotene supplements are associated with lung cancer in current smokers50,53, which has been classified as “convincing” in the second expert report of the World Cancer Research Fund and the American Institute for Cancer Research50. Among other carotenoids, dietary lycopene (mainly found in tomatoes) has been inversely associated with prostate cancer risk, but the level of evidence has been downgraded from probable50 to limited54.\n\nVitamin A or retinol is the best known retinoid. Retinoids are required for the maintenance of normal cell growth and differentiation; together with their dietary precursor (beta-carotene), they were some of the first agents to be tested in large population-based trials55. The CARET trial in the United States studied beta-carotene along with retinol among smokers and did not show benefit from retinol (nor for beta-carotene) supplementation52. Both the ATBC and the CARET trials found a significant increase in lung cancer incidence in the retinol/beta-carotene-containing arms56.\n\nFolic acid or folate, a water-soluble vitamin B, is an important cofactor in one-carbon metabolism. Folate appears to possess dual modulatory effects on colorectal carcinogenesis depending on timing and dosage. In normal colorectal mucosa folate deficiency appears to enhance neoplastic transformation, modest levels of folic acid supplementation appear to suppress, whereas high supplemental doses appear to enhance the development of cancer. Of note, folate deficiency appears also to inhibit whereas folate supplementation has a promoting effect on the progression of established colorectal neoplasms57. On the basis of a lack of compelling supportive evidence from studies in humans and its potential tumor-promoting effect, folic acid supplementation cannot currently be recommended for colorectal cancer chemoprevention. The evidence concerning the inverse association of dietary folate intake with pancreatic cancer has been downgraded from probable50 to limited58.\n\nVitamin C is a water-soluble antioxidant and enzyme cofactor. Humans do not have the ability to synthesize it and must obtain it through diet. Vitamin C has two chemical forms, one reduced (ascorbic acid) and one oxidized (dehydroascorbic acid) form.\n\nIn 1997, expert panels at the World Cancer Research Fund (WCRF) and the American Institute for Cancer Research had concluded that dietary vitamin C could reduce the risk of the stomach (probably) as well as mouth, pharynx, esophagus, lung, pancreas, and cervical cancers (possibly), but in their updated report in 2007, only the evidence with respect to esophageal cancer was considered probable and there was no evidence that vitamin C supplementation modifies the risk of cancer50,59. Recently, in a large-scale clinical trial in men, vitamin C supplementation had no immediate or long-term effects on the risk of prostate or other site-specific cancers or total cancer60.\n\nVitamin D plays an important role in calcium metabolism but also exerts various other physiological functions. Experimental studies have shown that many cell types, including colorectal cells, express vitamin D receptors, and activation of these receptors by 1,25(OH)2D (1,25-dihydroxycholecalciferol or calcitriol) has been reported to exert antitumor effects61.\n\nIn 2008, the International Agency for Research on Cancer (IARC) Working Group on Vitamin D, having examined the evidence on various cancer sites, concluded that evidence from observational studies for an inverse association between serum 25-hydroxyvitamin D levels and the incidence of colorectal cancer and sporadic colorectal adenomas was consistent and persuasive, but there was only limited evidence of a causal association and this was due to possible confounding by other dietary or lifestyle factors. Results from randomized trials to that date had not demonstrated an effect of vitamin D supplementation on colorectal cancer risk but could not be judged as contradictory to the evidence from observational studies, either62. Hence, there have been suggestions for a minimum vitamin D intake in the context of colorectal cancer prevention61.\n\nVitamin E is a fat-soluble vitamin with antioxidant activity. It refers to a group of compounds that include both tocotrienols and tocopherols, among which α-tocopherol is the most biologically active.\n\nIn the ATBC trial, an inverse association between vitamin E supplementation and prostate cancer was reported but it disappeared post-interventionally63,64. Null results for vitamin E supplementation were also reported in the Physicians’ Health Study with respect to prostate as well as overall cancer60. In the Selenium and Vitamin E Cancer Prevention Trial (SELECT) in men, vitamin E, alone or in combination with selenium, was not associated with a reduction in prostate cancer risk; a subsequent report even noted an increase in the risk of prostate cancer among those who received vitamin E65,66. Hence, current evidence does not support the use of vitamin E for cancer prevention67.\n\nCalcium is an essential nutrient and plays an important role in muscular contraction, cellular growth, cell adhesion, and bone formation. Results from large observational studies support a relatively consistent inverse association of calcium intake with colorectal adenomas and colorectal cancer61,68. This effect is thought to be exerted by binding to toxic secondary bile acids and ionized fatty acids to form insoluble soaps in the lumen of the colon or by directly reducing proliferation, stimulating differentiation, and inducing apoptosis in the colonic mucosa69.\n\nIn a systematic review and meta-analysis, supplemental calcium was reported to be effective for the prevention of adenoma recurrence in populations with a history of adenomas, but no association was found for colorectal cancer70.\n\nThere is some concern that a high calcium intake may increase prostate cancer incidence. The evidence on diets high in calcium or dairy products is considered as limited but suggestive, whereas the evidence on calcium supplements has been downgraded from probable to limited, on the basis of which no conclusions can be drawn50,54.\n\nSelenium is an essential cofactor for the major antioxidant enzyme glutathione peroxidase, which protects against oxidative damage to lipids, lipoproteins, and DNA50,71. The results of the SELECT clinical trial did not provide encouraging data for prostate cancer prevention65. Also, a more recent phase III study of selenium versus placebo in patients with high-grade prostatic intraepithelial neoplasia found no benefit in the prevention of progression to prostate cancer and even suggested that higher intake might increase the risk of cancer72. In its updated report of 2014 on prostate cancer, the WCRF concluded that there is limited suggestive evidence that low plasma concentrations of selenium are associated with increased prostate cancer risk, but no conclusions can be drawn on the basis of the existing evidence for selenium supplementation54.\n\nFlavonoids are polyphenolic compounds that inhibit carcinogen-activating enzymes and possess various antioxidant properties. More than 5,000 individual flavonoids have been identified and have been classified into subclasses on the basis of their range and structural complexity. Fruits and vegetables, along with tea and wine, are the main dietary sources of flavonoids73.\n\nEpidemiologic data, though not conclusive, suggest a protective role of flavonoids on particular cancer types, such as lung, breast, colon, and prostate73–78. In a meta-analysis of observational studies, flavonol and flavone intake, but not other flavonoid subclass or total flavonoid intake, were associated with a decreased breast cancer risk, especially among post-menopausal women79. With respect to colorectal cancer, evidence on the role of flavonoid intake was judged to be insufficient and conflicting80. Evidence on a potential chemopreventive role of various polyphenols, such as isoflavones, on prostate cancer risk has also been reported81–83.\n\nIn 2007, the National Institutes of Health, in a State-of-the-Science statement on multivitamin/multimineral supplements and chronic disease prevention, indicated that data are scarce on the efficacy and safety of multivitamin and mineral supplement use in primary prevention of chronic diseases in the general adult population. Specifically for cancer, though the statement recognized a potential benefit among persons with poor nutritional status or suboptimal antioxidant intake, it concluded that use of multivitamin supplements by the general population was not supported by the existing scientific evidence84. In 2013, a meta-analysis of randomized controlled trials concluded that multivitamin/multimineral use had no effect on cancer prevention85. Of note, the expert panels of the WCRF report concluded that the evidence from its review of trials did not show that micronutrient supplements have any benefits in cancer survivors, whereas high-dose supplements may even be harmful50.\n\n\nD. Vaccines for cancer prevention\n\nSeveral infections have been linked to increased cancer risk; however, only two vaccines against infectious agents are currently used in clinical practice for the prevention of cancer: the vaccine against the hepatitis B virus (HBV) and the vaccine against human papilloma virus (HPV)86,87.\n\nThe HBV vaccine was developed in the late 1960s. The first commercial vaccine was circulated in the early 1980s; genetically engineered vaccines were developed in the late 1980s and these vaccines are the ones currently used. Chronic HBV infection is a major cause of hepatocellular carcinoma, and by preventing the infection and chronic carriage state, the HBV vaccine provides protection against hepatocellular carcinoma88.\n\nHPV vaccination was introduced much later than HBV vaccination. The first HPV vaccine was approved by the FDA in the mid-2000s. There are more than 40 HPV types that infect human mucosal surfaces, but most infections are asymptomatic and transient. However, certain oncogenic types that persist can cause cervical cancer and other, less common, cancers, including cancers of the anus, penis, vulva, vagina, and oropharynx. Other, non-oncogenic HPV types can cause genital warts89,90. Two preventive HPV vaccines, one quadrivalent (which protects against types 16, 18, 6, and 11) and one bivalent (which protects against types 16 and 18), are currently used, but research for new vaccines that will protect against more oncogenic types of the virus is ongoing91. Although history of use of this vaccine is not long, current evidence suggests that it is both effective and safe91,92.\n\n\nConclusions\n\nThe concept of chemoprevention is intuitively attractive as it implies avoidance of suffering caused by the diagnosis of cancer, the disease itself, and its treatment. There are, however, serious constraints in the documentation of effectiveness of chemopreventive agents for cancer11,95, mainly stemming from the long latency of the condition they are addressing. Cancer latency is in the range of years and is certainly longer than the duration of the clinical trials designed to address the effectiveness of cancer chemopreventive agents. The problem could be bypassed with the use of intermediate biomarkers, but these are frequently lacking. Of note, trials assessing the effectiveness of chemopreventive agents have to rely on changes in the incidence of cancer in a population, and this is a rare event, even among high-risk population groups. Other limitations in the documentation of effectiveness of chemopreventive agents are related to the preservation of bioactivity of the various compounds following digestion as well as to the determination of the appropriate effective dose. Furthermore, because chemoprevention refers to the widespread and long-term use of compounds by the general “healthy” population, safety is an issue of paramount importance that needs to be addressed in studies with long follow-up in large segments of the population in order to be able to identify even rare side effects.\n\nAlthough the opinions about the potential of cancer chemoprevention vary widely in the literature, there is little disagreement about the role of aspirin, whereas a diet rich in vegetables and fruits appears to convey more protection than individual micronutrients11,93–95. Among categories of chemopreventive agents, hormonal ones and vaccines used for cancer prevention might hold more promise for the future. Also, the potential of differential effectiveness of chemopreventive agents by particular genotypes96 is extremely intriguing and could open new prospects for cancer chemoprevention.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have 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\nWorld Cancer Report 2014. Stewart BW and Wild CP, Eds. IARC (WHO). Lyon, France: 2014. Reference Source\n\nFerlay J, Soerjomataram I, Ervik M, et al.: GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013.\n\nBiomarkers in Cancer Chemoprevention. IARC Scientific Publications No. 154. Eds Miller AB, Bartsch H, Boffeta P, Dragsted L, and Vainio H. International Agency for Cancer Research. Lyon, 2001. Reference Source\n\nSporn MB, Dunlop NM, Newton DL, et al.: Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Fed Proc. 1976; 35(6): 1332–8. 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Edited by Adami HO, Hunter D & Trichopoulos D. Oxford: University Press; 2008; 517–555. Publisher Full Text\n\nThompson IM, Goodman PJ, Tangen CM, et al.: The influence of finasteride on the development of prostate cancer. N Engl J Med. 2003; 349(3): 215–24. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMadersbacher S: Words of wisdom. Re: Effect of dutasteride on the risk of prostate cancer. Andriole GL, Bostwick DG, Brawley OW, et al. REDUCE Study Group. N Engl J Med 2010;362:1192–202. Eur Urol. 2010; 58(2): 312. PubMed Abstract | Publisher Full Text\n\nTheoret MR, Ning YM, Zhang JJ, et al.: The risks and benefits of 5α-reductase inhibitors for prostate-cancer prevention. N Engl J Med. 2011; 365(2): 97–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWilt TJ, Macdonald R, Hagerty K, et al.: 5-α-Reductase inhibitors for prostate cancer chemoprevention: an updated Cochrane systematic review. BJU Int. 2010; 106(10): 1444–51. 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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nIkeda Y, Shimada K, Teramoto T, et al.: Low-dose aspirin for primary prevention of cardiovascular events in Japanese patients 60 years or older with atherosclerotic risk factors: a randomized clinical trial. JAMA. 2014; 312(23): 2510–20. PubMed Abstract | Publisher Full Text\n\nChan KK, Oza AM, Siu LL: The statins as anticancer agents. Clin Cancer Res. 2003; 9(1): 10–9. PubMed Abstract\n\nSingh PP, Singh S: Statins - the Holy Grail for cancer? Ann Transl Med. 2013; 1(1): 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonovas S: Statins: do they have a potential role in cancer prevention and modifying cancer-related outcomes? Drugs. 2014; 74(16): 1841–8. PubMed Abstract | Publisher Full Text\n\nGazzerro P, Proto MC, Gangemi G, et al.: Pharmacological actions of statins: a critical appraisal in the management of cancer. Pharmacol Rev. 2012; 64(1): 102–46. 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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDale KM, Coleman CI, Henyan NN, et al.: Statins and cancer risk: a meta-analysis. JAMA. 2006; 295(1): 74–80. PubMed Abstract | Publisher Full Text\n\nNielsen SF, Nordestgaard BG, Bojesen SE: Statin use and reduced cancer-related mortality. N Engl J Med. 2012; 367(19): 1792–802. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nQuinn BJ, Kitagawa H, Memmott RM, et al.: Repositioning metformin for cancer prevention and treatment. Trends Endocrinol Metab. 2013; 24(9): 469–80. PubMed Abstract | Publisher Full Text\n\nDeCensi A, Puntoni M, Goodwin P, et al.: Metformin and cancer risk in diabetic patients: a systematic review and meta-analysis. Cancer Prev Res (Phila). 2010; 3(11): 1451–61. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEvans JM, Donnelly LA, Emslie-Smith AM, et al.: Metformin and reduced risk of cancer in diabetic patients. BMJ. 2005; 330(7503): 1304–5. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSehdev A, Shih YC, Vekhter B, et al.: Metformin for primary colorectal cancer prevention in patients with diabetes: a case-control study in a US population. Cancer. 2015; 121(7): 1071–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZhang ZJ, Zheng ZJ, Shi R, et al.: Metformin for liver cancer prevention in patients with type 2 diabetes: a systematic review and meta-analysis. J Clin Endocrinol Metab. 2012; 97(7): 2347–53. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWorld Cancer Research Fund/American Institute for Cancer Research. Food, Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective. Washington DC: AICR, 2007. Reference Source\n\nAlbanes D, Heinonen OP, Huttunen JK, et al.: Effects of alpha-tocopherol and beta-carotene supplements on cancer incidence in the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study. Am J Clin Nutr. 1995; 62(6 Suppl): 1427S–1430S. PubMed Abstract\n\nOmenn GS, Goodman GE, Thornquist MD, et al.: Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med. 1996; 334(18): 1150–5. PubMed Abstract | Publisher Full Text\n\nCortés-Jofré M, Rueda JR, Corsini-Muñoz G, et al.: Drugs for preventing lung cancer in healthy people. Cochrane Database Syst Rev. 2012; 10: CD002141. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWorld Cancer Research Fund/American Institute for Cancer Research. Food, Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective. Washington DC: AICR, 2007. Updated 2014. Reference Source\n\nPapas AM: Antioxidant Status, Diet, Nutrition and Health. Boca Raton, FL: CRC Press, 1999. Reference Source\n\nThe effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. N Engl J Med. 1994; 330(15): 1029–35. PubMed Abstract | Publisher Full Text\n\nKim YI: Folate and colorectal cancer: an evidence-based critical review. Mol Nutr Food Res. 2007; 51(3): 267–92. PubMed Abstract | Publisher Full Text\n\nWorld Cancer Research Fund/American Institute for Cancer Research. Food, Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective. Washington DC: AICR, 2007. Updated 2012. Reference Source\n\nWorld Cancer Research Fund/American Institute for Cancer Research. Food research and the prevention of cancer: a global perspective. Washington, DC: American Institute for Cancer Research. 1997. Reference Source\n\nWang L, Sesso HD, Glynn RJ, et al.: Vitamin E and C supplementation and risk of cancer in men: posttrial follow-up in the Physicians' Health Study II randomized trial. Am J Clin Nut. 2014; 100(3): 915–23. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nZhang X, Giovannucci E: Calcium, vitamin D and colorectal cancer chemoprevention. Best Pract Res Clin Gastroenterol. 2011; 25(4–5): 485–94. PubMed Abstract | Publisher Full Text\n\nIARC Working Group on Vitamin D. Vitamin D and cancer/a report of the IARC Working Group on Vitamin D. IARC Working Group Reports. Lyon, France, 2008; 5. Reference Source\n\nHeinonen OP, Albanes D, Virtamo J, et al.: Prostate cancer and supplementation with alpha-tocopherol and beta-carotene: incidence and mortality in a controlled trial. J Natl Cancer Inst. 1998; 90(6): 440–6. PubMed Abstract | Publisher Full Text\n\nVirtamo J, Pietinen P, Huttunen JK, et al.: Incidence of cancer and mortality following alpha-tocopherol and beta-carotene supplementation: a postintervention follow-up. JAMA. 2003; 290(4): 476–85. PubMed Abstract | Publisher Full Text\n\nLippman SM, Klein EA, Goodman PJ, et al.: Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA. 2009; 301(1): 39–51. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKlein EA, Thompson IM Jr, Tangen CM, et al.: Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA. 2011; 306(14): 1549–56. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBennett LL, Rojas S, Seefeldt T: Role of antioxidants in the prevention of cancer. J Exp Clin Med. 2012; 4(4): 215–222. Publisher Full Text\n\nWorld Cancer Research Fund/American Institute for Cancer Research. Food, Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective. Washington DC: AICR, 2007. Updated 2011. Reference Source\n\nChan AT, Giovannucci EL: Primary prevention of colorectal cancer. Gastroenterology. 2010; 138(6): 2029–2043.e10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarroll C, Cooper K, Papaioannou D, et al.: Supplemental calcium in the chemoprevention of colorectal cancer: a systematic review and meta-analysis. Clin Ther. 2010; 32(5): 789–803. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRayman MP: The importance of selenium to human health. Lancet. 2000; 356(9225): 233–41. PubMed Abstract | Publisher Full Text\n\nMarshall JR, Tangen CM, Sakr WA, et al.: Phase III trial of selenium to prevent prostate cancer in men with high-grade prostatic intraepithelial neoplasia: SWOG S9917. Cancer Prev Res (Phila). 2011; 4(11): 1761–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRomagnolo DF, Selmin OI: Flavonoids and cancer prevention: a review of the evidence. J Nutr Gerontol Geriatr. 2012; 31(3): 206–38. PubMed Abstract | Publisher Full Text\n\nMursu J, Nurmi T, Tuomainen T, et al.: Intake of flavonoids and risk of cancer in Finnish men: The Kuopio Ischaemic Heart Disease Risk Factor Study. Int J Cancer. 2008; 123(3): 660–3. PubMed Abstract | Publisher Full Text\n\nCutler GJ, Nettleton JA, Ross JA, et al.: Dietary flavonoid intake and risk of cancer in postmenopausal women: the Iowa Women's Health Study. Int J Cancer. 2008; 123(3): 664–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeterson J, Lagiou P, Samoli E, et al.: Flavonoid intake and breast cancer risk: a case--control study in Greece. Br J Cancer. 2003; 89(7): 1255–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang L, Lee IM, Zhang SM, et al.: Dietary intake of selected flavonols, flavones, and flavonoid-rich foods and risk of cancer in middle-aged and older women. Am J Clin Nutr. 2009; 89(3): 905–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRossi M, Bosetti C, Negri E, et al.: Flavonoids, proanthocyanidins, and cancer risk: a network of case-control studies from Italy. Nutr Cancer. 2010; 62(7): 871–7. PubMed Abstract | Publisher Full Text\n\nHui C, Qi X, Qianyong Z, et al.: Flavonoids, flavonoid subclasses and breast cancer risk: a meta-analysis of epidemiologic studies. PLoS One. 2013; 8(1): e54318. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJin H, Leng Q, Li C: Dietary flavonoid for preventing colorectal neoplasms. Cochrane Database Syst Rev. 2012; 8: CD009350. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHwang YW, Kim SY, Jee SH, et al.: Soy food consumption and risk of prostate cancer: a meta-analysis of observational studies. Nutr Cancer. 2009; 61(5): 598–606. PubMed Abstract | Publisher Full Text\n\nLall RK, Syed DN, Adhami VM, et al.: Dietary polyphenols in prevention and treatment of prostate cancer. Int J Mol Sci. 2015; 16(2): 3350–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTing H, Deep G, Agarwal C, et al.: The strategies to control prostate cancer by chemoprevention approaches. Mutat Res. 2014; 760: 1–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNational Institutes of Health State-of-the-Science Panel. National Institutes of Health State-of-the-Science Conference Statement: multivitamin/mineral supplements and chronic disease prevention. Am J Clin Nutr. 2007; 85(1): 257S–264S. PubMed Abstract\n\nMacpherson H, Pipingas A, Pase MP: Multivitamin-multimineral supplementation and mortality: a meta-analysis of randomized controlled trials. Am J Clin Nutr. 2013; 97(2): 437–44. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCancer vaccines. National Cancer Institute. Assessed June 2015. Reference Source\n\nLiu JK: Anti-cancer vaccines - a one-hit wonder? Yale J Biol Med. 2014; 87(4): 481–9. PubMed Abstract | Free Full Text\n\nHepatitis B Vaccine. Hepatitis B Foundation. Doylestown, Pennsylvania. Updated 2013-02-26. Assessed June 2015. Reference Source\n\nMonographs on the Evaluation of Carcinogenic Risks to Humans: HPV. IARC Scientific Publications No. 90. International Agency for Cancer Research. Lyon, 2005. Reference Source\n\nMuñoz N, Bosch FX, de Sanjosé S, et al.: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003; 348(6): 518–27. PubMed Abstract | Publisher Full Text\n\nHPV vaccines. Centers for Disease Control and Prevention. Assessed June 2015. Reference Source\n\nHerrero R, González P, Markowitz LE: Present status of human papillomavirus vaccine development and implementation. Lancet Oncol. 2015; 16(5): e206–16. PubMed Abstract | Publisher Full Text\n\nSteward WP, Brown K: Cancer chemoprevention: a rapidly evolving field. Br J Cancer. 2013; 109(1): 1–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSerrano D, Lazzeroni M, Bonanni B: Cancer chemoprevention: Much has been done, but there is still much to do. State of the art and possible new approaches. Mol Oncol. 2015; 9(5): 1008–17. PubMed Abstract | Publisher Full Text\n\nLandis-Piwowar KR, Iyer NR: Cancer chemoprevention: current state of the art. Cancer Growth Metastasis. 2014; 7: 19–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu AH, Tseng CC, Van Den Berg D, et al.: Tea intake, COMT genotype, and breast cancer in Asian-American women. Cancer Res. 2003; 63(21): 7526–9. PubMed Abstract"
}
|
[
{
"id": "10573",
"date": "28 Sep 2015",
"name": "Michihiro Mutoh",
"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",
"responses": []
},
{
"id": "10574",
"date": "28 Sep 2015",
"name": "Hasan Mukhtar",
"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",
"responses": []
},
{
"id": "10575",
"date": "28 Sep 2015",
"name": "Janusz Jankowski",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-916
|
https://f1000research.com/articles/4-914/v1
|
28 Sep 15
|
{
"type": "Review",
"title": "Recent advances in echocardiography for valvular heart disease",
"authors": [
"Rebecca Hahn"
],
"abstract": "Echocardiography is the imaging modality of choice for the assessment of patients with valvular heart disease. Echocardiographic advancements may have particular impact on the assessment and management of patients with valvular heart disease. This review will summarize the current literature on advancements, such as three-dimensional echocardiography, strain imaging, intracardiac echocardiography, and fusion imaging, in this patient population.",
"keywords": [
"echocardiography",
"valvular heart disease",
"three-dimensional echocardiography",
"strain imaging",
"intracardiac echocardiography",
"fusion imaging"
],
"content": "Introduction\n\nThe American Heart Association/American College of Cardiology Guidelines for the Management of Patients with Valvular Heart Disease1 state that echocardiography (transthoracic [TTE] or transesophageal [TEE]) is the imaging modality of choice for the assessment of patients with valvular heart disease. Numerous less invasive therapies, such as percutaneous or transcatheter interventions, have recently been introduced for the treatment of structural heart disease. Many of these procedures require extensive multi-modality imaging guidance and have increased interest in advancements in echocardiography. Recent advancements in echocardiography are of particular relevance to valvular disease. This review will discuss the application of these new technologies to diagnose and manage various types of valvular heart diseases.\n\n\nThree-dimensional echocardiography\n\nThe echocardiographic advancement that has had the most impact on the diagnosis of valvular heart disease is real time three-dimensional (RT3D) echocardiography. The advantages of three-dimensional (3D) imaging over two-dimensional (2D) imaging has been well described in the most recent societal guidelines: “Recommendations for cardiac chamber quantification by echocardiography in adults” update2 and “Recommendations for image acquisition and display using 3D echocardiography”3. These guidelines review the significant data supporting the improved accuracy and reproducibility of 3D imaging for ventricular volumes and mass, as well as valvular morphology and function. Initially introduced in the year 2000, the continued improvement of 3D technology has led to its widespread availability and its growing utility, particularly for valvular heart disease4.\n\nRT3D TEE has significantly changed the assessment of valvular pathology and has revolutionized patient selection not only for surgical repair but also for newer transcatheter procedures, discussed in a subsequent section.\n\nRT3D TEE is not only more accurate than 2D techniques in identifying specific mitral valve pathology in the setting of complex disease but the diagnosis can be made more rapidly, which is of particular use in the intraoperative evaluation of patients undergoing mitral valve repair5–9. RT3D echo improves the accuracy and reproducibility of planimetry measurements of mitral valve area in the setting of rheumatic disease by ensuring on-axis imaging of the short-axis view10–13. This technology has also been integral to our understanding of the dynamic nature of the mitral valve complex in normal patients, as well as in primary and secondary mitral valve disease14–18.\n\nRecent RT3D TEE studies have shown a coupling of mitral and aortic valve dynamic anatomy. Mitral valve diseases may affect normal mitral-aortic coupling and aortic valve function; different patterns of abnormal mitral-aortic coupling are associated with different Carpentier types of mitral regurgitation19. Conversely, changes in aortic morphology may affect mitral valve function, particularly in the setting of aortic stenosis and calcification of the aortic-mitral fibrous continuity20. RT3D TEE has shown changes in mitral valve morphology following surgical aortic valve replacement21 as well as transcatheter aortic valve replacement (TAVR)22. Notably, a decrease of tenting area predicted those patients whose mitral regurgitation improved following TAVR.\n\nAortic valve morphology as well as aortic root measurements are more accurate and reproducible with RT3D imaging. Inter-commissural distance and free leaflet edge lengths can be measured by 3D echocardiography and are used to choose the tube graft size in valve-sparing root operations23. Larger left ventricular outflow tract areas and calculated aortic valve dimensions and areas are obtained by RT3D TEE24. Planimetry of the aortic valve and left ventricular outflow tract area by RT3D has been shown to be accurate and reproducible25–27 and may influence surgical decision-making in the setting of moderate-to-severe aortic stenosis28. With accurate measurement of the left ventricular outflow tract, geometric assumptions used in the continuity equation are avoided, resulting in more precise estimations of aortic valve areas using 3D echocardiography over traditional 2D methods.\n\nProsthetic valve function can also be accurately assessed using RT3D TEE. With transcatheter solutions to bioprosthetic valve failure29–31 and paravalvular regurgitation32–34 RT3D TEE has become an important tool for intra-procedural guidance during percutaneous interventions. TEE can depict not only the relevant cardiac landmarks adjacent to the sites of paravalvular leaks but also wires, delivery catheters, and closure devices35. RT3D TEE imaging results in a more accurate localization of paravalvular defects and an estimation of the size of the defect that correlated better with surgical findings when compared with 2D TEE36.\n\nThree-dimensional color Doppler may overcome the limitations of 2D and standard Doppler measurements for quantifying regurgitation3,37,38. Studies have shown the feasibility of measuring the 3D vena contracta (narrowest portion of the regurgitant jet) on RT3D echocardiography to assess the severity of regurgitation for native regurgitant valve disease37,39–41, as well as following surgical42 or transcatheter interventions43.\n\nCalculation of regurgitant volume in native valvular disease using the proximal isovelocity surface area (PISA) method44 has known technical limitations, primarily the geometric assumptions of PISA shape required to calculate effective regurgitant orifice area. Multiple studies have validated the use of single-beat RT3D echocardiographic color Doppler imaging allowing the direct measurement of PISA without geometric assumptions for aortic, mitral, and tricuspid regurgitation assessment45–48.\n\nNewer methods of determining relative flows within the heart make use of the velocity and direction of flow information which can be derived from color Doppler. Off-line software has been developed which uses 2D color Doppler images to determine the velocity, flow rate, and flow volume in any given region of the heart49. Extension of this technology to 3D color Doppler volume sets is now possible and allows rapid, accurate, and reproducible quantitation of relative stroke volumes50,51. Thavendiranathan et al.51 used the velocity information encoded in the volume color Doppler data, targeting the appropriate region of interest by using the simultaneous 3D imaging of the mitral annulus and left ventricular outflow tract. Color Doppler velocity is multiplied by a known area of this cross-section (a voxel area), and the resulting spatially averaged flow rates are used to generate flow-time curves that resemble those obtained by magnetic resonance imaging. The temporal integration of the flow-time curve generates the stroke volume. There was excellent correlation between the automated measured mitral inflow and aortic stroke volumes, and magnetic resonance imaging stroke volume (r = 0.91, 95% confidence interval [CI], 0.83–0.95, and r = 0.93, 95% CI, 0.87–0.96, respectively, P<0.001) and very low interobserver variability. Automation of the measurement process allowed calculations of mitral inflow and aortic stroke volumes to be performed very rapidly. This methodology will likely become the standard for measurement of regurgitant volumes in the future.\n\nTAVR has become an acceptable alternative treatment for high-risk or inoperable patients with severe symptomatic aortic stenosis52–55. Three-dimensional echocardiography has been shown to improve sizing of the transcatheter valve56–58. RT3D TEE is comparable to computed tomography for annular assessment and prediction of paravalvular regurgitation due to oversizing59,60, as well as measurement of coronary artery height61. RT3D TEE has been shown to provide superior spatial visualization and anatomic orientation, optimizing procedural performance, and RT3D TTE can be used to assess the severity of paravalvular regurgitation following TAVR62. Further study of this technique for quantifying regurgitant severity is warranted in addition to a unified scheme for grading paravalvular regurgitation following TAVR63. Newer devices, with features such as external skirts or the ability to reposition, may reduce the incidence of post-TAVR complication.\n\nThree-dimensional TEE may also improve procedural success and shorten procedure time for the MitraClip™ device (Abbott Vascular Structural Heart, Menlo Park, CA) (Figure 1)64–66. Altiok et al.65 performed a structured analysis to compare information and guidance capability provided by RT3D TEE compared to 2D TEE and found 3D TEE advantageous in 9 of 11 steps of the percutaneous mitral repair procedure, including optimizing trans-septal puncture site, guidance of the clip delivery system, precise positioning of the clip delivery system simultaneously in anterior-posterior and lateral-medial direction, valvular regurgitation jet position, adjustment and visualization of the clip position relative to the valvular orifice, and assessment of remaining regurgitant jets65. Following MitraClip, assessment of residual regurgitation could also be assessed by 3D color Doppler43. A >50% reduction in regurgitant volume using the product of vena contracta areas defined by direct planimetry of RT3D color Doppler and velocity time integral using continuous-wave Doppler was associated with greater left atrial and ventricular remodeling.\n\nPanel A shows the baseline mitral valve morphology with a very large prolapsing and partially flail P2 (middle) scallop (red arrows). Panel B shows positioning of the MitraClip device (blue arrow). Panel C is a dual plane three-dimensional image (ventricular and atrial views) of the final, 2-clip (yellow arrows) resulting double orifice. There was trivial residual mitral regurgitation.\n\n\nStrain imaging\n\nRecent American Society of Echocardiography Chamber Quantification guidelines strongly recommend routine assessment of ventricular systolic function by quantification of ventricular volumes and calculation of ejection fraction (EF)2. Cardiac mechanics, however, can now be assessed with the use of both tissue Doppler and speckle tracking for the measurement of myocardial displacement67. The measurement of myocardial deformation or “strain” is the fractional change in the length of a myocardial segment (expressed as a percentage of the baseline length). Strain rate is the rate of change in strain. The deformation of the myocardium is directional: lengthening would be represented by positive strain, and shortening by negative strain. Systolic strain can be measured along the anatomic coordinates of the cardiac chambers: longitudinal (negative strain), radial (positive strain), and circumferential (negative strain). The strengths and weaknesses of strain measurement have been well described67; however, the recent standardization of strain Digital Imaging and Communications in Medicine (DICOM) format will reduce inter-vendor variability which, along with improved software analysis and automation packages, will likely increase the clinical acceptability and use of this powerful technique.\n\nNumerous studies have shown the utility of strain imaging for assessing left ventricular function in aortic valve disease. In the presence of normal EF, increasing severity of aortic stenosis was associated with reduced global longitudinal strain (GLS)68,69. Subclinical improvement in global and regional systolic function by tissue Doppler and speckle strain also occurs following TAVR (Figure 2)70–72. In low flow, low gradient, severe aortic stenosis with normal EF, strain parameters improved following TAVR, even in the absence of significant change in EF73. Regional strain abnormalities in patients with severe aortic stenosis may be able to further sub-stratify patients with concomitant infiltrative diseases, such as amyloid as well as coronary disease. In patients with cardiac amyloid, relative apical sparing (with preserved apical longitudinal strain) was sensitive (93%) and specific (82%) in differentiating amyloid from controls, some of whom had severe aortic stenosis. In patients with moderate or severe aortic stenosis and concomitant coronary disease, on the other hand, worse apical and mid longitudinal strain parameters were predictive of significant coronary artery stenosis74.\n\nPanel A shows a global circumferential strain (GCS) of -11% prior to TAVR. Panel B shows a GCS of -18% following TAVR. This represents an improvement (greater shortening) in ventricular mechanics.\n\nBecause mortality is significantly associated with symptom development75, strain has been postulated as a possible early marker of ventricular dysfunction in asymptomatic patients with severe aortic stenosis and thus may be a useful tool in determining the timing of intervention in this population. In fact, Carasso et al.76 showed that longitudinal strain was low in asymptomatic patients with severe aortic stenosis with supernormal apical circumferential strain and rotation. In symptomatic patients, however, longitudinal strain was significantly lower with no compensatory circumferential myocardial mechanics. Other investigators suggest that, after adjusting for aortic stenosis severity and EF, only basal longitudinal strain (and not GLS) was an independent predictor of symptomatic status77. In fact, following TAVR, the improvement in GLS may be a result of basal and mid segment improvement only78.\n\nStrain imaging may be particularly useful in predicting outcomes in patients with severe aortic stenosis. In patients with low flow, low gradient, aortic stenosis with normal EF, a recent study showed both stroke volume index (≤35 ml/m2) and GLS (>-15%) are independently associated with worse survival79. In patients with low flow, low gradient, aortic stenosis with reduced EF, GLS is independently associated with mortality and dobutamine stress GLS may provide incremental prognostic value beyond GLS measured at rest80. Three-dimensional GLS may be a better predictor of outcome compared to 2D strain81. Finally, Kusunose et al.82 studied 395 patients with moderate-severe aortic stenosis (aortic valve area <1.3 cm2) and found that GLS was an independent predictor of mortality in this population. A GLS >-12% was associated with the lowest survival82.\n\nDeformation characteristics have also been studied in patients with aortic regurgitation83–87. In a prospective study of young patients (<18 years old) with aortic regurgitation, the only significant predictor of progression of disease on multi-variable analysis was GLS (P=0.04, cut-off value of >-19.5%, sensitivity of 77.8%, specificity of 94.1%, and area under the curve of 0.89)83. Prospective studies of adult patients have also shown that strain parameters by speckle-tracking could detect early myocardial systolic and diastolic dysfunction, and lower strain values were associated with disease progression in medically managed patients, or impaired outcomes in surgically treated patients85. A systolic radial strain rate of <1.82/sec was a good predictor of postoperative left ventricular dysfunction86. Finally, in a prospective study, 60 patients with chronic aortic regurgitation were followed for 64 months and global longitudinal strain (four-chamber view only) was an independent predictor of mortality (hazard ratio 1.313, 95% CI 1.010-1.706, P=0.042)87.\n\nChronic mitral regurgitation is associated with complex left ventricular adaptive remodeling, eccentric hypertrophy, and, eventually, reduced EF. Current guidelines recommend intervening on severe, asymptomatic mitral regurgitation in the setting of reduced EF because of a high incidence of persistent or worsening dysfunction88. In chronic severe degenerative mitral regurgitation, numerous studies have shown that a reduced baseline GLS signifies a maladaptive preload-related change that is associated with a reduction in left ventricular EF immediately after mitral valve repair89–91. A GLS cutoff of >-19.9% was a strong independent predictor of long-term left ventricular dysfunction and may become an appropriate indication for intervention in the setting of normal EF90.\n\n\nIntra-cardiac echocardiography\n\nAlthough TEE imaging is well established and provides exceptional images, particularly for intra-procedural guidance, it most commonly requires general anesthesia and may be associated with intermittent obstruction of fluoroscopic viewing92. With the current move toward conscious sedation for structural heart disease interventions, intra-cardiac echocardiography (ICE) may be an acceptable alternative in some patients with no other adequate intra-procedural imaging options. Evidence that ICE guidance can improve safety and outcome of interventional procedures is still lacking; however, ICE imaging for paravalvular leak closure has been reported to be feasible and advantageous32,93. A reduction in contrast use has also been reported with 2D ICE when used in TAVR (Figure 3)94. The recently introduced AcuNav® V catheter (Siemens Inc. Mountain View, USA) represents the only commercially available RT3D ICE system. The 10F catheter carries a matrix transducer providing a 22° × 90° real-time volume image. This small volume represents the main limitation, particularly in near field applications, such as structural heart disease interventions.\n\nPanel A is the two-dimensional ICE image with panel B showing the simultaneous three-dimensional volume during positioning of the transcatheter aortic valve (yellow arrows).\n\n\nFusion imaging\n\nCombining images from two or more different imaging techniques, or fusion imaging, has been accomplished most recently with real-time echocardiography and fluoroscopy95–97. This technology, which co-registers the TEE probe position with the intervention table and the angulation of the fluoroscopy C-arm, allows for relatively accurate placement of the TEE image onto the fluoroscopic image. This integration eliminates the need for two different image display monitors and the mental integration of two very different imaging datasets by the operator of structural heart disease interventions.\n\nThe ability to define targets on echocardiographic images (whether 2D or 3D), and co-register these targets onto the fluoroscopic images, should improve guidance of structural heart disease interventions (Figure 4). This technology has been shown to be safe and feasible for the transcatheter mitral repair procedure with the MitraClip™ device (Abbott Vascular Structural Heart, Menlo Park, CA) and shows a trend towards reduction of fluoroscopy and procedure time98.\n\nAfter coregistration of the transesophageal echocardiographic probe with the fluoroscopic image, the two images can be fused to allow a more comprehensive understanding of anatomy. Localizing the regurgitant orifice on transesophageal echo imaging can then be translated to the corresponding position on the fluoroscopic image.\n\n\nConclusion\n\nEchocardiography is the primary imaging modality for the diagnosis and management of patients with valvular heart disease. Improvement in surgical outcomes and advances in interventional techniques require further refinements in echocardiographic imaging. Three-dimensional echocardiography, strain imaging, intracardiac echocardiography, and fusion imaging have significant application in advancing our understanding of pathophysiology and anatomy, as well as the diagnosis and management of patients with valvular heart disease.\n\n\nAbbreviations\n\n2D, two-dimensional; 3D, three-dimensional; confidence interval, CI; EF, ejection fraction; GLS, global longitudinal strain; ICE, intra-cardiac echocardiography; PISA, proximal isovelocity surface area; RT3D, real time three-dimensional; TAVR, transcatheter aortic valve replacement; TEE, transesophageal echocardiography; TTE, transthoracic echocardiography.",
"appendix": "Competing interests\n\n\n\nRebecca T. Hahn has received speaker honoraria from Edwards Lifesciences, St. Jude Medical and Boston Scientific, and a research grant from Philips Healthcare.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNishimura RA, Otto C: 2014 ACC/AHA valve guidelines: earlier intervention for chronic mitral regurgitation. Heart. 2014; 100(12): 905–7. PubMed Abstract | Publisher Full Text\n\nLang RM, Badano LP, Mor-Avi V, et al.: Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. J Am Soc Echocardiogr. 2015; 28(1): 1–39.e14. PubMed Abstract | Publisher Full Text\n\nLang RM, Badano LP, Tsang W, et al.: EAE/ASE recommendations for image acquisition and display using three-dimensional echocardiography. J Am Soc Echocardiogr. 2012; 25(1): 3–46. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDahou A, Bartko PE, Capoulade R, et al.: Usefulness of global left ventricular longitudinal strain for risk stratification in low ejection fraction, low-gradient aortic stenosis: results from the multicenter True or Pseudo-Severe Aortic Stenosis study. Circ Cardiovasc Imaging. 2015; 8(3): e002117. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNagata Y, Takeuchi M, Wu VC, et al.: Prognostic value of LV deformation parameters using 2D and 3D speckle-tracking echocardiography in asymptomatic patients with severe aortic stenosis and preserved LV ejection fraction. JACC Cardiovasc Imaging. 2015; 8(3): 235–45. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKusunose K, Goodman A, Parikh R, et al.: Incremental prognostic value of left ventricular global longitudinal strain in patients with aortic stenosis and preserved ejection fraction. Circ Cardiovasc Imaging. 2014; 7(6): 938–45. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDi Salvo G, Rea A, Mormile A, et al.: Usefulness of bidimensional strain imaging for predicting outcome in asymptomatic patients aged ≤ 16 years with isolated moderate to severe aortic regurgitation. Am J Cardiol. 2012; 110(7): 1051–5. PubMed Abstract | Publisher Full Text\n\nIida N, Seo Y, Ishizu T, et al.: Transmural compensation of myocardial deformation to preserve left ventricular ejection performance in chronic aortic regurgitation. J Am Soc Echocardiogr. 2012; 25(6): 620–8. PubMed Abstract | Publisher Full Text\n\nOlsen NT, Sogaard P, Larsson HB, et al.: Speckle-tracking echocardiography for predicting outcome in chronic aortic regurgitation during conservative management and after surgery. JACC Cardiovasc Imaging. 2011; 4(3): 223–30. PubMed Abstract | Publisher Full Text\n\nOnishi T, Kawai H, Tatsumi K, et al.: Preoperative systolic strain rate predicts postoperative left ventricular dysfunction in patients with chronic aortic regurgitation. Circ Cardiovasc Imaging. 2010; 3(2): 134–41. PubMed Abstract | Publisher Full Text\n\nPark SH, Yang YA, Kim KY, et al.: Left Ventricular Strain as Predictor of Chronic Aortic Regurgitation. J Cardiovasc Ultrasound. 2015; 23(2): 78–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNishimura RA, Otto CM, Bonow RO, et al.: 2014 AHA/ACC guideline for the management of patients with valvular heart disease: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014; 63(22): 2438–88. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nde Isla LP, de Agustin A, Rodrigo JL, et al.: Chronic mitral regurgitation: a pilot study to assess preoperative left ventricular contractile function using speckle-tracking echocardiography. J Am Soc Echocardiogr. 2009; 22(7): 831–8. PubMed Abstract | Publisher Full Text\n\nWitkowski TG, Thomas JD, Debonnaire PJMR, et al.: Global longitudinal strain predicts left ventricular dysfunction after mitral valve repair. Eur Heart J Cardiovasc Imaging. 2013; 14(1): 69–76. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPandis D, Sengupta PP, Castillo JG, et al.: Assessment of longitudinal myocardial mechanics in patients with degenerative mitral valve regurgitation predicts postoperative worsening of left ventricular systolic function. J Am Soc Echocardiogr. 2014; 27(6): 627–38. PubMed Abstract | Publisher Full Text\n\nSmith LA, Monaghan MJ: Monitoring of procedures: peri-interventional echo assessment for transcatheter aortic valve implantation. Eur Heart J Cardiovasc Imaging. 2013; 14(9): 840–50. PubMed Abstract | Publisher Full Text\n\nOsman F, Steeds R: Use of intra-cardiac ultrasound in the diagnosis of prosthetic valve malfunction. Eur J Echocardiogr. 2007; 8(5): 392–4. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBartel T, Bonaros N, Edlinger M, et al.: Intracardiac echo and reduced radiocontrast requirements during TAVR. JACC Cardiovasc Imaging. 2014; 7(3): 319–20. PubMed Abstract | Publisher Full Text\n\nCorti R, Biaggi P, Gaemperli O, et al.: Integrated x-ray and echocardiography imaging for structural heart interventions. EuroIntervention. 2013; 9(7): 863–9. PubMed Abstract | Faculty Opinions Recommendation\n\nGao G, Penney G, Ma Y, et al.: Registration of 3D trans-esophageal echocardiography to X-ray fluoroscopy using image-based probe tracking. Med Image Anal. 2012; 16(1): 38–49. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKaiser M, John M, Heimann T, et al.: 2D/3D registration of TEE probe from two non-orthogonal C-arm directions. Med Image Comput Comput Assist Interv. 2014; 17(Pt 1): 283–90. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nQuaife RA, Salcedo EE, Carroll JD: Procedural guidance using advance imaging techniques for percutaneous edge-to-edge mitral valve repair. Curr Cardiol Rep. 2014; 16(2): 452. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10566",
"date": "28 Sep 2015",
"name": "Judy Hung",
"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",
"responses": []
},
{
"id": "10567",
"date": "28 Sep 2015",
"name": "Jeroen J. Bax",
"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",
"responses": []
},
{
"id": "10568",
"date": "28 Sep 2015",
"name": "Takahiro Shiota",
"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 by Dr. Hahn from Columbia University covers major areas of the topic (recent advances in echocardiography in valvular heart disease) and is well written. With this, readers can appreciate her daily clinical experiences with new modalities such as three dimensional color Doppler echocardiography. In addition, recent major publications, including her own, are well chosen for the educational purpose.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-914
|
https://f1000research.com/articles/4-912/v1
|
28 Sep 15
|
{
"type": "Case Report",
"title": "Case Report: Compound heterozygous nonsense mutations in TRMT10A are associated with microcephaly, delayed development, and periventricular white matter hyperintensities",
"authors": [
"Mohan Narayanan",
"Keri Ramsey",
"Theresa Grebe",
"Isabelle Schrauwen",
"Szabolcs Szelinger",
"Matthew Huentelman",
"David Craig",
"Vinodh Narayanan",
"C4RCD Research Group",
"Keri Ramsey",
"Theresa Grebe",
"Isabelle Schrauwen",
"Szabolcs Szelinger",
"Matthew Huentelman",
"David Craig"
],
"abstract": "Microcephaly is a fairly common feature observed in children with delayed development, defined as head circumference less than 2 standard deviations below the mean for age and gender. It may be the result of an acquired insult to the brain, such prenatal or perinatal brain injury (congenital infection or hypoxic ischemic encephalopathy), or be a part of a genetic syndrome. There are over 1000 conditions listed in OMIM (Online Mendelian Inheritance in Man) where microcephaly is a key finding; many of these are associated with specific somatic features and non-CNS anomalies. The term primary microcephaly is used when microcephaly and delayed development are the primary features, and they are not part of another recognized syndrome. In this case report, we present the clinical features of siblings (brother and sister) with primary microcephaly and delayed development, and subtle dysmorphic features. Both children had brain MRI studies that showed periventricular and subcortical T2/FLAIR hyperintensities, without signs of white matter volume loss, and no parenchymal calcifications by CT scan. The family was enrolled in a research study for whole exome sequencing of probands and parents. Analysis of variants determined that the children were compound heterozygotes for nonsense mutations, c.277C>T (p.Arg93*) and c.397C>T (p.Arg133*), in the TRMT10A gene. Mutations in this gene have only recently been reported in children with microcephaly and early onset diabetes mellitus. Our report adds to current knowledge of TRMT10A related neurodevelopmental disorders and demonstrates imaging findings suggestive of delayed or abnormal myelination of the white matter in this disorder. Accurate diagnosis through genomic testing, as in the children described here, allows for early detection and management of medical complications, such as diabetes mellitus.",
"keywords": [
"Primary microcephaly",
"Delayed development",
"Periventricular leukomalacia",
"White matter hyperintensities",
"Diabetes mellitus",
"TRMT10A",
"tRNA methyltransferase"
],
"content": "Clinical summary\n\nWe present case histories of siblings (a sister and brother) born to healthy non-consanguineous parents, with microcephaly and delayed development.\n\nThe first patient is a 12 year old girl who was born at 39 weeks gestation weighing 4 lb 10 oz, without perinatal difficulties. She was healthy as an infant, except for recurrent ear infections, which were treated with tympanostomy tubes. Hypotonia was noted during infancy, but she reached her early motor milestones, including crawling and walking, at the normal ages. Speech delay and behavioral problems were noted by 4 years of age. There was no regression. Ophthalmological evaluation was normal. Physical exam at age 4.5 years showed microcephaly, low anterior hair line, deep set eyes with mild hypotelorism, shortened forehead, and her neurological exam was normal. Head circumference was 3.5 SD below the mean for age in girls. These features have remained constant at subsequent examinations.\n\nMRI scan of the brain showed mild FLAIR/T2 hyperintensities in the periatrial white matter (Figure 1A); a CT scan did not show calcifications. Skeletal x-rays did not show any abnormality of her bones. Hearing tests, renal ultrasound, and echocardiogram were all normal. High resolution karyotype and array comparative genome hybridization (CGH) were normal. Carbohydrate deficient transferrin study, lysosomal enzymes, and 7-dehydrocholesterol level were also normal. She has not had documented hypoglycemia or hyperglycemia. Psychometric testing led to the diagnosis of mild intellectual disability. Speech and occupational therapy were provided through school and she has continued to make steady developmental progress.\n\n(1-A) – Axial MRI FLAIR image from the affected female child done at 4.5 years of age shows hyperintensities in the periventricular (primarily periatrial) white matter (arrows). (1-B) Axial FLAIR and sagittal T1 MRI images from the affected male child at 2.5 years of age. This shows bilateral periventricular (periatrial) and subcortical white matter hyperintensities consistent with dysmyelination (arrows). Brain architecture (corpus callosum, cerebellum, and cortical gyration) was normal.\n\nThe second patient is a 10 year old boy who was born at 36 weeks gestation weighing 5 pounds, and developed mild neonatal jaundice. The pregnancy was complicated by oligohydramnios and decreased fetal movement. He was diagnosed with hypotonia during infancy. He too achieved motor milestones at appropriate times (walked at 15 months) but speech was delayed. He had an episode of febrile status epilepticus at 17 months, and subsequent epileptic seizures without fever. Seizures have been controlled with levetiracetam (50 mg/kg/day, corresponding to 500 mg twice daily). A recent EEG showed symmetric, bilateral, frontally dominant polyspike and wave discharges, consistent with generalized epilepsy. He has a history of recurrent pulmonary infections and reactive airway disease; initial testing by CFTR gene sequencing was not conclusive and revealed only benign variants. Sweat chloride testing was abnormal on one occasion and then borderline, pancreatic elastase was normal, and a clinical diagnosis of cystic fibrosis was made. He also had recurrent otitis media that required tympanostomy tubes.\n\nPhysical examination at age 2.5 years showed small stature (5% for height and weight) microcephaly (head circumference 3 SD below mean for age), low anterior hair line, deep set eyes with mild hypotelorism, and a normal neurological exam. His phenotype was similar to his older sister, although he seemed to be more severely affected. MRI of the brain at age 2.5 years showed extensive FLAIR/T2 hyperintensities in the periventricular and subcortical white matter (Figure 1B).\n\nThe following biochemical and genetic tests were normal: array CGH, fragile X test, TORCH titers, quantitative plasma amino acids, quantitative urine organic acids, fluorescent in-situ hybridization (FISH) test for Pelizaeus-Merzbacher disease, very long chain fatty acids, enzyme assays for CLN1 (PPT1 – palmitoyl-protein thioesterase 1) and CLN2 (TPP1-tripeptidyl-peptidase 1), and 7-dehydrocholesterol. He has not had documented hypoglycemia or hyperglycemia.\n\nHis features have remained constant on follow-up examinations, with persistent microcephaly and mild hypotelorism. He is making slow progress in school with speech, occupational and physical therapy.\n\n\nEthics\n\nPatients and parents were enrolled into a clinical research protocol sponsored by the Translational Genomics Research Institute (TGen) approved by the Western Institutional Review Board, Protocol Number 20120789. After informed consent, blood samples were collected for DNA and RNA extraction. A separate buccal swab was sent for Sanger sequencing and independent confirmation of selected variants in a clinical laboratory (GeneDx, Gaithersberg, MD).\n\n\nExome sequencing\n\nLibraries were prepared using the Illumina’s TruSeq DNA sample preparation kit and the TruSeq exome enrichment kit (Illumina, Inc., San Diego, CA, USA), following the manufacturer’s protocol. Sequencing was done by 100-bp paired-end sequencing on a HiSeq2000 instrument (Illumina, Inc., San Diego, CA, USA). Reads were aligned to the Human Genome (Hg19/GRC37) using Burrows-Wheeler transform alignment (BWA v.0.7.5)1. PCR duplicates were removed using Picard v.1.922, and base quality recalibration, indel realignment and single nucleotide polymorphism (SNP) and indel discovery were performed using the Genome Analysis Toolkit (GATK v.2.5-2)3. Variants were annotated with SnpEff 3.2a and selected (SnpSift) for protein-coding events. Prediction scores were loaded from dbNSFP and used for filtering.\n\n\nResults\n\nExome sequencing identified 33,640 SNPs and indels common to the two affected individuals. The majority of these variants were common in the population, and only 4,355 were considered either novel, private or rare variants. An annotated variant file was created that included variants in any of the four family members (male child, female child, father, and mother). Excluding UTR (untranslated region) and synonymous variants left 452 candidate variants.\n\nThere were no variants consistent with de novo mutation in both children (with presumed germline mosaicism in a parent). Four variants were identified consistent with a homozygous recessive model: SERINC2, PTH2R, SGK223, and PMP22. None of these were consistent with the clinical phenotype.\n\nWe considered an X-linked recessive model (female child less severe than male) and identified variants were in NHS (Nance-Horan syndrome), DDX53, WAS, and TREX2; four variants in RPGR. The TREX2 variant has been reported in 83 hemizygotes in the ExAC Browser database v 0.3 (Broad Institute). The DDX53 variant is rare and has been implicated as a potential X-linked autism locus. X chromosome inactivation studies in the carrier mother have not been done.\n\nWhen we considered compound heterozygous model, we identified only two variants in a single gene, TRMT10A. Both children have inherited a maternal variant encoding a nonsense mutation, c.277C>T (p.Arg93*) and a paternal variant encoding a nonsense mutation, c.397C>T (p.Arg133*). The Arg133* variant has been observed in one of 120,000 alleles in the ExAC Browser database, while the Arg93* allele has never been reported before. Based on the damaging nature of these mutations (protein truncation), consistency with an autosomal recessive model, and two previous reports of homozygous TRMT10A mutations in children with primary microcephaly4,5, we concluded that the TRMT10A mutations were causal for our patients’ phenotype.\n\n\nDiscussion\n\nMicrocephaly is a clinical condition, rather than a specific diagnosis, defined as having a small brain size, measured by head circumference. It is generally accepted that head circumference less than 2–4 standard deviations below the mean for age and gender is considered to be microcephaly6,7.\n\nPrimary microcephaly is either congenital and present at birth, or progressive and caused by postnatal decrease in head circumference growth rate (as in Rett syndrome, for instance). Congenital microcephaly occurs more frequently in patients born to consanguineous parents. Congenital microcephaly can be further classified by clinical and imaging features set forth by Barkovich and others8,9. Patients with autosomal recessive primary microcephaly (MCPH) have microcephaly at birth and non-progressive mental retardation10,11. Microcephaly with normal or simplified gyration (MSG) is thought to be a result of abnormal neuronal and glial proliferation or apoptosis12.\n\nMany genes have been implicated in the development of microcephaly (summarized in Table 1) including ASPM, MCPH1, CDK5RAP2, CEP152, CENPJ, WDR62, STIL, CASC5, CEP135, ZNF335, PHC1, CDK6, with many of them contributing to centrosome and spindle protein dysfunction during mitosis6,10,13–17. Mutations in the ASPM gene are the most common in this group, and account for 30–40 percent of these cases18. Genes implicated in primary microcephaly with associated features such as cognitive and motor impairment and epilepsy include SLC25A19, ATR, ARFGEF2, and RAB3GAP119. PYCR2 mutation described by Nakayama causes dysfunctional proline synthesis and leaves affected patients more susceptible to oxidative stress induced apoptosis, leading to postnatal microcephaly from cell death in the developing brain20.\n\nThis table contains a summary of selected primary microcephaly syndromes, including MCPH 1–15. Information provided includes a brief description of the phenotype, gene that is mutated, and pathogenic mechanism.\n\nPrimary microcephaly is a result of a defect in the generation of the appropriate number of neurons during early neural development. Cell division of neural progenitors in the neuroepithelium can be symmetric (generating two progenitors) or asymmetric (generating one progenitor and one post-mitotic neuron). Microcephaly has been associated with microtubule, centrosome, and mitotic dysfunction. Dysfunction in the mitotic machinery may cause inappropriate asymmetric cell division during neurogenesis in the developing cortex and subventricular zone21,22. Abnormal control of apoptosis during the neurogenesis phase of development can also lead to microcephaly. Studies in mice have demonstrated microcephaly due to an increase in neuronal apoptosis and depletion of the neural progenitor population23. Specifically, endoplasmic reticulum stress-induced apoptosis has been shown to cause microcephaly and early onset diabetes from beta cell ER stress-induced apoptosis.\n\n\nTRMT10A and microcephaly\n\nTRMT10A (also known as RG9MTD2 – RNA (guanine 9)-methyltransferase domain-containing protein 2) is homologous to a yeast tRNA methyltransferase (Trm10) and methylates tRNAs at the m1G9 position. TRMT10A is expressed in several tissues (liver, kidney, spleen, lung, fat) but at especially high levels in brain and pancreatic islet cells4. Mutation of another tRNA methyltransferases, NSUN2, has been associated with microcephaly, short stature, intellectual disability, and facial dysmorphism24,25.\n\nThere are two previous reports of patients with homozygous nonsense or missense mutation of TRMT10A resulting in microcephaly. In the first report, Igoillo-Esteve and colleagues described 3 siblings, from a large consanguineous Moroccan family, with microcephaly, intellectual disability, short stature, and early-onset diabetes4. Whole exome sequencing identified a homozygous p. Arg127* nonsense mutation in the TRMT10A gene in the 3 affected patients; both parents and an unaffected brother were heterozygous for this mutation. Features in the oldest patient (at age 26), in addition to microcephaly and intellectual disability, included short stature, absence seizures, development of diabetes at age 22, and a variety of dysmorphic features. Brain MRI showed normal architecture and gyration. The other two children developed diabetes at age 19 and 14 respectively. The TRMT10A Arg127* mutation resulted in greatly reduced mRNA levels (presumably by nonsense-mediated decay) and absent protein levels. Loss of TRMT10A protein induced apoptosis in rat β-cells and increased stress-induced apoptosis by many endogenous molecules including high levels of glucose4.\n\nIn a second report, Gillis and colleagues described 3 siblings (one female and two male) born to healthy, non-consanguineous parents from a small, inbred Jewish community of Uzbekistan5. All 3 patients suffered from microcephaly, intellectual disability, short stature, seizures, and altered glucose metabolism. The female child had delayed pubertal development and was amenorrheic at age 19 years. Exome sequencing in one affected patient followed by targeted genotyping in the rest of the family (parents, two affected sibs and 9 unaffected sibs) defined a homozygous p. Gly206Arg missense mutation in TRMT10A as causing disease in this family. All three affected patients had episodes of hypoglycemia, hyperinsulinemia, and abnormal response to glucose. Brain imaging in two of these patients was normal. The Gly206Arg mutation resulted in almost complete loss of methyltransferase activity5. This suggested that decrease in methylated tRNA is responsible for growth failure, developmental delay and impaired glucose metabolism.\n\nTRMT10A microcephaly, in contrast to primary microcephaly linked to centrosome and spindle function during mitosis, is likely related to the loss of brain volume due to increased apoptosis. TRMT10A deficiency increases the amount of beta cell apoptosis, and presumably neuronal apoptosis. The development of diabetes and abnormal glucose response may be related to endoplasmic reticulum stress-induced apoptosis induced by endogenous fatty acids and high levels of glucose4. Endoplasmic reticulum stress-induced apoptosis has also been implicated in a syndrome of primary microcephaly associated with simplified gyral pattern, epilepsy, and infantile diabetes caused by mutation of the IER3IP1 gene19.\n\nHere we report two additional patients with primary (congenital) microcephaly and intellectual disability caused by compound heterozygous nonsense mutations of TRMT10A. These patients have not displayed hypoglycemia nor early onset diabetes. Brain imaging did show abnormalities in the central white matter, suggesting delayed or abnormal myelination. TRMT10A is not currently included in microcephaly gene panels available from commercial laboratories, stressing the importance of exome sequencing in the genetic diagnosis of primary microcephaly. The correct genetic diagnosis in our patients allows for anticipation and early treatment of medical complications by institution of meticulous glucose monitoring and control from an early age.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the parent of the patients.",
"appendix": "Author contributions\n\n\n\nMN prepared the clinical summary and wrote the manuscript, including the discussion section, and prepared Table 1.\n\nKR was involved in patient enrollment in the research study, review of clinical records, data analysis, clinical testing, and manuscript review and editing.\n\nTG was involved in patient evaluation, clinical phenotyping, manuscript review, and editing. IS, SS, MH, and DC were involved in exome sequencing, data analysis, writing methods and results sections, and in manuscript review and editing.\n\nVN was involved in patient evaluation, enrollment in the research study, review of clinical records, analysis of exome sequencing data, manuscript review and editing, and prepared Figure 1. All authors have agreed to the final content of this manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by donations to the Center for Rare Childhood Disorders. These include private anonymous donations. TGen also receives support from the State of Arizona.\n\nI confirm that the 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 acknowledge the help and cooperation of the many families involved with the Center for Rare Childhood Disorders, members of the Advisory Committee of the Center for Rare Childhood Disorders, and the TGen Foundation.\n\nThe authors acknowledge the contributions of all members (current and past) of The C4RCD Research Group.\n\nC4RCD Research Group:\n\nThis group includes the clinical team and laboratory research team involved in patient enrollment, sample processing, exome sequencing, data processing, preparation of variant annotation files, data analysis, validation of data, and return of research data to families. Candidate genes are identified and discussed at data analysis meetings of the entire group. The following members of the group (listed in alphabetical order) have contributed significantly to this work:\n\n1. Newell Belnap (nbelnap@tgen.org)\n\n2. Jason J. Corneveaux (deceased)\n\n3. Amanda Courtright (acourtright@tgen.org)\n\n4. David W. Craig (dcraig@tgen.org)\n\n5. Matt de Both (mdeboth@tgen.org)\n\n6. Brooke Hjelm (bhjelm@uci.edu)\n\n7. Matthew J. Huentelman (mhuentelman@tgen.org)\n\n8. Ahmet Kurdoglu (akurdoglu@tgen.org; akurdoglu@ashiondx.com)\n\n9. Vinodh Narayanan (vnarayanan2@tgen.org)\n\n10. Keri M. Ramsey (kramsey@tgen.org)\n\n11. Sampathkumar Rangasamy (srangasamy@tgen.org)\n\n12. Ryan Richholt (rrichholt@tgen.org)\n\n13. Isabelle Schrauwen (ischrauwen@tgen.org)\n\n14. Ashley L. Siniard (asiniard@tgen.org)\n\n15. Szabolcs Szelinger (sszelinger@tgen.org)\n\n\nReferences\n\nLi H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010; 26(5): 589–595. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarbelanne M, Tsang WY: Molecular and cellular basis of autosomal recessive primary microcephaly. Biomed Res Int. 2014; 2014: 547986. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWaters AM, Asfahani R, Carroll P, et al.: The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes. J Med Genet. 2015; 52(3): 147–156. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartin CA, Ahmad I, Klingseisen A, et al.: Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy. Nat Genet. 2014; 46(12): 1283–1292. PubMed Abstract | Publisher Full Text\n\nNicholas AK, Swanson EA, Cox JJ, et al.: The molecular landscape of ASPM mutations in primary microcephaly. J Med Genet. 2009; 46(4): 249–253. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoulton CJ, Schot R, Kia SK, et al.: Microcephaly with simplified gyration, epilepsy, and infantile diabetes linked to inappropriate apoptosis of neural progenitors. Am J Hum Genet. 2011; 89(2): 265–276. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakayama T, Al-Maawali A, El-Quessny M, et al.: Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination. Am J Hum Genet. 2015; 96(5): 709–719. PubMed Abstract | Publisher Full Text\n\nNigg EA, Raff JW: Centrioles, centrosomes, and cilia in health and disease. Cell. 2009; 139(4): 663–678. PubMed Abstract | Publisher Full Text\n\nBettencourt-Dias M, Hildebrandt F, Pellman D, et al.: Centrosomes and cilia in human disease. Trends Genet. 2011; 27(8): 307–315. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMao H, Pilaz LJ, McMahon JJ, et al.: Rbm8a haploinsufficiency disrupts embryonic cortical development resulting in microcephaly. J Neurosci. 2015; 35(18): 7003–7018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbbasi-Moheb L, Mertel S, Gonsior M, et al.: Mutations in NSUN2 cause autosomal-recessive intellectual disability. Am J Hum Genet. 2012; 90(5): 847–855. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhan MA, Rafiq MA, Noor A, et al.: Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability. Am J Hum Genet. 2012; 90(5): 856–863. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "10548",
"date": "13 Oct 2015",
"name": "Gihan Tennekoon",
"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\nTRMT10A is an tRNA (guanine) methyltransferase that is highly expressed in the pancreatic islet cells and brain. In the brain it appears expression occurs early during gestation and expression is in the dorsal telencephalon with high expression in the ventricular zone. Later there is expression in the cerebellum. This case report describes 2 siblings where the male is more severely affected. From previous reports and these 2 cases it appears to be an autosomal recessive disorder that is characterized by mild dysmorphic features including short stature, microcephaly, non-progressive intellectual disability predominantly affecting speech, no gross for fine motor difficulties and no cerebellar dysfunction. Previous reports have not described any specific changes on the imaging studies whereas this report shows periatrial and periventricular white matter changes consistent with delayed myelination. Furthermore, neither of these children had difficulties with glucose metabolism. In term of understanding the pathophysiology of how TRMT10A brain development there is more work needed. Nevertheless, in the setting of primary microcephaly with speech delay and abnormal glucose homeostasis, TRMT10A mutations should be considered.",
"responses": []
},
{
"id": "10547",
"date": "19 Oct 2015",
"name": "Ganeshwaran H. Mochida",
"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 two siblings with microcephaly and developmental delay, who were found to have compound heterozygous variants in the TRMT10A gene. Biallelic mutations of TRMT10A have recently been reported in individuals with microcephaly and abnormalities in glucose homeostasis. Seeing that only a small number of individuals reported with this condition to date, this paper adds additional insights into this condition. The technique used in this paper is standard whole-exome sequencing. Though the manuscript does not include functional studies of the identified TRMT10A variants, these variants are predicted to lead to a premature truncation of the protein and are therefore likely to be pathogenic.I have following comments and suggestions:It would be helpful to include the actual physical measurements (not just standard deviations) for the affected individuals. It is not explicitly stated which individuals in the family had exome sequencing done and if the TRMT10A variants were confirmed by Sanger sequencing. Even though there seems to be only one protein isoform for TRMT10A in the public databases, the ID of the reference sequence used for annotation of the mutations should be included. There are several differences in clinical manifestation between the affected individuals described in this manuscript and those previously reported. Specifically, the individuals described herein do not have obvious alteration of glucose homeostasis. Also, the cerebral white matter signal abnormalities on brain MRI have not been reported previously. These features are clinically important, and it would be helpful to discuss these points more in depth.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-912
|
https://f1000research.com/articles/4-485/v1
|
05 Aug 15
|
{
"type": "Software Tool Article",
"title": "iCTNet2: integrating heterogeneous biological interactions to understand complex traits",
"authors": [
"Lili Wang",
"Daniel S. Himmelstein",
"Adam Santaniello",
"Mousavi Parvin",
"Sergio E. Baranzini",
"Lili Wang",
"Daniel S. Himmelstein",
"Adam Santaniello",
"Mousavi Parvin"
],
"abstract": "iCTNet (integrated Complex Traits Networks) version 2 is a Cytoscape app and database that allows researchers to build heterogeneous networks by integrating a variety of biological interactions, thus offering a systems-level view of human complex traits. iCTNet2 is built from a variety of large-scale biological datasets, collected from public repositories to facilitate the building, visualization and analysis of heterogeneous biological networks in a comprehensive fashion via the Cytoscape platform. iCTNet2 is freely available at the Cytoscape app store.",
"keywords": [
"big data integration",
"heterogeneous network",
"drug re-purposing",
"disease ontology"
],
"content": "Introduction\n\nIn the past decade, an exponential increase in the amount and variety of publicly available genomic, transcriptomic, proteomic and other ‘omics’ data has occurred, altogether encompassing a wide range of biological interactions. Each dataset captures distinct features of molecular functions involved in complex traits, with the goal of describing and ultimately understanding biological complexity. However, these datasets are mostly used in isolation, and even the integration of any two of them would take a significant effort for the average biological investigator.\n\nPrevious work in this area is largely limited to merging data of only two types. Goh et al.1 built the first “Diseasome”, a bipartite network of diseases and their associated genes. Lage et al.2 merged protein-protein interaction networks with disease-gene associations. Similar approaches have been taken to integrate genes (transcripts) with tissue3 and miRNA4. More recently, drug-target (and drug-side effects) networks have attracted attention due to the potential of this approach to illuminate on candidates for drug repositioning5,6. While the integration of heterogeneous biological interactions would be key in fueling practical applications of systems biology from rational drug discovery to disease risk prediction, dedicated approaches and tools to accomplish this task are only starting to emerge.\n\nHeterogeneous data sets can be joined based on common keys (i.e., identifiers or ontology terms), but the integration of large-scale biological interactions is time-consuming, and particularly hampered by the lack of universal identifiers in different repositories. We previously described the integrated Complex Trait Networks (iCTNet) as an attempt to capture multiple biological relationships available in the public domain. In the original version of iCTNet7, five types of biological interactions (protein-protein, disease-gene, drug-gene, tissue-gene, disease-tissue) were integrated in a graph fashion, allowing for practical and intuitive integration of those data sources within the Cytoscape 2 environment. We argue that incorporating an expanded roster of popular databases would maximize the utility of this tool in many ways. Such integration of heterogeneous interactions would further accelerate our understanding of complex traits, and ultimately enable development of predictive disease models and facilitate drug discovery and repositioning. In this study, we present iCTNet2, a Cytoscape 3 app and database incorporating nine different types of interactions among six different types of entities: phenotypes, genes (proteins), miRNAs, tissues, drugs, and drug side effects. In addition to increasing the size of the database by a factor of 10, a central and distinctive feature of iCTNet2 is the incorporation of disease and anatomical ontologies as scaffolds onto which the different data types are integrated.\n\n\nMaterial and methods\n\niCTNet2 app is an update to the iCTNet plugin for Cytoscape2. The app was developed in Java version 7 for Cytoscape 3. The core of iCTNet2 is the iCTNet2 database, which can be accessed via the iCTNet2 app from Cytoscape8, through a user-friendly graphical interface (Figure 1). iCTNet2 app uses the Model-view-controller (MVC) pattern, dividing the app into three parts. The Model objects represent the data structures of a variety of biological entities and interactions. The View objects include three panels, where the user can search and select entities. The Control objects inherit org.cytoscape.work.AbstractTask class, implementing the database connection and the communication between the Model and the View.\n\nAll data resources have been processed and stored in a relational MySQL (http://www.mysql.com) database system. Currently, the iCTNet2 app is the only available access to the iCTNet2 database. The database schema has been designed using MySQL Workbench 5.2 (http://www.mysql.com/products/workbench). All the queries are executed in terms of stored procedures through JDBC API. Once the user clicks the “Load” button, the data is queried and loaded into Cytoscape. The iCTNet2 database collects a variety of large-scale biological datasets from public repositories to facilitate the building, visualization and analysis of heterogeneous biological networks. Additionally, iCTNet2 incorporates the disease ontology (DO)9 as the primary vocabulary for cataloguing phenotypes in a tree-like structure. Table 1 lists the publicly available resources used to build the iCTNet2 database.\n\nPhenotypes/diseases. In addition to DO, we also included two other disease vocabularies: the Experimental Factor Ontology (EFO) and MEDIC. EFO is an ontology developed by the European Bioinformatics Institute (EBI) with a detailed disease component10. MEDIC is a list of vocabularies produced by the Comparative Toxicogenomics Database (CTD)11 which incorporates disease terms from the Online Mendelian Inheritance in Man (OMIM)12 and the U.S. National Library of Medicine’s Medical Subject Headings (MeSH) (http://www.nlm.nih.gov/mesh/). The DO includes OMIM cross-references, thus providing the mapping for our network. DO cross-references were stacked onto OMIM and MeSH to provide mappings to MEDIC. Since the DO did not include direct mappings to the EFO, relevant EFO terms were manually mapped. Our mapping currently only covers the subset of EFO disease terms available in the GWAS catalog as of Dec 201413 (We submitted these 137 mappings to the DO, which now includes them as cross-references). In total, there are 6,338 phenotype records in the iCTNet2 database.\n\nGene. Gene names are obtained from the HUGO Gene Nomenclature Committee’s list of human genes (HGNC). iCTNet2 only includes currently valid genes, but also incorporates outdated gene symbols and synonyms into an alias table for reference. Non-protein coding genes are included as well. In order to map symbols or ids across different data resources, genes are identified using the integer portion of their HGNC IDs14. The iCTNet database includes 38,079 gene records.\n\nmiRNAs. miRNAs and their targets are collected from an online database miRCat (http://www.mirrna.org), which in turn, assembles data from five databases: microRNA.org, miRTarBase, tarbase, microT (v3.0) and miR2Disease.\n\nTissues. Tissue types were taken from BRENDA tissue ontology15. We rooted the ontology at ‘whole body’ (BTO:0001489) to exclude the non-animal tissue portions of the ontology.\n\nDrugs. The CTD (12) provides the primary resource for drugs and references to DrugBank16 identifiers providing the mapping between the two resources. Thus, iCTNet2 contains information of 151,378 drugs in total. However, the function of only 10% of them is currently associated (mapped) to genes. Mapped drugs in iCTNet include over 13,000 curated chemicals and associations with several other major chemical databases. DrugBank 3.0 contains fewer entries, but has extensive information on most FDA approved therapeutics.\n\nSide effects. The side effect ontology is retrieved from the Medical Dictionary for Regulatory Activities (MedDRA) (http://www.meddra.org). While providing a high quality and widely adopted vocabulary, the commercial nature of this resource prevents large-scale republication of its terms. Instead, our database reports the Unified Medical Language System (UMLS) (http://www.nlm.nih.gov/research/umls/) concepts for side effects. Since MedDRA is a source vocabulary for the UMLS, the mapping is straightforward and reversible. Nonetheless, upon request we will provide researchers who have a valid MedDRA license with an untranslated version of our database which includes the hierarchical relationships between side effects.\n\nPhenotype-gene. The phenotype-gene associations are the primary resources to study the genetic factors of complex traits. iCTNet2 merges phenotype-gene associations from three online databases: GWAS Catalog, OMIM and CTD. Only CTD relationships with direct evidence of \"marker/mechanism\" were included. To convert from SNP to gene associations, we combined overlapping loci for each GWAS Catalog disease17. The mode author reported gene for each loci was selected as primary.\n\nPhenotype-tissue. These edges represent physiopathological information (i.e. which tissues/organs are likely affected by each disease). To identify tissue relationships with diseases and side effects, we used an ontology inference method. Anatomical disease and side effect terms were manually mapped to their affected tissues in the BTO. For example, connective tissue disease (DOID:65) was mapped to connective tissue (BTO:0000421). Affected tissues were propagated to more specific terms, so only high-level DO and MedDRA terms required manual mapping. See Data S1–S3 for the complete mappings.\n\nGene-tissue. iCTNet2 collects an extensive atlas of tissue-specific gene expression from the GNF gene atlas18. The expression patterns of 79 human tissues are available that can provide important clues about gene functions.\n\nDrug-disease. The drug-disease interactions (indications) are collected from CTD, which in turn, are manually curated from the literature.\n\nDrug-gene. The drug-gene interactions are assembled from CTD and DrugBank, two major databases containing drug information.\n\nDrugs-side effects. The side effects of drugs in humans are an essential source to understand human phenotypes. iCTNet2 collects the information of 888 drugs and 1,450 side effect terms from the side effect resource (SIDER)19, with available side effect frequency.\n\nProtein-protein interactions (PPI). PPIs are among the most studied interactions in network biology, although the known interactions may present only one tenth of the entire interactions. PPIs are collected from ppiTrim20, which further curates iRefIndex21, a master database consolidating interactions from 15 different sources (including BIND, HPRD, etc).\n\nmiRNA-gene. MicroRNAs (miRNAs) are short RNA sequences that regulate the expression of target genes. 2,457miRNA-gene interactions are collected from the online database miRCat.\n\nAll data resources have been processed and stored in a relational MySQL (http://www.mysql.com) database system. Currently, the iCTNet2 app is the only available access to the iCTNet2 database.\n\nOnce installed, iCTNet2 will show up automatically on the left hand side of the Cytoscape window. So through the Cytoscape platform, networks constructed via iCTNet2 can be visualized in different layouts with many visualization features. Cytoscape built-in functions or analysis apps can be easily applied as well.\n\n\nResults\n\nThere are three options to start building networks with iCTNet2. As the metagraph can be cyclic (Figure 1), we simplified the construction process by enabling the user to select the starting node type as being a disease, gene or drug. Different types of networks (e.g. disease, gene or drug) offer complementary views from different perspectives. Next, a case study is presented starting with the network from disease nodes as an example.\n\nStarting from any phenotype(s) in the database, users can add gene, drug and tissue directly (if connections among them exist), and secondly add miRNA, side effects and PPIs to further grow the network. As an example, we created three disease (phenotype)-gene networks by selecting all data available in the GWAS Catalogue (threshold p-value 1E-7), CTD and OMIM databases. The connected component of each network was markedly different in size and topological properties. The GWAS network was comprised of 1547 nodes (82 diseases + 1465 genes) connected through 2010 edges (ratio N/E = 0.77), the CTD network included 5166 nodes (1168 diseases + 3998 genes) and 12657 edges (N/E = 0.41) and the OMIM network was formed by 2265 nodes (699 diseases + 1566 genes) and 2228 edges (N/E = 1.01). Upon layout within Cytoscape (spring embedded) a clearly distinct topology emerged for each network, with the GWAS network displaying a wheel and spike pattern with most diseases at the center (Figure 2A), and the OMIM network displaying a circular symmetric pattern, with most diseases towards the periphery (Figure 2C). The CTD network displayed a pattern that resembled an aggregate of the other two, an expected outcome given that this database includes information on both common and rare diseases (Figure 2B). The different topology between GWAS and OMIM networks clearly reflects the type of information each database contains. The central disposition of most diseases in the GWAS network (and the larger proportion of genes to diseases) highlights their polygenic nature and reflects the large amount of gene (locus) sharing among common diseases, consistent with our current understanding of their pathogenesis. On the other hand, the peripheral disposition of diseases in the OMIM network is a reflection of the limited genetic sharing characteristic of monogenic diseases, which dominate this database. Consistent with these observations, a network analysis conducted within Cytoscape showed differences between GWAS and OMIM networks in several parameters, including centrality, neighborhood connectivity and shortest path length distributions (Figure 2).\n\nNetworks were generated using iCTNet 2.0 for diseases represented in the GWAS Catalog (A), the Comparative Toxicogenomics Database (B) and OMIM (C). Note the different topological characteristics (described below each network), particularly between A and C. Topological analysis was performed with Network analysis (a Cytoscape Core app).\n\nWe next downloaded the GWAS disease-gene network for 18 common autoimmune diseases (and their first degree protein interactions) (Figure 3A). A clear pattern of gene sharing can be observed (green triangles in the center of the network represent shared genes between at least two diseases), consistent with our understanding on the genetic commonalities among autoimmune diseases. Using standard Cytoscape procedures, we further filtered this network to obtain only the protein interactome associated with more than one autoimmune disease (Figure 3B). A highly connected component (n=98) emerged (N/E = 0.60) with several key genes of known immunological function (e.g. STAT1, STAT3, NFKB1, RELA and MAPK1) at its center. To further explore the biological relevance of these nodes, a gene ontology analysis was performed on this network using the BiNGO App22 and results were displayed as a new network (Figure 3C). Confirming our previous observations, the set of genes associated with multiple autoimmune diseases is highly enriched (as indicated by the orange colored nodes) in immunological processes ranging from levels as general as leukocyte proliferation, and regulation of immune response, to as specific as regulation of MAPKKK and JAK-STAT cascades.\n\n(A) Common autoimmune diseases and their associated genes (according to the GWAS catalog) are displayed. (B) Genes associated with multiple autoimmune diseases form a densely connected network at the protein level. (C) Gene ontology analysis of the genes in (B) shows over-representation of immune related proteins.\n\nIn an attempt to evaluate the current pharmacological landscape in autoimmune disease treatment, we added all drugs known to be used to treat each autoimmune disease in the network according to CTD. As observed for genetic associations, while most treatments are disease-specific, there is substantial sharing of treatment modalities among multiple diseases (Figure 4). This suggests that drug repurposing is a plausible strategy for diseases with shared genetic susceptibility and pathophysiological mechanisms.\n\nIncreased sharing of indications can be readily detected among diseases of similar etiology. Drugs are represented by blue squares, and the opacity of the square is proportional to its degree, thus shared drugs appear darker. Diseases are represented as circles.\n\n\nConclusion\n\nThe iCTNet2 database and Cytoscape app are a systematically-developed resource and tool for studies requiring integration of multi-domain biological information. iCTNet2 illustrates how powerful the integration of heterogeneous biological interactions can be, through a simple and user-friendly interface. Comprehensive views of a given disease, including its genetic risk, gene expression profile, biological pathways affected, and actual and potential therapeutic options are just a few clicks away. Similarly, global landscapes of entire groups of diseases (i.e. malignancies, autoimmune disorders, etc) and their relevant “data neighbourhoods” can be easily created. Being a Cytoscape app, iCTNet2 also provides flexibility to conduct further analysis on the generated networks for further exploration, such as disease gene prediction, module detection, and topological network analysis.\n\n\nSoftware availability\n\nThe App is available via the Cytoscape App Store.\n\nThe source code can be accessed at https://github.com/LiliWangQueensu/iCTNet2_v2\n\nhttps://zenodo.org/record/21386#.VbIoo_JzbIU\n\nDOI 10.5281/zenodo.21386\n\nMIT license",
"appendix": "Author contributions\n\n\n\nLW developed and implemented the app. DSH and AS contributed to develop the database and mapped data types. PM provided supervision and funding. SEB conceived the idea, provided funding and supervision and wrote the manuscript. All authors have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported from grants from the National Multiple sclerosis Society (AN085369) and the National Institutes of Health (R01NS088155) to SEB.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary Material\n\nSupplementary Data S1.\n\nMapping file between Genomics Institute of the Novartis Research Foundation (GNF) and Brenda Tissue Ontology (BTO).\n\nClick here to access the data.\n\nSupplementary Data S2.\n\nMapping file between Brenda Tissue Ontology (BTO) and the Human disease ontology (DO).\n\nClick here to access the data.\n\nSupplementary Data S3.\n\nMapping file between Brenda Tissue Ontology (BTO) and the Medical Dictionary for Regulatory Activities (MedRA).\n\nClick here to access the data.\n\n\nReferences\n\nGoh KI, Cusick ME, Valle D, et al.: The human disease network. Proc Natl Acad Sci U S A. 2007; 104(21): 8685–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLage K, Hansen NT, Karlberg EO, et al.: A large-scale analysis of tissue-specific pathology and gene expression of human disease genes and complexes. Proc Natl Acad Sci U S A. 2008; 105(52): 20870–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuan Y, Gorenshteyn D, Burmeister M, et al.: Tissue-specific functional networks for prioritizing phenotype and disease genes. PLoS Comput Biol. 2012; 8(9): e1002694. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKozomara A, Griffiths-Jones S: miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 2014; 42(Database issue): D68–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYamanishi Y, Kotera M, Moriya Y, et al.: DINIES: drug-target interaction network inference engine based on supervised analysis. Nucleic Acids Res. 2014; 42(Web Server issue): W39–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchadt EE, Friend SH, Shaywitz DA: A network view of disease and compound screening. Nat Rev Drug Discov. 2009; 8(4): 286–95. PubMed Abstract | Publisher Full Text\n\nWang L, Khankhanian P, Baranzini SE, et al.: iCTNet: a Cytoscape plugin to produce and analyze integrative complex traits networks. BMC Bioinformatics. 2011; 12: 380. 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): 2498–504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchriml LM, Arze C, Nadendla S, et al.: Disease Ontology: a backbone for disease semantic integration. Nucleic Acids Res. 2012; 40(Database issue): D940–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMalone J, Holloway E, Adamusiak T, et al.: Modeling sample variables with an Experimental Factor Ontology. Bioinformatics. 2010; 26(8): 1112–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavis AP, Murphy CG, Johnson R, et al.: The Comparative Toxicogenomics Database: update 2013. Nucleic Acids Res. 2013; 41(Database issue): D1104–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamosh A, Scott AF, Amberger JS, et al.: Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic Acids Res. 2005; 33(Database issue): D514–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWelter D, MacArthur J, Morales J, et al.: The NHGRI GWAS Catalog, a curated resource of SNP-trait associations. Nucleic Acids Res. 2014; 42(Database issue): D1001–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHGCN Hugo Gene Nomenclature Committee. Reference Source\n\nGremse M, Chang A, Schomburg I, et al.: The BRENDA Tissue Ontology (BTO): the first all-integrating ontology of all organisms for enzyme sources. Nucleic Acids Res. 2011; 39(Database issue): D507–13. PubMed Abstract | Publisher Full Text | Free 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): D1035–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHimmelstein DS: Extracting disease-gene associations from the GWAS Catalog. In ThinkLab. 2015. Publisher Full Text\n\nSu AI, Wiltshire T, Batalov S, et al.: A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci U S A. 2004; 101(16): 6062–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuhn M, Campillos M, Letunic I, et al.: A side effect resource to capture phenotypic effects of drugs. Mol Syst Biol. 2010; 6(1): 343. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStojmirovic A, Yu YK: ppiTrim: constructing non-redundant and up-to-date interactomes. Database (Oxford). 2011; 2011: bar036. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRazick S, Magklaras G, Donaldson IM: iRefIndex: a consolidated protein interaction database with provenance. BMC Bioinformatics. 2008; 9: 405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 2005; 21(16): 3448–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "9869",
"date": "24 Aug 2015",
"name": "Amitabh Sharma",
"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\nBaranzini et al. updated the iCTNet database from the data collected from public repositories to facilitate the building, visualization and analysis of heterogeneous biological networks in a comprehensive fashion via the Cytoscape platform. I like the manuscript and source developed by Baranzini group. The resource update is important and timely, and is well-done and clearly described. It is freely available and provides a good resource for the community to understand the connections between different omics or the big data in disease medicine.A few minor comments would improve the manuscript:Add some text in the conclusion about how version 2 is better than version 1. The Phentoypes/diseases vocabulary sources do not overlap much, did this result in a lot of data loss while integrating? Add some description regarding the edges in the Figure 2 legend. What are different node colors? GWAS network is much sparse because of the incompleteness of the interactome and also we have literature bias for the OMIM data. In figure 3, is the network PPI only or aggregated network of all sources? Also, Figure 3C should include only those terms that are below specific thresholds, like p<0.05.Overall, an excellent work.",
"responses": []
},
{
"id": "9868",
"date": "02 Sep 2015",
"name": "Gary D Bader",
"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 Cytoscape app that provides the entry point to the iCTNet resource, containing networks connecting a variety of concepts, including genes, drugs, side effects, tissues, miRNAs and phenotypes. This resource is very useful for users wishing to start with one of these information types and navigate to others e.g. to find all genes and drugs involved in a disease of interest. In general the app works very smoothly. My comments relate mainly to missing details and text to be clarified, as detailed below. Major points:P5 “Next, a case study is presented starting with the network from disease nodes as an example.” It would be useful to describe the full workflow, including the scientific question, rationale and end goal.There is a “Create similarity network” feature present in the App menu, but this is not described in the manuscript. It would be useful to add a section describing the feature and a use case. If the user loads too much data, the app will take a long time to respond and the process can’t easily be canceled. The user should be warned in the manuscript or via the app that large queries may take a long time. Search starting points can be gene, disease or drug. Why can’t users search by other starting points e.g. tissue? The last update date of the ICT database and the date and version used for each resource should be clearly communicated to the user e.g. via the manuscript, app and/or ICT website. Minor points:Page 2: clarify “stacked onto”. P2: “ids” -> identifiers P3: “The CTD (12)” – what does ‘(12)’ mean? P3 – drugs paragraph. This section is a bit unclear. CTD provides references to drugbank identifiers? Should it be that CTD provides references to drugbank records? How is the ‘function’ of drugs defined – is this just the drug target? Drugbank contains fewer entries compared to what? P3 – “phenotype-gene” section. “To convert from SNP to gene associations, we combined overlapping loci for each GWAS Catalog disease17.” How were the loci combined?“The mode author reported gene for each loci was selected as primary.” – what is a mode?Page 3 and 4 – in the “Types of interactions” section, all sub-sections should include the number of interactions e.g. how many gene-tissue interactions are there?P5 – what is a metagraph?P5 – “spike” -> “spoke”? P5 – “Using standard Cytoscape procedures, we further filtered this network to obtain only the protein interactome associated with more than one autoimmune disease (Figure 3B)” – the Cytoscape procedures should be detailed to make it easier for users to replicate the results in the manuscript.Figure 1 – the tissues circle of nodes is covered by edges from other circles – can it be moved out a bit to show how it connects to other circles?",
"responses": [
{
"c_id": "1610",
"date": "28 Sep 2015",
"name": "Sergio Baranzini",
"role": "Author Response",
"response": "Thank you for your comments!Please see a point-by point answer belowMajor points:P5 “Next, a case study is presented starting with the network from disease nodes as an example.” It would be useful to describe the full workflow, including the scientific question, rationale and end goal.This has been added to the revised manuscriptThere is a “Create similarity network” feature present in the App menu, but this is not described in the manuscript. It would be useful to add a section describing the feature and a use case. We have expanded the manuscript to describe this feature in detail and have modified Figure 3 to include an example of this feature. If the user loads too much data, the app will take a long time to respond and the process can’t easily be canceled. The user should be warned in the manuscript or via the app that large queries may take a long time. We have introduced a warning message for large networks. Specifically, the message will be shown if: (1) the size of query diseases > 50 and PPI depth>0; or the size of query diseases > 100;(2) the size of query genes >500 and PPI depth >0; or the size of query genes > 1000;(3) the size of query drugs > 100 and PPI depth >0; or the size of query drugs > 200;Search starting points can be gene, disease or drug. Why can’t users search by other starting points e.g. tissue? Technically, this should be possible. however, with the three provided starting points, all other searches are technically possible using basic Cytoscape functions. The last update date of the ICT database and the date and version used for each resource should be clearly communicated to the user e.g. via the manuscript, app and/or ICT website. Table 1 has been updated.Minor points:Page 2: clarify “stacked onto”. doneP2: “ids” -> identifiers doneP3: “The CTD (12)” – what does ‘(12)’ mean? removed (12)P3 – drugs paragraph. This section is a bit unclear. CTD provides references to drugbank identifiers? Should it be that CTD provides references to drugbank records? How is the ‘function’ of drugs defined – is this just the drug target? Drugbank contains fewer entries compared to what? This paragraph has been re-writtenP3 – “phenotype-gene” section. “To convert from SNP to gene associations, we combined overlapping loci for each GWAS Catalog disease17.” How were the loci combined?We have provided a reference detailing how this was done. Basically, we proceeded as follows:Lead-SNPs were assigned windows—regions wherein the causal SNPs are assumed to lie—retrieved from the DAPPLE server. Windows were calculated for each lead-SNP by finding the furthest upstream and downstream SNPs where r2 > 0.5 and extending outwards to the next recombination hotspot. Associations were ordered by confidence, sorting on following criteria: high/low confidence, p-value (low to high), and recency. In order of confidence, associations were overlapped by their windows into disease-specific loci. By organizing associations into loci, associations from multiple studies tagging the same underlying signal were condensed.“The mode author reported gene for each loci was selected as primary.” – what is a mode?CorrectedPage 3 and 4 – in the “Types of interactions” section, all sub-sections should include the number of interactions e.g. how many gene-tissue interactions are there?The numbers of interactions are now specified.P5 – what is a metagraph?We refer to a metagraph as the graph describing the interactions among the different node types.P5 – “spike” -> “spoke”? doneP5 – “Using standard Cytoscape procedures, we further filtered this network to obtain only the protein interactome associated with more than one autoimmune disease (Figure 3B)” – the Cytoscape procedures should be detailed to make it easier for users to replicate the results in the manuscript.DoneFigure 1 – the tissues circle of nodes is covered by edges from other circles – can it be moved out a bit to show how it connects to other circles?Done"
}
]
}
] | 1
|
https://f1000research.com/articles/4-485
|
https://f1000research.com/articles/4-367/v1
|
24 Jul 15
|
{
"type": "Review",
"title": "Intracellular Dynamics of the Ubiquitin-Proteasome-System",
"authors": [
"Maisha Chowdhury",
"Cordula Enenkel",
"Maisha Chowdhury"
],
"abstract": "The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body’s cells.",
"keywords": [
"Proteasome",
"Storage Granules",
"Dynamics",
"Nuclear Transport",
"Ubiquitin System",
"Blm10",
"Importin",
"Karyopherin",
"Quiescence"
],
"content": "Introduction\n\nProteolysis determines the half-life of proteins and thus controls protein homeostasis. If protein homeostasis is disrupted, the incidence of protein misfolding and neurodegenerative diseases such as Huntington’s, Parkinson’s and Alzheimer’s increases (Ciechanover & Brundin, 2003).\n\nIn eukaryotic cells two highly conserved degradation pathways exist: long-lived proteins are degraded within the lysosome, an organelle with membranes which protect the surrounding cytoplasm against lysosomal hydrolases (Rendueles & Wolf, 1988); short-lived proteins are degraded by proteasomes, multimeric protease complexes which move between the nucleo- and cytoplasm (Hershko & Ciechanover, 1998). Proteasomal substrates are often nuclear proteins such as proteins regulating cell cycle progression (cyclin-dependant kinases and their inhibitors), gene expression (transcriptions factors), DNA damage and stress response; although, misfolded proteins occurring during protein synthesis in the cytoplasm are also rapidly degraded by the proteasome (Kirschner, 1999; Vabulas & Hartl, 2005; von Mikecz, 2006). As a result proteasomal proteolysis serves to eliminate obsolete proteins which compete with functional proteins for binding partners and are prone to associate with irreversible and toxic protein aggregates (Goldberg, 2003).\n\nHere, we want to address the dynamics of proteasomes, which select their substrates by specific determinants such as poly-ubiquitylation, a covalently linked chain of ubiquitin molecules (Finley, 2009). This ubiquitin-dependent proteolysis undertakes up to 90% of protein degradation in growing yeast and cultured mammalian cells and consumes considerable amounts of ATP, since the activation and conjugation of ubiquitin to the protein substrate as well as the unfolding and translocation of the protein substrate into the proteasome is ATP-dependent (Coux et al., 1996). Natively-disordered proteins also qualify as proteasome substrates and are cleaved without post-translational ubiquitin modification (Fishbain et al., 2015).\n\nThe advent of live cell imaging and GFP-labelling technologies in the 1990s (Tsien, 1998) have greatly facilitated the study of proteasome dynamics in yeast and mammalian cells. Through these non-invasive techniques, the localization of the proteasome in growing yeast and highly proliferating cancer cells has been elucidated to be primarily nuclear (Enenkel et al., 1998; Laporte et al., 2008; McDonald & Byers, 1997; Russell et al., 1999). In line with this finding, increasing evidence in the literature suggests that certain misfolded proteins are imported from the cytoplasm into the nucleus solely for proteasomal degradation (Park et al., 2013; Prasad et al., 2010). Conversely, transient nuclear proteins are exported into the cytoplasm for proteolysis, indicating a dynamic shift of proteasomal substrates between the nucleus and cytoplasm (Chen & Madura, 2014a). Under nutrient deprivation and during transition from proliferation to quiescence, yeast proteasomes gather in proteasome storage granules (PSGs) at the nuclear envelope (NE) and endoplasmic reticulum (ER) membrane (Enenkel, 2014; Knecht & Rivett, 2000; Wojcik & DeMartino, 2003). PSGs are membraneless organelles which seem to pinch off the NE/ER and are retained as stable entities in the cytoplasm. When cells resume growth, PSGs dissipate and proteasomes are rapidly imported into the nucleus to contribute their function in cell proliferation (Laporte et al., 2008). The mechanism of PSG formation and clearance is still unknown but seems to be conserved, since PSG-like structures are observed in primary cell lines of non-dividing neuronal cells and in immortalized cell lines of cancer cells, if they are chemically arrested in cell cycle progression (Kaganovich et al., 2008).\n\nOur knowledge about proteasome dynamics in mammalian cells is poor. Thus, the focus of this review will be to critically integrate the literature about the dynamics of the proteasome, particularly based on studies in yeast. In our overview of the ubiquitin-proteasome system and common principles of nuclear transport, we cite and refer to original work and review articles written by investigators who did seminal work on these topics. In the paragraphs addressing detailed knowledge about proteasome dynamics we cite the original work.\n\n\nDiscussion/analysis of the literature\n\nUbiquitylation is a post-translational modification commonly associated with proteasomal protein degradation. At least four ubiquitin molecules, conserved peptides of 76 amino acids, are required for a poly-ubiquitin chain to be recognized by the proteasome. Hershko and colleagues in the early 1980s showed that poly-ubiquitylation requires three ATP-dependent enzymes: the ubiquitin activation enzyme (E1), a family of ubiquitin conjugating enzymes (E2) and a family of ubiquitin protein ligases (E3) (Hershko & Ciechanover, 1998). First, ATP hydrolysis is required to activate the AMP linkage to the C-terminal glycine of ubiquitin which enables the transfer of the ubiquitin moiety to the active site cysteine of the E1. Second, the E1-bound ubiquitin is linked to the active site cysteine residue of an E2 by transesterification. Finally, the E3 transfers the ubiquitin onto the substrate depending on the class of the E3 enzyme (RING, HECT and U-box ligases) (Finley et al., 2012; Harper & Schulman, 2006). Upon transfer of ubiquitin onto the substrate, the C-terminal glycine forms an isopeptide bond with an ε-amino group of a lysine residue in the substrate. Elongation of the ubiquitin chain is achieved as succeeding ubiquitin form isopeptide linkages with specific lysines of the preceding ubiquitin (Hershko & Ciechanover, 1998). Prior to degradation, deubiquitinating activities within the proteasome cleave and recycle the ubiquitin molecules from the substrates (Crosas et al., 2006; Hanna et al., 2006; Lam et al., 1997; Verma et al., 2002). Deubiquitinating enzymes in the cyto- and nucleoplasm provide an additional level on the plasticity on the repertoire of proteasomal substrates (Sahtoe & Sixma, 2015). Intriguingly, GFP-labelled ubiquitin and the E1, named UBA1, is primarily nuclear in growing yeast and mammalian cells suggesting that ubiquitin-dependent proteolysis mainly occurs in the nucleus (Huh et al., 2003; Salomons et al., 2010; Sugaya et al., 2014; Sugaya et al., 2015).\n\nComposed of over 40 subunits, the proteasome is a protein complex of 2.5 MDa which consists of two main components: the 20S core particle (CP) and the 19S regulatory particle (RP) (Coux et al., 1996).\n\nProteasome configurations centered on the CP can have either one or two RPs but also one or two alternative proteasome activating complexes giving rise to a variety of proteasome complex configurations. Proteasome holo-enzymes engaged in the degradation of poly-ubiquitylated proteins require the RP, thus occur either as RP-CP or RP-CP-RP, also termed the 26S and the 30S proteasome, respectively (Eytan et al., 1989).\n\nThe proteasome belongs to the family of threonine proteases and its maturation follows the concept of zymogen activation upon which proteases are activated, once they arrive at their destination. With a molecular mass of 700 kDa, the CP is composed of seven distinct α and β subunits, each of which form heptameric rings stacked into a barrel composed of two outer α rings and two inner β rings (Groll et al., 1997). The maturation of the CP involves the dimerization of two inactive precursor complexes, resembling two half-CPs. Half-CPs consist of an α ring and β ring with five of the seven β subunits synthesized with propeptides. With the dimerization of two half-CPs into the pre-holo CP, the autocatalytic processing of the propeptides is triggered and three β subunits contribute an active site threonine with different peptide cleavage specificities (Li et al., 2007; Ramos et al., 1998). CP-dedicated chaperones, namely Pac/Pba/Poc 1-4 and Ump1, assist in CP assembly. Ump1 is a natively-disordered protein, which is buried inside the pre-holo CP and later on becoming the first substrate of the nascent CP (Kusmierczyk et al., 2008; Ramos & Dohmen, 2008). The α rings are the key players in CP gating. Normally CP α rings are closed, unless they are opened by the RP to allow access of protein substrates into the proteolytic cavity (Groll et al., 2000).\n\nAs “gate keeper” of the CP, the RP is the best understood proteasome activator (Rechsteiner & Hill, 2005). The RP is divided into two parts, the base and the lid subcomplexes. The RP base is composed of six ATPases of the triple A family (ATPases Associated with diverse cellular Activities), named Rpt1-6, and five non-ATPases, Rpn1, Rpn2, Rpn10, Rpn13 and Ubp6. The base Rpn subunits are involved in the recognition of the poly-ubiquitin chain and the Rpt ATPase subunits guide the unfolding and translocation of the polypeptide substrate into the CP (Finley et al., 1998). In contrast to the RP base subunits, the subunits comprising the RP lid are only of the non-ATPase class: Rpn3, Rpn5-9, Rpn11 and Rpn12 (Glickman et al., 1998). The main known function of the RP lid is the processing of poly-ubiquitin chains. Rpn11 contributes isopeptidase activity to recycle ubiquitin moieties from the protein substrates. Ubp6 also has ubiquitin hydrolase activity and assists in trimming poly-ubiquitin chains (Crosas et al., 2006; Hanna et al., 2006; Lam et al., 1997; Verma et al., 2002). In principle, the RP ensures that only targeted substrates are degraded by the proteasome, thereby conferring the ubiquitin- and ATP-dependence towards proteasomal protein degradation.\n\nTwo competing models exist for RP assembly (Funakoshi et al., 2009; Le Tallec et al., 2009; Park et al., 2009; Roelofs et al., 2009). The first posits that RP assembly occurs in modules independent of the CP with the help of four RP-dedicated chaperones, named Hsm3, Nas2, Nas6 and Rpn14 (Funakoshi et al., 2009). In contrast, the second model proposes that the CP serves as a scaffold for the heterohexameric ATPase ring of the RP base (Park et al., 2009). The second model, however, appears less likely with regard to X-ray structure analysis showing that the RP-dedicated chaperones hinder the association between the RP base and CP α ring (Barrault et al., 2012). The CP-independent assembly model is also supported by the finding that the assembly of RP base and lid can be reconstituted from recombinant proteins with the assistance of RP-dedicated chaperones but without the CP template (Beckwith et al., 2013). However, the CP could serve as a platform for RP base assembly, if RP-dedicated chaperones are limiting.\n\nAt this point, it is important to acknowledge the importance of GFP labelling and the ease with which it has allowed localization studies to be conducted (Enenkel, 2014; Groothuis & Reits, 2005). In our species of interest, Saccharomyces cerevisiae, which is an excellent model organism for eukaryotic cells, GFP labelling of proteasomes is achieved by homologous recombination techniques into the chromosomal locus to convert an endogenous proteasomal subunit to a GFP-tagged version (Enenkel et al., 1999; Laporte et al., 2008; McDonald & Byers, 1997). Nearly all proteasomal genes are essential and could be modified by GFP fusions without interfering with their function; we prefer the CP subunits α4 and β5, the CP-dedicated chaperone Ump1, and the RP subunits Rpn1, Rpt1 and Rpn11 as GFP-labelled reporters, because their GFP fusion proteins are fully incorporated into the proteasomal subcomplexes. So far, ~30 subunits of the yeast proteasome were labelled with GFP. All of them reveal the same subcellular localization as thoroughly investigated by direct fluorescence microscopy in living yeast (Laporte et al., 2008).\n\nSeminal studies on proteasome localization in vertebrate cells were performed by Werner Franke’s and Wolfgang Baumeister’s laboratories in the early 1990s. Proteasomes were mainly detected in the nuclei of Xenopus laevis oocytes and cultured mammalian cells (Amsterdam et al., 1993; Hügle et al., 1983; Kleinschmidt et al., 1983). Later investigations reported a shift towards cytoplasmic proteasomes dependent on the type of the cell line and the density of the cells (Wojcik & DeMartino, 2003). Proteasome localization also varies with the growth phase in yeast (Laporte et al., 2008; Weberruss et al., 2013). In growing yeast at logarithmic phase (OD~1), proteasomes are primarily nuclear. During the transition from proliferation to quiescence and the entrance into stationary phase (OD>3), proteasomes deplete from the nucleus and accumulate at the NE/ER in membraneless organelles. These enigmatic structures of proteasomes were initially observed by Isabelle Sagot and her co-workers, who coined the term proteasome storage granules (PSGs) (Laporte et al., 2008). With prolonged quiescence, one to two PSGs with a diameter of ~0.2 to 0.5 µm pinch off the NE into the cytoplasm. The PSGs are motile and stable in yeast cultures and are kept in quiescence for several weeks. If quiescent yeast cells are allowed to resume growth by replacing the glucose-depleted medium with glucose-rich medium, the PSG rapidly clears and the proteasomes are relocated into the nucleus within a few minutes.\n\nStudies with mammalian cancer cell lines also exploited GFP-labelling techniques and fluorescence recovery after photobleaching experiments. The experiments suggested that nuclear transport of GFP-labelled CP across the NE was inefficient. Only the mitotic breakdown of the NE and its reassembly after mitosis allowed nuclear uptake of proteasomes (Reits et al., 1997). However, this nuclear uptake mechanism cannot explain the predominant nuclear localization of proteasomes in yeast cells which divide without mitotic breakdown of the NE. Proteasomes are the second most abundant protein complexes in eukaryotic cells and require continuous synthesis within the cytoplasm and nuclear import during cell division (Weberruss et al., 2013). The most common route for protein complexes to cross the NE in an organism with closed mitosis is through nuclear pore complexes (NPC). Before we address this pathway for yeast proteasomes, we will shortly summarize the concept of nuclear transport through the NPC, a pathway conserved from yeast to human.\n\nThe NE is embellished with NPCs which regulate the entry of molecules into and out of the nucleus. Their principal function is to allow free diffusion of small molecules, such as water/ions/peptides, and to block non-specific translocation of macromolecules that exceed 40kDa or a diameter larger than 5nm (Aitchison & Rout, 2012; Wente & Rout, 2010). Translocation of larger macromolecules requires specific interactions with the NPC. Protein cargoes therefore associate with soluble transport factors, called karyopherins/importins/exportins, that themselves interact with phenylalanine-glycine rich nucleoporins (FG-Nups) decorating the NPC (Wozniak et al., 1998). Importins and exportins identify their protein cargoes by nuclear localization sequences (NLSs) and nuclear export signals (NESs), which ensure their nuclear import and export, respectively. In the literature, there are variations of nuclear import and export signals, only some of which comply with the classical import/export concept. The classical concept applies for nuclear import of proteasomes. Thus, we will focus on the key components required for the classical pathway (Gorlich & Kutay, 1999).\n\nThe classical nuclear import cycle starts with the association of the importin/karyopherin αβ heretodimer, called Srp1/Kap95 in yeast, with the cargo NLS. Two types of classical NLSs exist: the monopartite NLS which contains five basic amino acid residues and the bipartite NLS in which two clusters of basic residues are spaced by 10–12 indifferent residues. Importin α has the NLS-binding grooves, and importin β mediates the interaction with FG-Nups. The directionality of nuclear transport is dictated by the Ran-GTP/GDP gradient across the NE. Ran is a small GTPase, named Gsp1 in yeast. Ran exists in its GTP-bound state in the nucleus and in its GDP-bound state in the cytoplasm due to the actions of the Ran guanine nucleotide exchange factor (RanGEF) and the RanGTPase activating protein (RanGAP) in the nucleo- and cytoplasm, respectively (Gorlich & Kutay, 1999; Moore & Blobel, 1993). In the nucleus, the cargo-importin αβ complex encounters RanGTP, which results in the release of the cargo (Rexach & Blobel, 1995). Cargo-free importin αβ is recycled into the cytoplasm for the next round of nuclear import.\n\nOur studies in yeast strongly suggest that newly synthesized proteasomes are imported from the cytosol into the nucleus as inactive precursor complexes and that the maturation of nuclear CP proceeds to completion post-import (Lehmann et al., 2002). Although electron microscopy studies have shown that the NPC could expand to accommodate the longitudinal passage of the 30S proteasome, the permeability barriers towards macromolecules such as CP precursor complexes and RP assembly modules must be overcome by specific importins/karyopherins (Pante & Kann, 2002). Several classical NLSs exist within the N-termini of distinct α subunits which were proposed to be either accessible rendering the CP in an import-competent conformation, or to be masked rendering the CP in an import-incompatible conformation (Tanaka et al., 1990). Indeed, recent cryo-EM structure analysis revealed flexible and less structured α ring surfaces in Ump1-associated CP precursor complexes (Kock et al., 2015), in compliance with our finding that importin α recognizes CP precursor complexes but not mature CP with closed α rings (Lehmann et al., 2002). Our model upon which CP precursor complexes are imported into the nucleus was supported by the following observations (Figure 1). First, when tagged with GFP, Ump1 is predominantly nuclear in spite of the fact that CP precursor complexes are assembled from nascent subunits in the cytoplasm. Second, in importin α mutants namely srp1-49 but not in srp1-31, several groups found that the CP is mislocalized to the cytoplasm, providing another piece of evidence for the classical import pathway of proteasomes. Unprocessed and incompletely processed β5 subunits, crucial determinants of CP precursor complexes and pre-holo-CP, respectively, accumulate in srp1-49 mutants, while precursors of β5 subunits are hardly detectable in wild type cells (Lehmann et al., 2002). Third, when CP maturation is delayed by UMP1 deletion, all CP reporter proteins accumulate in the nucleus, although half of the CP is not fully matured and most likely exists as pre-holo-CP (Fehlker et al., 2003; Lehmann et al., 2008).\n\nThe α rings with the classical NLS are depicted in red. The β rings with propeptides are depicted in blue. The CP-dedicated chaperone and maturation factor Ump1 is depicted in yellow. The completion of CP maturation occurs in the nucleus with the degradation of Ump1.\n\nIn the case of the RP, functional NLSs were identified in RP base subunits Rpn2 and Rpt2 and are recognized by importin α (Figure 2). The deletion of the Rpn2 NLS caused a temperature sensitive phenotype and mislocalizations of the RP base into cytosolic foci, whereas the deletion of the Rpt2 NLS was compensated by the presence of the Rpn2 NLS. At permissive temperatures, neither the Rpn2 nor the Rpt2 NLS deletion had severe impact on nuclear proteasome localization suggesting a redundancy of proteasomal NLSs (Wendler et al., 2004). Isono et al. (2007) later confirmed that Rpn2 provides a crucial NLS to aid nuclear import of the RP base and that the lid is separately imported. The nuclear import of the RP lid also requires importin α, though no classical NLS has been identified within RP lid subunits; rather Sts1, a short-lived protein that itself contains a classical NLS, associates with Rpn11 to facilitate nuclear import of the RP lid by importin αβ (Chen et al., 2011). In accordance, deletion of the Sts1 NLS has downstream effects on the nuclear localization of RP lid in addition to RP base and CP, which suggests that proteasomes could also be transported as holo-enzymes (Chen & Madura, 2014b). In order to ensure comparable stoichiometry of proteasomal subcomplexes in the nucleus and similar kinetics by which they are imported into the nucleus, it is reasonable that importin αβ is used as common nuclear import receptor.\n\nRpn2/Rpt2 and Sts1 confer classical NLS to the RP base and lid complex, respectively. Sts1 is short-lived and most likely degraded with RP-CP assembly.\n\nRecent fluorescence correlation spectroscopy studies also support the conclusion that proteasomes can be imported into the nucleus as holo-enzymes (Pack et al., 2014; Figure 3). However, the maturation state of the GFP-labelled proteasomes was unclear. Possibly, pre-holo-CP are the real nuclear transport intermediates which degrade Ump1 and Sts1 upon the arrival in the nucleus with the completion of proteasome maturation.\n\nWhen cells experience nutrient exhaustion or enter quiescence, a drastic change in proteasome localization is observed. In prolonged quiescence, proteasomes deplete from the nucleus and reside in motile and reversible PSGs in the cytoplasm (Laporte et al., 2008). Upon addition of glucose, cells receive the signal to resume proliferation, and PSGs dissolve rapidly, where proteasomes are relocated in the nucleus. How PSGs are organized is not understood. Premature PSG formation in proliferating cells was found to depend on vacuolar ATPases and linked premature PSG formation with disregulation of the intracellular pH. In view of that, PSGs could serve as storage depots for mature proteasomes in quiescence, to protect the proteasome from cellular stress and to be eliminated by autophagocytosis (Peters et al., 2013). The storage of proteasomes during quiescence would also alleviate energy-consuming synthesis of new proteasomes with cell proliferation (Laporte et al., 2008).\n\nThe formation of PSG-like structures is also observed by chemical inhibition of proteasomes in mammalian cells or temperature sensitive proteasome mutants in yeast, conditions which result in cell cycle arrest. In spite of the differences between chemically-induced cell cycle arrest and quiescence, inhibited proteasomes are sequestered into juxta nuclear quality control compartments (JUNQs), situated at the cytoplasmic side of the NE and behaving similar to PSGs (Kaganovich et al., 2008; Weberruss et al., 2013). When the cell cycle-arrested mutants were allowed to resume growth at permissive temperatures or upon withdrawal of proteasome inhibition, JUNQs were seen to dissolve like the PSG. In the context of these studies in cell-cycle arrested yeast and mammalian cells, poly-ubiquitylated proteins were found to be accumulated in the JUNQ as well, suggesting that the JUNQ provides a major place for proteolysis (Kaganovich et al., 2008). All studies on the JUNQ and PSGs agree that these enigmatic organelles serve protective functions. Their presence protects cells against proteo- and genotoxic stress and confers cell fitness during aging. Post-translation modifications such as N-acetylation also play a role in PSG organization, but their targets are unknown (Saunier et al., 2013; van Deventer et al., 2015; Weberruss et al., 2013).\n\nThough the CP and RP co-localize in the PSG, they seem to be loosely associated. Conflicting reports exist about the stability of RP-CP assemblies in lysates of quiescent cells (Bajorek et al., 2003; Hanna et al., 2012; Weberruss et al., 2013). The finding that RP-CP assemblies are less stable coincides with the decline in ATP during quiescence as well as the reduced proclivity of the proteasome to degrade poly-ubiquitinated substrate. Instead of an association of the CP with the RP, most CP is seen interacting with Blm10, a conserved 240 kDa HEAT repeat protein (Weberruss et al., 2013). Upon exit from quiescence, the PSGs rapidly clear and mature proteasomes are imported into the nucleus within a few minutes. The imported proteasomes must be matured and assembled, as time does not permit the new synthesis of precursor complexes (Laporte et al., 2008). Here, Blm10 plays an important role and represents the first characterized nuclear transporter which particularly facilitates nuclear import of mature CP (Figure 4). Quiescent blm10Δ mutants exhibit a significant delay in resuming cell growth due to the deficit in mature CP in the nucleus. Furthermore, Blm10 binds FG-Nups and GTP-bound Ran and dissociates from the CP upon interaction with RanGTP, suggesting that Blm10 shares functional similarities with Kap95, the classical importin β (Weberruss et al., 2013). Along this line, Blm10 belongs to the HEAT repeat family with α-solenoid fold, a structural feature shared by β karyopherins/importins (Huber & Groll, 2012). During cell proliferation, Blm10 is also expressed but to a much lesser extent (Weberruss et al., 2013). Only a minor fraction of the CP, pre-holo-CP and CP precursor complexes is associated with Blm10 in growing yeast. The Blm10-bound fraction significantly increases under geno-and proteotoxic stress suggesting a high demand for nuclear proteasomes under these growth conditions (Doherty et al., 2012; Fehlker et al., 2003; Lehmann et al., 2008). Since Blm10 associates with constitutively open or disordered CP α rings, Blm10 also plays a role in regulating α-ring gating during CP maturation (Lehmann et al., 2008). The wider α ring conformation of CP-precursor complexes seems to be preferentially bound to Blm10 and importin αβ by representing import intermediates. Thus, the Blm10-dependent import pathway complements the canonical nuclear import pathway.\n\nIn quiescent yeast cells mature CP is stored in reversible and motile granules in the cytoplasm, which rapidly clear with the resumption of growth. Blm10 mediates the nuclear import of mature CP.\n\nFor the RP, the import pathway upon exit from quiescence is yet to be solidified. A possible candidate for a RP-dedicated nuclear import receptor is Rpn2 which exhibits a similar α-solenoid fold as Blm10 and importin β, all of which belong to the family of HEAT-repeat proteins (Huber & Groll, 2012; Kajava, 2002).\n\n\nConclusions\n\nIn this review, we discussed the recent literature on the dynamics of the ubiquitin-proteasome system with a major focus on the proteasome. During cell proliferation a high traffic volume of proteasomes and proteasomal substrates arises between the cyto- and nucleoplasm. In cell-cycle arrested and quiescent cells, proteasomes exit the nucleus and accumulate with poly-ubiquitylated proteins in motile and reversible PSGs in the nuclear periphery. While the basic concepts of nuclear import of proteasomes during cell proliferation and upon exit from quiescence are understood, little is known about the nuclear export of proteasomes during the transition from proliferation to quiescence. We may wonder why proteasomes exit the nucleus during quiescence. Which kind of substrates will be available in the cytoplasm, once proteasomes are sequestered into the PSG? Possibly, PSG-resident proteasomes are starving for newly synthesized proteins which arise with the resumption of cell proliferation.\n\nThe dynamics of proteasomes and their substrates are fascinating and will inspire our discussions and experiments in the future.",
"appendix": "Author contributions\n\n\n\nM.C. prepared the first draft of 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\nThis work was supported by grants from NSERC (4422666-2011) awarded to M.C. and C.E. and from CIHR (325477) awarded to C.E.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nWe thank Zhu Chao (Jerry) Gu and Julianne Burcoglu for critical reading of the manuscript.\n\n\nReferences\n\nAitchison JD, Rout MP: The yeast nuclear pore complex and transport through it. Genetics. 2012; 190(3): 855–883. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmsterdam A, Pitzer F, Baumeister W: Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control. 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}
|
[
{
"id": "9632",
"date": "31 Jul 2015",
"name": "Petra Wendler",
"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 by Chowdhury and Enenkel nicely summarizes the current knowledge on the trafficking of proteasomes between cyto- and nucleoplasm. It's a very well written manuscript that gives a balanced and comprehensive view on localization studies on both, yeast and mammalian, proteasomes. I only have a few minor comments:The figures should be more informative. I would suggest to summarize all four figures in one or two figures, and to include/highlight all potential importers mentioned in the text (Kap95/Blm10/Rpn2). It would be tremendously helpful to add a figure summarizing the proteasomal localization data from yeast and mammalian cells. This figure should include which proteasomal subunits have been found where in the cell during which growth phase. It should also indicate the location of JUNQ and PSGs. Although the 20S chaperones Pba1/2 are mentioned, I am missing a discussion of their role in proteasomal localization. Binding of Pba1/2 blocs the access to the NLS of the alpha ring, as shown by Stadtmueller et al. (2012) and Kock et al. (2015). They thus seem to prevent nuclear import of proteasomal precursors. The authors incorrectly referred to \"cryo-EM structure analysis of Ump1-associated CP precursor complexes\" (Kock et al., 2015). This should be changed to \"EM structure analysis…\". Furthermore this point can be strengthened by also citing Wani et al. (2015).",
"responses": [
{
"c_id": "1547",
"date": "28 Sep 2015",
"name": "Cordula Enenkel",
"role": "Author Response",
"response": "Thanks for your comments which were very supportive of our review.According to your suggestions we renewed the figures and added more information about PSG and JUNQ. We also discussed possible functions of Pac1/2 in CP assembly and nuclear import.Although it would be very helpful to summarize proteasomal localization data from yeast and mammalian cells in a table, it is difficult to reconcile the data in the mammalian system. Proteasome localizations depend on the mammalian cell line, the growth conditions, the fixation conditions and antibodies used in these studies. We prefer to mentioned these inconsistencies in the text. In yeast, proteasome localizations are independent of the reporter subunit as monitored by direct and indirect fluorescence microscopy."
}
]
},
{
"id": "9988",
"date": "14 Aug 2015",
"name": "Philip Coffino",
"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\nScope of this review and its intended readership\n\nThe proteasome is a big and complex protein machine. This review centers on questions of its subcellular localization and where various steps in its assembly process happen. The basic question is inescapable and interesting: Proteins are made in the cytoplasm, but many or most proteasomes are found in the nucleus. How do large proteasome assemblies or subassemblies cross the nuclear membrane and where do they get put together (or sometimes taken apart)? Presenting these matters requires background in two areas: 1. The structure and assembly of proteasomes and 2. Nuclear pore trafficking. Both are well-explored subjects, and some background on these matters needs to be presented. However, most readers of this review will bring some knowledge of these subjects, and wish to learn here about the specifics of the core subject, proteasome intracellular trafficking. I and perhaps other readers would prefer more on this and less (if space limitations require) of general background information. Additionally, how about comparing proteasome nuclear traffic and that of ribosomes? Ribosomes and proteasomes are the big machines found ubiquitously in eukaryotes. What common or distinct themes govern their subcellular movement and assembly? A Technical QuestionMuch of the primary data supporting the conclusions described here depend on using various proteasome proteins fused to GFP in budding yeast. In interpreting these data it is critical to know whether or not such fusions alter kinetics or invoke alternate pathways compared to native controls. The paragraph headed Localization of the proteasome provides insufficient information related to this question. Minor considerationsIn the second paragraph of the Introduction it is said that “… long-lived proteins are degraded within the lysosome … short-lived proteins are degraded by proteasomes …” This seems too broad a claim- see Fuertes et al. (2003)[ref 1] for example- and should be modified or justified. In the third paragraph of the Introduction: “Natively-disordered proteins also qualify as proteasome substrates and are cleaved without post-translational ubiquitin modification.”, the reference which follows is about something quite different and another more relevant reference should be used. In the first paragraph under Discussion/analysis of the literature it is said that “At least four ubiquitin molecules … are required for a poly-ubiquitin chain to be recognized by the proteasome.” No longer regarded as so categorically true, but in any case reference should be made to Thrower et al. (2000)2 for this specific claim. Figure 3 conveys no information and can be dropped. Page 3, first full paragraph, right column: Change “in compliance with our finding” to “consistent with our finding”. In that same paragraph, perhaps the last sentence, “Third, when CP maturation is delayed by UMP1 deletion, all CP reporter proteins accumulate in the nucleus, although half of the CP is not fully matured and most likely exists as pre-holo-CP.” could be rewritten to clarify the intended meaning and how this supports the model of nuclear import of CP precursor complexes. Page 5, left column, second full paragraph: “ …where proteasomes are relocated in the nucleus.” change to : “ …and proteasomes are relocated to the nucleus.” Page 5 same paragraph: “ …and to be eliminated by autophagocytosis” change to “ …and elimination by autophagocytosis”.",
"responses": [
{
"c_id": "1548",
"date": "28 Sep 2015",
"name": "Cordula Enenkel",
"role": "Author Response",
"response": "Thanks for your constructive criticism of our review which addressed the intracellular dynamics of the ubiquitin-proteasome system. We reduced some background information, though nuclear transport of proteasomes will not be intelligible without profound knowledge of proteasome structure and assembly. We also omitted detailed informations about technical approaches, which were addressed in related reviews (Burcoglu et al., 2015). We answered your question with regard to direct and indirect localization studies using GFP technologies in living cells and antibodies in fixed cells. In yeast, the studies are highly consistent, while the data in mammalian cells are still difficult to reconcile.We also appreciate your idea to compare nuclear transport and assembly of ribosomes and proteasomes. A chapter was added to address this point. However, we apologize that we can only refer to reviews about ribosome assembly and transport, because the citation of the original work would be beyond the scope of our review.Your minor considerations were easily addressed."
}
]
},
{
"id": "9634",
"date": "20 Aug 2015",
"name": "Paula C. Ramos",
"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 dynamics of proteasome composition and subcellular distribution is an important subject with far-reaching implication. Recently, substantial progress has been made on this subject, while many open questions remain. It is therefore of considerable value that Chowdhury and Enenkel have summarized and commented the current literature on this subject in this nice and timely review. I have only a few comments that should be easy to address:In the second paragraph of the introduction it is stated that long-lived proteins are degraded in the lysosome, whereas short-lived proteins are degraded by the proteasome. While experimental data suggest that this is true for the bulk of the proteins in S. cerevisiae (e.g. Lee and Goldberg,1996), there is evidence from inhibitor studies with mammalian cells indicating that also long-lived proteins are among the proteasome substrates (Rock et al. 1994). Thus, while the statement in the introduction goes in the right direction, it would be better to soften it a bit. The sentence mentioning that Ump1 is a natively-disordered protein refers to papers from 2008. Reports that provided the first data supporting this notion, however, where published in 2013 by two independent groups (see below). Therefore, I would suggest to refer to these references in this contextRef.1: Sá-Moura B, S.A., Fraga J, Fernandes H, Abreu IA, Botelho HM, Gomes CH, Marques AJ, Dohmen RJ, Ramos PC, Macedo-Ribeiro S. Biochemical and Biophysical Characterization of Recombinant Yeast Proteasome Maturation Factor Ump1. Computational and Structural Biotechnology Journal 7, e201304006 (2013)Ref 2: Uekusa, Y. et al. Backbone ¹H, ¹³C and ¹⁵N assignments of yeast Ump1, an intrinsically disordered protein that functions as a proteasome assembly chaperone. Biomol NMR Assign (2013). The structural data on 15S precursor complex were not obtained via cryo-EM but rather by negative stain EM (reference Kock et al. 2015). When the authors discuss that the alpha ring of the 15S precursor complex is more relaxed and that would support the idea that the NLS would be more available for transportation from the cytoplasm into the nucleus, the authors have not considered or discussed the location of the chaperones Pba1-Pba2, that covers the surface corresponding to the pore of the 20S proteasome and thus would hide the NLS in this precursor form. I suggest to consider this point in the respective discussion and possibly to add Pba1-Pba2 to the 15S precursor cartoon shown in figure 1. It is mentioned (in the paragraph on “Nuclear import of proteasomes during proliferation”) that several groups found mislocalization of the CP to the cytoplasm, but no references are provided to support this notion. It does not become entirely clear whether the authors propose that PSG and JUNQ are different names for one and the same compartment. Some of the remarks seem to suggest that, but this question is not explicitly raised and addressed. Maybe the authors could clarify this issue and their view on it a bit more. While the authors describe PSGs as follows: “yeast proteasomes gather in proteasome storage granules (PSGs) at the nuclear envelope (NE) and endoplasmic reticulum (ER) membrane”, from which “with prolonged quiescence… pinch off the NE into the cytoplasm”, in the study that originally coined the term PSG (Laporte et al. 2008), they were defined as “motile cytoplasmic structures” that are “not associated with specific organelles or any detectable membrane”. In fact, these PSGs were distinguished from locations of proteasomes at or close to the nuclear periphery (e.g. Figure 2B of that study). It thus appears as if Chowdhury and Enenkel use a different or wider definition of PSGs than the one used originally by Laporte et al., which is relevant to the previous issue (6.). If so, it would be helpful to propose and explain the reasons for such an expansion of the definition of PSG. It is probably not ideal to describe PSGs as organelles. While the entire process of ubiquitylation is ATP-dependent, of the three types of enzymes involved, only E1 is an ATP-dependent enzyme, E2 and E3 are not.",
"responses": [
{
"c_id": "1546",
"date": "28 Sep 2015",
"name": "Cordula Enenkel",
"role": "Author Response",
"response": "Thanks for the helpful comments which were all considered in the revised version of our review.With regard to point 7, Laporte et al. (2008) report the depletion of nuclear proteasomes during the transition from logarithmic to stationary phase and their accumulation at the NE / ER in dots close to the nuclear periphery. After 4-5 days in stationary phase the dots are mainly found in the cytoplasm and coined PSG (Figure 1 of that work).I also clarified that PSG and JUNQ may be one and the same structure as shown by supplementary data in our work (Weberruss et al. 2013)."
}
]
}
] | 1
|
https://f1000research.com/articles/4-367
|
https://f1000research.com/articles/4-902/v1
|
25 Sep 15
|
{
"type": "Antibody Validation Article",
"title": "Reactivity of vertebrate-directed phospho-eEF2 antibody against the Caenorhabditis elegans orthologue phospho-EEF-2",
"authors": [
"Viviane Alves"
],
"abstract": "Eukaryotic protein translation is divided into three mains stages: initiation, elongation and termination. Regulation of this process occurs at the initiation and elongation step. eEF2 kinase phosphorylates eEF2 factor, blocking its ribosome interaction and thus translation elongation. This kinase activity can be detected by measuring eEF2 phosphorylation status. Here I show that vertebrate-specific antibody against phospho-eEF2 has excellent reactivity against C. elegans orthologue protein phospho-EEF-2.",
"keywords": [
"EFK-1",
"EEF-2",
"translation regulation",
"Caenorhabditis elegans"
],
"content": "Introduction\n\nProtein synthesis determines the cellular proteome and its regulation is pivotal to maintaining cellular homeostasis. This process is divided into three mains stages: initiation, elongation and termination1. The most studied step in eukaryotes is translation initiation and its regulation is critical to cell survival under stress conditions. Elongation regulation is also important in modulating translation. During this process, eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to the P site on the ribosome. The phosphorylation of eEF2 at threonine 56 by eEF2 kinase (eEF2K) inhibits its binding to ribosome and thus its activity2–5 in stress- or starvation-related conditions6–8. eEF2 kinase is an atypical α-kinase, normally dependent on Ca2+ ions and calmodulin, and apparently has only one substrate, the elongation factor eEF29. eEF2 phosphorylation by eEF2K results in a reduction in translation rate. C. elegans processes eEF2 and eEF2K orthologues, named as EEF-2 (94.8 kDa) and EFK-1 (87.8 kDa), respectively. As in other eukaryotes, the Thr56 residue and adjacent sequences in EEF-2 are conserved. The only described data about efk-1 shows that it is important to nutrient deprivation resistance10. My lab studies the role of efk-1 in various aspects of C. elegans survival and the simplest way to measure its activity is to determine the EEF-2 phosphorylation status. Here we show that phospho-eEF2 antibody from Cell Signaling Technology Inc., specific to vertebrates, has excellent reactivity against C. elegans phospho-EEF-2 and this property is preserved after freeze/thaw cycles.\n\n\nMaterial and methods\n\nThe standard C. elegans strain N2 Bristol (Caenorhabditis Genetic Center – CGC- wild isolate) and knockout strain efk-1 (CGC#RB2588), were maintained at 15°C and propagated on E. coli strain OP50 (CGC) using established procedures11,12. Gene knockout was verified by Polymerase Chain Reaction (PCR) using GoTaq® DNA polymerase (Promega) and specific primers to efk-1 (Fw- ATGACGATCGACACAACAAA/Rv- AGATCACCAACTCCTTGAATATCG) and act-1 (Fw-ACCATGTACCCAGGAATTGC/Rv- TGGAAGGTGGAGAGGGAAG) (Figure 1).\n\nAn efk-1 fragment (~790 bp) was amplified by PCR using specific primer pair to verify the knockout background (efk-1 KO) using N2 worms as positive control and act-1 amplification as load control.\n\nWorms (n= ~10) were collected in M9 buffer12 and washed three times by centrifugation at 1000 rpm for 1 min (RT) in M9 Buffer to remove bacterial cells. Worm pellets were heated (95°C) in 2X sodium dodecyl-sulphate (SDS) sample buffer (62.5mm Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 100mM DTT) for 10 min. Samples were loaded in a gradient gel (8–16% - GE Healthcare Lifesciences) using the ECL® gel box system (GE Healthcare Lifesciences) at 150V (as per manufacturer’s protocol). Separated proteins were transferred to an ECL®-Hybond (GE Healthcare Lifesciences) membrane using semi-dry transfer system (Bio-Rad). The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-Buffered Saline (TBS-50mM Tris, 150mM NaCl) containing 0.5% Tween 20 (TBS-T) for 1 h at room temperature, then incubated overnight at 4°C with primary monoclonal antibodies raised against vertebrate phospho-eEF2 (Thr56) (1:1000 in TBS plus 2,5% BSA – 94.8 kDa - Cell signaling Technology Inc. #2331 (Danves, MA – USA) – reactivity: human, mouse, rat, hamster, monkey, chicken) and a monoclonal anti-α-Tubulin produced in mouse (1:1000 – Sigma-Aldrich Co. LCC #T6047 - reactivity: human, chlamydomonas, African green monkey, chicken, mouse, bovine, rat, kangaroo rat, sea urchin) simultaneously. After washes, membrane was the incubated with secondary anti-rabbit/anti-mouse IgG horseradish peroxidase (HRP) antibody for 40 minutes at room temperature, subject to new washes and incubated with anti-mouse IgG HRP antibody (HRP – 1:2000 – Sigma-Aldrich Co. LCC). Signal detection was performed with Luminata® forte HRP Western substrate (Millipore). Mixed primary antibodies were stored at -20°C until needed and thawed at room temperature when necessary. Reagents are listed in Table 1 and Table 2 and the WB protocol is given in Table 3.\n\n\nResults\n\nUsing the described protocol, I could specifically detect the phosphorylated form of EEF-2, eEF-2 orthologue in C elegans since in efk-1 knockout worms, used as a negative control of efk-1 activity, there is no equivalent signal in western blots (Figure 2 and Figure 3). Detection of α-tubulin was used as a western blot load control. Some differences can be observed in α-tubulin detection when comparing lanes (Figure 2 and Figure 3), despite the use of approximately the same number of nematodes. This can be explained by the fact that some worms remain attached to the pipette tip after washes, and this effect can be circumvented adding 0.01% Triton X-100 (Sigma Aldrich) to C. elegans wash buffer (M9 buffer). In addition, both antibodies (directed to target and load control) can be used and detected simultaneously, reducing analysis time (Figure 3).\n\nWestern blot showing that phospho-eEF2 antibody recognizes C. elegans (N2 Bristol) phospho-EEF-2, since protein detection signal is absent in efk-1 knockout worms. A) phospho-EEF-2 detection at the first time antibody dilution in TBS-T-BSA. B) phospho-EEF-2 detection after five freeze/thaw cycles of the antibody in TBS-T-BSA. SE- short exposure (5 sec); LE-long exposure (1 min).\n\nWestern blot showing that phospho-eEF2 and α-tubulin antibodies can be incubated and developed simultaneously. efk-1 knockout worms were used as negative control to phospho-EEF-2 detection showed in wild-type worms (N2). A) Western blot short exposure (1 min). B) Western blot short exposure (3 min). Indicated molecular weight based on Precision Plus Protein™ Dual Color Standard (Bio-Rad).\n\nEEF-2 phosphorylation is not observed in efk-1 knockout C. elegans, indicating that EFK-1 is the sole EEF-2 kinase in my tested conditions. Surprisingly, primary antibodies diluted in TBS-T-BSA (either phospho-eEF2 and α-tubulin, together or individual dilutions), stored at -20°C, can be reutilized at least five times (by thawing at room temperature) without losing specificity/reactivity (Figure 2B).\n\n\nConclusion\n\nIn this work, I show that vertebrate-directed phospho-eEF2 antibody specifically recognizes C. elegans orthologue phospho-EEF-2. This finding is very useful to those who work with translation elongation regulation using C. elegans as a model and I recommend this antibody to detect EFK-1 activity in C. elegans.",
"appendix": "Competing interests\n\n\n\nThe author declares no competing interests.\n\n\nGrant information\n\nThis work was supported by CNPq – Centro Nacional de Desenvolvimento Cientifico e Tecnologico, Capes – Coordenaçao de Aperfeiçoamento de Pessoal de nível Superior, and PRPq-UFMG – Pro-reitoria de Pesquisa da Universidade Federal de Minas Gerais.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe author thanks the Caenorhabditis genetic stock center (CGC) funded by NIH Office of Research Infrastructure Programs (P40 OD010440) for the C. elegans strains and Dr. Beatriz A. Castilho for the phospho-eEF2 antibody.\n\n\nReferences\n\nMerrick WC: Eukaryotic protein synthesis: still a mystery. J Biol Chem. 2010; 285(28): 21197–201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNairn AC, Palfrey HC: Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2. J Biol Chem. 1987; 262(36): 17299–303. PubMed Abstract\n\nRyazanov AG, Shestakova EA, Natapov PG: Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translation. Nature. 1988; 334(6178): 170–3. PubMed Abstract | Publisher Full Text\n\nNairn AC, Hemmings HC Jr, Greengard P: Protein kinases in the brain. Annu Rev Biochem. 1985; 54: 931–76. PubMed Abstract | Publisher Full Text\n\nCarlberg U, Nilsson A, Nygård O: Functional properties of phosphorylated elongation factor 2. Eur J Biochem. 1990; 191(3): 639–45. PubMed Abstract | Publisher Full Text\n\nWang X, Regufe da Mota S, Liu R, et al.: Eukaryotic elongation factor 2 kinase activity is controlled by multiple inputs from oncogenic signaling. Mol Cell Biol. 2014; 34(22): 4088–103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnight JR, Bastide A, Roobol A, et al.: Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation. Biochem J. 2015; 465(2): 227–38. PubMed Abstract | Publisher Full Text\n\nMoore CE, Mikolajek H, Regufe da Mota S, et al.: Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia. Mol Cell Biol. 2015; 35(10): 1788–804. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKenney JW, Moore CE, Wang X, et al.: Eukaryotic elongation factor 2 kinase, an unusual enzyme with multiple roles. Adv Biol Regul. 2014; 55: 15–27. PubMed Abstract | Publisher Full Text\n\nLeprivier G, Remke M, Rotblat B, et al.: The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation. Cell. 2013; 153(5): 1064–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrenner S: The genetics of Caenorhabditis elegans. Genetics. 1974; 77(1): 71–94. PubMed Abstract | Free Full Text\n\nStiernagle T: Maintenance of C. elegans. WormBook. 2006; 1–11. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10543",
"date": "05 Oct 2015",
"name": "Richard Silva",
"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\nViviane Alves shows that a phospho antibody raised against mammalian eEF2 can specifically detect the phosphorylation status of its counterpart in C. elegans, EEF-2. This is conceptually a very important validation in the field, especially because antibodies produced to detect specific phosphorylation sites in mammalian cells very often do not cross-react with non-mammalian organisms. The challenge in making use of these antibodies is that successful detection may depend on the specificity and affinity of the antibody for the phospho-protein of interest and on the amino acids sequences surrounding the specific phospho site. To make matters worse, the sequence and size of phosphopeptides employed to raise these antibodies are not always provided by companies (including the anti phospho eEF2K antibody validated in this manuscript), making it hard to in-silico predict their cross reactivity before acquisition. Therefore, this manuscript will be highly valuable to prevent researchers interested in studying the regulation of elongation during protein synthesis in C. elegans to go through the laborious validation to detect the inhibitory activity of EEF2 in C. elegans under different cues. The author also provides the conditions and a very neat protocol for the simultaneous detection of phospho EEF2 with a loading control that will prove quite helpful for researchers, saving time and resources. The manuscript is clearly written and easy to read. The experiments are generally carefully performed with appropriate controls. Only minor suggestions and changes should be made (See below) Consider revising or changing the following points (minor suggestions)Inhibitory phosphorylation of eEF2K is elicited in response to different cues. Therefore, it would be really informative to emphasize that the phosphorylation signal observed in figures 2 and 3 is basal. Maybe this could be included within the results section. In addition, basal phosphorylation signal is quite high and possibly out of linear range, even with short exposures, which could be troublesome for those aiming to detect subtle differences in phosphorylation among different samples submitted or not to different stress conditions. Perhaps, it would be helpful to add a suggestion in the manuscript for the use of a higher anti-phospho EF2K dilution or alternatively, to make use of a weaker chemiluminescent substrate solution. Conclusion could emphasize the important validation that the load control (tubulin) can be specifically and simultaneously probed in the same membrane. The confirmation of the knockout for efk-1KO C. elegans strains is important to prove the specificity of the antibody against EEF2. However, the PCR result (figure 1) was not addressed in the results section, which starts with figure 2. Consider including a simple paragraph with this result in this section. Although the author made use of a semi-dry transfer apparatus and membranes are only rinsed in the buffer when this system is employed, it has been previously shown that the composition of buffers may influence the semi-dry transfer efficiency (www.nature.com/protocolexchange/protocols/2925). Therefore, the author could consider including the recipe of the buffer in table 3. Maybe I missed the point, but why were membranes first incubated with both anti-rabbit/anti mouse IgG-HRP and then incubated a second time with anti IgG-HRP against mouse only? (See material and methods section). In figure 3B, substitute short for long In the sentences:C. elegans processes eEF2 and eEF2K orthologues = substitute processes for possess.After washes, membrane was the incubated with secondary” = substitute the for then.Used as a negative control of efk-1 activity = Change efk-1 for EF2K (the activity refers to the enzyme not the gene itself).Antibody for 40 minutes at room temperature, subject to new washes and incubated = substitute subject for subjected.Can be circumvented adding = substitute with: can be circumvented by addingMembrane using semi-dry transfer system = substitute with: membrane using a semi-dry transfer system",
"responses": []
},
{
"id": "10541",
"date": "05 Dec 2016",
"name": "Kevin N. Dalby",
"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 the use of a commercial vertebrate-specific antibody available from Cell Signaling Technologies to detect the site-specific (Thr56) phosphorylated form of the C. elegans ortholog of elongation factor 2 in lysates generated from the worms by western blotting.\nThe specificity of the antibody is demonstrated using efk-1 knock out worms.\n\nImportantly, it is also established that no other kinase in C. elegans phosphorylates eEF2 on Thr56, thus, its phosphorylation can be used as a proxy for elk-1 (the eEF2K ortholog) activity.\n\nIt should be noted that this is a proxy however and does not directly measure elfk-1 activity. Changes in phospho-eEF2 can come about because of changes in phosphatase levels.\nThe technical aspects are well described.\n\nThis appears to be a very useful tool for analyzing phospho-eEF2 levels in C. elegans.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-902
|
https://f1000research.com/articles/4-901/v1
|
25 Sep 15
|
{
"type": "Research Note",
"title": "Ask a clearer question, get a better answer.",
"authors": [
"Dominic Henri",
"Lesley Morrell",
"Graham Scott",
"Lesley Morrell",
"Graham Scott"
],
"abstract": "Many undergraduate students struggle to engage with higher order skills such as evaluation and synthesis in written assignments, either because they do not understand that these are the aim of written assessment or because these critical thinking skills require more effort than writing a descriptive essay. Here, we report that students who attended a freely available workshop, in which they were coached to pose a question in the title of their assignment and then use their essay to answer that question, obtained higher marks for their essay than those who did not attend. We demonstrate that this is not a result of latent academic ability amongst students who chose to attend our workshops and suggest this increase in marks was a result of greater engagement with ‘critical thinking’ skills, which are essential for upper 2:1 and 1st class grades. The tutoring method we used holds two particular advantages: First, we allow students to pick their own topics of interest, which increases ownership of learning, which is associated with motivation and engagement in ‘difficult’ tasks. Second, this method integrates the development of ‘inquisitiveness’ and critical thinking into subject specific learning, which is thought to be more productive than trying to develop these skills in isolation.",
"keywords": [
"assessment",
"essay",
"critical thinking",
"critical writing",
"critical synthesis",
"inquisitiveness"
],
"content": "Introduction\n\nSupporting the development of critical thinking skills in students can be considered to be one of the key goals of most higher education institutions (ten Dam & Volman, 2004). Critical thinking skills such as analysis, evaluation and synthesis represent the highest levels of learning and literacy capabilities, and are highly sought after by employers (CBI, 2009; Krathwohl, 2002; Miller & Tanner, 2015). Despite the focus on teaching critical thinking skills at university, only ~2/3 of UK graduates (lower than the global average) were capable of exhibiting them during a recent literacy skills survey, which is disappointing given the strong correlation between high level skills and employment among graduates (OECD, 2013; OECD, 2015). This paper evaluates a simple method of encouraging students to engage with these higher level skills in their written assessments.\n\nChanock (2010) outlines five goals that an essay should fulfil. The first is “presenting a question/problem to the reader” and forms the focus of this study. In our experience, students who fail to achieve high grades on written assignments do so because they write descriptive essays lacking a question or problem to solve; i.e they do not understand goal one (Cottrell, 2011). By defending a position or hypothesis using understanding drawn from wider literature, students can provide evidence of high-level literacy and critical thinking (Kellogg & Raulerson, 2007; Miller & Tanner, 2015). Previous studies support the idea that encouraging questioning behaviour promotes the exhibition of critical thinking by students at a range of levels (Commeyras & Summer, 1998; Keeley et al., 1998; Tsui, 2002). In fact, lack of practice performing critical thinking is thought to be a particularly important barrier to the development of higher-level literacy skills (Cottrell, 2011; Kellogg & Raulerson, 2007). We hypothesise that by making the first objective of an essay more obvious to students, and by encouraging them to approach written assignments as questions that need to be answered, students are more likely engage with higher level learning outcomes (Krathwohl, 2002).\n\nA second cited barrier to the development of higher-level literacy skills is reticence on the part of the student, as essays containing evaluation and synthesis are more difficult to write than descriptive essays (Cottrell, 2011; Keeley et al., 1995). An important aspect of our method involves allowing the students to choose what question they are most interested in within the defined subject area. In this study each student was asked to explore a broader concept associated with a specific animal behaviour they chose to study earlier in the year (for details see Methods section). We believe that the sense of ownership of the task could help to improve engagement with ‘difficult’, higher-learning outcomes, as motivation to engage with studying is thought to be positively associated with personal interest in the topic (Pintrich, 2003).\n\nWe propose that coaching students to pose a question in their essay title can integrate ‘inquisitiveness’ development into written assessments. Integration of the development of these skills into the context of the course has been argued as being more effective than trying to teach these skills in a separate course (ten Dam & Volman, 2004; Wingate, 2006). We suggest that students who start by posing a question in the title are more likely to understand the first of Chanock’s (2010) aims of an essay as well as being more likely to exhibit higher-level literacy skills throughout their writing. Thus, we hypothesise that students who pose a question in their title will obtain higher assessment scores than those who do not, as evidence of higher-level skills are essential in obtaining higher marks.\n\n\nMethods\n\nProject participants were students enrolled on a second year undergraduate module Behavioural Ecology (UK level 5, 20 credits). We believe this group of 55 individuals to be typical of the wider population of UK undergraduate students enrolled on Honours Degree Programs in the Biological Sciences. 23 of the students were male and 32 were female.\n\nAssessment of the module was by an end of module written examination (50%) and summative coursework (50%). This coursework comprised three tasks (A, B and C) worth 10%, 10% and 30% respectively. Task A required pairs of students to work together to find a short (3 minute) video clip of animals performing a behaviour that interested them and to complete a written assessment in the form of briefing notes for a film crew interested in recreating the video as part of a wildlife documentary. This task encourages observation and description. Task B required individual students to self-assess task A and reflect (in writing) upon their use of the assessment criteria in doing so. This task encourages students to think about the assessment criteria and the way in which they are applied. Task C required students to write a detailed essay exploring the underlying principles and wider context of the behaviour chosen for assessment A. The notes provided to students to explain these tasks are available as supplementary material (Appendix 1).\n\nThis project investigates the impact of an optional workshop-based intervention that took place after the students had received grades and feedback on assessments A & B and before they completed assessment C. All students were invited to attend a workshop led by DH and LM as preparation for assessment C with a focus on improving essay writing skills. All students were provided equal opportunity to attend; multiple timeslots were available for students with other commitments. At the workshop, DH & LM explained the function of a good essay in that it should outline a problem that needs to be solved, then present and evaluate the various solutions using wider literature. We suggested that in order to help the students do this they should present a question that needs answering as the essay title, and then use the essay to answer that question with reference to the broader literature (see Appendix 2 for essay titles). We then helped students create a relevant question to ask, suggesting they avoid descriptive ‘how’ questions, and focused on evaluative ‘why’ or ‘to what degree’ type questions.\n\nThis activity was not conceived as a research project and because attendance at the workshop was optional student attendance was not monitored. For the purpose of this study we assumed that students who posed a question in the title of their essay had attended the workshop and understood the underlying concepts of the workshop, and this has been used as the independent factor in our analysis. We acknowledge that this lack of certainty in the allocation of students to the did/did not attend category does need to be borne in mind when interpreting our results. Another possible confounding factor is that voluntary workshop attendance may be skewed towards individuals who are more engaged or motivated with the module; and these individuals are more likely to obtain higher grades because of this higher engagement with the module content (Pintrich, 2003). We have controlled for inherent capability or engagement of the student in this study by including the previous mark on Assessment A of the student as an independent factor in our statistical analyses (see Statistics). Students’ essays were marked by an assessor who was not involved in the delivery of the module or aware of the purpose of the workshops but who does have the relevant disciplinary expertise (GS), so as to not influence student grades.\n\nEthical approval for publication of our study was obtained from the University of Hull, SoBBEs ethics board (Code H038). As the significance of the results presented here was only noted after marking had taken place, it was not possible to obtain student approval. However, students cannot be identified individually from the study results or data set, which was deemed sufficient by the ethics board.\n\nA generalised linear mixed effect model (GLMM) was used to test for an effect of posing a question as the essay title on the percentage mark awarded to the essay. The student’s mark on assignment A was included as a second independent variable to control for the effect of inherent capability. As assignment A was written in pairs, the pair groups were included as a random factor (random intercepts) to control for non-independence of the marks. An observation level, random factor was included to account for overdispersion (Harrison, 2014). As the dependent factor was a percentage, the GLMM was run with a Binomial error structure. All statistics were performed in R using the glmer function in the lme4 package (Bates, 2010) of R v3.1 (R Development Core Team, 2014). The Minimum Adequate Model was established via log-likelihood ratio comparisons using Maximum Likelihood approximation, for which X2 results indicating significance are reported (Bates, 2010).\n\n\nResults\n\nEssays with a question in the title scored significantly higher than those without (X21= 4.62, P= 0.03; Figure 1 & Table 1). There was no significant effect of score of previous assignment (X21= 3.02, P= 0.10), or interaction between the two independent variables (question in title:previous score, X21= 0.81, P= 0.36).\n\n\nDiscussion & Conclusion\n\nOur results support our original hypothesis that students who posed a question in the title of their essay would obtain higher grades than those who did not. We suggest that this is because the process of coaching students to use questions to think ‘inquisitively’ improves the likelihood they will engage with critical thinking skills, such as analysis, evaluation and synthesis. Our results support this because evidence of these skills is necessary for work to be awarded 1st class grades (70% or higher), and we note the much higher proportion of students posing a question who obtained a 1st class grade (44% [asked a question] vs. 22% [did not ask a question]). Given the importance of critical thinking skills for obtaining higher degree classifications, better literacy scores and gaining employment following graduation, we suggest this outlook may be added to the methods of developing student essay skills (CBI, 2009; OECD, 2013; OECD, 2015). Our method is particularly advantageous because it can be easily integrated into the curricula; as opposed to needing to be taught separately (ten Dam & Volman, 2004; Wingate, 2006). Furthermore, the workshop method in our study focused on helping students develop their own questions to answer, encouraging student ownership and motivation in order to overcome any reticence to engage in ‘difficult’ higher-level literacy skills (Cottrell, 2011; Keeley et al., 1995).\n\nIt is important to state that we believe the whole process of teaching students to think in terms of questions/problems and how to answer them is important; as opposed to merely the act of placing a question in the title. We also do not suggest that this is a blanket method of encouraging students to develop high-level literacy/critical thinking skills; evidently, some of our students who attended the workshop and used the method did poorly (hence did not grasp the underlying concept) and other students did well despite not using the method detailed herein (Figure 1). This is to be expected where students construct their knowledge base and its application individually and thus respond differently to instruction based on their prior experiences and learning preferences, and does not undermine its validity as a potential tool for broader teaching strategies (ten Dam & Volman, 2004). We concede that a more extensive study, including more students across multiple assessments, is required to resolutely confirm the trends found herein. Further work should focus on helping students to distinguish between descriptive ‘how’ questions and evaluative ‘why’ questions to see if this further improve the efficacy of the method.",
"appendix": "Author contributions\n\n\n\nDH & LM taught the workshops and performed the statistical analysis. GS performed the marking. All authors equally shared the conceptualisation and development of the project. DH wrote the manuscript with significant input from LM & GS.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work was unfunded and produced while employed by the University of Hull.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nAppendix 1. Assessment notes provided to students.\n\nAssessment title: Assessment A: Documentary Briefing Notes\n\nWeighting: 10%\n\nSubmission: electronic submission only\n\nWord count (or equivalent): 1000\n\nAssessment overview:\n\nYour task is to work in pairs to produce a briefing paper for a wildlife documentary maker who is making a film about behavioural ecology. You should think of your work as satisfying two needs; it should provide all of the information needed by the film maker; and, it should enable another film crew to replicate the filming should they need to at a future time.\n\n1. Locate a short piece of video footage (<3 min) that clearly shows wild animals carrying out a particular behaviour. Your video clip should show a single behaviour, not a series of different behaviours performed by the same animal. In choosing your clip, you should think particularly how you would develop your work on the topic for Assessment C.\n\na. Background: Provide a link to the footage (if it is a short section of a longer piece remember to explain where the section of interest starts/stops). Write a brief description of the species involved, geographical location and the best time/location to film the behaviour in question\n\nb. Description: Write a description of the footage - what does the viewer see? This section should describe what is going on (“the squirrel is burying nuts under dry leaves”), not explain what is happening.\n\nc. Explanation: Here, you should explain what the behaviour is and why the animal is carrying it out (“the squirrel is caching nuts ready for the winter”, and then explain why the squirrel does this)\n\nd. Wider concepts: Summarise the wider scientific principles underlying the behaviour that you will write about in Assessment C. Think about the general area in which the behaviour lies, that could form the focus of Assessment C. Examples might include optimal foraging, or intersexual selection, or animal migration. This section is not about identifying as many concepts as you can, but about highlighting the topic that you will develop further in Assessment C.\n\nRemember to carefully read the assessment criteria for the task – they provide you with a lot of potentially useful guidance.\n\nAssessment A marking criteria\n\nAssessment title: Assessment B-self assessment of the briefing notes\n\nWeighting: 10%\n\nSubmission: electronic submission only\n\nWord count (or equivalent): 500\n\nAssessment overview: You should write a short individual reflection that explains to the assessor what mark you think your briefing notes (assessment a) will be awarded:\n\n1. State what grade (% mark) you think the work is worth (refer to the assessment criteria for this task).\n\n2. Write a short justification of that mark. In doing so it is important that you refer to the assessment criteria for assessment A and that you provide evidence to support your claim, (you might for example include extracts from the submitted piece as evidence, but pasting in large chunks of your assignment A is a waste of words). In particular, you should focus on the differences between the classification boundaries and explain why your assignment falls into a particular category. Use the assessment criteria for assessment B to guide you in the types of areas you should include in your self-assessment.\n\nAssessment B marking criteria\n\nAssessment title: Assessment C\n\nWeighting: 30%\n\nSubmission: electronic submission only\n\nWord count (or equivalent): 2000\n\nAssessment overview: Individually write a detailed essay on the scientific principles underlying the behaviour you chose to write about in assessments A and B. You should not simply expand the work you have already done, but focus on explaining the wider scientific concept underlying the behaviour you chose. While your essay should not focus on the particular species in your video clip, you may of course use it as an example within the essay.\n\nWe expect you to make extensive use of the published literature (preferably articles from peer reviewed journals) in writing this essay.\n\nAssessment C marking criteria\n\nAppendix 2. Data table used to explore the primary hypothesis.\n\n\nReferences\n\nBates DM: lme4: Mixed-effects modeling with R. Springer. 2010. Reference Source\n\nCBI [Confederation of British Industry]: Future Fit: Preparing graduates for the world of work. Beacon Press, London. 2009. Reference Source\n\nChanock K: The right to reticence. Teaching in Higher Education. 2010; 15(5): 543–552. Publisher Full Text\n\nCommeyras M, Summer G: Literature questions children want to discuss: What teachers and students learned in a second-grade classroom. Elem Sch J. 1998; 99(2): 129–152. Publisher Full Text\n\nCottrell S: Critical thinking skills: Developing effective analysis and argument. Palgrave Macmillan, London. 2011. Reference Source\n\nHarrison XA: Using observation-level random effects to model overdispersion in count data in ecology and evolution. PeerJ. 2014; 2: e616. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKeeley SM, Shemberg KM, Cowell BS, et al.: Coping with student resistance to critical thinking: What the psychotherapy literature can tell us. College Teaching. 1995; 43(4): 140–145. Publisher Full Text\n\nKeeley SM, Ali R, Gebing T: Beyond the sponge model: Encouraging students’ questioning skills in abnormal psychology. Teach Psychol. 1998; 25(4): 270–274. Publisher Full Text\n\nKellogg RT, Raulerson BA 3rd: Improving the writing skills of college students. Psychon Bull Rev. 2007; 14(2): 237–242. PubMed Abstract | Publisher Full Text\n\nKrathwohl DR: A revision of bloom’s taxonomy: an Overview. Theor Pract. 2002; 41(4): 212–218. Publisher Full Text\n\nMiller S, Tanner KD: A portal into biology education: an annotated list of commonly encountered terms. CBE Life Sci Educ. 2015; 14(2): 14:fe2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOECD [Organisation for Economic Cooperation and Development]: OECD Skills Outlook 2013. OECD Publishing, Paris. 2013. Publisher Full Text\n\nOECD [Organisation for Economic Cooperation and Development]: Graph 2.3. The effect of education and literacy proficiency on labour market participation: Adjusted odds ratios showing the effect of education and literacy on the likelihood of participating in the labour market among adults not in formal education, 2012. In OECD Skills Outlook 2015. OECD Publishing, Paris. 2015. Publisher Full Text\n\nPintrich PR: A motivational science perspective on the role of student motivation in learning and teaching contexts. J Educ Psychol. 2003; 95(4): 667–686. Publisher Full Text\n\nten Dam G, Volman M: Critical thinking as a citizenship competence: teaching strategies. Learn Instr. 2004; 14(4): 359–379. Publisher Full Text\n\nTsui L: Fostering critical thinking through effective pedagogy: Evidence from four institutional case studies. J High Educ. 2002; 73(6): 740–763. Publisher Full Text\n\nWingate U: Doing away with ‘study skills’. Teaching in Higher Education. 2006; 11(4): 457–469. Publisher Full Text"
}
|
[
{
"id": "10704",
"date": "07 Oct 2015",
"name": "Rachel Stubbington",
"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 considers a workshop conducted to support students preparing a summative written assignment, which had the unplanned benefit of improving critical thinking skills, as evidenced by higher assignment grades. The development of higher-order thinking skills in undergraduate students is of considerable relevance in relation to the employment prospects of the increasingly diverse student body; this study is therefore a useful contribution to the literature. I have some suggestions below, consideration of which may improve the paper. Hypothesis 1. There is overlap between hypotheses 1 (“…by encouraging [students] to approach written assignments as questions that need to be answered, students are more likely to engage with higher level learning outcomes”) and 2 (“…students who pose a question in their title will obtain higher marks, as evidence of higher-level skills”). Hypothesis 1 is not tested by any analyses, and I can’t think of an appropriate approach to do. Since demonstrating higher-order learning outcomes and gaining higher marks are so closely related, perhaps no further analysis is warranted and H1 should be lost. If H1 is retained, the analyses and the Results will need expansion. Learning outcomes. Achieving higher-level learning outcomes by demonstrating critical thinking skills is at the heart of this paper. However, whilst the Assessment C marking criteria allude to higher-level learning outcomes (e.g. “shows convincing evidence of understanding” compared to “fails to show evidence of understanding”), no specific words (e.g. evaluate compared to describe) are used. Therefore, evidence that higher learning outcomes have been achieved could be more explicit; perhaps some descriptive text (including essay extracts) could be added to the Results to provide qualitative evidence that higher level learning outcomes were met. Participants. More detail of the cohort characteristics would be useful to justify the “We believe this group” statement, in particular in relation to ethnicity. At my own institution, BME groups are poorly represented on the Ecology pathway of BSc (H) Biological Sciences (who would take a Behavioural Ecology module) compared to, for example, those studying Biomedical Sciences. In the context of ‘Narrowing the Gap’ initiatives, the ethnic composition of the cohort could usefully be clarified. Methods. The Methods are clear and supplementary material is very useful. More detail could be provided on some aspects to meet the journal guideline that the work should be repeatable by others. For example, clarify how the “function of a good essay” was “explained” (could further supplementary material usefully be provided)? The statistical approach used is robust - would a brief description of / introduction to GLMMs (or perhaps a reference to further information) be useful? Expanding on my ‘Participants’ comments, consider whether analyses exploring differences between cohort sub-groups (e.g. ethnicity groups) would be useful Results. A few basics need adding e.g. how many students were in the “with” and “without” groups. Also, as mentioned in the Learning outcomes section above, consider adding qualitative evidence that higher level learning outcomes were met. Finally, the information presented in Table 1 is very limited and replicates Figure 1; I suggest Table 1 be lost and the mean / SD included in the text. Discussion. As well as recommendations for future research, the Discussion could usefully clarify how you might adapt the seminar in future, to maximize benefits and reduce the proportion of students who do “not grasp the underlying concept”. Study limitations. The lack of certainty about which students did / did not attend the workshop is unfortunate, but the limitation is recognised and this limitation does not undermine the usefulness of the study. Similarly, the lack of replication does reduce confidence in the results, but recommendations for a more extensive study are appropriate. Minor points I cannot spot <8 key words, have these been provided?“presenting a question/problem to the reader” isn’t a direct quote from Chanock, so I’m not sure quote marks are suitable.Use the past tense to describe work done: allowed in the Abstract; investigated in the Methods.Information about all methods is under the “Participants” subheading.It’s odd to specify “examination… summative coursework” – both assessments are summative.Avoid undefined abbreviations e.g. “SoBBEs”The semi-colon is used incorrectly e.g. “module; and these” “important; as opposed”; the clause after the semi-colon must be capable of standing alone as an independent sentence.Ensure phrases are sufficiently specific e.g. “to help the students do this” is a bit vague.Look for opportunities to be more concise e.g. remove “in order to” / “it is important to state that”.The ampersand is informal so should be replaced with and (e.g. “DH & LM”)Hyphenate adjectival phrases e.g. subject-specific.",
"responses": [
{
"c_id": "1650",
"date": "28 Oct 2015",
"name": "Dominic Henri",
"role": "Author Response",
"response": "Thank you very much, some really helpful points here. We will create a full response when we hear back from the third referee."
}
]
},
{
"id": "10553",
"date": "13 Oct 2015",
"name": "Kay Yeoman",
"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\nCritical thinking is certainly a key skill which we would like our students to develop during their time in Higher Education. The paper points to the OECD studies showing that students tend to lack these critical thinking skills. I think it would also be good to highlight the evidence for this in the education literature.The paper looks at an intervention through an optional workshop to coach students to think about framing essay titles into questions. The hypothesis being that those who formulate questions achieve a higher mark when compared against those who did not.In the methods it would be good to clarify if the gender ratio of that particular module is typical of the wider cohort, it is suggested, but not implicitly stated. It would also be good to know if there were any mature students in this cohort (maturity being another confounding factor). The workshop was optional, giving rise to a group without the intervention, and a group with the intervention (control). It would be good to have a table here showing the numbers in each group, and the gender and age split. The authors suggest a confounding factor in that more motivated students (possibly achieving higher grades) were more likely to opt in. It would be interesting to look at gender and maturity as factors as well within the self-selecting group vs control group. I would like to see more information about the intervention workshop, how was it run? How long was it? Did the workshop only cover the framing of titles as questions, or did it also cover the structured argument required to answer the question? Did students who attend this follow up conversations within the workshop with further questions? If so, how would this have influenced their final grade? Do you have any qualitative data from student feedback to show you what was valued within the workshop? The paper states that the independent assessor who marked the essays was unaware of the study being conducted. Were the essay titles removed prior to marking? I think it would be good for the discussion to examine the recent rise in the number of A-levels students taking the extended project qualification (approx. 33,000-data can be obtained from the Joint Councils for Qualifications). This dissertation requires students to formulate a research question, and then to investigate and critically analyse sources. This is becoming an increasingly popular and important qualification, and is set to rise with the removal of the AS examination. It would be good for the authors to discuss what future impact this might have on the quality of critical thinking of our students as they enter into higher education. I think this paper hints at straight forward intervention within the curriculum which could help develop critical thinking skills, but I think the needs more evidence over at least another cohort of students on the same module before the influence of the workshop can really be shown on student attainment.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-901
|
https://f1000research.com/articles/4-140/v1
|
04 Jun 15
|
{
"type": "Method Article",
"title": "Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP",
"authors": [
"David Pellerin",
"Hugo Gagnon",
"Jean Dubé",
"Francois Corbin",
"David Pellerin",
"Hugo Gagnon",
"Jean Dubé"
],
"abstract": "Sample preparation is a crucial step for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Sodium dodecyl sulfate (SDS) is a powerful denaturing detergent that allows for long-term preservation of protein integrity. However, as it inhibits trypsin and interferes with LC-MS/MS analyses, it must be removed from samples prior to these experiments. The Filter-Aided Sample Preparation (FASP) method is actually one of the preferred and simplest methods for such purpose. Nonetheless, there exist great disparities in the quality of outcomes when comparing FASP to other protocols depending on the authors, and recent reports have pointed to concerns regarding its depth of proteome coverage. To address these issues, we propose an Amicon-adapted in-solution-based enhanced FASP (eFASP) approach that relies on current best practices in comprehensive proteomics sample preparation. Human megakaryoblastic leukaemia cancer cells’ protein extracts were treated in parallel with both Amicon-adapted eFASP and FASP, quantified for remaining SDS and then analyzed with a 1-hr gradient LC-MS/MS run. The Amicon-adapted eFASP utilizes a passivated low molecular weight cut-off Amicon filter, and incorporates a cleaning step with a high-content deoxycholate buffer and a ‘one-step-two-enzymes’ trypsin/Lys-C in-solution digestion. Amicon-adapted eFASP was found more reproducible and deepened proteome coverage, especially for membrane proteins. As compared to FASP, Amicon-adapted eFASP removed much of SDS from high-protein samples and reached a notable depth of proteome coverage with nearly 1,700 proteins identified in a 1 hr LC-MS/MS single-run analysis without prior fractionation. Amicon-adapted eFASP can therefore be regarded as a simple and reliable sample preparation approach for comprehensive proteomics.",
"keywords": [
"bottom-up proteomics",
"Amicon",
"eFASP/FASP",
"biomarker",
"MEG-01",
"sample preparation",
"tandem mass spectrometry",
"in-solution digestion"
],
"content": "Introduction\n\nProteomics plays an increasingly greater role in diverse clinical settings provided that proteins give an integrative picture of patients’ phenotype and an accurate representation of changes in the status of an organism1. As such, intense efforts are now directed toward proteomics-based biomarker discovery, disease screening, and medical diagnosis2. Accuracy and reliability of bottom-up mass spectrometry (MS)-based proteomics analyses are undoubtedly dependent on the quality of prior sample preparation, which remains very challenging3. In fact, sodium dodecyl sulfate (SDS) is a powerful anionic detergent and one of the most widely used reagents for solubilisation and denaturation of proteins4. By binding amino acids through hydrophobic and ionic interactions, SDS alters proteins’ spatial conformational structure and inhibits proteases activity, thus enabling long-term conservation of structurally preserved proteins5. In spite of all its advantages, SDS significantly suppresses analyte ion signals in electrospray ionization (ESI)-MS, alters the chromatographic separation of peptides during liquid chromatography (LC)4,6, and strongly inhibits trypsin activity7, even at very low concentrations, which considerably limits protein identification. Samples therefore require to be completely depleted of SDS prior to digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, removing SDS while achieving a high recovery rate is a challenging task and several approaches have been proposed over the years. Methods such as gel electrophoresis separation along with in-gel digestion, protein precipitation and dialysis have shown variable efficacy, low reproducibility and limited routine applicability. Among the different protein preparation techniques recently developed, spin filter-based sample preparation, a concept first introduced by Manza et al.8 and further optimized for better detergent removal by Wisniewski et al.9 – Filter-Aided Sample Preparation (FASP) –, emerged as a promising approach and has already been successfully applied in diverse experimental settings9–12. In fact, FASP is among the simplest sample preparation methods for SDS-containing extracts. Operating in an ultrafiltration spin filter device, the method allows samples to be cleaned of SDS with urea prior to on-filter tryptic digestion13. Moreover, urea enables usage of filter membrane with a molecular weight cut-off of 30 kDa (30k MWCO), while not compromising depth of proteome coverage, as it ensures unfolding of proteins during the workflow14. FASP is also described as a universal sample preparation method as it allows for unbiased proteome representation.\n\nDuring past years, some groups have raised concerns regarding the depth of proteome coverage achieved with FASP15–20. Furthermore, there exist great disparities in the quality of outcomes when comparing FASP to other protocols depending on the authors and also as noted in practice by laboratories attempting to use FASP. Actually, Liebler and Ham21 first pointed out the difficulty of removing SDS by filter-aided buffer exchange, an observation further supported by Hustoft et al.16. The uncharged chaotrope urea may in fact have a limited capacity to deplete SDS from proteins as it may not completely dissociate negatively charged sulfate heads from basic amino acids. Disruption of these powerful ionic interactions may in fact require substitution of SDS by another anionic surfactant, such as sodium deoxycholate (NaDoc). Moreover, Proc et al.22 have shown that NaDoc was the most effective detergent to deepen proteome coverage and enhance digestion reproducibility when included in the digestion buffer. As long as its concentration remains below 1%, NaDoc promotes trypsin activity by easing contact between enzyme and cleavage sites23–26. This acid-insoluble surfactant is also MS-compatible as it is readily removed following acidification by either precipitation or phase transfer in organic solvent such as ethyl acetate24,25. Among the other factors possibly explaining the difficulties reported with FASP is the on-filter digestion, as it may have a more limited efficacy compared to the traditional in-solution approach27,28. These issues emphasize the need to fine-tune the FASP protocol with current state-of-the-art techniques in comprehensive proteomics in order to improve its robustness and reliability. As such, Erde et al. (2014) have recently proposed an enhanced FASP (eFASP) workflow to improve proteome coverage of SDS-solubilized protein samples18. This protocol, which is currently the only other equivalent alternative to FASP for preparing SDS-denatured proteins, incorporates a passivation step of the Microcon filter device, deoxycholic acid-based buffer exchanges, and a post-digestion phase transfer cleaning of peptides prior to LC-MS/MS analysis. These modifications led to a significant 14-fold increase in proteome coverage as compared to FASP18. Although the FASP was designed for use with the flat bottom filter Microcon device (EMD Millipore), several laboratories instead use the Amicon unit with V-shaped oriented filters (EMD Millipore)15,16,19,29. Difference between filters may in part explain why Nel et al. (2015) failed to show a significant difference in proteome coverage between eFASP and FASP while using Amicon devices29. In light of the striking discrepancy of proteome coverage between these two studies, an enhanced FASP workflow specifically tailored for Amicon filters was required in order to allow more laboratories to benefit from FASP advantages and ease-of-use in proteomics.\n\nHerein, we report a reproducible Amicon-adapted enhanced FASP (eFASP) workflow for comprehensive proteomics that is based on the best practices in sample preparation of SDS-containing protein extracts. The Amicon-adapted eFASP method presents key modifications to the FASP protocol and addresses the above-raised concerns. We compared both methods for SDS removal efficacy, depth of proteome coverage and reproducibility. Our results show that Amicon-adapted eFASP compares favourably to FASP and can therefore be regarded as a new alternative sample preparation approach for proteomics.\n\n\nExperimental section\n\nAmmonium bicarbonate (ABC), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), ethyl acetate, iodoacetamide, methanol, brilliant blue R250, phenylmethanesulfonyl fluoride (PMSF), potassium chloride (KCl), sodium chloride (NaCl), sodium deoxycholate (NaDoc), sodium dodecyl sulfate (SDS), stains-all, trizma base and urea were purchased from Sigma-Aldrich (St. Louis, MO) and were of the highest quality available. DL-Dithiothreitol (DTT), formamide, isopropanol, sodium hydroxide (NaOH), thiourea and Tween-20 were obtained from Thermo Fisher BioReagents (Ottawa, ON). OptimaTM LC/MS grade formic acid (FA) and water were obtained from Thermo Fisher chemicals (Ottawa, ON). Unless indicated, all other chemicals were from Sigma-Aldrich (St. Louis, MO) and minimally of ACS grade. RPMI-1640 (#350–007-CL), heat-inactivated fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin were obtained from Wisent Bioproducts (St-Bruno, QC). Amicon Ultra-0.5 ml centrifugal filters of 30 kDa (#UFC503096) and 10 kDa (#UFC501096) molecular weight cutoff (MWCO) were obtained from EMD Millipore Corporation (Billerica, MA). Trypsin Gold (#V5280) and Trypsin/Lys-C Mix (#V5073), both of Mass Spectrometry Grade, were from Promega (Madison, WI). BCA protein assay kit (#23227) and NanoDrop 2000c (#ND-2000c) were purchased from Thermo Scientific Pierce (Rockford, IL). Eppendorf Protein LoBind tubes were from Eppendorf (Hamburg, Germany).\n\nMEG-01 cells (CRL-2021, ATCC, Manassas, VA) were grown (37°C/5% CO2) in a medium containing 90% RPMI-1640, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Adherent cells were scraped into the medium and the resulting suspension was centrifuged at 430 g (5 min/4°C) for pelleting cells. Ice-cold 0.9% NaCl-resuspended cells were again pelleted by centrifugation. Cells were then lysed with a buffer consisting of 2% SDS, 0.1 M Tris-HCl pH 8 and 10 mM DTT, and heated for 7 min at 98°C. Nucleic acids were sheared by probe sonication (Misonix Sonicator 3000, Cole-Parmer, Hills, IL). Unbroken cells and debris were cleared by centrifugation at 16,000 g for 10 min. Sonication and centrifugation were repeated once. The final supernatant was carefully withdrawn, transferred in a LoBind tube and its protein concentration quantified by bicinchoninic acid (BCA) protein assay according to manufacturer’s instructions. “Non-SDS” control lysates were prepared with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT and 0.5 mM PMSF. Lysates were centrifuged at 16,000 g for 20 min (4°C). Supernatants were removed and transferred in LoBind tubes. Disulphide bridges were reduced following a 1h incubation at 37°C and protein concentration quantified by the BCA protein assay.\n\nImpact of increasing SDS concentrations (0.01% to 0.30% w/v) on trypsin (FASP) and one-step trypsin/Lys-C (Amicon-adapted eFASP) overnight digestion completion was assessed by Coomassie-stained 12% and 18% SDS-polyacrylamide gel electrophoresis (PAGE). SDS-free cell lysates were prepared by probe sonication of 50 mM ABC-resuspended MEG-01 pellet followed by 16,000 g centrifugation (10 min). Sonication and centrifugation were repeated once on collected supernatant. Lysates were solubilized in 6 M urea, reduced for 60 min at 37°C with 10 mM DTT and alkylated by 30 mM iodoacetamide for 30 min. One hundred-microgram whole cell lysate aliquots were next diluted six-fold in ABC buffer and digested in increasing concentrations of SDS5. Half of samples were afterward incubated for 12h at 37°C in the presence of (1) trypsin (enzyme-to-protein ratio 1:100 w/w) or (2) trypsin/Lys-C Mix (ratio 1:25 w/w) for proteolysis. Digested samples were next mixed with SDS loading buffer, heated and half the volume loaded onto a 12% SDS-PAGE while the other half was loaded on an 18% SDS-PAGE. Gels were subsequently stained for 3h in a Coomassie solution and scanned with an Odyssey® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).\n\nThe SDS concentration was measured according to Rusconi et al.30 by recording the absorbance (438 nm) (Ultrospec 2100 pro, Biochrom, Holliston, MA) of a stains-all dosage solution mixed with 1 µl of sample (Supplementary Table 1). To assess the assay’s specificity, we performed interference studies with all solutions and SDS-free biological samples used in FASP and Amicon-adapted eFASP protocols. The complete list of tested compounds is presented in Supplementary Table 2.\n\nRefer to Figure 1 for a detailed study flowchart (see also Supplementary Table 3).\n\nA total of two FASP and eight Amicon-adapted eFASP protocols were compared on the basis of several performance characteristics. Apart from the purification protocol itself, protocols differed in regard to the type of filter unit used, the passivation of filter device, the enzymatic digestion and following cleanup. n=3 for FASP and Amicon-adapted eFASP without passivation; n=2 for Amicon-adapted eFASP with passivation.\n\nPrior to protein purification, every non-passivated filter unit has been thoroughly rinsed following manufacturer’s instructions. A first cleaning spin (14,000 g/15 min [30k]/25 min [10k]) with 0.05 M NaOH was followed by a final spin with 500 µl of MS-grade H2O.\n\nThirty-microliter aliquots (150 µg proteins) of MEG-01 total extract were purified by the FASP protocol9. Aliquots were either dispensed to prerinsed Amicon Ultra-0.5 ml 10k filters (A10k) or Amicon Ultra-0.5 ml 30k filters (A30k). Every experiment was performed in triplicate (n=3). The FASP protocol was followed exactly as described by Wisniewski et al.9. Briefly, samples were washed twice with 200 µl of 8 M urea in 0.1 M Tris-HCl pH 8.5 and spun at 14,000 g for 15 min (30k) or 30 min (10k). Flow-through was discarded from collection tube and 100 µl of 50 mM iodoacetamide solution in 8 M urea, 0.1 M Tris-HCl pH 8.5 was added to each filter unit. After mixing filter content well for one minute, cysteines were alkylated at room temperature in the dark for 20 min without shaking. Excess iodoacetamide was then removed by spinning at 14,000 g for 10 min (30k) or 30 min (10k). Samples were further washed thrice with 100 µl of 8 M urea in 0.1 M Tris-HCl pH 8.5 and centrifugation resumed each time for 15 min (30k) or 30 min (10k) at 14,000 g. Three final buffer exchanges were performed by adding 100 µl 50 mM ABC followed by a 14,000 g spin for 10 min (30k) or 30 min (10k) and filtrates were discarded. Classically, FASP would have included an on-filter digestion. However, since our preliminary experiments using Amicons have shown suboptimal protein identifications and reproducibility with this approach, it was not further investigated and digestion has instead been performed in-solution. Precisely, concentrated samples were first recovered through a quick inverse spin of Amicons (1,000 g/5 min), thereafter transferred in a LoBind tube, diluted with 40 µl of ABC, dosed for residual SDS, and trypsin-digested (ratio 1:100 w:w) overnight (37°C) in solution. Proteolysis was quenched with 2% final (v:v) FA. Peptide yields were assessed before and after solid phase extraction (SPE) with the BCA assay on a NanoDrop-2000c.\n\nIn parallel, 150-µg aliquots of “non-SDS” lysate were also FASP-processed to confirm that nucleic acids and proteins do not interfere with the SDS colorimetric assay.\n\n(see Supplementary Protocol)\n\nWhen required by the protocol, filter units and collection tubes were filled with a 5% (v/v) Tween-20 solution in MS-grade water and soaked overnight at room temperature for passivation. Before use, passivated materials were twice immerged in water for 10 min with low-speed shaking. Filters and tubes were next twice filled with water and centrifuged at 14,000 g for 15 min (30k) or 25 min (10k).\n\nA total of 20 fifty-microliter aliquots (150 µg of proteins) of MEG-01 lysate were purified by the Amicon-adapted eFASP protocol, corresponding to 10 samples per type of filter (A10k and A30k) (see Supplementary Table 3). Lysates were mixed with iodoacetamide (50 mM final concentration), diluted to a volume of 500 µl with 8 M urea in 0.1 M Tris-HCl pH 8 and kept in dark for a 30-min alkylation. Samples were then deposited onto filter devices for centrifugal concentration at 14,000 g for 15 min (30k) or 30 min (10k). The flow-through was discarded, as was systematically the case following each subsequent centrifugation. SDS removal was initiated by adding 500 µl of 4% (w/v) NaDoc in 8 M urea to filter units, inverting devices thrice to mix the content well and centrifuging for 30 min (30k) or 45 min (10k). Two more washing steps were performed with urea by filling devices to completeness, inverting filters and spinning for 15 min (30k) or 30 min (10k). Excess urea was then removed with two buffer exchanges during which devices were filled with ABC and spun for 20 min (30k) or 30 min (10k). Protein recovery prior to in-solution digestion was performed by an inverse spin of Amicons in new collection tubes (1,000 g/5 min), either passivated or not. Proteins were diluted to a final volume of 150 µl with ABC, mixed with trypsin/Lys-C Mix (ratio 1:25 w:w) in a LoBind tube and digested for 12 h at 37°C.\n\nSamples were cleaned from residual SDS and NaDoc using either precipitation or phase transfer as described below. Yields were calculated with the BCA assay next to (1) precipitation or phase transfer and (2) SPE.\n\nThe concentration of SDS in purified samples was measured prior to digestion and next to precipitation or phase transfer. Before digestion, a first measurement of SDS was carried out after addition of 40 µl of ABC and a second after addition of the remaining ABC (150 µl final volume). Again, 150-µg aliquots of “non-SDS” control lysate were processed by Amicon-adapted eFASP to assess the stains-all colorimetric method’s specificity.\n\nFollowing Amicon-adapted eFASP, two methods for simultaneous SDS and NaDoc removal were compared: precipitation and phase transfer. The precipitation protocol is based on Zhou et al.5 and Zhou et al.24, with minor modifications. Briefly, samples were mixed with an equal volume of 4 M KCl, acidified with 2% FA and peptides were collected in the supernatant after centrifugation at 15,700 g for 15 min to pellet the potassium dodecyl sulfate (KDS) and deoxycholate precipitates.\n\nThe phase transfer procedure has been adapted from Masuda et al.25 and Yeung and Stanley31. First, an equal volume of 4 M KCl was added to each sample, as well as 1 ml of water-saturated ethyl acetate. Samples were then centrifuged at 16,000 g for 1 min to allow separation of organic and aqueous phases. Part of the KDS-containing organic phase was discarded, leaving approximately 1 mm of the ethyl acetate layer to avoid loss of aqueous-soluble peptides. The extraction was repeated once with 600 µl of ethyl acetate before acidification with 2% FA, after which deoxycholic acid was removed by three rounds of ethyl acetate extraction. Samples were next vacuum dried for 10 min to remove the remaining organic solvent.\n\nUnlike SDS, NaDoc cannot be quantified with the stains-all colorimetric assay. We therefore estimated its residual concentration in Amicon-adapted eFASP-prepared samples by visually comparing their deoxycholate acid-precipitated pellet with various concentrations of acid-precipitated NaDoc (standard curve).\n\nTo evaluate the processing capacities of Amicon-adapted eFASP regarding the highest amount of protein and the largest volume of sample that can be purified with no decrease in the effectiveness of SDS cleaning, we treated nine different samples containing various quantities of proteins and an equal number of volumes of lysate with A10k- and A30k-based Amicon-adapted eFASP. The remaining SDS concentration was quantified by the stains-all assay30. ‘Protein’ experiments were carried out with the lowest volume of lysate possible (<60 µl) and amounts of proteins ranged between 100 and 600 µg as initially measured by BCA protein assay. For ‘volume’ experiments, 50 µg of protein extract solubilized in lysis buffer volumes varying between 30 and 300 µl were used.\n\nRecovered digested peptide mixtures were cleaned by SPE prior to LC-MS/MS analysis. SPE was carried out on an Oasis HLB 96-well µElution Plate containing 2 mg Sorbent (Waters, Milford, MA). Elution was performed with 75% acetonitrile containing 2% FA.\n\nAcquisition was performed with a TripleTOF 5600 from AB SCIEX (Framingham, MA) equipped with an electrospray interface with a 25 μm I.D. capillary and coupled to an Eksigent μUHPLC (AB SCIEX). Analyst TF 1.6 software was used to control the instrument and for data processing and acquisition. The source voltage was set to 5.2 kV and temperature maintained at 375°C, curtain gas was set at 27 psi, gas one at 17 psi and gas two at 17 psi. Acquisition was performed in Data Dependent Acquisition (DDA) mode where a first period experiment was set for high resolution positive time-of-flight (TOF) MS in the m/z range 350–1,250 with a 25 ms accumulation time with declustering potential at 90 V and collision energy at 10. Top 40 ions were next triggered for 35 ms MS/MS experiment. Selected ions were next excluded after two occurrences for 45 sec. Separation was performed on a reversed phase HALO C18-ES column 0.3 μm i.d., 2.7 μm particles, 150 mm long (Advanced Materials Technology, Wilmington, DE) which was maintained at 50°C. Samples were injected by loop overfilling into a 5-μl loop that represented 3 µg of peptides. For the 60 min LC gradient, the mobile phase consisted in the following solvent A (0.2% v/v FA and 3% DMSO v/v in water) and solvent B (0.2% v/v FA and 3% DMSO in ethanol) at a flow rate of 5 μl/min. The gradient was the following: 0–45 min 2% B to 50% B, 45–49 min 50% B to 95% B, 49–53 min 95% B, 53–56 min 95% B to 2% B, 56–60 min 2% B and followed by a 2 min post-flush at 6 µl/min at final condition.\n\nProtein identification was performed with ProteinPilot V4.5 beta (AB SCIEX) with the instrument pre-set for TripleTOF 5600, iodoacetamide as cysteine alkylation and urea denaturation as special factor. Thorough search with false discovery rate analysis was performed with biological modification emphasis against Human UniProtKB/Swiss-Prot Release 2014_01. For protein identification and data analysis global false discovery rate was set at 1% and local false discovery rate was set at 5%. A combined ProteinPilot analysis of data acquired by each replicate of a specific protocol was also performed to generate a separate list of confidently identified proteins. Both combined and individual analyses of replicates are reported in the manuscript, the results of the latter being expressed unless otherwise indicated as: average ± standard deviation (SD) for the replicates. Note that in FASP and Amicon-adapted eFASP without passivation, data were summarized from triplicate experiments. In Amicon-adapted eFASP protocols with passivation, data were summarized from duplicate experiments. Chromatogram review and peak area information was performed with PeakView Software (AB SCIEX). Gene Ontology (GO) analyses were based on the PANTHER Classification System (v9.0) described by Mi et al.32. A Homo sapiens genome-wide data analysis was performed on every protein ID lists generated by combined ProteinPilot analysis of replicates and accessions searched for ‘Molecular Function’, ‘Biological Process’ and ‘Cellular Component’ classes and subclasses.\n\nWe assessed the reproducibility of FASP and Amicon-adapted eFASP by manually inspecting the total ion currents (TIC) chromatograms of each protocol. For quantitative results, we extracted and cumulated, for each replicate, the intensity (area under the peak) of the five most intense peptides from the five most abundant identified proteins (25 peaks total) per protocol. Likewise, peptide retention time was compared between replicates. The identity of these five proteins was determined by the combined Protein Pilot analysis of each replicate’s data. We then averaged the total cumulated intensity of the 25 peaks for every replicate, and compared protocols on the basis of the average cumulated peaks intensity and the resulting coefficient of variation (see Equation 1 below). We also performed the same analysis with the average peak height. Briefly, a cumulated peak height for each of the five proteins was calculated by adding up the peak height of each of its five most intense peptides. For every single replicate, a global peak height was obtained by averaging the cumulated peak height of the five proteins. Then, the global peak heights were averaged for all protocols (see Equation 2 below).\n\nEquations 1: Average cumulated signals intensity. The five most intense proteins along with their five most intense peptides for a given protocol were selected according to the combined Protein Pilot analysis of each of its replicate’s data.\n\n(1) Protein signal intensity = intensity of peptide 1 + intensity of peptide 2 + intensity of peptide 3 + intensity of peptide 4 + intensity of peptide 5\n\n(2) Replicate cumulated signal intensity = Protein signal intensity 1 + Protein signal intensity 2 + Protein signal intensity 3 + Protein signal intensity 4 + Protein signal intensity 5\n\n(3) Protocol average cumulated signals intensity = (cumulated signal intensity of R1 + cumulated signal intensity of R2 + cumulated signal intensity of R3)/3\n\nEquations 2: Average peak height. The five most intense proteins along with their five most intense peptides for a given protocol were selected according to the combined Protein Pilot analysis of each of its replicate’s data.\n\n(1) Protein cumulated peak height = peak height of peptide 1 + peak height of peptide 2 + peak height of peptide 3 + peak height of peptide 4 + peak height of peptide 5\n\n(2) Replicate global peak height = (Cumulated peak height of protein 1 + protein 2 + protein 3 + protein 4 + protein 5)/5\n\n(3) Protocol global peak height = (global peak height of R1 + global peak height of R2 + global peak height of R3)/3\n\n\nResults and discussion\n\nFew reliable and reproducible sample preparation methods are available for proteomics-based biomarker discovery, especially when conducted with SDS-solubilized biological samples. FASP is certainly one of the most efficient and simple available methods for such experiments, especially as it has been successfully applied to a variety of different samples including clinical material10–12. Nevertheless, as is the case with every other sample preparation method, FASP is not a processing method that works optimally with all biological samples and in every experimental setting15–20. In light of the very extensive literature on ‘best practices’ in sample preparation for proteomics experiments, we sought to tailor the FASP workflow for the Amicon filter device in order to improve its analytical performances. In this regard, we designed the Amicon-adapted eFASP, a complementary sample preparation method that introduces key modifications to the FASP and that is intended to improve proteome coverage and reproducibility. Among these modifications are: a passivation of the filter unit for improving SDS removal and peptide yields, a critical cleaning step with a high-content NaDoc buffer, and the substitution of on-filter for in-solution ‘one-step-two-enzymes’ trypsin/Lys-C digestion33,34. In this study, we extensively characterized the analytical properties of the Amicon-adapted eFASP by comparing it to the classic one-step digestion FASP approach9 during shotgun proteomics experiments. The extensive characterization of the human cancer cell MEG-01 proteome (a model of human leukemic megakaryoblast) is also described for the first time herein.\n\nIn agreement with Zhou et al.5, we confirmed the extreme sensitivity of trypsin to low concentrations of SDS, with complete digestion being achieved for samples containing ≤0.05% SDS (Figure 2A and Supplementary Figure 1A). The upper tolerable SDS limit is however not known for combined trypsin and Lys-C. Specifically, we found that optimal trypsin/Lys-C digestion efficacy, in a ratio of 1:25 w/w as is used in the Amicon-adapted eFASP protocol, was achieved for SDS concentrations ≤0.07% and that this combined proteolysis performed better than trypsin alone in higher SDS concentrations (Figure 2B and Supplementary Figure 1B), likely due to the greater stability of Lys-C in denaturing conditions25. The enzyme-to-protein ratio of 1:25 w/w in Amicon-adapted eFASP, representing a four-fold increase as compared to FASP, was chosen because it was previously shown preferable in comprehensive proteomics16.\n\nEffect of increasing SDS concentrations on the completion of overnight (A) trypsin (ratio 1:100 w/w) and (B) trypsin/Lys-C (ratio 1:25 w/w) digestion of MEG-01 total cell lysates assessed by a Coomassie-stained 12% SDS-PAGE. (C) Concentrations of residual SDS prior to digestion and (D) peptide yields in FASP (n=3) and Amicon-adapted eFASP (n=10). Nonparametric Mann-Whitney U test was performed for comparison of mean SDS concentrations and peptide yields: **p<0.01, *p<0.05. Mean (±S.D.) Statistical comparisons were made between the same filters as used in FASP and Amicon-adapted eFASP. TCL: Total cell lysate.\n\nSDS is not easily removed from proteins to which it binds strongly through ionic and hydrophobic interactions5. In FASP, usage of a washing buffer composed solely of urea has been assumed to be sufficient to completely deplete the SDS from up to 30 µl of a small protein extract (≤250 µg)9,13,14. However, this assumption was supported by the measurement of the residual detergent concentration in a FASP-cleaned 100-µl 2% SDS protein-free lysis buffer14. Rationally, the processing of a cell lysate would have been more appropriate for this purpose, especially as proteins are strongly bonded to SDS and may lower its critical micelle concentration (CMC ~6–8 mM)35, which can greatly impede its elimination (http://www.millipore.com/techpublications/tech1/6djp7f, EMD Millipore). Indeed, SDS micelles are not readily filtered by 30k and 10k MWCO membranes as their molecular weight is ~18–40 kDa14. Furthermore, urea is an uncharged chaotrope that disrupts hydrogen bonds and hydrophobic molecular interactions36. It enables the dissociation of SDS nonpolar tails from proteins but may not be as effective for disrupting the ionically bonded sulfate heads. We reasoned that introduction of the weak anionic surfactant NaDoc in the Amicon-adapted eFASP workflow would enhance the disruption of these powerful ionic interactions. Therefore, we measured for the first time the residual SDS concentration in Amicon-adapted eFASP- and FASP-processed 150-µg protein extracts. Owing to incorporation of a cleaning step with a high-concentration 4% (w/v) NaDoc buffer, Amicon-adapted eFASP significantly improves the removal of SDS from high-content protein samples independently of the filter used, and diluting proteins in a final volume of digestion buffer of 150 µl was sufficient to lower SDS concentration below the trypsin-activity-compatible 0.05% threshold for all conditions (Figure 2C and Supplementary Table 4). We also found that 4% NaDoc as used in the present Amicon-adapted eFASP version was more effective than 0.2% deoxycholic acid as is used in Erde’s eFASP18 for removing the SDS (Supplementary Table 5). In fact, among the ten different NaDoc concentrations (ranging from 0.1 to 5%) tested during Amicon-adapted eFASP designing, a single wash with 4% NaDoc was the most effective approach to deplete the SDS. Moreover, as exemplified by the complete depletion of SDS from FASP-processed samples containing <100 µg of proteins, the efficiency of filter-based SDS removal is tightly related to the initial amount of protein in the sample. This is further supported by the fact that recent experiments in which FASP led to lower-than-expected protein identifications have used >100 µg of proteins15–19. Equally important for efficient depletion of SDS is the starting volume of the sample, which was different in Amicon-adapted eFASP (50 µl) and FASP (30 µl). In fact, samples must be sufficiently diluted during buffer exchanges to minimize formation of unfilterable SDS micelles. This is more readily done with Amicon-adapted eFASP as the buffer volume is beneficially increased to 500 µl throughout the protocol, relative to the 200 µl in the FASP. Thereupon, when purifying <30 µl of a 150-µg protein extract with Amicon-adapted eFASP, SDS is almost completely (<0.05%) removed with both 10k and 30k filters. Of note, modifications brought by the Amicon-adapted eFASP protocol extend the processing limits to 50 µl/400 µg and 125 µl/600 µg for 10k and 30k filters, respectively (volume/protein) (Supplementary Table 6, Supplementary Table 7).\n\nMinimizing nonspecific protein and peptide binding to filter device’s plastic walls and membrane by passivation is possible with Tween-20 and SDS18. This reasonably implies that SDS present in the sample may bind to non-passivated filter reservoir’s surfaces and may not be completely removed despite buffer exchanges, further explaining the difficulty of removing it from spin filter-processed proteins. This may however be prevented by pre-treating filters with Tween-20. As such, lower concentrations of SDS were achieved in samples eFASP-processed with passivated Amicon filters (Supplementary Table 4). In sum, Amicon-adapted eFASP efficiently eliminates SDS by combining a passivation of the filter device, an increase of exchange buffer volume and an incorporation of a high-concentration NaDoc-based cleaning step. These features respectively prevent non-specific binding of SDS to device’s surfaces, minimize formation of unfilterable SDS micelles and ease dissociation of ionically bonded SDS from basic amino acids.\n\nAmicon-adapted eFASP provided 10% higher peptide recoveries for each type of filter as compared to FASP (Figure 2D and Supplementary Figure 2). The better yields of Amicon-adapted eFASP may be attributed to the filter passivation (Supplementary Table 4) and to its reduced number of centrifugations.\n\nWe next aimed to evaluate if Amicon-adapted eFASP compares favourably to FASP in regard to depth of proteome coverage. We found that Amicon-adapted eFASP noticeably deepened the MEG-01 cell’s proteome coverage relative to FASP (Figure 3A, B and Table 1). The lower concentrations of SDS remaining in samples after Amicon-adapted eFASP protocols, which were lower than the trypsin/Lys-C activity-compatible threshold, may in part explain the difference in proteome coverage. A considerable number of peptides/proteins were confidently identified (1% false discovery rate) in the three replicates of the Amicon-adapted eFASP/A10k/PPT protocol: 6,479/1,595, 6,567/1,596 and 6,424/1,603. The delivery rate was accordingly very high, with 27 proteins identified per minute, and the combined peptides-to-protein ratio was close to 6.0, an increment of 1 peptide per protein relative to FASP. This Amicon-adapted eFASP protocol (i.e. A10k/PPT) is the least variable regarding the number of identified peptides (coefficient of variation [CV] = 1.1%) and proteins (CV = 0.28%) per replicate. Combined analysis led to the identification of 12,431 peptides and 2,177 proteins, a significant increase of 200% for peptides and 150% for proteins compared to FASP with a 30k MWCO filter. Moreover, the protein overlap is ≥70% between the Amicon-adapted eFASP/A10k/PPT protocol and every other single one (Figure 3C and Supplementary Table 8). Along with improvements in SDS removal and peptide recoveries, passivation increased peptide and proteins identifications by 7% and 6%, respectively. There is also ≥80% homology among the proteins identified by a passivated Amicon-adapted eFASP approach and its non-passivated equivalent. In conjunction with the Amicon-adapted eFASP/A10k/PPT protocol, passivation yields identification of 1,662 and 1,698 proteins, for the two replicates. In light of the previous results, it seems clear that passivation of filter, simultaneous trypsin/Lys-C in-solution digestion and incorporation of NaDoc to enhance both removal of SDS and enzyme activity, were beneficent for the workflow. In fact, the concentration of residual NaDoc in Amicon-adapted eFASP digestion buffer as assessed by precipitation ranged between 0.20% and 0.35%. This concentration is known to significantly improve digestion reproducibility and to facilitate proteolysis of membrane and hydrophobic proteins22,25,37. As logically expected with a lower MWCO filter, fewer proteins were lost in A10k-based Amicon-adapted eFASP protocols, enabling the identification of more proteins. The opposite finding in FASP might be explained by the lower digestion efficiency achieved for A10k-processed samples due to higher concentrations of residual SDS, leading to loss of incompletely cleaved polypeptides during SPE.\n\n(A) Peptide and (B) protein identifications achieved by Amicon-adapted eFASP/precipitation and FASP protocols at a 1% global false discovery rate. Displayed are the results of the combined (green) and individual (blue) analyses of replicates. For individual analyses, data corresponding to the mean ± standard deviation were summarized from triplicate experiments. Comparison of peptide and protein total identifications between Amicon-adapted eFASP and FASP was performed with the nonparametric Mann-Whitney U test. (C) Protein identifications overlap from combined analysis of Amicon-adapted eFASP/precipitation and FASP protocols. The total number of proteins identified per protocol is indicated in the corresponding box. (D) Comparison of Gene Ontology annotations for specific cellular component classes in Amicon-adapted eFASP/precipitation- and FASP-processed and analyzed SDS lysates. Bars represent the percentage of identified proteins with the indicated annotations. PPT: Precipitation.\n\na. Results of the combined analysis of replicates.\n\nb. Average ± standard deviation of data acquired by individual analysis of replicates.\n\nn=3 for FASP and Amicon-adapted eFASP without passivation; n=2 for Amicon-adapted eFASP with passivation.\n\nPhase, Phase transfer; PPT, Precipitation; PT20, 5% Tween-20 passivation.\n\nFinally, it is worth noting that phase transfer-based Amicon-adapted eFASP protocols yielded 20% lower protein identifications as compared to precipitation-based ones (Table 1). This is in good agreement with Lin et al.37 and might have been caused by loss of ethyl acetate-miscible hydrophobic peptides and incorporation of peptide-containing water droplets in the organic phase during the extractions31. Moreover, there is no significant difference in sequence coverage and mean molecular weight of identified proteins among protocols (Supplementary Figure 3–Supplementary Figure 5 and Supplementary Table 4).\n\nAmicon-adapted eFASP is the first method to enable the identification in only one hr of nearly 1,700 proteins in a single analysis and 2,200 proteins in three separate runs from minimally prepared and non-fractionated SDS-solubilized lysates. To our knowledge, this is also the first study to achieve an identification yield this high in one hr with a 15-cm column, favourably comparing to Hebert et al.38 that reached 3,977 identifications with a 35-cm column.\n\nThe complete list of peptides and protein IDs identified by each protocol with both combined and individual analyses of replicates can be found in Supplementary Dataset 1–Supplementary Dataset 11.\n\nSupplementary Dataset 1: Peptides and proteins identified by combined analysis of replicates for each of the ten protocols of the study\n\nSupplementary Dataset 2: Peptides and proteins identified by individual analysis of each replicate for the FASP, Amicon 10k protocol\n\nSupplementary Dataset 3: Peptides and proteins identified by individual analysis of each replicate for the FASP, Amicon 30k protocol\n\nSupplementary Dataset 4: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, Amicon 10k, phase transfer protocol\n\nSupplementary Dataset 5: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, Amicon 10k, precipitation protocol\n\nSupplementary Dataset 6: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, passivated Amicon 10k, phase transfer protocol\n\nSupplementary Dataset 7: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, passivated Amicon 10k, precipitation protocol\n\nSupplementary Dataset 8: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, Amicon 30k, phase transfer protocol\n\nSupplementary Dataset 9: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, Amicon 30k, precipitation protocol\n\nSupplementary Dataset 10: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, passivated Amicon 30k, phase transfer protocol\n\nSupplementary Dataset 11: Peptides and proteins identified by individual analysis of each replicate for the Amicon-adapted eFASP, passivated Amicon 30k, precipitation protocol39\n\nVisual appraisal of TIC identified Amicon-adapted eFASP/A10k/PPT as the most reproducible protocol, which is further improved for significant signal enhancement throughout the gradient by passivation (Figure 4, Figure 5 and Supplementary Figure 6D). Noteworthy, the TIC spectra of each replicate are almost perfectly superposed one to another in these two protocols as exemplified by the ~6.5% CV in the cumulated intensity and global peak height. In fact, combination of precipitation and passivation always provided the strongest signal and the highest reproducibility for both Amicon 10k and 30k when used in the Amicon-adapted eFASP workflow (Supplementary Figure 6–Supplementary Figure 8). Signal suppression from remaining SDS seems to be present in both FASP/A10k and FASP/A30k chromatograms, which might also have contributed to the lower identification yields of FASP (Figure 4C and Supplementary Figure 6A). Indeed, it was previously shown that ≥0.01% (w/v) SDS significantly suppresses analyte ion signals and dramatically reduces protein identification in bottom-up proteomics approaches4.\n\nTotal ion current (TIC) chromatograms of the three replicates from (A) Amicon-adapted eFASP Amicon 10k precipitation (maximal signal intensity: 4.0e7) and (C) FASP Amicon 10k (maximal signal intensity: 1.5e7) within the total 1-hr analytical liquid chromatography gradient. Extracted-ion chromatograms of Histone H2B peptide LLLPGELAK from (B) Amicon-adapted eFASP Amicon 10k precipitation (maximum signal intensity: 2.5e6) and (D) FASP Amicon 10k (maximum signal intensity: 7.5e5). (B, D) The retention time (minutes) of the peptide is also indicated for each replicate.\n\nBars show the average cumulated MS signals intensities from each replicate for the five most intense peptides from the five most abundant identified proteins per protocol. Error bars are representative of the standard deviation of the mean associated with each replicate analysis. Data were summarized from triplicate experiments and duplicate experiments for the Amicon-adapted eFASP passivated Amicon 10k and Amicon 30k precipitation (Amicon-adapted eFASP/A10k/PT20/PPT and Amicon-adapted eFASP/A30k/PT20/PPT) protocols. Numbers above error bars indicate the coefficient of variation in the average cumulated MS signal intensities. PPT: Precipitation. PT20: 5% Tween-20 passivation.\n\nBroad and unbiased proteomics representation of subcellular compartments is a prerequisite for successful protein biomarker discovery. There were some differences in Gene Ontology (GO) cell component annotations between FASP and Amicon-adapted eFASP protocols, with more membrane and mitochondrion proteins, as well as more protein complex annotated in the latters (Figure 3D). On the other hand, the Amicon-adapted eFASP/passivated A10k/PPT protocol provided the highest combined GO annotations for nuclear and membrane proteins (Supplementary Figure 9), likely owing to the presence of NaDoc in the digestion buffer. Indeed, NaDoc helps to solubilize and expose membrane proteins’ cleavage sites to trypsin26, leading to an increase of their identification as previously shown18,23–25,37.\n\nIn summary, our Amicon-adapted eFASP protocol is an optimized Amicon unit-based sample preparation approach that provides efficient removal of SDS, high reproducibility, deep proteome coverage and enhanced identification of membrane proteins. Amicon-adapted eFASP enabled the identification of 1,700 proteins from SDS-solubilized proteins with minimal sample preparation and no fractionation, within a single run one-hr liquid chromatography and using a 15-cm column. Its implementation in proteomics may therefore improve the identification of high-confidence and significant biomarkers.\n\nF1000Research: Dataset 1. Supplementary Dataset 1–Supplementary Dataset 11, 10.5256/f1000research.6529.d4891339",
"appendix": "Author contributions\n\n\n\nDP designed and developed the method, designed and interpreted the experiments, performed the experiments and the bioinformatics analyses, and wrote the manuscript. HG contributed in the development of the method, designed and interpreted the experiments, performed the mass spectrometry experiments, assisted in data analysis and reviewed the manuscript. JD and FC supervised the work, designed and interpreted the experiments, assisted in data analysis and reviewed the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Université de Sherbrooke and the CHUS Research Center. DP holds a Frederick Banting and Charles Best Canada Graduate Scholarship Master Award from the Canadian Institutes of Health Research (CIHR) and an M.D.-M.Sc. Graduate Scholarship Master Award from the Fonds de Recherche du Québec – Santé (FRQS). HG holds a MITACS accelerated postdoctoral scholarship.\n\nI confirm that the 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 thank Dr. Robert Day for his participation in the elaboration and critical review of the manuscript. We also thank Sandra Gagnon for technical support, Simon Pellerin for data processing, and Artuela Çaku and Paméla Bouvier for helpful comments and suggestions on this manuscript.\n\n\nSupplementary material\n\nSupplementary data for 'Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP'.\n\n\n\n- Supplementary Figure 1. Effect of increasing concentrations of SDS on FASP (trypsin) and Amicon-adapted eFASP (trypsin/Lys-C) overnight digestion efficacy\n\n- Supplementary Figure 2. Peptide yields\n\n- Supplementary Figure 3. Sequence coverage by intervals\n\n- Supplementary Figure 4. Miscleavage frequencies\n\n- Supplementary Figure 5. Frequency of identified proteins’ theoretical molecular weight (kDa) for each protocol investigated\n\n- Supplementary Figure 6. Total ion current chromatograms\n\n- Supplementary Figure 7. Quantitative assessment of reproducibility: average cumulated signals intensity\n\n- Supplementary Figure 8. Quantitative assessment of reproducibility: average peaks height\n\n- Supplementary Figure 9. Distribution of Gene Ontology (GO) annotations of human leukemic megakaryoblast (MEG-01) proteins identified by Amicon-adapted eFASP and FASP protocols (combined analysis of replicates)\n\n- Supplementary Table 1. Spectrophotometric quantification of SDS with the stains-all colorimetric assay\n\n- Supplementary Table 2. Minimal effect of solutions and reagents used in FASP and Amicon-adapted eFASP protocols on stains-all dosage solution absorbance at 438 nm\n\n- Supplementary Table 3. Summary of the 10 protocols compared in this study\n\n- Supplementary Table 4. LC-MS/MS results of FASP and Amicon-adapted eFASP protocols\n\n- Supplementary Table 5. Concentration of residual SDS in 0.2% deoxycholic acid-based enhanced FASP (eFASP)-processed MEG-01 whole cell lysates\n\n- Supplementary Table 6. Volume processing capacity of Amicon-adapted eFASP\n\n- Supplementary Table 7. Protein processing capacity of Amicon-adapted eFASP\n\n- Supplementary Table 8. Protein identifications overlap\n\n- Supplementary Protocol. Amicon-adapted enhanced Filter-Aided Sample Preparation (eFASP)\n\nClick here to access the data.\n\n\nReferences\n\nAnderson NL: The clinical plasma proteome: a survey of clinical assays for proteins in plasma and serum. Clin Chem. 2010; 56(2): 177–185. 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PubMed Abstract | Publisher Full Text\n\nWiśniewski JR, Zielinska DF, Mann M: Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method. Anal Biochem. 2011; 410(2): 307–309. PubMed Abstract | Publisher Full Text\n\nLeón IR, Schwämmle V, Jensen ON, et al.: Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics. 2013; 12(10): 2992–3005. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHustoft HK, Reubsaet L, Greibrokk T, et al.: Critical assessment of accelerating trypsination methods. J Pharm Biomed Anal. 2011; 56(5): 1069–1078. PubMed Abstract | Publisher Full Text\n\nBereman MS, Egertson JD, MacCoss MJ: Comparison between procedures using SDS for shotgun proteomic analyses of complex samples. Proteomics. 2011; 11(14): 2931–2935. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErde J, Loo RR, Loo JA: Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J Proteome Res. 2014; 13(4): 1885–1895. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVowinckel J, Capuano F, Campbell K, et al.: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics. F1000Res. 2013; 2: 272. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAtrih A, Mudaliar MA, Zakikhani P, et al.: Quantitative proteomics in resected renal cancer tissue for biomarker discovery and profiling. Br J Cancer. 2014; 110(6): 1622–1633. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiebler DC, Ham AJ: Spin filter-based sample preparation for shotgun proteomics. Nat Methods. 2009; 6(11): author reply 785–786. PubMed Abstract | Publisher Full Text\n\nProc JL, Kuzyk MA, Hardie DB, et al.: A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J Proteome Res. 2010; 9(10): 5422–5437. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLin Y, Zhou J, Bi D, et al.: Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins. Anal Biochem. 2008; 377(2): 259–266. PubMed Abstract | Publisher Full Text\n\nZhou J, Zhou T, Cao R, et al.: Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane. J Proteome Res. 2006; 5(10): 2547–2553. PubMed Abstract | Publisher Full Text\n\nMasuda T, Tomita M, Ishihama Y: Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008; 7(2): 731–740. PubMed Abstract | Publisher Full Text\n\nChen EI, Cociorva D, Norris JL, et al.: Optimization of mass spectrometry-compatible surfactants for shotgun proteomics. J Proteome Res. 2007; 6(7): 2529–2538. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStrader MB, Tabb DL, Hervey WJ, et al.: Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems. Anal Chem. 2006; 78(1): 125–134. PubMed Abstract | Publisher Full Text\n\nKlammer AA, MacCoss MJ: Effects of modified digestion schemes on the identification of proteins from complex mixtures. J Proteome Res. 2006; 5(3): 695–700. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNel AJ, Garnett S, Blackburn JM, et al.: Comparative reevaluation of FASP and enhanced FASP methods by LC-MS/MS. J Proteome Res. 2015; 14(3): 1637–1642. PubMed Abstract | Publisher Full Text\n\nRusconi F, Valton E, Nguyen R, et al.: Quantification of sodium dodecyl sulfate in microliter-volume biochemical samples by visible light spectroscopy. Anal Biochem. 2001; 295(1): 31–37. PubMed Abstract | Publisher Full Text\n\nYeung YG, Stanley ER: Rapid detergent removal from peptide samples with ethyl acetate for mass spectrometry analysis. Curr Protoc Protein Sci. 2010; Chapter 16: Unit 16.12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMi H, Muruganujan A, Casagrande JT, et al.: Large-scale gene function analysis with the PANTHER classification system. Nat Protoc. 2013; 8(8): 1551–1566. PubMed Abstract | Publisher Full Text\n\nSaveliev S, Bratz M, Zubarev R, et al.: Trypsin/Lys-C protease mix for enhances protein mass spectrometry analysis. Nat Methods. 2013. Reference Source\n\nGlatter T, Ludwig C, Ahrné E, et al.: Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion. J Proteome Res. 2012; 11(11): 5145–5156. PubMed Abstract | Publisher Full Text\n\nChattopadhyay A, London E: Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge. Anal Biochem. 1984; 139(2): 408–412. PubMed Abstract | Publisher Full Text\n\nCañas B, Piñeiro C, Calvo E, et al.: Trends in sample preparation for classical and second generation proteomics. J Chromatogr A. 2007; 1153(1–2): 235–258. PubMed Abstract | Publisher Full Text\n\nLin Y, Liu Y, Li J, et al.: Evaluation and optimization of removal of an acid-insoluble surfactant for shotgun analysis of membrane proteome. Electrophoresis. 2010; 31(16): 2705–2713. PubMed Abstract | Publisher Full Text\n\nHebert AS, Richards AL, Bailey DJ, et al.: The one hour yeast proteome. Mol Cell Proteomics. 2014; 13(1): 339–347. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPellerin D, Gagnon H, Dubé J, et al.: Dataset 1 in: Amicon-adapted enhanced FASP: an in-solution digestion-based alternative sample preparation method to FASP. F1000Research. 2015. Data Source"
}
|
[
{
"id": "9056",
"date": "16 Jun 2015",
"name": "Lloyd D. Fricker",
"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 reading the manuscript, I initially thought that the authors had demonstrated that their modification of a technique for proteomics was a modest improvement over previously published methods. However, when I opened the supplemental datasets, I found some issues with the data. For example, in the FASP A10k data, there are 1199 proteins listed as “identified” but 401 of these were identified from a single peptide, and 5 identified from “0” peptides. How can a protein be identified without at least one confident peptide? Some laboratories that perform proteomic studies require 2 or more distinct peptides for a positive identification; some require 5 or more. If the latter, only 332 of the 1199 proteins would make the cut-off. In addition, there are columns in the supplemental dataset with no data (log precursor, fraction of the total signal, Apex retention time). What’s the point of including a column with no data? The supplemental datasets are not helpful in understanding the results, and should be modified. The main issue is whether the increased number of “identified” proteins reflects a real improvement in the technique, or just more noise in the system that gives tentative identifications that may not be real. In looking through the supplemental data, it was not clear to me that this is the case. At a minimum, the supplement needs to be edited and proteins based on “0” peptides should be removed. This will change many of the other figures/tables, which should only reflect proteins identified from 1 or more peptide! (2 would be preferable, but “0” is not acceptable)",
"responses": [
{
"c_id": "1602",
"date": "24 Sep 2015",
"name": "David Pellerin",
"role": "Author Response",
"response": "Reply from authors Thank you very much for your valuable input on our paper and datasets. Below you will find detailed explanations to all of your comments. We would also like to apologize for the delay in making this response. Unfortunately, we were waiting for the comments of the second reviewer before making changes to our paper and we are still waiting for them. First, the datasets have been restructured and modified according to the proteomic publication criterion of the Molecular and Cellular Proteomics journal (MCP publication standards http://www.mcponline.org/site/misc/Required_Manuscript_Guidelines_06-01-15.pdf). We are now providing new valuable information in the datasets, which may help to understand the purpose of our work. Precisely, protein identification reports, peptide summaries and peptide to spectra identification reports as well as the False Discovery Rate (FRD) tables (FDR analysis for protein, peptide and peptide to spectra match) are now presented in our supporting information. We also corrected the columns with no data, which have been inadvertently introduced in the dataset. In fact, there was probably a problem with data transfer from our analysis software. Everything is now corrected. Second, we apologize for the possible lack of clarity regarding the type of data analysis we performed in this study, which may have shed some confusion on the few proteins listed as identified with “0 peptide”. ProteinPilot unlike Mascot and Sequest reports 2 types of FDR score. The standard FDR analysis, termed global FDR, with a cut-off set at 99% (1% of the list would be accepted as a bad identification) is what we have used because we were not looking at particular and precise protein within the list. We were indeed performing shotgun and large-scale analysis for looking at the general tendency of protein identification between the different methods. However, when you look at a particular protein within the data set, it is recommended to use the local FDR (with a cut-off set at 95%) analysis that use a non-linear fitting model as published in Tang et al. (2008). Also we would like to provide additional information regarding the different FDR analysis using Proteomics System Performance Evaluation Pipeline Software (PSPEP) from AB SCIEX:About Global and Local False Discovery RatesOne of the main benefits of the PSPEP analysis is that it provides error estimation using a variety of methods and for a range of thresholds. FDRs can be divided into two categories – local and global. The local FDR estimates the “local” error rate around a given identification, which indicates the likelihood that that the specific identification is incorrect. The global FDR estimates the error rate of the whole “global” set of answers defined by a threshold value. That is, the global FDR tells you the likely error rate of the entire set of identifications with scores as good as or better than the threshold.PSPEP calculates the global FDR in two different ways:Using the conventional q-value approachUsing a fitted curve as a smoothing tool, to provide a more reproducible estimateFor global FDRs, evaluate the quality of the global FDR from fit using the Nonlinear Fitting plot. If the fit is good, the global FDR from fit is probably more accurate than the conventional global FDR. But if the fit is poor (or fails), the global conventional FDR is clearly better. It is important to understand that in the set with 5% global FDR, there are proteins that may have local error rates much higher than 5%. The type of FDR you use and the threshold setting you select, depend on your purpose. In general, local error rates are more meaningful, but global error rates can generally be determined with higher precision, so global FDRs may be better for some comparison purposes.Even though using the results obtained with the local FDR setting would change the total number of proteins in each group, this change should be coherent with the data quality of each group. Below is an example showing that changing the FDR cut-off does not really change the “tendency” and the “general meaning” of the data. Figure 1 (link)Concerning the “0 peptide-based” protein identification, these results are based on unique and over 95% confidence ProteinPilot peptide specific score (see below for explanation). One should also look at protein coverage based on confidence since ProteinPilot reports three score-based coverage (95%, 50% peptide confidence-based coverage, as well as overall coverage). Example of a “0 peptide” identified protein. The 95% local peptide FDR for FASP A10k indicates that 5363 peptides are valuable (N=5363). This corresponds to a ProteinPilot score of 94%. In the other hand, the 95% global peptide FDR indicates that 5755 peptides are valuable (N=5755). This corresponds to a protein pilot score of 89%. If we look at protein # 1174, which falls within the global protein 99% FDR cut-off but presents a “0 peptide” feature, and look at its corresponding peptide we see that the ProteinPilot score of this corresponding peptide is 94.92. This score is below both local and global FDR cut-off, meaning that we have actually identified the protein with one good peptide. Also, if you look at the “% coverage (50%)” column you can see that we can identify 2,4% of the protein; it correspond to the peptide itself. However, doing this exercise is exactly what should not be done with protein that fall only within the 99% global protein FDR cut-off and it is advised to use the 95% local protein FDR cut-off if the intention is to look at particular/precise protein identification. Within the 95% local protein FDR cut-off you will find most of the proteins with one high quality peptide, redundant acquisition of that peptide or more than one peptide.Figure 2 (Link)Figure 3 (Link)"
}
]
}
] | 1
|
https://f1000research.com/articles/4-140
|
https://f1000research.com/articles/4-409/v1
|
28 Jul 15
|
{
"type": "Research Article",
"title": "What’s in a Name? Exploring the Nomenclature of Science Communication in the UK",
"authors": [
"Sam Illingworth",
"James Redfern",
"Steve Millington",
"Sam Gray",
"James Redfern",
"Steve Millington",
"Sam Gray"
],
"abstract": "This study, via a consideration of the literature, and a survey of science communicators, presents concise and workable definitions for science outreach, public engagement, widening participation, and knowledge exchange, in a UK context.\n\nSixty-six per cent of participants agreed that their definitions of outreach, public engagement, and widening participation aligned with those of their colleagues, whilst 64% felt that their personal definitions matched those of their institute. However, closer inspection of the open-ended questions found the respondents often differed in the use of the nomenclature. In particular, the respondents found it difficult to define knowledge exchange in this context.",
"keywords": [
"Science Communication",
"Public Engagement",
"Outreach",
"Widening Participation",
"Knowledge Exchange"
],
"content": "Introduction\n\nBurns et al. (2003, pp. 183) define science communication as:\n\n“…the use of appropriate skills, media, activities, and dialogue to produce one or more of the following personal responses to science (the AEIOU vowel analogy): Awareness, Enjoyment, Interest, Opinion-forming, and Understanding.”\n\nThis robust definition covers most aspects of communicating science to the public under a number of different guises. Where things start to get complicated is in the semantics regarding the different types of science communication, and in their appropriate use and classification.\n\nScience communication is often analogously and interchangeably referred to as science outreach, public engagement, widening participation, and/or knowledge exchange, but what do these terms actually mean? As well as institutional biases towards the ‘correct use’ of these terminologies there exists personal nuances in terms of their interpretation, which oftentimes depend upon the role of the person in question and how they perceive science communication to fit into their research & teaching practices, and beyond.\n\nIt can be argued that these definitions are simply a matter of semantics, but with science communication becoming more prevalent in grant applications and income generation (see e.g. the Research Council UK’s ‘Pathway to Impact’ report [http://www.rcuk.ac.uk/ke/impacts/]), it is important for there to be consistency in what is a developing field. The advent of ‘Science 2.0’ (see e.g. Nattkemper, 2012) and what it entails is also an important driver behind having a clear and consistent nomenclature associated with science communication. Science 2.0 proposes a systemic change in the modus operandi of doing research and organising science, in which science communication will play a key part. With potentially large pots of money available in future grants, under specific terms and conditions, there needs to be a consistent terminology that can be drawn upon by the academic community and beyond.\n\nAccording to the European Commission public consultation into Science 2.0 (http://ec.europa.eu/research/consultations/science-2.0/background.pdf), something else that requires careful consideration is “the need to develop researcher and researcher reward schemes that reflect this (new) approach”. With potential reward schemes attributed to science communication activities, as well the creation of new job positions to fill these roles, it is important for all concerned to ensure that the language used in science communication is consistent.\n\nThis study, therefore, begins by discussing some of the definitions for science outreach, public engagement, widening participation, and knowledge exchange in the UK, derived from the common usage of these terms in the literature, and from the experiences of the authors. It then compares these definitions with the results of a survey of practicing science communicators from across the UK, and comments on the similarities and differences between the two, before identifying some suggestions for future nomenclature definitions within the field.\n\n\nLiterature analysis\n\nThe term ‘science outreach’ has been commonplace in research literature since the early 1990s, at which time the number of research articles on science communication started to increase. Many of these early articles describe science outreach as a school/education-linked activity, whereby academics are engaging with different groups of people such as the general public, students and teachers (see e.g. Greenler et al., 1993; Kelter et al., 1992). The term science outreach, which included activities such as mentoring, tutoring, giving presentations, supporting teachers and involvement with after-school clubs and summer schools, continued to become synonymous with school-related activity in to the 2000s, (e.g. Andrews et al., 2005; Krasny, 2005). Recently Ecklund et al. (2012) suggested that scientists involved in science outreach are often also engaged in some type of outreach involving school-aged children, demonstrating that the connection between school-related activity and science outreach remains strong.\n\nAlthough much of the literature using the term ‘science outreach’ is based on work carried out in North America, this definition is similar to that used in the UK. Many organisations in the UK who fund science communication (e.g. Royal Society, Royal Society of Chemistry, Society of Biology, and the Wellcome Trust) use science outreach when explicitly discussing science communication with school children.\n\nAlthough this link to school activity is present in the UK, there is some overlap with other commonly used science communication terms, in particular, public engagement; with some science communication practitioners using both terms together, e.g. schools outreach and public engagement.\n\nIn recent years there has been a shift from the deficit model of the ‘Public Understanding of Science’ towards a dialogue-based approach, which can be referred to as a ‘Public Engagement with Science and Technology’ (Schäfer, 2009, and references therein).\n\nPublic engagement can be thought of as a way to restore public trust in science, by developing a two-way dialogue between the general public and the scientific community (Wynne, 2006). Public engagement can foster global communication, enable shared experiences and methodology, standardize strategy, and generate shared viewpoints (Cohen et al., 2008). Furthermore, it can be defined as a deliberative process, promoted in both academic and policy circles, as a potential means to build public trust in risk decisions and decision-makers (Petts, 2008). With regards to policy makers, public engagement can be viewed as both relevant and useful in a regulatory context (see e.g. O’Doherty & Hawkins, 2010), with the results of public discussions with scientists being a worthwhile process in scientific development (Jones, 2007).\n\nRecent years have seen increasing encouragement by research institutions and funding bodies for scientists to actively engage with the public, who ultimately finance their work (Bowler et al., 2012), and whilst many research institutions now have dedicated resources for public engagement activities, such activities are not yet considered essential (Neresini & Bucchi, 2011). It is also unclear as to whether the institutional approach to public engagement is to focus on engaging with the public to promote their research and raise understanding, or if it is to open up a two-way dialogue in order to get their opinion on scientific research and protocol, especially in relation to potential political and ethical ‘hot potatoes’, e.g. geoengineering (Parkhill & Pidgeon, 2011) and nanotechnology (Jones, 2007).\n\nThe American National Centre for Media Engagement (http://mediaengagedev.org/engagement/why-engage/difference-between-outreach-and-engagement) defines outreach as “a mechanism for delivering value-added content”, whereas engagement means, “collaboratively addressing community concerns.” This would seem to be consistent with the UK-centric arguments that have been laid out above, i.e. that outreach is a means of educating the general public (in particular school children), whereas public engagement involves a two-way dialogue in which the general public can offer advice and opinions as to the current state of scientific research. This approach to defining public engagement as something different from outreach is corroborated by Holliman et al. (2009, pp. 56) who state that:\n\n“There is a heterogeneous community of practice operating in the space between what can be characterized as deficit-informed ‘science outreach’—aimed primarily at increasing scientific literacy—and dialogue-informed ‘public engagement’ seeking to foster productive exchanges between scientists and other stakeholders (including members of the public).”\n\nHowever, there still appears to be some uncertainty as to the difference between these approaches, and also to potential overlaps with regards to audiences; it is also unclear as to whether these definitions are consistent at an institutional level. As Rowe & Frewer (2005, pp. 251) remark:\n\n“Imprecise definition of key terms in the ‘public participation’ domain have hindered the conduct of good research and militated against the development and implementation of effective participation practices.”\n\nWidening Participation involves interventions targeted at social groups under-represented in Higher Education (HE), in order to encourage them to attend university. According to the Office of Fair Access (OFFA; http://www.offa.org.uk/) this includes:\n\nStudents from disadvantaged backgrounds\n\nStudents with disabilities\n\nStudents from some ethnic minority backgrounds\n\nCare leavers\n\nPart-time and mature students\n\nWith graduates benefitting from higher levels skills, knowledge, and access to the networks that are necessary to find higher paid work, the affordances of higher education are clear. Assuming disadvantaged social groups are afforded the same opportunities of access to employment through their university education, widening participation can help reduce social exclusion. It is not surprising, therefore, that the New Labour government largely reshaped the UK HE landscape in alignment with this ambition, with activity co-ordinated through Aimhigher, Lifelong Learning Networks, and the National Academy of Gifted and Talented Youth (see e.g. Frost, 2005).\n\nHowever, the institutional landscape has since changed, with a greater onus now on the universities to independently deliver these objectives. In addition, university widening participation activity has come under greater scrutiny by the Higher Education Funding Council England (HEFCE), whereby universities opting to charge over £6k annual tuition fees, must also agree to Fair Access Agreements (McCaig & Adnett, 2009).\n\nIn practice, widening participation aligns with the Pipeline or Learner Pathway model (see e.g. Clewell & Villegas, 1999); involving interventions designed to raise awareness and expectations of HE at various points within a learner’s education. With the emphasis on social mobility, widening participation focuses largely on targeting younger students from disadvantaged backgrounds utilising quantitative measures of poverty and deprivation, for example, the Index of Multiple Deprivation (Deas et al., 2003) and the eligibility for Free School Meals datasets.\n\nIn addition, widening participation can also be thought of as a consideration of the student lifecycle, beyond pre-entry and transition, to include university curriculum design, student support and employability. This follows concern that students from disadvantaged backgrounds perceive universities as ivory towers, i.e. places that are beyond their reach and are not for the likes of people like themselves (see e.g. Mangan et al., 2010). It is important, therefore, to consider the impact of traditional university practices or institutional culture, not only on access, but also on the retention and progression of students from non-traditional backgrounds through HE.\n\nVarious conceptualisations of knowledge exchange have been in UK higher education discourse since the late 90s, when the Higher Education Reach Out to Business and Community (HEROBC) initially emerged. HEROBC was initially part of the so-called ‘third stream’ of funding, designed to sit alongside institutions’ teaching and research activities, and to provide funds for universities and colleges to pursue interactions with business and the wider community. At the time these interactions were exclusively centred on knowledge and/or technology transfer (rather than exchange), with the purpose of HEROBC being to develop the capacity and capability for knowledge transfer between Higher Education Institutions (HEIs) and other sectors. Typical activities that were funded through HEROBC included skills matching between university and business, and the provision of gateways to enable business to access university expertise and employability initiatives.\n\nIn 2001, HEROBC evolved into the Higher Education Innovation Fund (HEIF), which focussed on funding activities designed to increase the capability of universities “to respond to the needs of business, especially in instances that would lead to identifiable economic benefits” (HEFCE, 2005, pp. 5) HEIF has since featured in four separate funding rounds, with explicit reference to knowledge exchange (rather than knowledge transfer) first emerging as a prominent part in December 2003 around the call for HEIF-2.\n\nHEFCE, through the annual Higher Education Business and Community Interaction Survey (HEBCIS), now leads the categorisation of knowledge exchange activities. HEBCIS requires universities to report expenditure across various knowledge exchange categories including contract research, consultancy, CPD, business start-up, employability programmes etc. As university HEIF allocations are tied to levels of expenditure reported through HEBCIS, this exercise has been a big influence on what UK universities prioritise, resource and define in terms of knowledge exchange.\n\nDespite the focus on expenditure, the important social, cultural and community role that universities play in wider society has not been entirely ignored. Influential voices have emerged around these concepts, most notably Professor David Watson (ex-Vice-Chancellor at Brighton University) who has been a champion of this societal agenda and the role that universities have to play within it, focusing on “civic and community” partnerships (Watson, 2007).\n\nWatson’s conceptualisation of knowledge exchange is rooted in a more engaged ‘two-way’ relationship between universities and external partners that sets out a much broader notion of knowledge transfer and knowledge exchange. John Goddard, the emeritus Professor of Regional Development Studies at Newcastle University UK, has also commented on the positions of universities as powerful engines of local and regional economic growth (see e.g. Goddard, 2009).\n\nThe most recent HEFCE definition states knowledge exchange “refers to HEIs’ engagement with businesses, public and third sector services, the community and wider public” (http://www.hefce.ac.uk/glossary/#letterK). This adoption of a more explicit referencing of engagement within the knowledge exchange landscape has largely come about through a subtle yet important shift within funding council priorities prefaced, for example, within the Beacons for Public Engagement initiative (2008–2012) and leading towards the uptake of the impact agenda within the UK’s Research Excellence Framework.\n\n\nSurvey\n\nIn order to assess the current opinion relating to the definitions of outreach, public engagement, widening participation, and knowledge exchange in UK HEIs, a survey conducted in the UK asked participants to relate their experience of science communication and its nomenclature.\n\nThe survey was conducted using Bristol Online Surveys (https://www.survey.bris.ac.uk/), and comprised 8 questions delivered with a mixed-method approach (i.e. qualitative and quantitative questions). The focus was to evaluate the participant’s views on what constituted outreach, public engagement, widening participation, and knowledge exchange. It also aimed to assess whether or not the participants felt as though their own opinions aligned with those of colleagues and their institution. A copy of the questionnaire is included as Supplementary material.\n\nThe survey was advertised via email, social media accounts, and the ‘psci-comm’ mailing list hosted by JISCMail. This study was carried out according to the British Educational Research Association’s (BERA) ethical guidelines for educational research, with all of the data in this study fully anonymised.\n\n\nResults & Discussion\n\nIn total, 47 people participated in the survey, and all bar one of them stated that they currently participated in outreach, public engagement, or widening participation events at their institute or company. Of the actively involved participants, 44 were located solely in UK, one in the Netherlands, and one was based in both the Netherlands and the UK.\n\nFigure 1 shows the results of the survey in relation to how the participant’s personal definitions differed from those of their colleagues and institutes/companies.\n\nSixty-six per cent of the participants agreed that their definitions of outreach, public engagement, and widening participation aligned with those of their colleagues, whilst 64% felt that their personal definitions matched those of their institute.\n\nOnly five of the participants felt as though the alignment of their definitions was different when comparing colleagues to institutes. Of these five, three of the participants felt as though their definitions matched those of their colleagues but not those of their institute, whilst two of the participants considered their definitions to be aligned with those of their institute, but not of their colleagues. These results suggest that in a still emergent field, the participants of this survey are likely to be the driving influence behind the definition of science communication at an institutional level.\n\nIt is also worth noting that whilst the majority of the participants felt as though their personal definitions of outreach, public engagement and widening participation matched those of their colleagues and institutes, there were approximately a third that did not feel as though this was the case. If the participants that took part in this survey represent a fair cross-section of people working in science outreach, public engagement, and widening participation across the UK then it is somewhat alarming that such a significant proportion of them feel as though the fundamental basis on which their work is founded lacks such clemency in its definitions.\n\nWhat is not clear from the above statistics is if there is any agreement between the participants’ personal definitions of outreach, widening participation, and public engagement. As such, in addition to the questions regarding how the participants felt their definitions matched those of their colleagues and institutes, the survey also contained the following questions, which aimed to further explore how these different facets of science communication are defined in the UK:\n\nHow would you define outreach?\n\nHow would you define public engagement?\n\nHow would you define widening participation?\n\nRespondents were also asked to define how knowledge exchange related to outreach, public engagement and widening participation\n\nFrom the responses to these open-ended questions, word clouds were created, in which an image was composed of the words used, for which the size of each word indicates its frequency. These word clouds are shown in Figure 2–Figure 5, all of which were produced using the web-based application Wordle (http://www.wordle.net). In the generation of these word clouds the key word(s) addressed in the question were removed, and all of the words were capitalised (so as to avoid repetition). For example, for the question concerning knowledge exchange, only the words ‘knowledge’ and ‘exchange’ were removed from the text used in the generation of the word cloud.\n\nRegarding the responses to the definition of outreach (Figure 2), what is immediately noticeable is the frequency with which the word ‘schools’ is used. Nearly half (46%) of the respondents made a direct association between science outreach and school education, whereby it was the job of the academic/scientist to translate their research/knowledge into an activity that would ensure a student would understand, become engaged and hopefully inspire into considering science at a higher level.\n\nOne respondent stated it was not an act solely for school students, but generally for young people under the age of 20. Others noted that outreach takes place outside the university campus (in any location) or specifically targeted communities that are considered ‘hard-to-reach’, or generally disengaged from university education.\n\nInterestingly, some respondents explicitly discussed the connection between outreach and public engagement. One respondent stated they believed outreach was “more educational than public engagement”, whilst two respondents believed outreach to be a subsection to public engagement, but did not elaborate on how the terms were differentiated. One respondent suggested that outreach was “More one-way focused, having a scientist talk to a non-expert, not necessarily in a two-way conversation.”\n\nFrom the results of the survey, the largest collective response to the issue of defining public engagement was that it was a two-way dialogue used to share information between two distinct groups, with 18 of the participants referring to something that was ‘two way’ or involved a sharing of ideas, rather than a one-way discourse. This is evidenced by the prominence of the words ‘two’, ‘way’ and ‘two-way’ in Figure 3. Some of the sample responses that matched this definition included: “Public engagement can be a two-way process, with academics learning and incorporating feedback from the public”, “Public Engagement is ideally a two-way process, by which information is shared between two different groups”, and “Activities in which members of public audiences communicate with specialists in a way that has the potential to influence the specialists' activities.”\n\nThis definition of public engagement being a two-way conversation between scientists and general members of the public would also seem to match that which was discussed in the introduction to this paper, and it is encouraging that the most popular philosophy matched the most common consensus of the literature. However, it should be noted that not all of the participants viewed public engagement as being defined in this manner. Some of the other definitions of public engagement included: “Audiences not associated with schools and colleges”, “Public engagement is meeting and engaging with the broader public to improve understanding and transparency of university teaching and research”, and “Any activity done with or to members of the public not in an organised educational group.”\n\nWhilst the most popular participant response to the nature of public engagement involved some sort of two-way communication, it is interesting to note that this accounted for only ~38% of the participants. Considering that 66% of the participants felt that their definitions of outreach, knowledge exchange, and public engagement agreed with those of their colleagues, this would appear to be counterintuitive. Instead, the results of this survey would seem to indicate that the participants’ definitions of public engagement are in fact many and far ranging.\n\nThe analysis of the results from responses to definition of widening participation, encouragingly reveal a broad consensus. A vast majority (89%), for example, articulated in various ways that widening participation refers to broadening access to university to include a wider range of social groups, in particular, people from groups currently under-represented in higher education, as evidence by the prominence of these terms in the word cloud shown in Figure 4. That said, three respondents claimed to have not have heard of the term widening participation before, whereas two expressed a rather cynical perception that widening participation had become “hijacked by university recruitment agendas”, and another respondent referred to “the annoying habit of targeting minority groups.”\n\nThe survey reveals less clarity, however, in terms of identifying specific target groups (some respondents referred to one or more target groups):\n\nTwelve respondents simply referred to a universal target group e.g. society, the public or simply engaging more people.\n\nNine referred to groups that were under-represented or hard-to-reach, but most were unable to provide specific examples. Only one respondent, for example, referred specifically to minority ethnic status, only one to gender, and another referred specifically to disability.\n\nNine understood widening participation in terms of relating specifically to schools or younger people\n\nEight referred to atypical social groups, i.e. groups who would not normally or traditionally attend university\n\nSix referred to groups experiencing some form of disadvantage\n\nThree understood widening participation targets in terms of targeting geographical areas\n\nThere were no references to mature or adult learners from non-traditional backgrounds\n\nA large number of responses refer to atypical social groups, people who express certain characteristics that appear to be defined against some notion of what constitutes a normal student, for example “those with different cultural attitudes and ideals.” This finding chimes with the concern that university staff continue to understand diversity in a way that reproduces the notion of universities as places for some normalized subject, defined against an atypical Other. Only one respondent, for instance, referred to widening participation in terms of curriculum support/design for widening participation of students already in HE.\n\nIn the survey, participants were asked how they felt knowledge exchange related to outreach, public engagement and widening participation, the results of which are shown in the word cloud in Figure 5. What is immediately clear is that there is no consistent definition or understanding of knowledge exchange amongst respondents. There are instead a broad range of concepts, definitions and views of knowledge exchange in evidence.\n\nSeventeen per cent of respondents admitted that they had not come across the term “knowledge exchange” before, with 19% identifying knowledge exchange as related to interactions between universities and business partners, leading to increased economic activities. By far the largest number of respondents (36%) felt that knowledge exchange was related to all three activities including outreach, public engagement and widening participation, often conceptualising it as (in the words of one respondent) “an essential part as it allows external stakeholders to influence our activities but also allows us to share expertise.”\n\nFrom the word cloud of these results, shown in Figure 5, it is perhaps surprising that the word outreach is so dominant, given that the earlier definitions in the survey (Figure 3) generally reference outreach activities as those within schools rather than commercial or income-generating partnerships with business or industry. The prevalence of public engagement (and widening participation) in the participants’ responses is perhaps to be expected, given the relationship between the professional support for knowledge exchange and public engagement within most institutions. Given the more business-oriented nature of knowledge exchange that was laid out previously, it was perhaps surprising to see less use of words such as commercialisation, HEIF, business, and enterprise.\n\n\nConclusion\n\nPerhaps the most noticeable result from this study is that the open-ended responses to the survey resulted in a wide range of definitions of outreach, public engagement, widening participation and knowledge exchange amongst the participants, despite the quantitative data indicating that two thirds felt that their definitions of outreach, knowledge exchange, and public engagement agreed with those of their colleagues. This would seem to indicate that further communication is required both within and between institutes to ensure a level of consistency amongst science communicators.\n\nBased on the current literature, and the results of this study, the following broad definitions are offered for each of the four considered topics:\n\nOutreach: a one-way discourse, in which scientists communicate their research to the general public.\n\nPublic Engagement: a two-way dialogue, in which scientists converse with members of the general public in a mutually beneficial manner.\n\nWidening Participation: any activity that engages with social groups under-represented in HE, in order to encourage them to attend university.\n\nKnowledge Exchange: any activity that involves engagement with businesses, public and third sector services, the community and the wider public, and which is monitored for funding purposes.\n\nIt is acknowledged that there is still some overlap between these definitions, for example a school assembly given by a university researcher at a local school might well be classed as being an outreach, widening participation, and knowledge exchange activity. In such instances it is important to consider the context of these classifications. In this example, the researcher’s faculty might classify the activity as outreach, the university’s widening participation team (or equivalent) may catalogue it as a widening participation activity, and the knowledge exchange offices (or equivalent) could acknowledge it in their records for HEFCE.\n\nIt is important for science communicators to consider the context in which their activity takes place, because depending on its classification, the activity may be eligible for different amounts of funding from different areas of resource. This consideration of context is especially important when applying for external funding, where science communicators will be expected to outline the specific area(s) in which their activity can be categorised.\n\nThe main limitations of this survey were the sample size, and also the fact that the participants were mainly active science communicators. For future work, it would be conducive to pursue responses from active researchers who did not consider themselves science communicators.\n\nThe results of the survey also indicate that the respondents were less comfortable defining terminology around knowledge exchange than they were about outreach, public engagement and widening participation. The job titles and functions of respondents may be an important factor here, and further work is needed to confirm this. A future study is planned which also aims to assess how the different perceptions of science communication nomenclature would break down according to stakeholders. For example, the ways in which an academic, museum and learned society view these definitions might be very different. An international study, with a much larger target audience, is also required so as to assess differences in perceptions of the science communication lexicon between countries, both those traditionally associated with the field and those that are not.\n\nIt is also worth noting that no mention has been given to the effect of science communication on the researchers, i.e. the sector of the field that is concerned with improving the effectiveness and engagement of scientists in communicating their research to a variety of audiences. A future study would also aim to assess how this aspect of science communication fitted in with the external facets discussed above.\n\nThis study, via a consideration of the literature, and a survey of science communicators, has presented concise and workable definitions for outreach, public engagement, widening participation and knowledge exchange. However, as with all names it is important that the people using them feel comfortable with them, and also that there is at least some form of consistency within the field (and beyond) as to their usage. This consistency will only come about by communication both within and between institutions, and this study aims to act as a starting point for such conversations, with planned future work aiming to further explore the perceptions of science communication and its nomenclature amongst a much wider target audience.\n\n\nData availability\n\nDataset 1. Answers to science communication questionnaire. These are the responses to the questionnaire that was used in this study to assess practitioner’s definitions of nomenclature in relation to science communication. http://dx.doi.org/10.5256/f1000research.6858.d97179 (Illingworth et al., 2015).",
"appendix": "Author contributions\n\n\n\nConceived and designed the experiments: SI. Performed the experiments: SI. Analysed the data: SI, JR, SM, SG. Wrote the paper: SI, JR, SM, SG.\n\n\nCompeting interests\n\n\n\nThe authors declare that there are no competing interests, either financial or otherwise.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe gratefully acknowledge the participation of all of the people who took the time to fill in the survey on science communication practices.\n\n\nSupplementary material\n\nScience communication questionnaire.\n\nClick here to access the data.\n\n\nReferences\n\nAndrews E, Weaver A, Hanley D, et al.: Scientists and public outreach: Participation, motivations, and impediments. Journal of Geoscience Education. 2005; 53(3): 281–293. Reference Source\n\nBowler MT, Buchanan-Smith HM, Whiten A: Assessing public engagement with science in a university primate research centre in a national zoo. PLoS One. 2012; 7(4): e34505. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurns TW, O'Connor DJ, Stocklmayer SM: Science communication: A contemporary definition. Public Underst Sci. 2003; 12(2): 183–202. Publisher Full Text\n\nClewell BC, Villegas AM: Creating a nontraditional pipeline for urban teachers: The pathways to teaching careers model. J Negro Educ. 1999; 68(3): 306–317. Publisher Full Text\n\nCohen ER, Masum H, Berndtson K, et al.: Public engagement on global health challenges. BMC Public Health. 2008; 8: 168. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeas I, Robson B, Wong C, et al.: Measuring neighbourhood deprivation: A critique of the Index of Multiple Deprivation. Environment and Planning Government and Policy. 2003; 21(6): 883–903. Publisher Full Text\n\nEcklund EH, James SA, Lincoln AE: How academic biologists and physicists view science outreach. PLoS One. 2012; 7(5): e36240. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrost P: The CTY summer school model: Evolvement, adaptation and extrapolation at the National Academy for Gifted and Talented Youth (England). High Ability Studies. 2005; 16(1): 137–153. Publisher Full Text\n\nGoddard J: Reinventing the Civic University. 2009. Reference Source\n\nGreenler RG, Lasca NP Jr, Brooks AS, et al.: The Science Bag™ at the University of Wisconsin–Milwaukee: A successful forum for science outreach. Am J Phys. 1993; 61(4): 326–329. Publisher Full Text\n\nHEFCE: Higher Education Innovation Fund round 3: Invitation and guidance for institutional plans and competitive bids. 2005. Reference Source\n\nHolliman R, Whitelegg L, Scanlon E, et al.: Investigating science communication in the information age: Implications for public engagement and popular media. Oxford University Press, 2009. Reference Source\n\nIllingworth S, Redfern J, Millington S, et al.: Dataset 1 in: What's in a Name? Exploring the Nomenclature of Science Communication in the UK. F1000Research. 2015. Data Source\n\nJones R: What have we learned from public engagement? Nat Nanotechnol. 2007; 2(5): 262–263. PubMed Abstract | Publisher Full Text\n\nKelter P, Hughes K, Murphy A: Science Outreach for the 1990s. School Science and Mathematics. 1992; 92(7): 365–369. Publisher Full Text\n\nKrasny ME: University K–12 Science Outreach Programs: How Can We Reach a Broad Audience? Bioscience. 2005; 55(4): 350–359. Publisher Full Text\n\nMangan J, Hughes A, Davies P, et al.: Fair access, achievement and geography: Explaining the association between social class and students' choice of university. Stud High Educ. 2010; 35(3): 335–350. Publisher Full Text\n\nMcCaig C, Adnett N: English universities, additional fee income and access agreements: Their impact on widening participation and fair access. Brit J Educ Stud. 2009; 57(1): 18–36. Publisher Full Text\n\nNattkemper TW: Are we ready for science 2.0? Paper presented at the KMIS 2012-Proceedings of the International Conference on Knowledge Management and Information Sharing. 2012. Reference Source\n\nNeresini F, Bucchi M: Which indicators for the new public engagement activities? An exploratory study of European research institutions. Public Underst Sci. 2011; 20(1): 64–79. Publisher Full Text\n\nO'Doherty KC, Hawkins A: Structuring public engagement for effective input in policy development on human tissue biobanking. Public Health Genomics. 2010; 13(4): 197–206. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParkhill K, Pidgeon N: Public engagement on geoengineering research: preliminary report on the SPICE deliberative workshops. Understanding Risk Working (2011–11). 2011; 29. Reference Source\n\nPetts J: Public engagement to build trust: False hopes? Journal of Risk Research. 2008; 11(6): 821–835. Publisher Full Text\n\nRowe G, Frewer LJ: A typology of public engagement mechanisms. Sci Technol Hum Val. 2005; 30(2): 251–290. Publisher Full Text\n\nSchäfer MS: From public understanding to public engagement: An empirical assessment of changes in science coverage. Sci Commun. 2009; 30(4): 475–505. Publisher Full Text\n\nWatson D: Managing civic and community engagement. McGraw-Hill International. 2007. Reference Source\n\nWynne B: Public engagement as a means of restoring public trust in science--hitting the notes, but missing the music? Community Genet. 2006; 9(3): 211–220. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "9684",
"date": "06 Aug 2015",
"name": "Laura Fogg Rogers",
"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 attempts to define common definitions for inter-changeable terms used in science communication through a literature review and survey of science communicators. It is a timely article, as this is a much sustained debate in both academic and practitioner circles, and relates to discussion in the literature about funding, incentivising and rewarding science communication. More particularly, it links directly to efforts to define the ‘impact’ of science communication through processes such as the Research Excellence Framework and the Public Engagement with Research agenda. The article is well grounded in this literature and refers to many of the current debates in this field.The article uses survey results to move forward the debate on these definitions, attempting to find consensus amongst science communication practitioners. However there were only 47 participants involved in the survey, which is a low sample size. The authors acknowledge this point however, and also note that it may be useful to repeat the survey with researchers and professionals who don’t consider themselves science communicators.Despite these limitations, the survey results indicate that while 66% of respondents state that their definitions match those of colleagues, the resulting qualitative data indicates wide differentiation. These results are presented through Wordle diagrams. While these are visually interesting, further qualitative thematic analysis, along with matrix analyses for coding frequency, could also have added to the results. Percentages are given for word frequency, but a methodology for how this was obtained would be useful.The resulting concluding definitions are a useful addition to the field, but still produce considerable overlap between activities. The article notes that further work is needed, and I would concur that this is a topic ripe for more in depth research.",
"responses": [
{
"c_id": "1596",
"date": "24 Sep 2015",
"name": "Samuel Illingworth",
"role": "Author Response",
"response": "Thank you for your comments, which are greatly appreciated, and which we have used to improve both the content and presentation of our study. As you have pointed out, we acknowledge that the number of participants in this survey is small, and the purpose of this study was to act as an initial scoping exercise amongst science communicators to try and determine how they would summarise the terms of outreach, public engagement, widening participation and knowledge exchange within the umbrella term of ‘science communication.’ The idea for this paper was to begin to further develop the conversation that needs to take place in regards to how science communicators use the terminology in their fields, initially on a UK-wide basis. Given that limited demographic data was taken in this study (which was in hindsight an oversight), the Wordle diagrams were simply a tool to indicate the frequency with which certain words occurred. However, it was not at all clear from the original paper how this had been done, and further thematic analysis has now been carried out using NVivo, with the text being rectified to reflect this. The word clouds have also been replaced with tables that outline the major themes for the thematic analysis, and the frequencies associated with them."
}
]
},
{
"id": "9683",
"date": "10 Aug 2015",
"name": "Paige Brown Jarreau",
"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\nSummary: A re-analysis of the data and a significant re-write is advised. The authors have provided a fine if brief literature overview of definitions of science outreach, public engagement, widening participation and knowledge exchange. This is also a timely topic, as the need for evidence-based science communication has grown in today's complex ecosystem of science new media and outreach efforts on the part of scientific institutions. However, there are major flaws in this paper as it is currently written and presented. The sample is extremely small for an online survey - this sample would have been more appropriate for in-depth interviews with each respondent. This is acceptable as long as the audience/population is clearly defined. Are those who answered the survey mostly active scientists, or mostly dedicated science communicators? This could make a big difference to participants’ definitions of science communication, outreach, public engagement, widening participation and knowledge exchange. Why only 47 respondents? The ‘psci-comm’ mailing list serves a much larger number of people than this (and the authors also don’t make any estimate of how many people they reached with their survey.) How long was data collected for? The authors should more clearly spell out their sampling goals and procedures. “These results suggest that in a still emergent field, the participants of this survey are likely to be the driving influence behind the definition of science communication at an institutional level.” How so? Please make this statement, and how/why this conclusion was reached, more clear. “If the participants that took part in this survey represent a fair cross-section of people working in science outreach, public engagement, and widening participation across the UK then it is somewhat alarming that such a significant proportion of them feel as though the fundamental basis on which their work is founded lacks such clemency in its definitions.” The authors should be VERY careful in making any statement regarding the implications of this survey representing a fair cross-section of people working in science outreach, etc., as per my comments on the sampling procedure above. The authors don’t present any data on the basic demographics of the respondents. This was a critical oversight. In the analysis of survey responses, I am not under the impression that Wordle is an academically rigorous method for mapping word and concept frequency. Other qualitative analysis tools such as AtlasTi would have been preferable, even if figures were created in Wordle for demonstration purposes only. Wordle images, while a visually appealing, make it very difficult to quickly gauge relative word frequency. The authors’ methodology in qualitatively analyzing, coding and interpreting the survey responses is very unclear, if not missing altogether. How were the survey responses approached during data analysis? Were the authors’ primarily looking for responses that corresponded with how the scientific literature defines concepts of outreach, engagement, etc., or were novel or conflicting definitions also coded and analyzed (e.g. open vs closed coding?)? From the description of survey response analysis, it is not clear if the data analysis reached saturation, or what theory or framework guided the textual analysis. It would be very useful to know the demographic information of those respondents who provided alternative definitions of outreach and engagement, and of those respondents who were unable to define widening participation, etc. Are these scientists? Are these professional science communicators? This type of basic information would enrich the meaning of these results. The authors state only in their conclusion section that the respondents were “active science communicators.” What type of science communicators? How many were involved in academic institutions? I think this type of information is paramount to the authors’ interpretation of their findings, and the lack of this data is a significant weakness in this paper. What insights did we glean from the authors’ survey of 47 science communicators regarding the definitions and terminology surrounding science outreach, public engagement, widening participation and knowledge exchange beyond what the authors presented in their literature review? What insights to the field and definition of science communication were uniquely contributed by this study? In this the authors are also unclear. It would have been useful for the authors to compare and contrast at greater length their respondents’ definitions of these concepts to published scientific literature definitions.",
"responses": [
{
"c_id": "1597",
"date": "24 Sep 2015",
"name": "Samuel Illingworth",
"role": "Author Response",
"response": "Thank you for your comments, which were very insightful, and which we have used to address these flaws, thereby helping to improve the academic rigour of the paper. Estimating the number of active science communicators in the UK is beyond the scope of this study. However, given that this study aims to provide an initial scoping exercise into the thoughts and consistency of active science communicators across the UK, and taking into account the limited time frame and zero budget of this study, an ideal sampling size of between 50 and 100 participants was chosen for the survey. Given the limitations in budget (which also precluded a more in-depth interviewing / focus group approach), a convenience sampling strategy was adopted, in which the survey was advertised using the ‘psci-comm’ mailing list hosted by JISCMail, as well through the Twitter accounts of the authors, all of whom are active participants in science communication networks across the Twittersphere. The target audience were people that identified themselves as being active UK science communicators, which is why this particular mailing list was adopted. Given that the psi-comm mailing list contains several hundred active science communicators, and that between them the authors have several hundred Twitter followers that identify themselves as UK-based science communicators, it is disappointing that more people were not able to participate in the survey, but we believe that the number of responses is still sufficient for the purposes of this study. This has now been made clearer in the text. Regarding the two statements relating to science communication at an institutional level and the cross-sections of science communicators that were represented, we agree that it was inappropriate to make such generalisations, and so these have been removed from the text. We agree that Wordle was not the most academically rigorous way of presenting our data, and that word frequencies could be potentially misleading. As such the word clouds have been replaced with tables that outline the major themes for the thematic analysis, and the frequencies associated with them. A far more detailed description of how this thematic analysis was carried out using NVivo is also given. Through an open coding approach, descriptive saturation was reached, and this is also now discussed in detail in the text. In terms of the lack of demographic data that was collected in regards to this survey, we realise that this was an oversight, and we have commented on this in the text. However, we still believe that this study is worthwhile, and that it has presented a relatively concise set of definitions that can be used for further discussions."
}
]
},
{
"id": "9685",
"date": "17 Aug 2015",
"name": "Sarah R. Davies",
"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 topic, and one that is ripe for study. As the authors point out, there is a growing amount of activity in and funding for various kinds of science communication (they point to the phenomenon of 'Science 2.0', but they may also like to think about this relates to scholarship on Mode 2 knowledge production - e.g. see Etzkowitz & Leydesdorff (2000)). There is definitely lots going on, a mass of different terms for related activities, and a degree of slippage between different terminologies.As the authors themselves point out, though, there are some problems with their methodology, and I also feel that the analysis and discussion would benefit from a fuller engagement with the literature. I'll take the latter point first.To start with, I think the authors need to more clearly make a case for their focus on the terms that their analysis is based on (that's rather clumsily put - but I hope my point will become clear). Their starting point, unreferenced, is that \"Science communication is often analogously and interchangeably referred to as science outreach, public engagement, widening participation, and/or knowledge exchange\". Yes - but many other terms are also used, including dialogue, PUS, scientific literacy, tech transfer, third stream activity, and co-enquiry. Why were these terms selected (and indeed why not 'science communication' itself)? This sense of arbitrariness is heightened by a literature analysis that bounces around in a somewhat haphazard way. The authors ignore, for instance, work that has attempted to define some of these terms (Burns et al. (2003), whom they cite at the very start of the paper, offer an extended discussion of terms they see as similarly related to science communication, including scientific literacy and PUS; the work of the NCCPE - http://www.publicengagement.ac.uk - is also a valuable resource for seeing how different forms of science communication have been framed). By interpreting the 'participation' that the science communication literature often refers to (e.g. in the Rowe & Frewer article they cite) as being about 'widening participation' initiatives they also make a bridge to an area of university outreach and external relations that is infrequently discussed in the main body of science communication research (wrongly so, I think - but that's another discussion); rather, the term 'participation' tends to be used in the tradition of deliberative theory and public participation in science research and policy (a randomly chosen example would be Bogner (2012)). All this is to say that things like 'public engagement' and 'widening participation' actually have rather different histories, and different communities around them - so it doesn't surprise me, for instance, that some respondents hadn't heard of widening participation initiatives. These are likely to be organised quite different within university systems. I was also surprised that the authors didn't spend more time considering other empirical work on practitioner definitions of these terms or of science communication generally. Some of my work has treated this (not that it's necessarily essential reading - but see, e.g., Davies (2013), but Jason Chilvers, John C Besley, and Kevin Burchell, among others, have also published analyses of how scientists and other practitioners of public communication tend to define and understand it (e.g. Chilvers (2008); also Besley (2010) ). So - in short, I think both the framing of the study, and the analysis and discussion, would benefit from a more thorough engagement with the qualitative literature that has built up around the meaning and practice of science communication. I also have some comments about the methodology and analysis - largely to do with the need for more explanation as to what the former was, and why it was chosen. For instance, the authors note that the survey was \"advertised via email, social media accounts, and the ‘psci-comm’ mailing list\"; earlier, they say that the study aimed to understand opinion on definitions of the four terms \"in UK HEIs\". What, exactly, is the target participant group? Everyone working in UK HEIs? A certain subset of this population, those who are interested in science communication? The HEIs themselves, as institutions (i.e. as organisations with particular brands)? How was the sampling strategy (the distribution of the survey) designed to reach the desired population? In terms of the survey advertisement via \"email and social media accounts\": email to whom, and why, and which social media accounts (and why - but you get the picture...)? The PSCI-COMM list is distributed to a large group of those interested or working in or researching science communication, in the UK but also internationally. Is the target population therefore 'science communicators'? (In practice almost all respondents had participated in science communication in some way, so perhaps so. But this needs to be clear.) [Apologies - I've just re-read your comment in the conclusion that respondents only being active science communicators is a weakness of the study. But in that case, if you're interested in everyone working in UK HEIs from chancellors to cleaners, you need to justify why you used a survey methodology, and why you thought your sampling strategy would reach everyone.]I would also like to know more about how the analysis, and particularly the qualitative analysis, was carried out. Using word clouds is a rather basic means of analysis, as it tells us only how often a word is cited, not the context in which it is used or the meaning that is attached to it; discussion of the qualitative responses is therefore important. Were these coded in some way? How were themes robustly identified? The data evidently did not reach saturation (to use a term from grounded theory), as the authors emphasise the diversity in the definitions given. Do you think you needed a larger sample size, or is there so much interpretative flexibility in these terms that saturation would never be reached?The authors do note, in the conclusion, that the sample size is a weakness of the study, and I would agree with this. As far as I can tell you were also not able to identify the role or situation of your respondents, only whether they had participated in science communication activities in the past. This, to me, also weakens the results significantly - or at least represents a lost opportunity. There are big differences between different kinds of communities within universities and as participants in communication (e.g. scientists, outreach officers, admin staff, PR teams, tech transfer offices...). It seems unlikely that these communities would have homogeneous definitions of the terms in question - or even have heard of them all (as your findings suggest).I want to close - and I do apologise for the lengthy review - with a broader point. The issues that you touch upon raise some fascinating questions. I would love to know more, for instance, about the relations between individuals and teams in universities working on 'widening participation' and on public engagement-type projects, and I would be very curious to know if academic staff give different kinds of definitions of these terms to those who organise science communication on a professional basis. In this regard I would encourage you to look beyond finding the 'right', or even commonly used, definitions of particular terms. Alan Irwin has talked about 'third order' studies of science communication, which explore how different terms are mobilised by different groups, and the kinds of effects that this has (see Irwin (2008) In: Bucchi M and Trench B (eds), Handbook of Public Communication of Science and Technology, London and New York: Routledge). Your study suggests interesting ways to explore how very different meanings can be applied to the same terms - it would be great to hear more, in the future, about how these different meanings are made to matter in particular contexts (such as, to go back to the very beginning, moves towards Mode 2 and entrepreneurial universities). I wish you all the best with this future research.",
"responses": [
{
"c_id": "1598",
"date": "24 Sep 2015",
"name": "Samuel Illingworth",
"role": "Author Response",
"response": "Thank you for your comments, which we found extremely useful, and which have helped us to reshape the paper into a more effective and considered study. The comment regarding the choice of outreach, public engagement, widening participation and knowledge exchange, and why these were chosen for selection is a pertinent one. However, we believe that in the context of UK institutions, these are the phrases that are most readily used in relation to science communication. PUS and scientific literacy are terms that are, in our experience, used more in an American and European context, whereas tech transfer and third stream activity would arguably be covered by knowledge exchange. However, we agree that the wording in the text was a bit strong, and has now been changed. We have also made a note in the conclusions regarding that future studies should also present participants with an open-ended question to define any further terms within the science communication lexicon which they believe to be important, and why. Regarding the bounciness of the literature analysis, we think that the literature that we have engaged with illustrates that there is no concise definition of the terms that we have chosen, and as such serves its purpose as a useful introduction and rationale for this study. It is understood that the selected terms have different historical and academic contexts, but we believe that the literature that we have engaged with illustrates this point.As noted in the response to the previous reviewer’s comments, we have now addressed the issue of the sampling size and the sampling strategy, and accept that not doing so in the previous version of this paper was an oversight. It should have also been made more apparent that only the responses from UK-based participants were included in the analysis, and this has now been corrected for in the text. In addition to this, only the participants who reported as being active in science communication were included in the analysis; again this has now been made clearer in the text. We agree that Wordle was not the most academically rigorous way of presenting our data, and that word frequencies could be potentially misleading. As such the word clouds have been replaced with tables that outline the major themes for the thematic analysis, and the frequencies associated with them. A far more detailed description of how this thematic analysis was carried out using NVivo is also given. Through an open coding approach, descriptive saturation was reached, and this is also now discussed in detail in the text. Again, we acknowledge that the lack of sufficient demographic data reduces the potential depth of the analysis, representing a lost opportunity that we hope to address in future studies. This will include, as noted in the conclusions, investigating how different stakeholders define different aspects of science communication, and how and why this changes depending on job title, and both across and between institutes. Thank you for your suggestions regarding future studies, especially your comments regarding how definitions of the nomenclature may change depending on job role. This has now been further discussed in the conclusions where we talk about future work, where with a broader study and more demographic data such questions will be targeted. We agree that there may well be no ‘right’ definition of particular terms, but having broad and workable descriptions might well help better communication between the different practitioners of science communication."
}
]
},
{
"id": "9682",
"date": "19 Aug 2015",
"name": "Cornelia Lawson",
"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 looks at a series of, perhaps, interchangeable terms used in the context of science communication and knowledge exchange and how they are understood by those working in academia. It provides an informative and brief literature review and concise definitions for each of the terms: outreach, public engagement, widening participation and knowledge exchange, and presents results of an original survey of science communicators.The research question is important and timely as concepts of science communication and knowledge exchange merge and the impact agenda gains in importance in the UK and elsewhere. However, I have some mayor concerns regarding the empirical analysis. The sampling framework and the sample are not sufficiently described making it difficult to interpret the results. In particular, at several points the authors mention science communicators but do not specify whether these were the target population. They also make no estimate as to how many people were reached and which sectors they work in. It is therefore not clear whether the final sample can be considered representative (as is claimed in making use of the phrase 'fair cross-section').The sample is very small and one is left wondering whether interviews with a similar number of respondents would not have enabled a better analysis and insights. The final sample also includes employees of universities and of companies but it is not clear how they are distributed and whether answers differ between the two. This is of importance as some of your conclusions seem to assume a homogeneous institutional environment, i.e an academic work environment. More over, we do not know whether respondents are academics, administrators, or professional science communicators. No collecting such information is a major flaw.The use of word clouds to analyse your data strikes me as very odd. The word clouds add no information what so ever and I would suggest removing them. First of all because it can only highlight word frequency, which it does not do very accurately (e.g. it does not look for word stems), and because it does not allow to reader to know the actual frequency or importance of words but only their relative frequency and importance. Other packages such as NVivo should be used as it allows to identify relationships between words and to deduce linkages between the different answers provided.Alternatively a comparison of definitions found in the literature and definitions provided by respondents would provide more interesting results and allow some important insights into how concepts are diffusing.A few final remarks on the literature review: The article does not specify when public engagement and widening participation were introduced as concepts and/or policies in the UK and I think this could be added. The impact agenda within the UK's REF is mentioned towards the end of the review and from there it seems that outreach, public engagement and knowledge exchange are part of this agenda, while widening participation is not. Perhaps this could be commented on here and in the conclusion.",
"responses": [
{
"c_id": "1599",
"date": "24 Sep 2015",
"name": "Samuel Illingworth",
"role": "Author Response",
"response": "Thank you for your helpful comments, which we have taken on board, and which have been used to improve the overall quality of the paper. Regarding the sampling framework and the sample size, we agree that this was not discussed in enough depth, but as discussed in the response to the previous comments this has now been rectified and discussed in the main body of the paper. Any claims that the paper makes in terms of generalisations have also been re-addressed, as we agree that given the sample size this is not appropriate.We also agree that not collecting further demographic data in terms of job titles and roles was an oversight, and as discussed in the responses to the previous comments, and also in the paper itself, this is something that we intend to do in future studies, using the findings of this study as a starting point for further investigation. We accept that the use of word clouds to illustrate the key points in the data was at best inconclusive and at worst potentially misleading. However, this has now been addressed, with a more in-depth analysis using NVivo now provided, which allows for a more considered examination in terms of the relative frequency and importance of the responses. Regarding the introduction of these terminologies in terms of policies, concerns about “access” to high education began to emerge alongside the expansion of the university sector in the latter part of 19th Century, but a research agenda on differential access only began to emerge following the recommendations of the Robbins Committee in 1963 to expand university attendance. These concerns resurfaced in 1990s when the divide between universities and polytechnics ended, ultimately leading to a commitment by the 1997 Labour Government to again expand by the sector by tackling barriers to higher education. Consequently, Labour established OFFA. This has now been commented on in the literature survey. In terms of the relationship between widening participation and REF, widening participation tends to align with teaching outcomes, rather than research. However, it is accepted that there is a blurring of the boundaries, e.g. a research-focused public engagement event may well inspire someone to attend university who wouldn't have otherwise. In addition to this, it is important to note that widening participation will likely become part of the Teaching Excellence Framework – an outcome based model that the UK government proposes to evaluate quality of teaching. This has also been commented on in the conclusions, as suggested."
}
]
}
] | 1
|
https://f1000research.com/articles/4-409
|
https://f1000research.com/articles/4-891/v1
|
23 Sep 15
|
{
"type": "Research Article",
"title": "Repository of mutations from Oman: The entry point to a national mutation database",
"authors": [
"Anna Rajab",
"Nishath Hamza",
"Salma Al Harasi",
"Fatma Al Lawati",
"Una Gibbons",
"Intesar Al Alawi",
"Karoline Kobus",
"Suha Hassan",
"Ghariba Mahir",
"Qasim Al Salmi",
"Barend Mons",
"Peter Robinson",
"Nishath Hamza",
"Salma Al Harasi",
"Fatma Al Lawati",
"Una Gibbons",
"Intesar Al Alawi",
"Karoline Kobus",
"Suha Hassan",
"Ghariba Mahir",
"Qasim Al Salmi",
"Barend Mons",
"Peter Robinson"
],
"abstract": "The Sultanate of Oman is a rapidly developing Muslim country with well-organized government-funded health care services, and expanding medical genetic facilities. The preservation of tribal structures within the Omani population coupled with geographical isolation has produced unique patterns of rare mutations. In order to provide diagnosticians and researchers with access to an up-to-date resource that will assist them in their daily practice we collated and analyzed all of the Mendelian disease-associated mutations identified in the Omani population. By the 1st of August 2015, the dataset contained 300 mutations detected in over 150 different genes. More than half of the data collected reflect novel genetic variations that were first described in the Omani population, and most disorders with known mutations are inherited in an autosomal recessive fashion. A number of novel Mendelian disease genes have been discovered in Omani nationals, and the corresponding mutations are included here. The current study provides a comprehensive resource of the mutations in the Omani population published in scientific literature or reported through service provision that will be useful for genetic care in Oman and will be a starting point for variation databases as next-generation sequencing technologies are introduced into genetic medicine in Oman.",
"keywords": [
"Genetic Disease",
"Birth Defects",
"disease-associated mutation data",
"Sultanate of Oman"
],
"content": "Introduction\n\nOman is situated in the South East of the Arabian Peninsula along the East coast of the Arabian Gulf (Figure 1). It has its borders with United Arab Emirates to the North, Saudi Arabia to the West and Yemen to the South West. Oman is the second largest territory in the Arabian Peninsula with an area of 82,000 square miles and a coastline length of 1,300 miles. The native Omani population comprises around 2.2 million inhabitants, and the rate of annual population increase is approximately 25 per 1000. Oman has a young population with nearly half of the population being under 15 years. The Omani population is characterized by a high growth rate, large family size, consanguineous marriages, and the presence of genetic isolates.\n\nClinical genetic services were introduced in the Sultanate of Oman in the past decade and they have become an important component of health care. This greatly facilitated the systematic collection of data on genetic diseases and birth defects in the past few decades. With the inauguration of the National Genetic Center in 2013, the existing clinical genetic services were supplemented by sophisticated genetic laboratory services.\n\nThe amount of published data available on genetic disorders in the Sultanate is considerable. There were a few previous attempts to list the genetic diseases reported in Oman1–4 and to link them to specific population groups and geographic locations5,6, analyze population structure7, and to estimate the impact of genetic disorders and birth defects on the community4 and summarize the genetic services available8. The advances in bioinformatics required to annotate human genomic variants and to place them in public data repositories have not kept pace with their discovery. The deposition of such data in the public domain is essential to maximize both their scientific and clinical utility9.\n\nHence, in the current study we present a comprehensive compilation of germline mutations in nuclear genes associated with human disease in the Omani population.\n\n\nMaterials and methods\n\nThe wealth of genetic variant data in Omani nationals was collected from multiple sources which form a basis for research into genetic conditions reported from Oman. Multiple sources of data were reviewed to form repository of mutations in Omani nationals introduced in this paper. The sources of data included:\n\n(1) 1993–2015 records of patients consulted by clinical geneticists of the Royal Hospital, the largest tertiary hospital in Oman;\n\n(2) 2008–2015 publications curated from PUBMED on birth defects and genetic conditions in Omani nationals. The keywords used were: “Oman”, “Genetic disorders”, “Birth defects”, “mutations”;\n\n(3) 2012–2014 commercial laboratories referral registry at the Royal Hospital for the samples tested overseas.\n\n(4) The internal genetic variant repository of the National Genetic Center <<HTTP://ogvd.net>>;\n\nThe data presented in this article was manually curated. The OMIM identifiers, Phenotype MIM accession numbers, Phenotype name (OMIM), mutation descriptions, and relevant publications with PMID numbers were all collected from the NCBI database repository. All unavailable through PubMed mutation details were checked with ClinVar, LOVD and CentoMD. The details of unpublished mutations are not included in the present study and feature in Table 2 as “Novel mutations”.\n\n\nResults\n\nIn this study, a wide range of genetic conditions with known mutations collected in Omani nationals were analysed. The disease classifications are comprised of 44 gene variants causing neurodevelopmental disorders, 21 inborn errors of metabolism, 13 endocrinopathies, 15 skeletal dysplasias, nine disorders of the immune system, four hereditary blood disorders as well as other National groups (Table 1).\n\nExtensive genetic studies were performed in Oman for Genetic Blood Disorders and various conditions leading to intellectual disabilities, mental and physical handicap.\n\nIn total more than 150 rare genetic disorders were listed in Table 2 and Table 3 with relevant OMIM numbers, PubMed ID (PMID), Gene/Locus name, nucleotide(s) change(s) and the source of the data (PubMed ID Number/ OMIM/ClinVar/LOVD/CentoMD). The names of genetic conditions in Table 2 are stated as found in OMIM “Phenotype-Gene Relationships” table as “Phenotype” arranged in alphabetical order. In Table 3, we present a separate list of 69 known mutations (11–15) that were collected through service provision at the Hemoglobinopathy Laboratory at the National Genetic Center in Oman.\n\nThe disorders are listed in alphabetical order along with the mutations detected in Omani patients. Novel genes and/or mutations identified for the first time in Omani nationals are marked by an asterisk (*). Unpublished mutation data referred as “Novel mutations” would be updated following publication, currently source stated as “NA”.\n\nThe different mutations reported by the National Genetic Center in patients with Hemoglobinopathies in Oman. Novel mutations are indicated by an asterisk (*) indicated to the left side of the mutation. Mutations are listed in ascending order based on nucleotide position.\n\nFor the majority (85%) of rare disorders presented in Table 2, data was derived from publications. The original mutations identified for the first time in Omani population constitute more than half of rare disease data presented in Table 2.\n\n\nDiscussion\n\nSoon after the completion of the Human Genome Project in 2003, it was clear that the genetic data collected until then presented only a glimpse of the complexity of the human genome and the significance of genetic variants in human disease. Since then, genetic researchers have unearthed innumerable variants that are not only individual-specific; but also ethnicity-, population- and country-specific. Human genetic variation databases have significant implications for both diagnostic and predictive medicine. Often, the pathogenicity of rare mutations is primarily assessed through multiple reports of occurrence in diseased patients that are documented and routinely updated in mutation databases. Given the fact that gene mutations and their frequencies in many Mendelian disorders differ widely between different ethnic groups, even within a country, national databases are highly valuable resources for studies on disease-gene associations, population diversity and genetic history10.\n\nThe catalog of Omani mutations presented here will therefore represent a valuable resource that may guide mutation analysis in Omanis suspected of having genetic disease. Unique circumstances in Oman with government-funded comprehensive healthcare throughout the country, and the national coverage for clinical genetics has made the present study possible. Future efforts will be required to extend this database to cover the full spectrum of mutations and population specific variants.\n\nThe disease-associated mutation data presented (Table 1, Table 2, Table 3) show a considerable proportion of novel disease genes as well as novel genetic variants within the Omani population. This was expected due to the presence of inbred and geographically isolated communities, the practice of consanguineous marriages, all of which have tended to skew the allelic spectrum toward rare and private variants within the Omani population. In addition to this, the list of genetic variants also reveals known mutations that were previously reported in certain non-Omani populations, thereby reflecting the historic genetic admixture that occurred in Oman, along the trade routes of a once powerful Omani empire and its foreign colonies. Many of the mutations reported are unique to the Omani population, suggesting a founder effect.\n\nThe interest in genetic testing is growing among physicians aiming to provide better medical care and genetic disease prevention. The data collected largely represent mutations of rare autosomal recessively inherited disorders in Oman. The mutation data in Table 2 can be searched by OMIM number, or by disease name. The names of diseases in Table 2 were chosen as described in OMIM in “Phenotype-Gene Relationships” table as “Phenotype” in order to ease finding specific genetic disorders by name.\n\nThe number of collected mutations among different disease groups (Table 1) reflect the frequency of disorders in the Omani population, the burden caused by genetic diseases4, and the interests of individual clinicians in genetic testing.\n\nThe knowledge of the genetics of Hemoglobin disorders is among the best in Oman due to national preventive programs and research starting from the 1990s. It is not surprising that around a third of all mutations known in Omani population to date are in four genes causing Hemoglobin disorders (Table 1, Table 3). The birth prevalence of infants with a hemoglobin disorder was recorded as 3.5–4.7/1,0007,11. The frequency of hemoglobin disorders in Oman is among the highest in the world, and may reflect natural selection due to advantage for survival, in the heterozygous state, against malaria. Around 10% of Omani nationals are carriers of the allele for sickle cell anemia, 2–3% carry an allele for Beta-thalassemia and 45% are carriers of an alpha-thalassemia allele12–15.\n\nGenetic disorders causing disabilities and handicap are of great concern. These are different groups of rare disorders leading to intellectual disability or physical handicap requiring detailed clinical classification, genetic testing, research and preventive measures. The high prevalence of birth defects and genetic conditions in Omani communities causes social, psychological and financial difficulties4. The development and use of national mutation data is of importance to Omani medical care because it not only allows the genetic burden of disease to be quantified, but also provides diagnosticians and researchers access to an up-to-date resource that will assist them in their daily clinical practice and biomedical research9. National databases for genetic variants are also significant from the perspective of preventive healthcare. There is a significant correlation between the occurrence of rare genetic variants associated with Mendelian disease and the burden of morbidity from complex diseases within a population. Heterozygous carriers for recessive disease genes do not manifest the recessive disease but may be at risk of developing complex trait conditions with some similarity in phenotype. For example, heterozygote carriers of mutations in the ataxia telangiectasia gene locus are reportedly susceptible to breast cancer16, and heterozygote carriers of mutations in the glucocerebrosidase (GBA) gene causing Gaucher disease are at an increased risk for Parkinson disease17,18. Hence, the collection of genetic variant data in national databases will contribute significantly to the prevention of genetic diseases in the population and might greatly impact the management of complex trait diseases in the future. Genetic scientists and international consortiums studying human genetic variation are increasingly interested in dissecting the interplay between genetic makeup and environmental influences on the pattern of diseases worldwide. Current research is expected to create a foundation for the national data online for the benefit of Oman Healthcare.\n\n\nData availability\n\nThis article was prepared to introduce the first Omani genetic variation database. This data is available online at HTTP://ogvd.net; raw datasets are not available for Royal Hospital laboratory and clinical data, as the registry contains confidential information that could not be deidentified.",
"appendix": "Author contributions\n\n\n\nRA and MB conceived the study. RA prepared the first draft of the manuscript. RA, AHS, ALF, GU, MH, HS and ASQ carried out the research. HN, AI, KK, MB and RP contributed to the preparation of the manuscript. All authors were involved in the revision of the draft.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no funding was involved in supporting this work.\n\n\nReferences\n\nTadmouri GO, Al Ali MT, Al Khaja N: Genetic disorders in Arab World: Oman. Publication of Center for Arab Genomic Studies, Dubai. 2008; 3. Reference Source\n\nRajab A: Genetic Disorders in Oman. In: Teebi AS (ed): Genetic Disorders Among Arab Populations. Berlin: Springer, 2010; 473–490. Publisher Full Text\n\nRajab A, Patton M: Genetic diseases in the Sultanate of Oman. In: Dhavendra Kumar (ed.), Genomics and health in the developing world; Oxford monograph on medical genetics. Oxford University Press, 2012; 678–693. Publisher Full Text\n\nRajab A, Al Salmi Q, Jaffer J, et al.: Congenital and genetic disorders in the Sultanate of Oman. First attempt to assess healthcare needs. J Community Genet. 2014; 5(3): 283–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRajab A, Bappal B, Al-Shaikh H, et al.: Common autosomal recessive diseases in Oman derived from a hospital-based registry. Community Genet. 2005; 8(1): 27–30. PubMed Abstract | Publisher Full Text\n\nAl-Thihli K, Al-Murshedi F, Al-Hashmi N, et al.: Consanguinity, endogamy and inborn errors of metabolism in Oman: a cross-sectional study. Hum Hered. 2014; 77(1–4): 183–8. PubMed Abstract | Publisher Full Text\n\nRajab A, Patton MA: Analysis of the population structure in Oman. Community Genet. 1999; 2(1): 23–5. PubMed Abstract | Publisher Full Text\n\nRajab A, Al Rashdi I, Al Salmi Q: Genetic services and testing in the Sultanate of Oman. Sultanate of Oman steps into modern genetics. J Community Genet. 2013; 4(3): 391–397. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatrinos GP, Smith TD, Howard H, et al.: Human Variome Project country nodes: documenting genetic information within a country. Hum Mutat. 2012; 33(11): 1513–9. PubMed Abstract | Publisher Full Text\n\nPatrinos GP, Cooper DN, van Mulligen E, et al.: Microattribution and nanopublication as means to incentivize the placement of human genome variation data into the public domain. Hum Mutat. 2012; 33(11): 1503–12. PubMed Abstract | Publisher Full Text\n\nRajab A, Patton MA: Major factors determining the frequencies of hemoglobinopathies in Oman. Letter to the Editor. Am J Med Genet. 1997; 71(2): 240–242. PubMed Abstract | Publisher Full Text\n\nAlkindi S, Al Zadjali S, Al Madhani A, et al.: Forecasting hemoglobinopathy burden through neonatal screening in Omani neonates. Hemoglobin. 2010; 34(2): 135–44. PubMed Abstract | Publisher Full Text\n\nWhite JM, Christie BS, Nam D, et al.: Frequency and clinical significance of erythrocyte genetic abnormalities in Omanis. J Med Genet. 1993; 30(5): 396–400. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaar S, Hussein HM, Merghoub T, et al.: Spectrum of beta-thalassemia mutations in Oman. Ann N Y Acad Sci. 1998; 850: 404–6. PubMed Abstract | Publisher Full Text\n\nDaar S, Hussain HM, Gravell D, et al.: Genetic epidemiology of HbS in Oman: multicentric origin for the βS gene. Am J Hematol. 2000; 64(1): 39–46. PubMed Abstract | Publisher Full Text\n\nAthma P, Rappaport R, Swift M: Molecular genotyping shows that ataxia-telangiectasia heterozygotes are predisposed to breast cancer. Cancer Genet Cytogenet. 1996; 92(2): 130–4. PubMed Abstract | Publisher Full Text\n\nGoker-Alpan O, Schiffmann R, LaMarca ME, et al.: Parkinsonism among Gaucher disease carriers. J Med Genet. 2004; 41(12): 937–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSidransky E, Samaddar T, Tayebi N: Mutations in GBA are associated with familial Parkinson disease susceptibility and age at onset. Neurology. 2009; 73(17): 1424–5, author reply 1425–6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10505",
"date": "09 Oct 2015",
"name": "Prajnya Ranganath",
"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 is well-written and though it does not present any new findings, it can be used as a reference database for Omani mutations. There are a few suggestions for the authors:a. Standard HGVS nomenclature should be followed for all the mutations and the authors should preferably stick to one format i.e. either DNA or protein notation. There is no uniformity in the present nomenclature followed in Table 2.b. Number of patients in whom each mutation was found, should be indicated – this would give an idea about any preponderance of specific mutations in this population.c. Functional validation studies, if available, or at least the mutation prediction scores should be mentioned for the novel sequence variants identified, which will help create a comprehensive database of new likely-pathogenic variants. Again, if the number of patients in whom each of these novel variants were identified is mentioned, we will get an idea as to whether these novel variants were present in more than one case and we can get further proof of the pathogenicity of these mutations.d. It would also be interesting to see if there is any ethnic group-wise preponderance of genetic diseases or mutations in the various subsets/ tribes/ regional groups that constitute the Omani population.",
"responses": []
},
{
"id": "10729",
"date": "14 Oct 2015",
"name": "Shaillay Dogra",
"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\nIts useful to have region/country specific databases that present a better representation of the prevalence and incidence of region specific diseases and health issues. Putting such data in context of whats known globally shall help in better interpreting whats peculiar to local population and possibly raise some interesting questions on why so? For example, is it the genetic structure or something in the environment?Some specific comments follow:Authors mention existence of tribal structures, consanguineous marriages etc in Omani society. Is it possible to delineate the effects of these factors on the reported mutations? For example, is this reported in the papers that the authors have collated data from and have the authors noted this aspect in their database? How does the mutation data reported here compare with mutation patterns or frequency seen in other populations? Do the authors provide information on this or link out to other similar resources from other countries? If a physician in Oman is looking up some mutation from this database and wants to know if this is something specific to Oman or is a more general mutation found in other populations too, would this information appear automatically in the database; or could they perform a manual search of the database? Do the authors want to comment about any data privacy issues that maybe associated with such a database, if any and to what extent? Are there any attributes on quality of data in the database? perhaps based on the technique used in the original paper or some other measure some quality metric can be assigned to the mutation information recorded in the database? It would be helpful if authors were able to illustrate out a case or two on how they expect a doctor in Oman to be able to use this database in a real clinical setting; to illustrate the usefulness of the database from a simple collection of data to something that can be used on a more regular basis by doctors in clinical setting.",
"responses": []
},
{
"id": "10917",
"date": "26 Oct 2015",
"name": "Milan Macek",
"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 pioneering piece of work which presents an authoritative overview of a representative mutation analysis (300 pathogenic variants in over 150 monogenic disorders) in a large cohort of Omani patients. Methodology is state of the art, collaboration with colleagues at Charite Berlin commendable and results which are important for DNA diagnostics in the Omani population, and beyond. The conclusions, are relevant and directly applicable. I fully approve this article without any comments to the Authors. Importantly, data are also freely accessible online.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-891
|
https://f1000research.com/articles/4-886/v1
|
22 Sep 15
|
{
"type": "Opinion Article",
"title": "A Taxonomy to Support the Statistical Study of Funding-induced Biases in Science",
"authors": [
"David Wojick",
"Patrick Michaels",
"Patrick Michaels"
],
"abstract": "The biomedical community is a leader in research on bias in science, including funding-induced bias. To facilitate this research, we have developed a taxonomy of fifteen different types of potential funding-induced bias. We describe each type of bias, as well as giving a snapshot of existing research and briefly discussing the potential for various forms of statistical analysis. We also introduce the concept of an amplifying bias cascade, wherein bias builds through successive iterations.",
"keywords": [
"Bias",
"Biased",
"Cascade",
"Funding",
"Scientometrics",
"Peer review",
"Paradigm protection",
"Taxonomy"
],
"content": "Introduction\n\nThe purpose of this report is to provide a framework for doing statistical research on the problem of funding-induced bias in science. In recent years the issue of bias in science has come under increasing scrutiny within the scientific community. The research question is does biased funding skew research in a preferred direction, one that supports an existing mission, practice, product, policy or paradigm?\n\nOur working definition of “funding-induced bias” is any scientific activity where the prospect of funding influences the result in a way that benefits the funder.\n\nWhile the basic concept of funding-induced bias is simple, the potential forms that this bias might take are far from simple. Science is a complex social system and funding is a major driver. In order to facilitate research into funding-induced bias it is necessary to isolate specific kinds of bias. Thus the framework presented here is basically a taxonomy of types of funding-induced bias.\n\nFor the purposes of future research the concept of funding-induced bias is analyzed in the context of the various practices in science where bias can occur. Fifteen different scientific practices are analyzed, ranging from the budgeting and funding for research to the publishing and communication of results. We make no distinctions regarding the source of the funding. The funding organization may be a commercial firm, a non-profit entity or a government agency, or even an individual.\n\nWhat we have found is that some types of bias are being studied extensively, including quantitatively. Various aspects of peer review and publication bias, especially in biomedicine, appear to be the most heavily researched types of bias. The role of funding in inducing bias is frequently alluded to as a potential financial conflict of interest. But the role of funding is not the focus of most research into bias, which tends to look more at the practice of bias than at its cause. Thus a new research thrust is likely needed.\n\nThe concept of funding-induced bias is one that specifically occurs in the discussion of and research into some of the fifteen bias research types that we have identified, but not in all of them. It tends to occur where specific funding is the issue. We are here using the concept more broadly, to include cases where the funding may be somewhat distant from the activity in question, such as in the communication of research results.\n\n\nCascading amplification of funding-induced bias\n\nIn this report we are mostly concerned with individual types of funding-induced bias. But there is an intrinsic sequence to the various biases we have identified and this raises the possibility of cascading amplification. By amplification we mean one biased activity is followed by another, such that the first bias is increased.\n\nA simple, and perhaps common, example of amplification might be when the hype in a press release is exaggerated in a news story. Let’s say the press release overstates the importance of the research result, but with some qualification. The news story then reports the result as a great breakthrough, far more strongly than the press release, ignoring the latter’s qualifications. In this way the original bias has been amplified.\n\nThere is also the possibility of cascading amplification. This is the case where one biased activity is followed by multiple instances of amplification. Using our example, suppose a single biased press release generates many different news stories, which vie with one another for exaggeration. This one-to-many amplification is properly termed a cascade.\n\nMoreover, there is the possibility of cascading amplification on a very large scale and over multiple biased stages. Here is an example of how it might work.\n\n1. A funding organization receives biased funding for research.\n\n2. They issue multiple biased Requests for Proposals (RFPs).\n\n3. Multiple biased projects are selected for each RFP.\n\n4. Many projects produce multiple biased articles, press releases, etc.\n\n5. Many of these articles and releases generate multiple biased news stories.\n\n6. The resulting amplified bias is communicated to the public on a large scale.\n\nOne can see how in this instance a single funding activity, the funder’s budget, might eventually lead to hundreds of hyperbolic news stories. This would be a very large scale cascading amplification of funding-induced bias.\n\n\nProtecting paradigms as a source of bias\n\nThomas Kuhn pointed out on his groundbreaking work “The Structure of Scientific Revolutions” that fundamental beliefs can take over a scientific field1. He called these entrenched beliefs “paradigms” and noted that they tend to direct scientific thinking in specific directions. Once these beliefs become entrenched they are difficult to dislodge, despite growing evidence that they may be incorrect. Moreover science, like any human endeavor, is subject to fads and fashions.\n\nClearly processes like paradigms and fashions can influence the funding of research. Kuhn notes that the paradigms tend to specify not only what the important questions are, but also what the answers are expected to look like, and these questions and answers are the focus of research funding. At some point this influence can become bias, especially when the paradigm becomes questionable. This may be especially true when the outdated paradigm is built into the mission, products or policies of a research funding organization.\n\nBiased funding in turn biases the funded research in complex and subtle ways. The purpose of this project is to systematically analyze many of these forms of funding-induced bias in science, in order to further future research. It should be noted however that this sort of bias need not be dishonest, and often is not, even though funding is involved. As Kuhn points out, defending a paradigm is the norm in scientific activity. Thus many of the biases are basically ideological in nature. Funding is simply part of the ideology, often a central part.\n\n\nIndicators of possible bias: controversy and allegations\n\n1) Bias does not go unnoticed, so controversy over specific aspects of the science related to a funding organization’s mission may be evidence of funding-induced bias. Controversy may include one or more of the following general aspects of the science.\n\na. Hypotheses being assumed or defended.\n\nb. Methods used in funded research.\n\nc. Assertions of what are claimed to be established facts.\n\nd. The use of specific models and assumptions in research.\n\n2) Allegations of specific practices of bias. The strongest evidence of bias may be specific allegations, along the lines of the fifteen practices of bias described at length below.\n\n\nApproaches to the research: test the practices in question for bias, preferably using quantitative statistical methods\n\nThe combination of specific sources and levels of bias, with aspects of controversy and allegations of biased practices yields a large number of specific possible cases that can be investigated individually. Thus the first step in research will often be to determine the specific case or cases in question. The goal is to be precise as to the possible source, scope, science and type of bias involved.\n\nFor example, the specific case may indicate which documents need to be analyzed for evidence of funding-induced bias. In particular, the mission aspect is a good starting point for bias research, because bias that is not related to organization’s mission is unlikely to be funding-induced.\n\nHowever, some of our fifteen bias practices are not directly funded by funding organizations. Rather the funding inducement is indirect, rendered by the community as it were. For example, publication in leading subscription journals is often not funded but it is an important indirect component of competing for future funding.\n\n\nAssessing the potential for quantification of each type of bias\n\nThe analysis below of each of the fifteen practices of bias includes a brief assessment of the potential for quantification. This assessment includes suggesting feasible approaches to successful quantification for each practice. This quantification assessment typically includes the following issues.\n\nIs there enough data to support quantification? For example, peer review and selection of proposals and journal articles tend to be black boxes, with little available data. Citations and co-authorship might be viable proxies for peer review. Persistence might be a proxy for selection.\n\nHow best to sample the available data? Computer based sampling methods, if feasible, are preferable to manual methods, because the latter are relatively expensive. Randomized sampling methods are preferable to convenience samples but are not always possible. Likewise, is the available data complete or partial? Partial data is often self sampled, which limits the scope of the resulting conclusions. Is a lot of processing of the data involved, before it can be analyzed? If so then how should it be done?\n\nWhat is the suggested best method of quantification? In particular is it subjective or objective, that is, is human judgment and classification of the data involved, or just simple counting of clearly defined instances. In the latter case the analysis might be done by computer, which is typically cheaper than manual analysis, depending on the amount of programming involved.\n\nAre indirect methods required? Where direct data is not available, using proxy data may be feasible, but this involves linking the proxy to the bias in question. Semantic approaches may also be feasible. For example, in the case of the hyping of research results in press releases, an approach to quantification might be by counting the occurrence of potentially hyperbolic words in a sample of press releases but not in the related abstracts or articles.\n\n\nPotential practices of funding-induced bias\n\nIn this section we briefly describe fifteen specific practices of bias in the context of funded scientific research. For convenience, as well as introductory discussion, these fifteen are first listed below as a kind of table of contents for this section:\n\n1. Funding agency programs that have a biased focus.\n\n2. Agency Strategic Plans, RFPs, etc., with an agenda, not asking the right questions.\n\n3. Biased peer review of research proposals.\n\n4. Biased selection of research proposals by the agency program.\n\n5. Preference for modeling using biased assumptions.\n\n6. Biased peer review of journal articles and conference presentations.\n\n7. Biased meta-analysis of the scientific literature.\n\n8. Failure to report negative results.\n\n9. Manipulation of data to bias results.\n\n10. Refusing to share data with potential critics.\n\n11. Asserting conjectures as facts.\n\n12. False confidence in tentative findings.\n\n13. Exaggeration of the importance of findings by researchers and agencies.\n\n14. Amplification of exaggeration by the press.\n\n15. More funding with an agenda, building on the above, so the cycle repeats and builds.\n\nWhile each of the biased practices listed above may occur in isolation, there is also a potential sequence to them, a cascade as it were. Science is a complex social system and funding is a major driver. Some of the practices listed above do not involve direct funding, but each can clearly be influenced by the existence or mere prospect of such funding.\n\nEach of these fifteen specific biased practices is discussed briefly below. Taken together they provide a kind of “field guide” to funding-induced bias.\n\n1. Funding agency programs that have a biased focus.\n\nIn some cases sponsors fund entire research programs that may be biased in their very structure. For example by ignoring certain scientific questions that are claimed to be important or by supporting specific hypotheses, especially those favorable to the organization’s goals.\n\nOrganizational funding requests and final research budgets are sometimes public documents, which are available for analysis. In many cases both the request and the funding occurs at the program level. Therefore, one basic approach to looking for bias is to examine how the funds are allocated to various research programs and questions. For example, suppose there are two competing hypotheses, one of which favors an organization’s mission, product or policy, while the other does not. Heavy funding of the former, compared to the latter, might be evidence of bias.\n\nGiven that this bias might be measured in dollars there is an excellent prospect for quantification. However, the budget documents might not break the dollars out into the funding categories needed to do the bias analysis, which presents a data problem. The use of proxies or estimation techniques may be necessary in such cases. Relatively subjective interpretation may also be required.\n\n2. Strategic Plans, RFPs, etc., with an agenda, not asking the right questions.\n\nResearch proposals may be shaped by Strategic Plans and Requests for Proposals (RFP’s). These documents often specify those scientific questions that the funding organization deems important, hence worthy of funding. Thus the resulting research proposals may be biased, speaking to what the funder claims is important rather than what the researcher thinks.\n\nThere is a small, but interesting, research topic called the “funding effect”. Full text Google Scholar search for 2010–2014 gives just 211 hits, of which just 5 find this term in the title. Expanding the period to 2002–2014 gives 329 full text hits and 8 in the title. It appears that the term “funding effect” was coined by Sheldon Krimsky around 2005 and most of the title occurrences are in papers by him. Thus there may be increasing attention to this concept but the literature is still very small. Moreover, most of the focus is on the biasing effect of commercial funding, such as by drug companies. For a Krimsky review article see “Do Financial Conflicts of Interest Bias Research? An Inquiry into the “Funding Effect” Hypothesis\"2.\n\nA much more common concept related to funding-induced bias is financial conflict of interest (FCOI). Google Scholar search for “FCOI” in titles for the period 2010–2014 gives zero hits. However it does find 186 occurrences in searching the full text, which suggests some research interest. Searching for the full phrase “financial conflict of interest” gives just 9 hits in titles, but over 5,000 in full text. These appear to be mostly research on either biomedical or professional activities.\n\nSearching on the broader concept phrase “conflict of interest” gives over 600 occurrences in titles. However, most the top hits appear to be either guidance or disclosures, not research on conflict of interest. Full text search gives over 240,000 hits. This very large number appears to be the effect of widespread conflict of interest policies, such that many articles include conflict disclosure clauses.\n\nThere are numerous ways in which the research funders can say what they are looking for. These are probably some of the best sources of evidence of bias in research funding.\n\nExamples include strategic plans, requests for proposals and clarifications or amendments thereto, scientific conference presentations by funding organization officials, pre-proposal conferences, as well as funding organization reports on the science, especially in relation to their mission.\n\nAnalysis of these sources is likely to be interpretative and opportunities for quantitative analysis may be limited. However, an example of a quantitative analysis might be the amount of text devoted to a hypothesis that supports an organization’s mission, product or policy, compared to the amount given to a competing hypothesis. Another might be patterns of occurrence of apparently biased statements in multiple sources. Where the documents include funding levels there is also the possibility of finding monetary measures of bias.\n\n3. Biased peer review of research proposals.\n\nThis bias may involve rejecting ideas that appear to conflict with the established paradigm, funding agency mission, or other funding interest. See also Bias #6: Biased peer review of journal articles and conference presentations.\n\nPeer review bias is the subject of considerable public discussion in the scientific community, as well as extensive scientific research. However, peer review is also used in the selection of papers to publish in scholarly journals and much of the discussion does not distinguish between peer review of proposals and articles. Thus there is some overlap between the literature snapshot provided here and that given under Bias #6 (Biased peer review of journal articles and conference presentations).\n\nA Google Scholar search on articles published 2010–2014 with “peer review” in the title gives about 3000 hits, which suggests a great deal of research. To be sure, some of these hits are false in the sense of not being analyses of peer review, but bias is mentioned frequently in the snippets so a lot of the research is focused on that topic. It appears that most of this research is focused on publications, not proposals.\n\nFull text search gives over 200,000 hits. This large number suggests that the term “peer review” probably occurs frequently in passing. A major review of peer review bias that covers both proposals and publications was published by Lutz Bornmann, entitled simply “Scientific peer review”3. Google Scholar lists 120 citations for this article so it is widely recognized.\n\nUnfortunately, the peer review process is typically not publicly available. This is especially true for those proposals that are rejected. Nether the proposals or the reviews, or even the names of the reviewers, are typically available for bias analysis.\n\nThus the prospects for bias research might be limited in this case, because of the secrecy, or they might involve indirect methods. For example, one might survey the researchers in the pool of candidates from which the reviews for a given program or project are likely to have been drawn, looking for evidence of bias.\n\nIn any case the prospects for simple quantification would seem to be limited, with a lot of interpretation required. Simply getting good data is the first research challenge.\n\n4. Biased selection of research proposals by the funding organization.\n\nThe selection of proposals is ultimately up to the funding program officers. As with the selection of peer reviewers, there is some concern that some funding organizations may be selecting research proposals specifically to further the organization’s agenda.\n\nA Google Scholar search on “biased funding of proposals” reveals some research on bias in proposal selection. However, it appears to be mostly focused on issues other than missions, products and policies. Topics include racial bias, gender bias and avoiding risky projects.\n\nGoogle Scholar gives about 25,000 hits for documents containing all three of the terms “proposal\", “funding” and “bias” in full text, for the five year period 2010–2014. Some of these relate to bias in selecting proposals for funding.\n\nWhen a proposal is selected for funding there may be some form of public notice, such as a press release, which can be used for bias research. However, the amount of information given may vary from case to case, ranging from a mere abstract to a detailed discussion of the technical proposal. The amount of funding may or may not be disclosed. Some funding organizations provide a compilation of funded proposals, which may facilitate comparisons and the search for funding patterns that might suggest bias in selection.\n\nUnfortunately the many proposals that are not funded are seldom made available. This secrecy makes it much more difficult to look for bias in proposal selection. After all, bias can be as much a matter of which proposals are not selected as it is about which are selected.\n\nGiven that dollar amounts are involved there is the potential for quantification of bias in funding. There is also the matter of the number of proposals funded and other measurable features of selection. This might include who receives how much funding, what the funding is for, etc. All things considered the potential for quantification is relatively high for some aspects of bias in proposal selection. The fact that there is little information available about the many proposals that are not selected is certainly a hindrance.\n\n5. Preference for modeling using biased assumptions.\n\nThe use of computer modeling is now widespread in all of the sciences. There is a concern that some funding agencies may be funding the development of models that are biased in favor of outcomes that further the agency’s policy agenda.\n\nUnfortunately, “bias” is a technical term in the modeling literature, making it difficult to find studies that are looking specifically at funding related bias. Google Scholar estimates about 230,000 hits in the five year period 2012–2014 for studies using both “bias” and “modeling” in their text.\n\nAdding the term “politically” reduces the hits to about 16,000 but these appear to be mostly modeling political processes, not looking at political bias in modeling itself. Many are focused on media bias. The same appears to be true for Google searches.\n\nBy the same token, Google Scholar search on “flaws” and “modeling” finds about 22,000 studies but most appear to be about modeling flaws, not flaws in modeling.\n\nGoogle Scholar full text search on “incorrect model” gives about 2,900 hits but these appear to be mostly technical discussions of modeling or specific models, unrelated to possible funding bias.\n\nThere appears to be very little scientific research on potential funding-induced bias in the construction or use of scientific models. This is surprising, given the extent to which models are used in developing and defending paradigms, products, missions and policies. This appears to be a major gap in policy related research. It is possible that a lot of research on biased models is being done in connection with providing comments on proposed regulations and similar policy efforts, where these are based on modeling. Apparently Google and Google Scholar do not cover these document domains.\n\nAssessing funding bias in computer models may be difficult, for several reasons. These models can be very complex and technical. They also may be proprietary, or only run on very large computers. These difficulties may explain the apparent lack of research on funding-induced bias.\n\nOne approach might be to mine the technical discussion of the model or models in question, as well as the model documentation. The modelers may even be openly biased. Here the primary challenge may be to relate the bias to the funding organization’s policy, product, mission or paradigm in question.\n\nWhere the model code is available it may be possible to run it using assumptions that differ from those supporting the agency mission or actions. Or it may be possible to develop an alternative model. Even a relatively simple model can indicate that a more complex model is biased. In some cases there may even be multiple models giving different results. It may also be possible to find modelers who are familiar with the code and who know where biases may be hidden. But due to the highly technical nature of models this may be a difficult line of research.\n\nNote too that modeling bias may be due to the selection or manipulation of input data, rather than to the construction of the model itself. Looking at the input data is a different research approach.\n\nAs for quantification, while computer models are mathematical, the assessment of model bias may not be statistical in nature. The goal may be to quantify the magnitude of the bias, rather than the frequency of its occurrence.\n\n6. Biased peer review of journal articles and conference presentations.\n\nThis issue is analogous to the potential bias in peer review of proposals, as discussed above. As in that case, this bias may involve rejecting ideas that conflict with the established paradigm, agency mission, or other funding interests.\n\nPeer review bias is the subject of considerable public discussion in the scientific community, as well as extensive scientific research. However, peer review is also used in the selection of proposals to fund and much of the discussion and research does not distinguish between peer review of proposals and articles. Thus there is some overlap between the snapshot provided here and that given under Bias #3 (Biased peer review of research proposals).\n\nA Google Scholar search on articles published 2010–2014 with “peer review” in the title gives about 3000 hits, which suggests a great deal of research. To be sure, some of these hits are false in the sense of not being analyses of peer review, but bias is mentioned frequently in the snippets so a lot of the research is focused on that topic. It appears that most of this research is focused on publications, not proposals. Full text search gives over 200,000 hits. This large number suggests that the term “peer review” probably occurs frequently in passing.\n\nMuch of the research into biased peer review occurs within the biomedical community. In part this is probably because issues affecting health and medicine can be quite serious. In addition, biomedicine is a very large research area, compared to the other specialties within science. For example, the US Federal basic research budget for the NIH is larger than the combined budgets for all other forms of basic research.\n\nThe biomedical community even has a regular gathering on the issue of peer review and publication. This is the “International Congress on Peer Review and Biomedical Publication” which is held every five years. The Seventh Congress was held in 2013, with 47 presentations and 63 posters4.\n\nBiased peer review of articles submitted to journals is already an active research area, so the primary challenge is to focus on the policy, product, mission or paradigm-supporting aspect. Unfortunately, just as with proposals, the journal peer review process is typically not publicly available. This is especially true for those articles that are rejected.\n\nNeither the submissions or the reviews, or even the names of the reviewers, are typically available for bias analysis. There are beginning to be exceptions to this secrecy. Some journals are even making the reviews public, especially for the accepted articles.\n\nOne might be able to arrange with the publisher to gain access to this secret data, especially if the scientific issue in question is quite narrow. Journals have probably become sensitive to the issue of bias. In many cases this issue might fall to the editorial board, not the publisher. They might even welcome some analysis.\n\nThus the prospects for bias research might be limited in this case, just as in the case of proposals, because of secrecy. Or the bias research might involve indirect methods.\n\nFor example, one might survey the researchers in the pool of candidates from which the reviews for a given journal are likely to have been drawn, looking for evidence of bias. A journal might even make its reviewer list available for analysis, when it will not do so for individual articles.\n\nSuppose one has the journal’s list of reviewers and there is a related controversy regarding an agency’s policy or paradigm. If the relevant reviewers can be classified according to their position on the controversy, then the list can be tested for its balance of representation. Of course this assumes that all reviewers carry equal weight so it is a relatively rough test. For example, in the case of the climate change debate one could look for skeptics versus warmers on the reviewer list.\n\nIn any case the prospects for simple quantification would seem to be limited, with a lot of interpretation required. Getting good data is the first research challenge.\n\n7. Biased meta-analysis of the scientific literature.\n\nMeta-analysis refers to studies that purport to summarize a number of research studies that are all related to the same research question. For example, meta-analysis is quite common in medical research, such as where the results of a number of clinical trials for the same drug are examined.\n\nThere is a sizeable literature in at least two fields on bias in meta-analysis. These fields are clinical medical trials and psychology. Some sample articles include work by Mueller et al.5 and by Ferguson and Brannick6.\n\nGiven that meta-analysis bias is already a significant research area, the challenge is primarily to adapt it to the realm of funding-induced bias. This would seem to be primarily a matter of doing three things. First, choose the meta-analytical document or documents to be analyzed. Second, identify the specific bias to be analyzed for, then compare the available literature with that chosen for the meta-analysis.\n\nThe first choice for meta-analyses to be analyzed might well be documents produced by, or funded by, the funding agency. This is especially true for documents specifically designed to support agency policies. Scientific review articles related to hypotheses which support agency policies are another likely candidate. In some cases the potential bias itself will dictate which documents should be analyzed for bias.\n\nIt is not clear that quantification can play a major role in this sort of bias research. For example, if a meta-analysis is found to be ignoring scientific papers reporting negative results, how many such papers there are may not be the issue. This may be more a matter of the strength of evidence, not a matter of counting up the sides.\n\n8. Failure to report negative results.\n\nThis topic has become the subject of considerable public debate, especially within the scientific community. Failure to report negative results can bias science by supporting researchers who promote questionable hypotheses.\n\nThere is a considerable literature on this topic, often under the heading of publication bias. Google Scholar full text search on “publication bias” for 2010–2014 gives over 22,000 hits, while title only search gives 236 hits. This bias is also termed “reporting bias” with 93 Google Scholar title hits and over 18,000 full text hits. These are relatively large numbers, indicating significant research activity.\n\nThere is a plain language listing of related bias types, with good references, from the blog Editage Insights: “Publication and reporting biases and how they impact publication of research” by Velany Rodriguez7.\n\nGiven that publication bias is already an active research area; the primary challenge is to look for bias that is related to funding or which supports funding organization needs. Thus the starting point is probably the agency policy or paradigm.\n\nA lot of this research is quantitative because it looks at bodies of research results, rather than at individual results. Publication bias is typically a pattern, not a single action. The scope may vary from a single journal up to an entire field.\n\n9. Manipulation of data to bias results.\n\nRaw data often undergoes considerable adjustment before it is presented as the result of research. There is a concern that these adjustments may bias the results in ways that favor the researcher or the agency funding the research.\n\nA full text Google Scholar search on “data manipulation” for the five year period 2010–2014 yields about 19,000 results. However, it appears that most of these are about tools and methods for benign data processing. A few address manipulation as a form of bias.\n\nThus there is an ambiguity in the use of the term data manipulation. Sometimes it refers to benign data processing but at other times it refers to questionable manipulation. However, it is clear that there is a significant body of research into the latter, which means the biased form of data manipulation.\n\nAnother approach to the literature is from the direction of scientific fraud, even though bias need not be fraudulent. A full text search on “fraud” and “data manipulation” for the period gives about 1,200 hits. Searching on “fraudulent” and “data manipulation” gives over 6,000 hits. Clearly the scientific community is concerned about fraudulent data manipulation and this is a significant research area.\n\nThe kind of funding-induced bias we are concerned with here falls somewhere in between benign data processing and outright fraud. While that middle ground exists in the literature it is not easy to find. Clearly this is a complex issue.\n\nGiven that there is already active research into possible bias in data manipulation, the principal challenge seems to be to focus some research on possible cases of funding-induced manipulation. It is likely that this research will involve specific cases, rather than statistical patterns. However, the manipulation itself will often be quantitative.\n\n10. Refusing to share data with potential critics.\n\nA researcher or their funding organization may balk at sharing data with known critics or skeptics, because of the negative effect it may lead to.\n\nData sharing is a major topic of research and discussion within the scientific community. Google Scholar returns about 29,000 full text hits for “data sharing” for the five year period 2101–2014. Searching on titles gives about 1,600 hits. These are relatively large numbers.\n\nMany of these articles are related to policy issues promoting data sharing, while many others are about specific cases, especially data repositories. (There is also a different use of the term, related to the design of computer network systems.)\n\nThere appears to be little work directly focused on not sharing data, especially for funding related reasons, although the general topic of not sharing data may be discussed in passing in articles promoting data sharing. In fact a full text search on “not sharing data” returns about 160 hits. Many of these articles are reporting surveys exploring researcher’s reasons for not sharing their data.\n\nThere are, however, some well known cases of scientists refusing to share policy relevant data. In the US one of the most prominent is the so-called Six Cities study regarding the long term health effects of airborne fine particulates. See for example the work of Kabat8. A Google search on “Six Cities study controversy” (without the quotation marks) provides many additional sources.\n\nIt appears that despite these prominent cases there is relatively little research into the practice of refusing to share data that is used to support funding organization policies, products, missions or paradigms.\n\nResearch in the area of refusing to share data because of its policy implications might be largely anecdotal. That is, one might look for allegations. Another possible approach might be to analyze agency Freedom of Information requests, to see how many pertained to attempts to get policy relevant data. Here the results might well be quantitative.\n\n11. Asserting conjectures as facts.\n\nIt can be in a researcher’s, as well as their funding organization’s, interest to exaggerate their results, especially when these results support an agency policy or paradigm. One way of doing this is to assert as an established fact what is actually merely a conjecture.\n\nSpeculation is a widely used term. Google Scholar lists over 1300 occurrences of “speculation” in titles for the period 2010–2014. These appear to be mostly studies related to forms of financial speculation. Search for the term occurring anywhere in the text during this period gives over 70,000 hits, many of which are probably incidental.\n\nNarrowing the term to “scientific speculation” gives about 800 full text hits, just 5 in titles. Here there is interesting work in the computational biology community using semantic analysis to try to identify speculative statements. These approaches may well be applicable to the problem of speculation presented as fact.\n\nMuch of this semantic research also uses the term “speculative statements” and Google Scholar search on that term gives about 240 occurrences in full text and 2 in titles, for the period. Many of these occurrences appear to be from this relatively small research community. A sample article is by Malhotra9.\n\nThe bias of asserting conjectures as facts is largely semantic in nature. It can occur anywhere in the life cycle of research, from agency documents to journal articles and media reports. It is basically a form of exaggeration, but with an epistemic dimension, claiming to know what is in fact not known.\n\nThere are various forms of semantic research that might be used to look for this bias in relation to agency policy. In a simple case one might first isolate key claims that are controversial, look for patterns of assertion that express them as settled. One might also look for the inclusion of policy prescriptions in the statement of the science.\n\nA broader analysis might look at the language being used in presenting the science. Good scientific writing is carefully crafted to be cautious. Terms like suggests, possibly, likely, may, might, etc. occur frequently when conclusions are stated. The lack of this sort of qualifying language might be diagnostic for the bias of asserting conjectures as facts. Semantic techniques like term vector similarity might be useful here.\n\nA lot of semantic analysis is quantitative in nature, especially when terms or occurrences are being counted. This is likely to be the case when one is gauging the level of confidence.\n\n12. False confidence in tentative findings.\n\nAnother way for a researcher, as well as their funding agency, to exaggerate their results is by claiming that they have answered an important question when the results actually merely suggest a possible answer. This often means giving false confidence to tentative findings.\n\nGoogle Scholar reports about 2500 articles using the exact term “false confidence” in the 2010–2014 time period. However, this term occurs just 5 times in article titles, suggesting that the concept per se is not a focal point for research.\n\nSome are using the term in passing, but in many cases this concept is the point of the analysis. However, these analyses appear to be mostly narrative, with little quantification. In many cases the article is of an editorial nature, see for example Michaels10.\n\nAll in all it seems that there is relatively little scientific research on the problem of false confidence, even though it is widely discussed.\n\nAs with the bias of asserting conjectures as facts, the bias of false confidence is semantic in nature. It can occur anywhere in the life cycle of research, from agency documents to journal and media reports. It is basically a form of exaggeration, but with an epistemic dimension, namely claiming an unjustified weight of evidence for a given finding.\n\nMoreover, as with exaggeration in general, one can look at how results are reported in the media or in press releases, compared to how they are stated in the journal.\n\nA lot of semantic analysis is quantitative in nature, especially when terms or occurrences are being counted. While this seems not to have been done for the problem of false confidence bias in the reporting of research, there is no obvious reason why it cannot be done.\n\n13. Exaggeration of the importance of findings by researchers and agencies.\n\nResearcher and agency press releases sometimes claim that results are very important when they merely suggest an important possibility, which may actually turn out to be a dead end. Such claims may tend to bias the science in question, including future funding decisions.\n\nFor “science” plus “hype” Google Scholar gives over 16,000 hits in a full text search for the period 2010–2014. Many are looking at specific cases where exaggeration may be an issue, often with a theme of “hope or hype”. However, the title search returns just 9 hits, a further indication that this language is primarily found in articles about specific cases of possible hype, not in studies of the occurrence of hype in science. A useful introductory article is by Rinaldi11.\n\nThen too, a Google Scholar full text search on “exaggeration” and “press releases” gives over 17,000 hits for the period 2010–2014. Oddly there are just two hits for the combination of these terms in titles, but many of the text hits are in fact on studies of press releases and exaggeration, including in science. Thus this is an active research area, including studies of press releases about scientific findings.\n\nNote that our different types of exaggeration-related bias are not always distinguished. Thus the number of articles related to each type may be greater than is indicated by the literature snapshots.\n\nExaggeration of importance is a third type of exaggeration, along with presenting speculation as fact and presenting tentative findings with false confidence. Unlike the other two, exaggeration of importance is about the future more than the findings. It is basically a claim about the future direction that science will take because of the findings being reported.\n\nAs with the other types of exaggeration, this type is also basically semantic in nature (but without so much of the epistemic dimension). Because it is forward looking it is likely to be characterized by future tense statements, which may even be a semantic basis for finding candidate statements. However, the prospects for quantification are unclear, because this seems to be more a case of specific instances, rather than a pattern of bias.\n\n14. Amplification of exaggeration by the press.\n\nThe bias due to exaggeration in press releases and related documents described above is sometimes, perhaps often, amplified by overly enthusiastic press reports and headlines.\n\nGoogle Scholar gives over 5000 hits for “media bias” 2010–2014 with 163 in the title. This literature appears to be found mostly political science, economics and communications journals, with a focus on political cases.\n\nHowever, a full text Google Scholar search on the co-occurrence of the three terms “exaggeration”, “science” and “news” for the same period gives over 18,000 hits (with just one occurrence in a title). A significant fraction of these many articles are exploring media exaggeration of scientific reports. Note too that some of the articles returned on searches related to our other types of exaggeration-related bias may address media bias as well.\n\nThe existing media bias research is a good model for research into funding related bias. What needs to be done in some cases is to change the focus from political bias to policy bias. This is not a stretch as the two are relatively closely related. Policy is often the outcome of the political process.\n\nLooking for paradigm supporting bias in scientific reporting may be more difficult. Here it will be necessary to carefully consider the scientific controversies that relate to a given agency’s policies. This sort of bias may be more subtle than overt political bias. Nevertheless, the existing research into media bias looks to be a good model.\n\nSome of the existing research is quantitative in nature, but much is not. A lot of it seems to be interpretative. An interesting issue here is whether bias and exaggeration come mostly from the media or from the original press releases. A recent quantitative study by Sumner illustrates a useful approach12.\n\n15. More funding with an agenda, building on the above, so the cycle repeats and builds.\n\nThe biased practices listed above all tend to promote more incorrect science, with the result that research continues in the same faulty direction. These errors may become systemic, by virtue of a biased positive feedback process. The bias is systematically driven by what sells, and critical portions of the scientific method may be lost in the gold rush.\n\nThere appears to be very little research looking at systematic linkages between combinations of the types of bias identified above, and subsequent funding. Some of these types of bias are attracting considerable research on an individual basis, but not in relation to subsequent agency funding.\n\nHowever, the concept that perverse incentives are damaging science is getting some discussion in the scientific community. See for example the work of Schekman13 and of Michaels14.\n\nIn this case one is probably looking for funding that occurs after the other types of bias, where the prior bias supported the funding agency’s mission, policy or paradigm. Quantification is certainly plausible, especially given that dollars is one of the measures.\n\n\nSome conclusions and observations\n\nSome types of bias are being studied extensively and quantitatively. Various aspects of peer review and publication bias, especially in biomedicine, appear to be the most heavily researched types of bias.\n\nThe role of funding in inducing bias is frequently alluded to as a potential financial conflict of interest. But it is not the focus of most research, which tends to look more at the practice of bias than at its cause. Thus a new research thrust is likely needed.\n\nThe role of government funding in inducing policy-driven bias seems to have received very little attention, even though it may be widespread. There are certain exceptions, most noticeably in the climate change debate and environmental policy in general. But here the attention is more a matter of public concern than one of quantitative scientific research.\n\nThe notion of cascading systemic bias, induced by funding, does not appear to have been much studied. This may be a big gap in the research on science policy. Moreover, if this sort of bias is indeed widespread then there is a serious need for new policies to prevent it, both at the funder level and within the scientific community itself.",
"appendix": "Author contributions\n\n\n\nPM conceived of this study, while DW carried out most of the detailed literature analyses. The taxonomy was developed jointly. DW wrote the first draft of this article, which PM then edited.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the Cato Center for the Study of Science. The authors declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Terence Kealey for his early support in endorsing the proposal for this project.\n\n\nReferences\n\nKuhn TS: The Structure of Scientific Revolutions. 1962. Reference Source\n\nKrimsky S: Do Financial Conflicts of Interest Bias Research? An Inquiry into the \"Funding Effect\" Hypothesis. In Science, Technology and Human Values. 2013; 38(4): 566–587. Publisher Full Text\n\nBornmann L: Scientific peer review. Ann Rev Info Sci Tech. 2011; 45(1): 197–245. Publisher Full Text\n\nSeventh International Congress on Peer Review and Biomedical Publication. 2013. Reference Source\n\nMueller KF, Meerpohl JJ, Briel M, et al.: Detecting, quantifying and adjusting for publication bias in meta-analyses: protocol of a systematic review on methods. Syst Rev. 2013; 2: 60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerguson CJ, Brannick MT: Publication bias in psychological science: prevalence, methods for identifying and controlling, and implications for the use of meta-analyses. Psychol Methods. 2012; 17(1): 120–8. PubMed Abstract | Publisher Full Text\n\nRodriguez V: Publication and reporting biases and how they impact publication of research. Editage Insights. 2013. Reference Source\n\nKabat G: What Is Really At Stake In The House Committee on Science, Space, and Technology Subpoena Of EPA Data. Forbes. 2013. Reference Source\n\nMalhotra A, Younesi E, Gurulingappa H, et al.: 'HypothesisFinder:' a strategy for the detection of speculative statements in scientific text. PLoS Comput Biol. 2013; 9(7): e1003117. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMichaels PJ: Decrying 'wishful science' on NPR. Cato Institute. 2014. Reference Source\n\nRinaldi A: To hype, or not to(o) hype. Communication of science is often tarnished by sensationalization, for which both scientists and the media are responsible. EMBO is the European Molecular Biology Organization. EMBO Rep. 2012; 13(4): 303–307. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSumner P, Vivian-Griffiths S, Boivin J, et al.: The association between exaggeration in health related science news and academic press releases: retrospective observational study. BMJ. 2014. 349: g7015. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchekman R: How journals like Nature, Cell and Science are damaging science. The Guardian. 2013. Reference Source\n\nMichaels PJ: Putting Headlines Ahead of Science. Cato Institute. 2014. Reference Source"
}
|
[
{
"id": "10827",
"date": "23 Oct 2015",
"name": "Ivan Oransky",
"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\nThank you for the opportunity to review this paper. While it tackles an important subject, we have serious reservations about its approach and conclusions, and do not approve it.The fact that this is to be published in an “Opinion” section does not absolve the writers from clearly expressing what text is actually opinion, what text is fact, and what facts are used to support their opinion. We would urge more scholarship in how the information is presented. The discussion within each category, what information is available, and how to get it appears very superficial. There is no information provided as to when the checks in Google Scholar were made, or how often they were made, or if the authors checked the “hits” to verify applicability to the topic at hand. The numbers are not static and can change in time. In Category 9, we get 1540 hits for “fraudulent” and “data manipulation” – they get “over 6000.” We get 3590 hits for “fraud” and “data manipulation” and they get “about 1200”.They refer to “Scientific Peer Review” by Lutz Bornmann and say that it is “widely recognized” because Google Scholar lists “120 citations for this article”. The authors not indicate whether the citations are used in a positive or negative light, or even in the same concept as these authors think. As of 10/17/15, the first 6 citations were by Bornmann himself. The authors make the statement: “ For the purposes of future research the concept of funding-induced bias is analyzed in the context of various practices in science where bias can occur.” There is little to no “analysis” provided, other than Google Scholar hits, which is meaningless for analysis as it has to many hidden variables to allow adequate evaluation, followed by suggestions as to how information may be obtained. The suggestions do not provide specific means to obtain information. The “taxonomy” has no information to justify the 15 categories chosen. They are not clearly defined as to meaning, context, and criteria. They supply no studies to support that any of these numbered items actually existed in form to affect any funding process. They may, but some type of evidence should be presented to provide justification for the category to exist and to be considered a discrete concept. List number 11 is : “Asserting conjecture as fact.” “It can be in a researcher’s, as well as their funding organizations, interest to exaggerate their results, especially when these results support an agency policy or paradigm, One way to do this is to assert as an established fact what is merely a conjecture.” The authors then list Google Scholar hits for the word “speculation.” There is no evidence offered for any associations with titles. There is no evidence offered that the hits for the word “speculation” were even associated with science. Google Scholar draws from a wide variety of indexing databases, and blogs, and various other items with internet access. The authors offer no evidence for statistically significant associations between the words they use for “hits” and any science-based studies. Some examples of statements meriting citation support (there are others): “The concept of finding-induced bias is one that specifically occurs in the discussion of and research into some of the fifteen bias research types that we have identified, but not in all of them. It tends to occur where specific funding is the issue”. What studies have been done that verify that this even occurs, that the fifteen bias types are indeed areas where bias is possible, and that the tendency to occur with specific funding issues is factual and not conjecture?“For example, the US Federal basic research budget for the NIH is larger than the combined budget for all other forms of basic research.” From where and when was this information obtained? Under the section “Quantification analysis method issues”, the authors write “What is the suggested best method of quantification? In particular is it subjective or objective, that is is human judgment and classification of the data involved, or just simple counting of clearly defined instances.” Qualitative studies (based in subjectively-derived data sets) would be the description of the former, and quantitative studies (based on analytical analysis of discrete data) would be the latter.\n\nA typical definition for general science can be found via internet sources:“qualitative research research dealing with phenomena that are difficult or impossible to quantify mathematically, such as beliefs, meanings, attributes, and symbols; it may involve content analysis.quantitative research research involving formal, objective information about the world, with mathematical quantification; it can be used to describe test relationships and to examine cause and effect relationships.” (Retrieved on 10/19/15 from http://medical-dictionary.thefreedictionary.com/Quantitative+research)The use of the term in subsequent discussion seems to indicate “comprehensive”, not “quantitative”. List number 12 is “False confidence in tentative findings.” “Google Scholar reports about 2500 articles using the exact term “false confidence” in the 2010-2014 time period. However, this term occurs just 5 times in article titles, suggesting that the concept per se is not a focal point for research.” This statement has several weaknesses in reasoning. 1. “false confidence” is a term that is also used to convey “bravado”, and thus the scholar reports may not be conceptually representative of the term as used by the authors. 2. Requiring the exact word usage to convey interest is misleading, as it may be called something different in researchers' vernacular. 3. Noting how many times the words are used in a title that is indexed with Google Scholar is misleading. The concept may be included under a greater concept, about which there may be numerous research studies published under a different database (Medline, Scopus, etc) 4. Using the term “confidence” in any search function related to research can be misleading as quantitative studies typically use “confidence intervals” in their statistical analysis. These are specifically defined mathematical concepts. The authors write that \"A meta-analysis refers to studies that purport to summarize a number of research studies that are all related to the same research question.” That is the wrong description of “meta-analysis”, which is not a “summary”. Meta-analyses take all the data from the studies they include, and perform analyses as if they are one huge data pool. Statistical results obtained are therefore not “cumulative”, as the creation of a large data pool may allow differences in means, trends and associations. Quite commonly, differences in effects are found because the size of the dataset will reduce bias found in smaller samples. \"Definition of META-ANALYSIS: a quantitative statistical analysis of several separate but similar experiments or studies in order to test the pooled data for statistical significance.” Retrieved on 10/19/15 from http://www.merriam-webster.com/dictionary/meta-analysis. The authors write that “It is not clear that quantification can play a major role in this sort of bias research. For example, if a meta-analysis is found to be ignoring scientific papers reporting negative results, how many such papers there are may not be the issue. This may be more a matter of the strength of evidence, not a matter of counting up the sides”. As discussed previously, it is in these meta-analyses that quantification can and will play a major role. (See definition as given above). If papers are “ignored’ without sound rationale, that is indeed an issue, as the sample set is incomplete. However, if papers are “ignored” and not included for issues of poor methodology, missing values, differences in measuring instruments, or inclusion of confounders not defined, then the exclusion decision can be considered reasonable. A good meta-analysis should consider all the available research, and provide sound reasoning for what studies are or are not included. Under item number 10, the authors write, “A researcher or their funding organization may balk at sharing data with known critics or skeptics, because of the negative effect it may lead to.” “There are, however, some well known (sic) cases of scientists refusing to share policy relevant data. In the US one of the most prominent is the so-called Six Cities study regarding the long term health effects of airborne fine particulates.”\n\nThe term “refusing” is connotatively misleading, as indicated in the first three articles (the third was actually from a book chapter) found in the Google search the authors said they run. The issue as described was in participant privacy, which might be considered a HIPPA issue in some instances. The participants had been promised privacy (confidentiality) and the researchers were (by what was in the articles) merely holding to their promise:“The year was 1997, and Dockery had arrived in Washington to tell Congress that because it had promised study participants confidentiality, Harvard couldn’t share the raw data from its federally funded Six Cities study” Retrieved on 10/19/15 from http://www.hsph.harvard.edu/news/magazine/f12-six-cities-environmental-health-air-pollution/. \"The authors of both studies have resisted demands to open up their data to public scrutiny. In the case of the Harvard study, for instance, they cite the need to keep the identities and health status of some 8,000 study subjects in six communities, including Watertown, Mass., confidential. They contend that, even if names and addresses are removed, it would be possible for someone to determine the identities of many subjects based on their age, hometown, and date of death. The controversy poses a test for government officials and scientific researchers, who increasingly are being asked to balance the health care privacy rights of individuals against demands for data from outside researchers, the public, and, politically motivated critics.” Retrieved on 10/19/15 from https://www.bostonglobe.com/news/nation/2013/09/06/landmark-harvard-study-health-effects-air-pollution-target-house-gop-subpoena/2K0jhfbJsZcfXqcQHc4jzL/story.html.“When Harvard researchers published the Six Cites Study suggesting that fine particulate pollution led to an unexpectedly high mortality rate, particulate-emitting industries were understandably concerned.7 A number of affected industries requested the original data supporting the study, but the Harvard researchers refused, because they were concerned that even the redacted data could be used to identify original study participants who had been assured of confidentiality.8” (The citation numbers go to references within the book.) Retrieved on 10/19/15 from https://books.google.com/books?id=Ah6-__otORAC&pg=PA263&lpg=PA263&dq=six+cities+study+controversy&source=bl&ots=TobrbzQ9mo&sig=NocuZxJ7my_4p_IfvRRWeG-NnR8&hl=en&sa=X&sqi=2&ved=0CCgQ6AEwAmoVChMIq4Luv5nPyAIVSoANCh2FIgiq#v=onepage&q=six%20cities%20study%20controversy&f=false. The authors write that “There appears to be very little scientific research on potential funding-induced bias in the construction or use of scientific models.” and “This appears to be a major gap in policy related research” Web of Science gave back 320 hits for “research funding”and “bias” and “modeling.\"",
"responses": [
{
"c_id": "1672",
"date": "28 Oct 2015",
"name": "David Wojick",
"role": "Author Response",
"response": "Reply to Reviewers by David Wojick and Patrick Michaels The Reviewers seem to have missed the point of our article, which is simply to provide a taxonomy of bias types, in order to facilitate future research. Instead they have focused on largely incidental statements made in our discussions of various types of bias. These discussions have no bearing on the validity or usefulness of our taxonomy. As a result, their criticism is not constructive.There are several ways in which a taxonomy can be criticized. It may contain categories which do not belong, or make false distinctions, or be incomplete. The Reviewers have not addressed these issues. In fact the word \"taxonomy\" occurs just once in the almost 2000 word review, where they claim we have not justified our categories. These categories are obviously basically the workflow steps of science, from initial budgeting for research funding through to the communication of reported results. In addition we have identified three distinct forms of exaggeration in communication. Allegations of bias have been made for all of these categories and our literature snapshots make clear that the subject of bias is an active research area for many of them. We cannot imagine a better justification than this. This is a very simple taxonomy, with well understood categories. One of us (DW) has been involved in the development of a number of very complex taxonomies. See for example http://scholarlykitchen.sspnet.org/2013/02/05/a-taxonomy-of-confusions/ and http://www.cendi.gov/presentations/KOS_OSTI_Energy_Taxonomy.pdf. Our bias taxonomy is rudimentary and transparent compared to these complex structures.In addition, we have two new results which we consider important. The first is that there seem to be several major gaps in the research on bias. The second is the potential for bias cascades, which is arguably our most important result. The Reviewers address neither of these findings. The word \"gap\" only occurs once, in a quote from our report, which is simply dismissed; ironically via an inconclusive three terms Google Scholar search. The number of hits is low and the bulk of hits from three term searches are unlikely to be related to the combined topic. The word \"cascade\" does not occur at all.The Reviewers first concern seems to be our use of Google Scholar (GS), which we consider to be a powerful scientometric tool. In their first comment, they claim that GS search results are volatile, offering several search results that are quite different from ours. However, we have rerun our searches and get the same results as before, so the Reviewers must simply be running different searches. The GS Advanced Search feature does allow for a certain amount of flexibility. We find no evidence of serious volatility in GS search results.Moreover, it is important to understand the purpose of our \"snapshot\" searches. This is merely to gauge the relative size of the research community for each of the fifteen bias types. We deem rough order of magnitude to be sufficient. That is, are there tens of hits, or hundreds, thousands, tens of thousands, or none? In this context the difference between, say, 3,000 and 6,000 is irrelevant.The GS searches also may facilitate future research, by pointing people to the relevant communities. In this sense a GS search is a reference to a community, just as a citation is a reference to a paper.Some of the Reviewer comments seem to suggest that our writing should be more technical, for example, in our brief explanation of meta-analysis. However, our results have implications for science policy, as well as for research, so we have elected to be as non-technical as possible. Policy makers are often not scientists. The Reviewers also raise a number of broad issues, based on incidental statements made in our discussions. These range from whether 120 citations indicate wide awareness of a paper, to how a taxonomy is constructed? These are indeed interesting questions in the study of science, but they have no bearing on our results. Moreover, the use of (1) citation based metrics and (2) taxonomies are standard practices, not things that we have to explain or justify.Given that the Reviewers are science writers and journalists, it is perhaps natural that they should raise these broad issues. There also seems to be a difference of opinion regarding the need for better bias research. We are surprised at this, given that the Reviewers are from Retraction Watch. However, it is well beyond the scope of our article to consider these issues. We therefore find no reason to revise our article."
}
]
},
{
"id": "11669",
"date": "12 Jan 2016",
"name": "Frederick Grinnell",
"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 Wojick and Michaels has interesting potential. As it stands, however, I suggest \"not approved\" for the reasons that follow. The underlying logic is flawed. On one hand, the authors write, “We make no distinctions regarding the source of funding.” On the other hand, they are aware of Krimsky’s findings about the biasing effect of commercial funding. Given the differences in funding goals by commercial and non-commercial funders and different types of non-commercial funders, failure to consider funder mission as a variable undermines the analysis. Second, the paper makes factual errors. For instance, the statement, “The selection of proposals is ultimately up to the funding program officers,” is somewhat correct at NSF but mostly false at NIH. Some fact checking with organizational representatives would be useful. The suggestion of a link between bias and Kuhnian paradigms is unconvincing given the differences in organizational missions and oversight mechanisms. Finally, the organization of the paper makes it hard to follow. The discussion of cascading effects would make much more sense at the end of the manuscript rather than at the beginning once the discussion of individual effects has been completed. I would find this paper more valuable if it developed the taxonomy in terms of funder-dependent differences in biases and their potential functions within the funding organizations. For instance, NIH intentionally is known to be biased towards new investigators but recently discovered to be unintentionally biased towards minority investigators. Once the funder-dependent biases have been documented, a discussion of how they might arise and how they might influence investigator behavior also would be interesting to develop further.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-886
|
https://f1000research.com/articles/4-884/v1
|
22 Sep 15
|
{
"type": "Method Article",
"title": "A citation-based, author- and age-normalized, logarithmic index for evaluation of individual researchers independently of publication counts",
"authors": [
"Aleksey V. Belikov",
"Vitaly V. Belikov",
"Vitaly V. Belikov"
],
"abstract": "The use of citation metrics for evaluation of individual researchers has dramatically increased over the last decade. However, currently existing indices either are based on misleading premises or are cumbersome to implement. This leads to poor assessment of researchers and creates dangerous trends in science, such as overproduction of low quality articles. Here we propose an index (namely, the L-index) that does not depend on the number of publications, accounts for different co-author contributions and age of publications, and scales from 0.0 to 9.9. Moreover, it can be calculated with the help of freely available software.",
"keywords": [
"Research",
"scientific",
"metrics",
"impact",
"weighted",
"bibliometric",
"scientometric",
"Publish or Perish"
],
"content": "Introduction\n\nThere is an ever-present need to evaluate researchers’ performance, because resources are limited and contenders are numerous. Before the advent of journal impact factors (JIFs,1), all evaluations were performed via peer review. Although JIFs were intended for librarians to decide which journals to subscribe to, they have become a commonly used proxy for the quality of journal articles2. However, the distribution of citations to individual articles within a journal is highly skewed. Twenty five percent of the most highly cited articles can account for 90% of a journal’s IF3. The rest of the articles receive a few citations each, if any. Thus, using JIFs for assessing the quality of individual articles and, further, for evaluating researchers is categorically not recommended4.\n\nThe rapid development of the internet and electronic citation databases, such as PubMed, Google Scholar, Web of Science, Scopus, CiteSeer and others, has made it easier to count citations of individual articles. It is now possible to automatically calculate the total number of citations that the publications of a given researcher have accumulated. However, these numbers can range from 1 to 100,000 and, obviously, do not represent the equal variation in researchers’ capabilities. For example, human IQ scores vary only about 2–4 fold5.\n\nThere are two main reasons why the total number of citations cannot be used to adequately compare individual researchers. First, there is usually more than one author for each publication. Some of the most highly cited articles, such as reports from experiments on particle accelerators6 or from genome sequencings7, and guidelines for medical practitioners8, have from tens to hundreds of co-authors, usually listed in alphabetical order. Thus, it is inadequate to assign all the thousands of citations to each of those authors. A similar situation is when a researcher operates some very expensive and thus rare equipment, and is listed on papers of all other researchers who perform experiments on that equipment9. It is clear that the researcher also does not deserve all citations of those papers, as his contribution is purely technical. Many ways to divide citations between co-authors have been proposed10,11, but the only practical way is to split them equally, i.e. to assign each co-author 1/n of the citations, where n is the total number of co-authors.\n\nAnother factor is that citations accumulate with time. A researcher who started his career 30 years ago will undoubtedly have more citations than a young postdoc, but this does not necessarily mean that the former is a better scientist. Moreover, a paper that has been highly cited is likely to be cited even more in the future12. Thus, citations exhibit the behavior of preferential attachment, which results in their distribution according to the power law13. These considerations make it necessary to adjust for the age of each publication, in order to properly assess current capabilities and impact of researchers, not their past successes, and to partially compensate for the preferential attachment. Dividing the number of citations by the age of the publication in years seems to be an adequate measure, as it mirrors the power law distribution that citations have.\n\nFinally, a large variety of individual citation metrics have been proposed14, the most widely disseminated of which is the h-index15. The drawback of the majority of these metrics is that they take into consideration the number of publications. For example, the h-index can never exceed the total number of publications a scientist has. However, several researchers of undisputed scientific merit, such as Sir Isaac Newton, Gregor Mendel or Peter Higgs, have published only a few, however significant, works. This lack in the number of publications leads them to have h-indices of 4, 1 and 9, respectively, which are disparagingly low. All other derivatives of the h-index, as well as all indices that take into account publication counts, suffer from the same drawback and hence should never be used for evaluation purposes. However, they are used, promoting a grueling and futile quest for quantity of publications, at the expense of quality, reflected in the infamous “publish or perish” catchphrase16. Fortunately, this issue has been recently called to public attention, most notably in the form of the San Francisco Declaration on Research Assessment, and some measures have been proposed17.\n\nOverall, there is an immense need for a simple but reliable indicator for individual researcher assessment. Here, we propose such an index, which accounts for different co-author contributions and age of publications, and does not depend on the number of publications. Moreover, it conveniently ranges from 0.0 to 9.9, and can be calculated with the help of freely available software.\n\n\nMethods\n\nTo address the concerns highlighted in the introduction of this article, we have set out to construct an index that accounts for different co-author contributions and age of publications. This has initially led us to the following formula:\n\n\n\nwhere I – preliminary index, ci – number of citations to i-th publication, ai – number of authors of i-th publication, yi – age in years of i-th publication, N – number of publications.\n\nWe then decided to estimate the range of values that our preliminary index can have. First, we calculated I for a hypothetical PhD student who recently received the first citation to his first paper, with 5 authors: I=15×1=0.2. Next, we calculated I for Albert Einstein and Charles Darwin, two of the most prominent and well-known scientists. To this aim, we utilized the freely available software Publish or Perish. This program imports citation data from Google Scholar and allows removal of irrelevant results, such as publications of homonym authors. A parameter AWCRpA (age-weighted citation rate per author) can be obtained from this program, and is equivalent to I from formula (1). The AWCRpA values for Einstein and Darwin, as calculated by Publish or Perish at the time of writing this article, were 6466 and 6178, respectively. Thus, even upon correcting for multiple authorship and age, citations vary by approximately 30,000 fold. As it is unlikely that the human brain can exhibit such a tremendous difference in its efficiency or talents, normalized citations should be mapped to a more appropriate scale, in order to make them more useful in meaningfully comparing researchers. It seems that a 1–10 scale is optimal, because it is closer to the true variation in human intellectual or other capabilities5, and is widely used in various metrics, thus being more intuitive. The natural logarithm function appears to be ideal for this scaling purpose. Compared to the square root or the cube root, the natural logarithm allows better resolution of differences between the majority of scientists, with the exception of the most prominent ones (Figure 1). To account for some of the negative values that arise when there is less than one normalized citation, the resulting index is increased by one point. In extreme cases where the index value still remains negative, it is advised to simply consider it as zero.\n\nThe natural logarithm (ln cn), cube root (cn3) and square root (cn) functions of normalized citations (cn) are shown.\n\nFinally, the formula for the Logarithm index (L-index) has become:\n\n\n\nwhere ci – number of citations to i-th publication, ai – number of authors of i-th publication, yi – age in years of i-th publication, N – number of publications.\n\nWhen ∑i=1Nciaiyi is calculated as AWCRpA in Publish or Perish, formula (2) takes the form:\n\n\n\nEquation 3 has been used to obtain all L-index values in this article.\n\nTo calculate typical L-indices for a PhD student, a postdoc and a principal investigator (PI), we averaged the L-index values for 5 PhD students, 10 postdocs and 15 PIs that we personally know (see Supplementary Table 1).\n\n\nResults and discussion\n\nFigure 2 shows the typical scale of the L-index with the indication of the values for ten of some of the most prominent and widely recognized scientists, as well as the typical values for a PhD student, a postdoc and a principal investigator (PI) (see Supplementary Table 1).\n\nThe typical range of the L-index is shown. The positions of 10 of the most famous scientists are indicated, along with their L-index scores. The positions of a typical PhD student, a postdoc and a principal investigator (PI) are also displayed. The raw data can be seen in Supplementary Table 1.\n\nIt can be seen from this figure that the L-index adequately captures the intuitive ranking, i.e. PhD student < Postdoc < PI < … < Albert Einstein. Moreover, it allows the objective (or, at least, statistically averaged collective subjective) quantitative assessment of researchers, which is a virtue that traditional peer review cannot accomplish. However, in cases where L-indices of the applicants are equal up to one decimal place, we strongly suggest the use of peer review, involving thorough examination of their publications, rather than differentiation of scientists based on the second decimal place, to avoid false precision and statistical bias. In case of young researchers that have only a few citations, it is also advisable to use peer review, as the limited data do not allow for the statistically robust calculation of the citation index.\n\nThe L-index can increase or decrease with time, as it depends on the age of publications. Thus, it favors the impact of recent publications and gives a much needed advantage to younger researchers. However, if a scientist has made such a significant discovery that its impact only increases with time, his L-index will stay high regardless of the age of the publication. Perfect examples of this are Albert Einstein and Charles Darwin. Despite them ceasing to publish original work decades ago, their L-indices are still higher than those of the absolute majority of current researchers (Figure 2).\n\nThe quantitative comparison of the L-index with other evaluation indices, such as the h-index, is purposefully avoided in this article, for the reason that those indices have been designed on different premises, such as to account for the number of publications. When evaluating the performance of a researcher, it should first be decided which parameter is considered adequate for the purpose – the number of publications, which does not tell anything about their quality, or the number of citations, which, however indirectly, indicates the impact that the publications have made. If the latter option is selected, the L-index can help to account for the effects of multiple co-authorship and aging of publications, and present the results in a simple and intuitive form.",
"appendix": "Author contributions\n\n\n\nAVB co-created the early version of the citation index formula, created the final L-index formula, collected and analyzed the data, interpreted the results and wrote the manuscript. VVB co-created the early version of the citation index formula, interpreted the results and revised the manuscript. All authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nThe authors are active researchers and hence are affected by current research evaluation practices. The authors have no professional or personal connections to Harzing.com, Google Scholar or any other citation database or citation analysis software developers.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors are grateful to Prof. A. Borthwick, Dr. A. Barmashov, and V. Hrubilov for the useful discussions.\n\n\nSupplementary material\n\nTable 1. The raw data that was used to calculate L-indices for Figure 2.\n\nAWCRpA were obtained from Publish or Perish, upon excluding publications of homonym authors. L-indices were calculated according to formula (3), using Excel formula editor and LN function. Average L-indices were calculated using AVERAGE function.\n\nClick here to access the data.\n\n\nReferences\n\nGarfield E: Citation analysis as a tool in journal evaluation. Science. 1972; 178(4060): 471–9. PubMed Abstract | Publisher Full Text\n\nAdam D: The counting house. Nature. 2002; 415(6873): 726–9. PubMed Abstract | Publisher Full Text\n\nNot-so-deep impact. Nature. 2005; 435(7045): 1003–4. PubMed Abstract | Publisher Full Text\n\nBeware the impact factor. Nat Mater. 2013; 12(2): 89. PubMed Abstract | Publisher Full Text\n\nHunt E: Human intelligence. Cambridge University Press, 2010. Reference Source\n\nAad G, Abajyan T, Abbott B, et al.: Observation of a new particle in the search for the Standard Model Higgs boson with the ATLAS detector at the LHC. Phys Lett B. 2012; 716(1): 1–29. Publisher Full Text\n\nVenter JC, Adams MD, Myers EW, et al.: The sequence of the human genome. Science. 2001; 291(5507): 1304–51. PubMed Abstract | Publisher Full Text\n\nPerk J, De Backer G, Gohlke H, et al.: European Guidelines on cardiovascular disease prevention in clinical practice (version 2012): The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts). Atherosclerosis. 2012; 223(1): 1–68. PubMed Abstract | Publisher Full Text\n\nAnderson C: Authorship. Writer's cramp. Nature. 1992; 355(6356): 101. PubMed Abstract | Publisher Full Text\n\nTscharntke T, Hochberg ME, Rand TA, et al.: Author sequence and credit for contributions in multiauthored publications. PLoS Biol. 2007; 5(1): e18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen HW, Barabási AL: Collective credit allocation in science. Proc Natl Acad Sci U S A. 2014; 111(34): 12325–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMerton RK: The Matthew Effect in Science: The reward and communication systems of science are considered. Science. 1968; 159(3810): 56–63. PubMed Abstract | Publisher Full Text\n\nPrice DDS: A general theory of bibliometric and other cumulative advantage processes. J Am Soc Inf Sci. 1976; 27(5): 292–306. Publisher Full Text\n\nVan Noorden R: Metrics: A profusion of measures. Nature. 2010; 465(7300): 864–6. PubMed Abstract | Publisher Full Text\n\nHirsch JE: An index to quantify an individual's scientific research output. Proc Natl Acad Sci U S A. 2005; 102(46): 16569–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarfield E: What is the primordial reference for the phrase ‘publish or perish’? The Scientist. 1996; 10(12): 11. Reference Source\n\nHicks D, Wouters P, Waltman L, et al.: Bibliometrics: The Leiden Manifesto for research metrics. Nature. 2015; 520(7548): 429–31. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10421",
"date": "16 Oct 2015",
"name": "Youngim Jung",
"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 proposes a novel index based on the number of citations, normalized by the number of authors and the date of publication, which is then scaled from 0.0 to 9.9 using the natural logarithm. The index proposed is intuitively designed in a simple way for readers to understand, with a fractional counting scheme adopted to normalize the number of citations, which appears reasonable. However, it will be necessary to adjust the index according to the relationship between the number of citations and the date of publication, since they are not precisely inversely proportional in every time window.Finally and fundamentally, whether the number of authors and the date of publication are truly indicative of publication quality remains open to speculation.",
"responses": []
},
{
"id": "10836",
"date": "17 Nov 2015",
"name": "Danielle M. Colbert-Lewis",
"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 title is appropriate for the content of the article and the abstract represents a suitable summary for the work. The design, methods and analysis used in the article represented a thorough approach to the subject. The conclusions are suitable, balanced and justified on the basis of the results of the study. The data represented allows enough information to replicate the experiment for further research. The data is usable with the structure that has been provided.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-884
|
https://f1000research.com/articles/4-883/v1
|
22 Sep 15
|
{
"type": "Research Article",
"title": "The ICR1000 UK exome series: a resource of gene variation in an outbred population",
"authors": [
"Elise Ruark",
"Márton Münz",
"Anthony Renwick",
"Matthew Clarke",
"Emma Ramsay",
"Sandra Hanks",
"Shazia Mahamdallie",
"Anna Elliott",
"Sheila Seal",
"Ann Strydom",
"Lunter Gerton",
"Nazneen Rahman",
"Elise Ruark",
"Márton Münz",
"Anthony Renwick",
"Matthew Clarke",
"Emma Ramsay",
"Sandra Hanks",
"Shazia Mahamdallie",
"Anna Elliott",
"Sheila Seal",
"Ann Strydom",
"Lunter Gerton"
],
"abstract": "To enhance knowledge of gene variation in outbred populations, and to provide a dataset with utility in research and clinical genomics, we performed exome sequencing of 1,000 UK individuals from the general population and applied a high-quality analysis pipeline that includes high sensitivity and specificity for indel detection. Each UK individual has, on average, 21,978 gene variants including 160 rare (0.1%) variants not present in any other individual in the series. These data provide a baseline expectation for gene variation in an outbred population. Summary data of all 295,391 variants we detected are included here and the individual exome sequences are available from the European Genome-phenome Archive as the ICR1000 UK exome series. Furthermore, samples and other phenotype and experimental data for these individuals are obtainable through application to the 1958 Birth Cohort committee.",
"keywords": [
"exome",
"exome sequencing",
"next-generation sequencing",
"NGS",
"population genetics",
"variant",
"gene variation"
],
"content": "Introduction\n\nThe advent of exome sequencing has increased our ability to comprehensively capture gene variation, furthering both discovery of genes predisposing to disease and the expansion of clinical genomic sequencing. In both contexts the spectrum and frequency of gene variation in the general population is a necessary consideration when evaluating potential association with disease. Although publicly accessible summary exome datasets such as the Exome Sequencing Project and Exome Aggregation Cohort (ExAC) series are available, these are compilations of exome data from disease cohorts rather than the general population, and individual level data is not provided, hampering utility1,2. The 1000 Genomes project has made individual population-based exome data available. However, the sample sizes for any given population range from 61 to 113 individuals, limiting detection of rarer variation3. Some larger population datasets are available; studies of 2,636 Icelanders and of 250 Dutch individuals have been recently published4,5, although individual sequences are not accessible, impeding direct comparative analyses of data from different populations. Moreover the spectrum of variation from founder populations, such as the Icelanders, is not representative of outbred populations, particularly in relation to rare variation. This is of crucial importance in clinical genomics because the majority of disease-causing gene variants are very rare.\n\nA further limitation of some of the available datasets is the sub-optimal detection of insertions and deletions (collectively termed ‘indels’). Many publications of these datasets have either excluded all/some indels, or have explicitly stated that the indel calling is not high-quality1,3,6–8. This has potential to severely compromise clinical genomic utility, as indels are a major class of pathogenic variant, and are routinely and robustly detected by pre-NGS mutation detection methods, such as Sanger sequencing.\n\nHere we have sought to enhance knowledge of rare gene variation in outbred populations and to provide an exome dataset with utility in research and clinical genomics. To this end we have generated exome data in 1,000 UK individuals from the general population (the ICR1000 UK exome series) and applied an analytical pipeline with high sensitivity and specificity for indel detection.\n\nWe included 1,000 samples from the 1958 Birth Cohort, selecting individuals with self-reported white British ancestry. The 1958 Birth Cohort is a population series of individuals born in the UK during one week in 1958. It is a longitudinal study collecting information on numerous aspects of development with the first sweep of data collection in 1965 and the most recent in 2013. Biomedical assessment was undertaken during 2002–2004, at which time blood samples and informed consent were obtained for creation of a genetic resource (www.cls.ioe.ac.uk). The advantages of this series are: first, it is population-based and unselected with respect to disease. Second, unlike most published exome series we are able to make the individual exome sequences available, greatly enhancing utility. Third, phenotype data, DNA and cell lines from individuals in whom we performed exome sequencing are available to researchers following a successful application to the 1958 Birth Cohort committee.\n\nWe performed exome sequencing using the Illumina TruSeq exome and Illumina instruments. Average coverage of the target across all individuals was 47×. Median overall coverage of the target at 15× was 91% across the 1,000 individuals, with a median of 47,240,000 reads mapping to the target. The FASTQ files for all individuals are available from the European Genome-phenome Archive (EGAD00001001021). We have called the dataset the ICR1000 UK exome series.\n\nWe analysed the data with the OpEx pipeline (version 1.0.0) which has high sensitivity (95%) and specificity (97%) for indels, with a low false discovery rate (3.4%). OpEx includes Stampy v1.0.14 for alignment, Platypus v0.1.5 for variant detection and CAVA v.1.1.1 for variant annotation9–11. Of particular note, the default OpEx indel calls are consistent with the clinical convention (i.e. called at the most 3’ position in the coding strand) but if alternative representation(s) are possible this is noted and the call at the most 5’ position is also provided11.\n\nTo confirm the series provides representation of individuals with similar ancestry, we performed principal component analysis with the HapMap data for the Central European, Han Chinese, Gujarati Indian, and Yoruban populations12. All individuals clustered tightly with the Central Europeans, with no evidence of ethnic outliers (Figure 1).\n\nPlot of first and second principal components from PCA using HapMap populations and the ICR1000 UK exome series showing that the series clusters with the Central European population, with no ethnic outliers.\n\nWe evaluated 19,922 genes from the Ensembl database, of which 17,588 passed our sequence performance criteria and were included in the analyses outlined below (Supplementary Table 1). We calculated the percentage of coding bases that achieved coverage of at least 15×, the same threshold at which variant calling was optimised. This allows one to place a gene’s observed variation in the context of what was successfully sequenced, an essential requirement in clinical genomics. The genes included and the percentage successfully covered is given in Supplementary Table 1. We observed variation in 17,464 of the 17,588 genes (Figure 2). The 124 genes with no variation had less than 1.1kb of coding sequence and/or less than 80% of coding sequence successfully sequenced at 15× (Supplementary Figure 1). The 538 genes with no protein-altering variation were similarly enriched for smaller genes (Supplementary Figure 1). 712 genes had no rare (0.1%) protein-altering variants. Conversely, 2,296 genes had only rare protein-altering variants. The remaining genes had variants across the frequency spectrum (Figure 2, Supplementary Table 2).\n\nProtein-altering variation includes nonsynonymous variants, inframe indels and protein-truncating variants (i.e. frameshifting indels or variants that alter essential splice-site residues). The majority of genes include variants across the frequency spectrum.\n\nWe outputted all variants in exons or within 10 bp of the intron-exon boundary and detected 295,391 distinct variants in total: 284,437 base substitutions, 7,378 deletions and 3,576 insertions (Figure 3). Summary information for all 295,391 variants is given in Supplementary Table 2. We stratified the variants by frequency and type. We term variants present in only one individual i.e. with frequency of 0.1%, ‘rare’, those with frequency between 0.2–5% (i.e. present in 2–50 individuals) ‘low-frequency’ and those with frequency >5% (i.e. present in >50 individuals) ‘common’. The majority of the 295,391 variants were rare (54%, n=159,073), with 30% (n=87,450) occurring at low frequency and 16% (n=48,868) being common (Figure 3, Supplementary Table 2).\n\nThe number and percentage of variants in each category is shown in white text. For all variant types, rare variants predominate, and the distribution of variants of different frequencies is similar.\n\nWe first considered the quality of the base substitution calls. The overall transition/transversion (Ti/Tv) ratio was 3.05, confirming our low false detection rate. The Ti/Tv ratio increased from 3.17 to 3.25 as frequency decreased from common to low as anticipated13. The Ti/Tv ratio of the rare variants was lower, as expected, at 2.91. This is because rare variants are enriched for nonsynonymous variants, a class more likely to contain transversions, thus resulting in a lower Ti/Tv ratio4,8. The nonsynonymous/synonymous ratios were 0.90, 1.46, and 1.86 for the common, low, and rare base substitutions respectively, showing good agreement with previous estimates3. In total we identified 284,437 base substitutions; 99,568 synonymous, 158,932 nonsynonymous, 22,613 in flanking intronic sequence and 3,324 affecting stop codons (3,210 stop-gain, 114 stop-loss). 54% (n=153,349) of the base substitutions were rare, 29% (n=83,891) were low frequency and 17% (n=47,197) were common (Figure 3, Supplementary Table 2).\n\nWe next considered the insertions and deletions. We identified 10,954 indels in total, of which 52% (n=5,724) were rare, 33% (n=3,559) were low frequency and 15% (n=1,671) were common (Figure 3, Supplementary Table 2). The type and length of indel detected influenced variant frequency and we detected twice as many deletions as insertions (P=8.68×10-289), in line with previous data (Figure 4)4,14. For coding indels the majority of common variants were inframe 3 bp variants or 1 bp deletions or insertions. However, we observed a different distribution for rare coding indels, with more frameshifting single base indels than inframe 3 bp indels (P=1.2×10-10; Figure 4). This likely reflects selection against frameshifting indels becoming common, as they are more often biologically deleterious than inframe mutations.\n\nVariant frequency varies with type and length of indel. Deletions are more common than insertions, particularly for rare variants. There is enrichment of indels of 3 bp, 6 bp and 9 bp in coding but not non-coding sequence, because these cause inframe variants.\n\nOur data allow us to describe the average spectrum of gene variation of a UK individual born in 1958, who has 21,978 gene variants (range=19,637–23,009). This includes 9,993 synonymous variants (range=8,921–10,406), 8,718 nonsynonymous variants (range 7,834–9,114), 418 deletions (range=290–476) and 289 insertions (range 234–333) (Table 1, Supplementary Table 3).\n\nThe ranges are given in Supplementary Table 3. Values are rounded to the nearest whole number. The functional impact class supplied by CAVA is given in parentheses. Full details of the CAVA classification system are given in Münz et al.11\n\nWe believe these results and the underlying raw ICR1000 UK exome data have considerable utility in scientific and translational research and in clinical genomics.\n\n\nMethods\n\nWe used lymphocyte DNA from 1,000 individuals with self-reported white British ancestry obtained from the 1958 Birth Cohort Collection, a continuing follow-up of persons born in the United Kingdom in one week in 1958. Biomedical assessment was undertaken during 2002–2004 at which blood samples and informed consent were obtained for creation of a genetic resource (www.cls.ioe.ac.uk).\n\nWe prepared DNA libraries from 1.5 µg genomic DNA using the Illumina TruSeq sample preparation kit. DNA was fragmented using Covaris technology and the libraries were prepared without gel size selection. We performed target enrichment in pools of six libraries (500 ng each) using the Illumina TruSeq Exome Enrichment kit. The captured DNA libraries were PCR amplified using the supplied paired-end PCR primers. Further details are given at http://images.illumina.com/documents/products/datasheets/datasheet_truseq_exome_enrichment_kit.pdf. Sequencing was performed with an Illumina HiSeq2000 (SBS Kit v3, one pool per lane) generating 2×101 bp reads.\n\nWe analysed sequencing reads with the OpEx pipeline. OpEx includes Stampy v1.0.14 for alignment, Platypus v0.1.5 for variant detection and CAVA v.1.1.1 for variant annotation9–11. The OpEx pipeline is available at http://www.icr.ac.uk/OpEx. Full details of the OpEx description and performance are being prepared for publication. In brief, we validated the OpEx pipeline using 142 independent samples for which we had previously generated extensive data from genotyping and sequencing studies. We evaluated 12,656 sites which included 11,926 common base substitutions with minor allele frequency (MAF) ≥5%, 409 variants validated by Sanger sequencing, and 321 sites known to be negative through variant caller comparison analyses. For base substitutions the sensitivity was 99.7% (12017/12049), specificity was 96% (44/46) and the FDR 0.02% (2/12019). For indels ≤10 bp, the sensitivity was 95% (256/269), specificity was 97% (266/275) and the FDR 3.4% (9/265). The detection of longer indels (>10 bp) was less robust, with higher false detection rates than for small indels, these were therefore excluded from the variant tables and analyses.\n\nIn the variant analyses we included base substitution calls for which at least one call across all samples had a QUAL score >100. For deletions we included calls for which at least one call across all samples had both PASS in the FILTER column and ≥20% of reads included the deletion. For insertions we only included calls that had both PASS in the FILTER column and ≥20% of reads included the insertion. Seven samples showed excess heterozygosity and were excluded from the variant analyses.\n\nWe selected 116 gene variants that were detected once in the series to further evaluate variant calling performance. This included 31 base substitutions, 36 deletions, and 49 insertions. The 31 base substitutions, 13 of the deletions, and 2 of the insertions occurred in genes for which we had Sanger sequencing primers available in-house. The remaining 23 deletions and 47 insertions were selected randomly from amongst 96 individuals whose DNA was included on the same plate to aid the laboratory work. For these 70 indels, we generated FASTA sequence for the 500 bp window surrounding the called variant using the BSgenome package in R15. We used BatchPrimer3 to design primers with minimum product size of 200 bp, optimal of 350 bp, maximum of 501 bp, max Tm difference of 5.0, and default for all other settings. We performed PCR reactions using the Qiagen Multiplex PCR kit, and bidirectional sequencing of resulting amplicons using the BigDye terminator cycle sequencing kit and an ABI3730 automated sequencer (ABI PerkinElmer). All sequencing traces were analysed with both automated software (Mutation Surveyor version 3.10, SoftGenetics) and visual inspection.\n\n113 of the 116 calls were detected by Sanger sequencing. Only one of the validated variants had a discrepant annotation; an insertion of 1 bp in a run of T’s in the OpEx call appeared to be a deletion of 1 bp in the Sanger sequence. There were three false positive OpEx calls (FDR = 0.03). Two were insertions of 6 bp and 8 bp present at the end of reads, a common site of false positive calls. The other was a 3 bp deletion in a region with poor mapping quality and poor coverage.\n\nWe selected transcripts from the Ensembl database (version 65) and evaluated the 19,922 genes present on chromosomes 1–22, X, or Y, that had both a start and stop codon, and were not known pseudogenes as specified in the default exome transcript database supplied with OpEx. The specific transcripts selected are provided in Supplementary Table 1 (see Supplementary Table 4 for descriptions of column headers).\n\nGene coverage was evaluated using the coding bases of the selected transcript; intronic and UTR sequence was excluded. 2,334 genes were excluded from the variant analyses because either <50% of coding bases were targeted by the pulldown (n=1,380) or <50% of coding bases were covered at ≥15× in 500+ individuals (n=954). Of the 1,380 genes that were not targeted by the pulldown, 207 had ≥50% of the gene covered at ≥15× in at least one individual and thus represented off-target effects (Supplementary Table 1).\n\nWe utilized the genotype data from the 397 unrelated individuals in the Central European, Han Chinese, Gujarati Indian, and Yoruban populations in Phase 3 of the HapMap project12 to perform PCA. The genotypes were evaluated for 2,577 base substitution variants on chromosome 1 which were called in both the exome data and the HapMap data. PCA was performed using the prcomp function in R. To allow confident imputation of reference homozygotes in the exome data, variants were required to have ≥13× coverage at the position for every individual. Ten individuals were excluded from PCA analysis as fewer than 90% of the variants met the coverage requirement.\n\nWe assessed variation in the 17,588 genes with good coverage. Variants in exons or flanking sequence up to ten bases into the intron were outputted and included in Supplementary Table 2 (see Supplementary Table 4 for descriptions of column headers). Variants detected in only one individual in fewer than 15 reads were excluded. Multi-allelic variants were separated into their constituent alleles for the individual-level and gene-level analyses. For the individual-level analyses, the most severe consequence was selected for variants that affected multiple genes based on the functional impact class supplied by CAVA11. For the gene-level analyses, variants that affected multiple genes were included as variants in each gene. We defined coding indels as those that affected any exonic or essential splice-site (+1, +2, -1, -2) residue, and non-coding indels as those that affected residues +3 to +10 or -3 to -10.\n\nThe Ti/Tv and nonsynonymous/synonymous ratios were calculated in R using the exonic base substitutions. The overall proportion of deletions amongst the 10,954 indels (0.67) was tested for significant difference from 0.5 using the prop.test function in R. The number of 1 bp and 3 bp coding deletions was compared between rare and common variants using a 2×2 contingency test with the chisq.test function in R.\n\nThe FASTQ files for all 1,000 individuals have been deposited in the European Genome-phenome archive (EGA). The accession number is EGAD00001001021.\n\nThe files are available at https://www.ebi.ac.uk/ega/datasets/EGAD00001001021.\n\nDetails of how to request access to the data are available at www.icr.ac.uk/ICR1000exomes.\n\nResearchers and authors that use the ICR1000 UK exome series should reference this paper and should include the following acknowledgement: \"This study makes use of the ICR1000 UK exome series data generated by Professor Nazneen Rahman’s Team at The Institute of Cancer Research, London”.",
"appendix": "Author contributions\n\n\n\nN.R. designed the experiment. A.R., E.Ra., and S.H. generated the exome data. E.Ru., and A.E. undertook data management. S.S. undertook sample management and Sanger validations. E.Ru., M.M., and G.L. undertook tool development. E.Ru., S.M., M.M., and N.R. performed data analyses. M.C. and A.S. undertook the data and administrative management required for data to be accessible. E.Ru. and N.R. wrote the manuscript. All authors contributed to the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe acknowledge the use of DNA from the British 1958 Birth Cohort collection funded at the Centre for Longitudinal Studies by the MRC grant G0000934 and the Wellcome Trust grant 068545/Z/02. We acknowledge support from the NIHR RM/ICR Specialist Biomedical Research Centre for Cancer. This study was funded by the Institute of Cancer Research, London.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nScatterplot of gene coverage in 90% of samples vs the coding length in base pairs.\n\nTranscript, coverage, and variation information for 19,922 genes from the Ensembl database (v65).\n\nClick here to access the data.\n\nVariant information for all 295,391 exome variants detected in the ICR1000 series.\n\nClick here to access the data.\n\nThe range and average number of variants in the ICR1000 series by variant type and frequency.\n\nClick here to access the data.\n\nDescriptions of column headers for Supplementary Table 1 and Supplementary Table 2.\n\nClick here to access the data.\n\n\nReferences\n\nFu W, O’Connor TD, Jun G, et al.: Analysis of 6,515 exomes reveals the recent origin of most human protein-coding variants. Nature. 2013; 493(7431): 216–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nExome Aggregation Consortium (ExAC). Cambridge, MA. Accessed: December 2014. Reference Source\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\nGudbjartsson DF, Helgason H, Gudjonsson SA, et al.: Large-scale whole-genome sequencing of the Icelandic population. Nat Genet. 2015; 47(5): 435–44. PubMed Abstract | Publisher Full Text\n\nGenome of the Netherlands Consortium. Whole-genome sequence variation, population structure and demographic history of the Dutch population. Nat Genet. 2014; 46(8): 818–25. PubMed Abstract | Publisher Full Text\n\nMacArthur DG, Balasubramanian S, Frankish A, et al.: A systematic survey of loss-of-function variants in human protein-coding genes. Science. 2012; 335(6070): 823–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLim ET, Raychaudhuri S, Sanders SJ, et al.: Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders. Neuron. 2013; 77(2): 235–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTennessen JA, Bigham AW, O'Connor TD, et al.: Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science. 2012; 337(6090): 64–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLunter G, Goodson M: Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 2011; 21(6): 936–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRimmer A, Mathieson I, Lunter G, et al.: Platypus: An Integrated Variant Caller. 2012. Reference Source\n\nMünz M, Ruark E, Renwick A, et al.: CSN and CAVA: variant annotation tools for rapid, robust next-generation sequencing analysis in the clinical setting. Genome Med. 2015; 7(1): 76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe International HapMap Consortium. The International HapMap Project. Nature. 2003; 426(6968): 789–96. PubMed Abstract | Publisher Full Text\n\nDePristo MA, Banks E, Poplin R, et al.: A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011; 43(5): 491–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMontgomery SB, Goode DL, Kvikstad E, et al.: The origin, evolution, and functional impact of short insertion-deletion variants identified in 179 human genomes. Genome Res. 2013; 23(5): 749–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPages H: BSgenome: Infrastructure for Biostrings-based genome data packages. R package version 1.16.5. Reference Source"
}
|
[
{
"id": "10592",
"date": "30 Sep 2015",
"name": "Gail Jarvik",
"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 executed and data are available. If anything the authors are modest about impact. My report is below:Dr Rahman and her colleagues have summarized both variant and in/del variation in 1000 UK Biobank participants and shared these data. Their analyses show the rate of common and rare variation in the participants. While these estimates are important and underscore that rare variation is very common, possibly the largest impact of this work is the ability to assess the frequency of in/dels in a population-based cohort. Allele frequency data are among the most important consideration in determining whether a variant is pathogenic or not. Clinical laboratories and researchers alike rely on public databases for minor allele frequency data. As the authors rightfully point out, the ExaC variant database is a compilation of individuals with various diseases, which can skew allele frequencies. The Exome Variant Server is limited to heart, lung, and blood phenotypes, but contains no in/del data. While there is limited data within ancestry groups here, the well-curated in/del data will be a boon to those classifying variant pathogenicity. Further, the ability to access the phased sequence data provided here adds utility. This is an important resource that adds to our understanding of rare human variation.",
"responses": []
},
{
"id": "10652",
"date": "29 Oct 2015",
"name": "Fiona Cunningham",
"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 ICR1000, a UK-based reference data set for variation. Using 1000 consented samples from the 1958 UK birth cohort, they performed deep exome sequencing to a median depth of 15X. The samples were chosen from those who self-reported as having white British ancestry though does not comprise samples from individuals of other ancestry in the general British population. All data have been deposited in the EGA, and so are available as a resource to other bona fide researchers.This is a thorough and well performed study that includes detailed methods and results. In particular the authors have developed a pipeline with high sensitivity and specificity for detecting indels. This results in findings of nearly 11,000 indels, over half of which are novel and in total over 159,000 rare variants were found (with frequency of 0.1%). It would be helpful if the authors could include a comparison to the UK10K project. This was a major UK-based initiative that sequenced 4000 genomes of individuals with deep phenotype data and these data are also available in the EGA.In conclusion, this is valuable work that found new rare variants that will improve exome analysis. In particular, identification of novel indels, which have not been well characterised in many other studies, is of importance to the field and will be of benefit to clinical genomics where correct interpretation of rare variants is key.---Here is a citation for the database mentioned in the paper. Including this is helpful for continued development and funding. European Genome-phenome Archive:Lappalainen, I., Almeida-King, J., Kumanduri, V., Senf, A., Spalding, J.D., ur-Rehman, S., Saunders, G., Kandasamy, J., Caccamo, M., Leinonen, R., et al. (2015). The European Genome-phenome Archive of human data consented for biomedical research. Nat Genet 47, 692–695.",
"responses": []
},
{
"id": "10831",
"date": "27 Nov 2015",
"name": "Klaudia 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 authors performed exome sequencing of 1000 UK individuals from the 1958 Birth Cohort who are representative of an outbred population. This data set will be useful for the research community and for clinical genomics, especially since it provides individual genotypes from high-coverage data with a high-quality INDEL calling pipeline.The study is very clear and well described and it will provide a good quality data set for further research. It would be interesting to compare the variant calls of this study to the population based UK10K-cohorts call set which is based on low-coverage whole-genome sequencing of 3781 samples (UK10K Consortium, 2015).The authors say low-frequency variants with allele frequencies between 0.2–5% are variants present in 2–50 individuals. It would have been more accurate to refer to allele counts of 4-100 instead, since the allele number is 2000 for 1000 individuals.I was wondering whether it would have been possible to keep all individuals in the principle components analysis if a different set of variants had been selected for analysis.In summary, the exome sequencing data set of 1000 UK individuals will provide the research community with a useful resource, a European based set of variants and especially a high- quality set of INDELs.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-883
|
https://f1000research.com/articles/4-625/v1
|
25 Aug 15
|
{
"type": "Correspondence",
"title": "Hippocampal development and the dissociation of cognitive-spatial mapping from motor performance",
"authors": [
"Bryan D. Devan",
"Christopher Magalis",
"Robert J. McDonald",
"Christopher Magalis",
"Robert J. McDonald"
],
"abstract": "The publication of a recent article in F1000Research has led to discussion of, and correspondence on a broader issue that has a long history in the fields of neuroscience and psychology. Namely, is it possible to separate the cognitive components of performance, in this case spatial behavior, from the motoric demands of a task? Early psychological experiments attempted such a dissociation by studying a form of spatial maze learning where initially rats were allowed to explore a complex maze, termed “latent learning,” before reinforcement was introduced. Those rats afforded the latent learning experience solved the task faster than those that were not, implying that cognitive map learning during exploration aided in the performance of the task once a motivational component was introduced. This form of latent learning was interpreted as successfully demonstrating that an exploratory cognitive map component was acquired irrespective of performing a learned spatial response under deprivation/motivational conditions. The neural substrate for cognitive learning was hypothesized to depend on place cells within the hippocampus. Subsequent behavioral studies attempted to directly eliminate the motor component of spatial learning by allowing rats to passively view the distal environment before performing any motor response using a task that is widely considered to be hippocampal-dependent. Latent learning in the water maze, using a passive placement procedure has met with mixed results. One constraint on viewing cues before performing a learned swimming response to a hidden goal has been the act of dynamically viewing distal cues while moving through a part of the environment where an optimal learned spatial escape response would be observed. We briefly review these past findings obtained with adult animals to the recent efforts of establishing a “behavioral topology” separating cognitive-spatial learning from tasks differing in motoric demands in an attempt to define when cognitive-spatial behavior emerges during development.",
"keywords": [
"Spatial navigation",
"Place response",
"Hippocampal development",
"Multiple memory systems",
"Latent learning",
"Learning/performance distinction",
"Individual differences",
"Strategy selection"
],
"content": "Summary of new target findings\n\nThe study by Comba et al.1 suggests a critical period of prenatal development (PND) in rodents during which neuronal mossy fiber growth in the hippocampus is associated with the emergence of spatial behavior. The researchers emphasize the PND 15–18 month period where this growth connecting the dentate gyrus to the CA3 cellular field is most prominent. It is also noted in the introduction that such growth may be related to the finding that neurogenesis-based processes specific to the hippocampus are also associated with the emergence of spatial learning. The researchers describe a “behavioral typology” control in their research design to dissociate effects based on non-cognitive motor demands from true cognitive information processing, which was supported by their data analyses of place learning in the Morris water maze versus spatial exploration in a dry land task (with swimming being more difficult than common ambulation). Despite the difference in motor demands, there was a common emergence of spatial behavior proficiency on each task at PND20. The researchers also found that enhanced mossy fiber projections, revealed by synaptophysin staining in the CA3 region, preceded the emergence of spatial behavior. Developmentally-dependent functional changes in cFOS positive cells were increased in all hippocampal subregions measured, while training-dependent changes were restricted to the CA3 and CA1 regions for groups trained in the water maze. The researchers conclude that mossy fiber connectivity along with enhanced function of the hippocampus precedes the emergence of spatial behavior at PND20, confirming their hypothesis of a sensitive period for hippocampal growth and the emergence of cognitive-spatial function.\n\n\nReview of the broader issue: “Behavioral typology”\n\nThis research is very important and exciting when considering past studies of place learning ability in adult rats and research attempts to dissociate motor performance from true cognitive processing. In previous water maze studies of latent learning2,3 using passive placement on the goal in the water maze to view distal cues to form a cognitive map of the environment, mixed results and individual differences seem to obscure matters4–8, leading to one interpretation that movement and cognitive mapping may necessarily occur simultaneously, a finding that has been replicated in humans using a virtual version of the water maze9. Hence, for the water maze at least, movement through the environment seems to be an important constraint on highly proficient spatial learning and navigation. The use of a separate dry land task by Comba et al. with less motoric demands seems to be in agreement with the difference in motor demands between the original rodent version of the task10 requiring swimming and the human virtual version9 using minimal hand/finger movements to navigate. The role of dynamic movement during spatial tasks and the motoric demands have been topics of intense interest with the role of the hippocampus as a substrate for cognitive mapping11,12, path integration13–17, or the conductor of a symphony of dynamic movement and mapping18 as part of a larger neural network of brain systems19 have all been hotly debated theoretically over the years. The separation of cognitive and motor performance associated with developmentally-specific changes in hippocampal circuitry by Comba et al. is an exciting finding that may have important implications for a central role of the hippocampus in cognitive-spatial information processing as it emerges early in development.\n\nConsequently, the “behavioral typology” issue in the Comba et al. study, along with other aspects of their research design, is of critical importance in assessing the emerging role of the hippocampus in cognitive-spatial behavior. The following matters should be considered by all interested in this fascinating area of research in general, and in the Comba et al. study in particular.\n\n\nSpecific considerations\n\n1) The level of spatial proficiency in escaping to a hidden platform for PND20 rats given only eight trials in the water maze is not comparable to the asymptotic level of escape latency performance (< 10 sec) observed in most water maze studies after considerably more extensive training. The 1 day water maze training paradigm is likely tapping into ventral hippocampal function in which the animals are just approaching the general location. After more training, dorsal hippocampus forms a more precise representation of the location. The authors should discuss this work20 and an analysis of their data (dorsal versus ventral) would be of interest.\n\n2) On a related matter, escape is not required, and exploration of an object at a novel “place” in the dry land task is very different from the typical water maze procedure, involving the presence of a local cue or familiar beacon (with different motivation). Some might argue that the lack of “true” spatial proficiency in the water maze is a flaw or weakness of the study; however, given the focus on the emergence of spatial behavior, and that well-learned escape responses are dependent on other brain regions21,22 that contribute/correlate with movement parameters19, it seems reasonable to expect less performance-wise using the escape latency measure than what is typically observed in most studies of this type.\n\n3) Consequently, spatial bias on a probe test might be considered as an alternative measure in future studies as it does not depend on a well-learned escape response that may be less hippocampal-dependent and more closely approximates the dwell time that is measured on the spatial exploration task.\n\n4) The object/place task is interesting. Integration with ideas about direct versus indirect measures of memory and the role of the hippocampus in one versus the other, and how these ideas relate to their different measures on this task would be of interest23.\n\n5) There are other obvious differences between the two tasks. Water maze for example is not disrupted by disorientation procedures but a dry-land version of spatial localization is disrupted by disorientation24. This work suggests that the representations are different as well, not just the behavioral topology. This should be discussed.\n\n6) The number of rats in each group for the water maze task is quite low (n = 5/group).\n\n7) A statement on the standardization of immunohistochemical procedures would be reassuring for those not familiar with the specific techniques used. Also, the use of an unbiased stereology technique should be considered.\n\n8) It appears that different behavioral procedures may have been conducted at different institutions. A statement on the time of day of testing and other procedural controls would provide reassurance that there are no threats to internal validity.\n\n9) More information on the recording and quantification of exploratory behavior (e.g., video recording, tracking, and interrater reliability) would be helpful for assessment and replication.\n\n10) It is interesting that the researchers note that PND18 rodents traveled a longer distance in the novel relocation task then the other groups (even though apparently PND20 rats exhibited more exploratory behavior). This finding may warrant further discussion to support the argument that both tasks assess potentially related cognitive functions.\n\n11) Reassurance that no statistical assumptions were violated (e.g., sphericity). Tukey HSDs are specifically based on studentized q-related statistics but t-tests were reported. Though this may have been simply an alternative method (e.g. regression-based) to report the Tukey post-hoc results, possibly Bonferroni t-tests with separate mean square error denominators may be optimal for potential corrections to assumption violations.\n\n\nNeural substrates of cognition and spatial performance\n\nFollowing the lead of prior studies of spatial memory and hippocampal function using the radial arm maze25, Morris initially used the approach of transecting the fornix/fimbria to disrupt hippocampal function26, but only observed modest impairments in the water maze. Only later studies showed that direct neurotoxin lesions of the hippocampus produced severe impairments27,28. Sutherland and Rodriguez22 showed that only complete transaction of the fornix/fimbria abolished both acquisition and retention of navigation to a hidden platform and detailed the effects of lesions to structures receiving input from the hippocampus via the fornix/fimbria, including severe impairments of postoperative acquisition produced by bilateral damage to the medial nucleus accumbens or bilateral damage to the anterior thalamic area with little effect on retention of preoperatively acquired place navigation. Also, damage to the medial septum or mammillary complex produced modest impairments evident only in postoperative acquisition. These findings were among the first to detail network connections involved in place navigation in the water maze.\n\nA subsequent study comparing preoperative fornix/fimbria versus caudate-putamen lesions revealed somewhat surprising results: place navigation escape latency performance was severely impaired by caudate-putamen lesions and only mildly impaired by fornix/fimbria lesions, manifesting on the latter trials of acquisition when controls had reached asymptotic performance (approximately under 10 sec/trial block). A detailed analysis of the animals’ behavior revealed that fornix/fimbria-lesioned rats used compensatory strategies, circumnavigating at a more-or-less constant distance from the pool wall until swimming into the platform, often without slowing down and anticipating climbing onto the refuge (failing to disinhibit the forepaws from the natural swimming posture). The fornix rats also showed evidence of using an angled trajectory from all start points that often led directly to the platform from one of the four start locations. The combination of these non-spatial strategies led to spared performance early in acquisition, consistent with the variable results obtained in previous studies. Despite the relatively less severe impairment on escape trials, fornix rats failed to show the normal spatial bias for the location of the platform when it was removed from the pool for the standard probe test. In contrast, the severe impairment in escape latency observed during acquisition trials for caudate-lesioned rats, which was due to prolonged thigmotaxis, did not interfere with their showing a near normal spatial bias on the probe test. These findings suggest that fornix rats were impaired at knowing where the platform was formerly located, while caudate-lesioned rats were impaired at implementing procedural aspects of the task (related to thigmotaxis) but not in knowing where. This conclusion directly contradicts the interpretation that fornix rats can learn a place response (knowing where) but have difficulty getting there (a motor impairment)17 and is supported by human virtual navigation studies19,29 detailing a network of interactive brain systems. In comparison to the Comba et al. study, these findings suggest that a single day of place training with the primary performance measure of escape latency is less hippocampal-dependent than a probe test measure of spatial bias. Hence, a more impressive demonstration of when hippocampal-related spatial-cognition emerges during development would involve the use a probe test of performance in the water maze, as noted above.\n\nThe issue of spared early water maze acquisition following hippocampal damage and severe impairment due to caudate lesions suggests that the initial trials/day(s) of water maze training may primarily involve procedural learning as animals get accustomed to navigating in water and learn about the task demands. This may be related to the greater sensitivity of probe test performance to hippocampal damage21, which typically occurs after considerable training. Analysis of micro-behaviors, such as the characteristics of swimming, pausing and path patterns may be more informative then the overall latency to escape, another reason why probe test behavior has the potential to reveal the specifics of spatial learning/memory, especially in a non-aggregated form that considers the temporal aspects of navigational behavior30,31.\n\n\nIndividual differences in spatial behavior as a function of other factors\n\nGiven the fact that fornix-lesioned rats may use compensatory strategies during escape acquisition21, and that a network of brain structures may contribute to spatial behavior19,22,29, it is possible that individual differences may emerge during development. Individual differences in adult rats during place navigation and following a latent learning test in a novel environment suggested differential influence of the stimulus control of spatial behavior, with entrance into a room representing a polarizing cue for some individuals4. Such changes may occur later in development and involve cholinergic markers in other brain structures such as the striatum, with stable measurements observed in the hippocampus32.\n\nA form of individual difference, sex/gender, is another area where mixed results have been observed in the water maze33 and have been shown to depend on strain and volumetric brain differences, including the hippocampus and its subregions, prefrontal cortex areas and the amygdala34. Behaviorally, the largest sex differences have been reported during the initial trajectory phase of a trial35, which may depend on effective processing of distal features of the environment in planning appropriate navigational behavior. In another study young male and female rats were equally proficient in finding the platform during training trials, however probe tests showed that young male rats had better knowledge of the platform's precise location and was correlated with larger basal forebrain cholinergic neurons compared to females36. Further, there was no sex difference in aged rats that exhibited an overall spatial learning impairment, however aged males now had smaller cholinergic neurons whereas no change was observed in females. These results reveal a complex interaction between sex, age and spatial behavior. Comparing performance on different spatial tasks in humans, including virtual water maze, Astur et al.37 concluded that even after equating factors such as motivation, stress and motor demands, procedural demands of the tasks may nevertheless lead to differential strategy selection during spatial memory, and suggested that researchers use caution when utilizing different tasks interchangeably as tests of spatial memory. Hence, assuming that differences in the motoric demands of two tasks isolates spatial cognition may not account for a myriad of other differences that could potentially influence performance.\n\n\nConclusion\n\nAlthough the main contribution of the Comba et al. study is in the potential advancement of our knowledge on hippocampal plasticity related to the emergence of cognitive-spatial behavior during a developmentally-specific sensitive period, the importance of integration across networks and neural systems should not be lost in the reductionism. For example, recent findings show that water maze performance may change the functional connectivity between subregions of hippocampus and striatum in female rats and humans38,39. Perhaps future work may focus on a comparison of sensitive periods across these systems to define functional connectivity among cellular networks of brain systems. Focus may also be expanded to different time points in the life cycle, to not only the developmental emergence of cognitive function but also the stabilization, maintenance and eventual decline, having potential translational value in the treatment of neurodegenerative disease.",
"appendix": "Author contributions\n\n\n\nBDD conceived the correspondence after being invited to referee the target research article cited within. All authors (BDD, CM and RJM) contributed significant parts of the draft manuscript, were involved in the revision 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\nComba R, Gervais NJ, Mumby DG, et al.: Emergence of spatial behavioral function and associated mossy fiber connectivity and c-Fos labeling patterns in the hippocampus of rats [v1; ref status: indexed, http://f1000r.es/5nr]. F1000Res. 2015; 4: 396. Publisher Full Text\n\nKeith JR, McVety KM: Latent place learning in a novel environment and the influences of prior training in rats. Psychobiology. 1988; 16(2): 146–51. Reference Source\n\nSutherland RJ, Linggard R: Being there: a novel demonstration of latent spatial learning in the rat. Behav Neural Biol. 1982; 36(2): 103–7. PubMed Abstract | Publisher Full Text\n\nDevan BD, Petri HL, Mishkin M, et al.: A room with a view and a polarizing cue: individual differences in the stimulus control of place navigation and passive latent learning in the water maze. Neurobiol Learn Mem. 2002; 78(1): 79–99. PubMed Abstract | Publisher Full Text\n\nChew GL, Sutherland RJ, Whishaw IQ: Latent learning does not produce instantaneous transfer of place navigation: A rejoinder to Keith and McVety. Psychobiology. 1989; 17(2): 207–9. Reference Source\n\nWhishaw IQ: Latent learning in a swimming pool place task by rats: Evidence for the use of associative and not cognitive mapping processes. Q J Exp Psychol B. 1991; 43(1): 83–103. PubMed Abstract\n\nJacobs WJ, Zaborowski JA, Whishaw IQ: Failure to find latent spatial learning in the Morris Water Task: Retraction of Jacobs, Zaborowski, and Whishaw (1989). J Exp Psychol Anim Behav Process. 1989; 15(3): 286. Publisher Full Text\n\nJacobs WJ, Zaborowski JA, Whishaw IQ: Rats repeatedly placed on a hidden platform learn but quickly forget its location. J Exp Psychol Anim Behav Process. 1989; 15(1): 36–42. Publisher Full Text\n\nHamilton DA, Driscoll I, Sutherland RJ: Human place learning in a virtual Morris water task: some important constraints on the flexibility of place navigation. Behav Brain Res. 2002; 129(1–2): 159–70. PubMed Abstract | Publisher Full Text\n\nMorris RGM: Spatial localization does not require the presence of local cues. Learn Motiv. 1981; 12(2): 239–60. Publisher Full Text\n\nO'Keefe J, Nadel L: The hippocampus as a cognitive map. Oxford: Clarendon Press. 1978. Reference Source\n\nTolman EC: Cognitive maps in rats and men. Psychol Rev. 1948; 55(4): 189–208. PubMed Abstract | Publisher Full Text\n\nWhishaw IQ, Jarrard LE: Evidence for extrahippocampal involvement in place learning and hippocampal involvement in path integration. Hippocampus. 1996; 6(5): 513–24. PubMed Abstract | Publisher Full Text\n\nWhishaw IQ, Maaswinkel H: Rats with fimbria-fornix lesions are impaired in path integration: a role for the hippocampus in “sense of direction”. J Neurosci. 1998; 18(8): 3050–8. PubMed Abstract\n\nWhishaw IQ: Place learning in hippocampal rats and the path integration hypothesis. Neurosci Biobehav Rev. 1998; 22(2): 209–20. PubMed Abstract | Publisher Full Text\n\nWhishaw IQ, McKenna JE, Maaswinkel H: Hippocampal lesions and path integration. Curr Opin Neurobiol. 1997; 7(2): 228–34. PubMed Abstract | Publisher Full Text\n\nWhishaw IQ, Cassel JC, Jarrad LE: Rats with fimbria-fornix lesions display a place response in a swimming pool: a dissociation between getting there and knowing where. J Neurosci. 1995; 15(8): 5779–88. PubMed Abstract\n\nSutherland RJ: The navigating hippocampus: An individual medley of movement, space, and memory. In: G. Buzsáki CHV, editor. Electrical activity of the archicortex. Budapest, Hungary: Akadémiai Kiadó;. 1985; 255–79.\n\nMaguire EA, Burgess N, Donnett JG, et al.: Knowing where and getting there: a human navigation network. Science. 1998; 280(5365): 921–4. PubMed Abstract | Publisher Full Text\n\nRuediger S, Spirig D, Donato F, et al.: Goal-oriented searching mediated by ventral hippocampus early in trial-and-error learning. Nat Neurosci. 2012; 15(11): 1563–71. PubMed Abstract | Publisher Full Text\n\nDevan BD, Goad EH, Petri HL: Dissociation of hippocampal and striatal contributions to spatial navigation in the water maze. Neurobiol Learn Mem. 1996; 66(3): 305–23. PubMed Abstract | Publisher Full Text\n\nSutherland RJ, Rodriguez AJ: The role of the fornix/fimbria and some related subcortical structures in place learning and memory. Behav Brain Res. 1989; 32(3): 265–77. PubMed Abstract | Publisher Full Text\n\nMoses SN, Sutherland RJ, McDonald RJ: Differential involvement of amygdala and hippocampus in responding to novel objects and contexts. Brain Res Bull. 2002; 58(5): 517–27. PubMed Abstract | Publisher Full Text\n\nGibson BM, Shettleworth SJ, McDonald RJ: Finding a goal on dry land and in the water: differential effects of disorientation on spatial learning. Behav Brain Res. 2001; 123(1): 103–11. PubMed Abstract | Publisher Full Text\n\nBecker JT, Walker JA, Olton DS: Neuroanatomical bases of spatial memory. Brain Res. 1980; 200(2): 307–20. PubMed Abstract | Publisher Full Text\n\nMorris RGM, Garrud P, Woodhouse IQ: Fornix Lesions Disrupt Location Learning by the Rat. Behav Brain Res. 1981; 2(2): 266–7. Publisher Full Text\n\nSutherland RJ, Kolb B, Whishaw IQ: Spatial mapping: definitive disruption by hippocampal or medial frontal cortical damage in the rat. Neurosci Lett. 1982; 31(3): 271–6. PubMed Abstract | Publisher Full Text\n\nMorris RG, Garrud P, Rawlins JN, et al.: Place navigation impaired in rats with hippocampal lesions. Nature. 1982; 297(5868): 681–3. PubMed Abstract | Publisher Full Text\n\nMaguire EA, Frith CD, Burgess N, et al.: Knowing where things are parahippocampal involvement in encoding object locations in virtual large-scale space. J Cogn Neurosci. 1998; 10(1): 61–76. PubMed Abstract | Publisher Full Text\n\nDevan BD, McDonald RJ: A cautionary note on interpreting the effects of partial reinforcement on place learning performance in the water maze. Behav Brain Res. 2001; 119(2): 213–6. PubMed Abstract | Publisher Full Text\n\nDevan BD, Stouffer EM, Petri HL, et al.: Partial reinforcement across trials impairs escape performance but spares place learning in the water maze. Behav Brain Res. 2003; 141(2): 91–104. PubMed Abstract | Publisher Full Text\n\nColombo PJ, Gallagher M: Individual differences in spatial memory and striatal ChAT activity among young and aged rats. Neurobiol Learn Mem. 1998; 70(3): 314–27. PubMed Abstract | Publisher Full Text\n\nJonasson Z: Meta-analysis of sex differences in rodent models of learning and memory: a review of behavioral and biological data. Neurosci Biobehav Rev. 2005; 28(8): 811–25. PubMed Abstract | Publisher Full Text\n\nKeeley RJ, Bye C, Trow J, et al.: Strain and sex differences in brain and behaviour of adult rats: Learning and memory, anxiety and volumetric estimates. Behav Brain Res. 2015; 288: 118–31. PubMed Abstract | Publisher Full Text\n\nWoolley DG, Vermaercke B, Op de Beeck H, et al.: Sex differences in human virtual water maze performance: novel measures reveal the relative contribution of directional responding and spatial knowledge. Behav Brain Res. 2010; 208(2): 408–14. PubMed Abstract | Publisher Full Text\n\nVeng LM, Granholm AC, Rose GM: Age-related sex differences in spatial learning and basal forebrain cholinergic neurons in F344 rats. Physiol Behav. 2003; 80(1): 27–36. PubMed Abstract | Publisher Full Text\n\nAstur RS, Tropp J, Sava S, et al.: Sex differences and correlations in a virtual Morris water task, a virtual radial arm maze, and mental rotation. Behav Brain Res. 2004; 151(1–2): 103–15. PubMed Abstract | Publisher Full Text\n\nWoolley DG, Laeremans A, Gantois I, et al.: Homologous involvement of striatum and prefrontal cortex in rodent and human water maze learning. Proc Natl Acad Sci U S A. 2013; 110(8): 3131–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWoolley DG, Mantini D, Coxon JP, et al.: Virtual water maze learning in human increases functional connectivity between posterior hippocampus and dorsal caudate. Hum Brain Mapp. 2015; 36(4): 1265–77. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10254",
"date": "10 Sep 2015",
"name": "Derek Hamilton",
"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 brief correspondence was motivated by a recent F1000Research report by Comba et al. in which the development of mossy fiber projections and associated functional alterations in the CA3 and CA1 subfields were argued to precede the emergence of spatial cognition as measured at PND16, 18, or 20 in two tasks that differ in the motor and motivational properties. Based on the observations that improved latencies in the Morris water task and detection of changes in object location were observed only at PND20 the authors of the original target article conclude that the aforementioned neurobiological development precedes emergence of the purely spatial cognitive abilities in question (without respect to specific motor skills). Devan et al. utilize this conclusion as a point of departure for discussion of a long-standing issue in the study of spatial navigation and its development concerning the relative contributions of cognitive and motor skills. This is perhaps best represented in the literature on “latent learning” in the water task as well as the broader literature on neural systems involved in motor and spatial learning to which the authors have made important contributions. The authors briefly provide a summary overview of the target article findings, a review of the broader issue of “behavioral topology”, an enumerated list of specific considerations (many related specifically to the target article), a brief review of some key neurobiological findings relevant to the central theme of the manuscript, and a final section on the significance of individual differences in performance. I am inclined to recommend approval because this is a brief correspondence piece that nicely frames what has been an important, if not central, thematic issue in the field of spatial behavior for many years in the context of the recent report by Costa et al. This is a reasonable opinion/correspondence article that emerged from a prior published F1000Research review which may influence subsequent thinking and research on the ontogeny of spatial navigation and its neural bases and does not include specific technical/methodological aspects that require review. I do, however, offer several recommendations for improving the manuscript that the authors could consider.Perhaps the most significant issue is that the basic premise is based on the conclusion that spatial proficiency emerges in tasks that vary in motor and motivational demands around PND20. As is noted, this conclusion is based on latency data in the water task that suggest improvement in performance at PND20 not observed at younger ages. The path length data, however, suggest that the animals are swimming roughly 4 times the diameter of the pool in order to escape, regardless of age. Thus, the suboptimal reductions in latency among the PND20 rats do not reflect direct trajectories to the escape platform characteristic of “optimal” spatial learning, which complicates interpretation. This is likely related to the limited number of training trials that were used, which the authors address. Given the importance of this conclusion, additional reference to prior work that used more training trials (e.g., 12-24) concentrated on a single day may provide better support for a developmental emergence of spatial learning around PND19-21.\n\nThe authors draw attention to several important aspects of the Costa et al. article that are worthy of consideration and frame these in the context of general points for which readers interested in the topic should be aware. This is an important component of the manuscript and, overall, these points are well reasoned and accurate. In the spirit of highlighting points of importance for studies of this type I would suggest the authors consider a few additional points (or modifications) that, in my opinion, are important for developmental studies of navigation in rats. These include : 1) considering development of thermoregulation abilities (e.g., Brown and Whishaw, 2000; Akers and Hamilton, 2007) which could potentially affect performance (in both motor and cognitive domains) differently in dry and wet tasks, 2) the apparatus size (e.g., Carman and Mactutus, 2001), and 3) to expand on point 3 from the authors’ enumerated list, the use of tasks/measures that are sensitive to the detection of constituent cognitive processes during development (see e.g., Akers et al., 2011, as data from that study suggest that distal visual cues can control some aspects of spatial behavior in the water task as early as PND17, but definitely not on PND16). Regarding point 4 in the authors’ list, I agree that this is interesting, however, some brief elaboration on this point could help frame this suggestion for readers unfamiliar with the direct/indirect distinction as described in Moses et al. Under “Neural substrates of spatial cognition”, the work described in the paragraph beginning “A subsequent study …” should include a citation for clarification. Perhaps the most important single statement in this correspondence is in the conclusion that attention to the development of functional interactions among the broader circuitry involved in spatial navigation not be lost in the context of conclusions about specific behavioral, cognitive, or neurobiological elements. The potential influence of this suggestion for subsequent research and thinking on this important issue could be strengthened by emphasizing this point earlier in the manuscript. Some minor issues In the first sentence of the “Summary of New Target Findings” the word “month” should be omitted “Typology” or “topology” : The authors use both “behavioral typology” and “behavioral topology” in the manuscript. In the sentence (page 4, col 2 of pdf) beginning “Analysis of micro …”, “then” should be “than”",
"responses": []
},
{
"id": "10249",
"date": "10 Sep 2015",
"name": "Jeffrey M. Long",
"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 article was inspired by the author’s recent review of an F1000Research report examining the temporal relationship between hippocampal anatomical development and spatial cognition. The first part of this opinion article summarizes their previous review and the second part nicely expands into a more theoretical discussion of the relationship between the cognitive and motor demands of spatial memory tasks. The manuscript calls attention to several important factors to be considered when critically evaluating performance in spatial memory tasks and I recommend approval.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-625
|
https://f1000research.com/articles/4-872/v1
|
18 Sep 15
|
{
"type": "Study Protocol",
"title": "A home-based comprehensive care model in patients with Multiple Sclerosis: A study pre-protocol",
"authors": [
"Lufei Young",
"Kathleen Healey",
"Mary Charlton",
"Kendra Schmid",
"Rana Zabad",
"Rebecca Wester",
"Kathleen Healey",
"Mary Charlton",
"Kendra Schmid",
"Rana Zabad",
"Rebecca Wester"
],
"abstract": "Background Disability is prevalent in individuals with multiple sclerosis (MS), leading to difficulty in care access, significant caregiver burden, immense challenges in self-care and great societal burden. Without highly coordinated, competent and accessible care, individuals living with progressive MS experience psychological distress, poor quality of life, suffer from life-threatening complications, and have frequent but avoidable healthcare utilizations. Unfortunately, current healthcare delivery models present severe limitations in providing easily accessible, patient-centered, coordinated comprehensive care to those with progressive MS. We propose a home-based comprehensive care model (MAHA) to address the unmet needs, challenges, and avoidable complications in individuals with progressive MS with disabling disease.Objective The article aims to describe the study design and methods used to implement and evaluate the proposed intervention. Method The study will use a randomized controlled design to evaluate the feasibility of providing a 24-month, home-based, patient-centered comprehensive care program to improve quality of life, reduce complications and healthcare utilizations overtime (quarterly) for 24 months. A transdisciplinary team led by a MS-Comprehensivist will carry out this project. Fifty MS patients will be randomly assigned to the intervention and usual care program using block randomization procedures. We hypothesize that patients in the intervention group will have fewer complications, higher quality of life, greater satisfaction with care, and reduced healthcare utilization. The proposed project is also expected to be financially sustainable in fee-for-service models but best suited for and gain financial success in valued-based care systems. Discussion This is the first study to examine the feasibility and effectiveness of a home-based comprehensive care management program in MS patients living with progressive disability. If successful, it will have far-reaching implications in research, education and practice in terms of providing high quality but affordable care to population living with severe complex, disabling conditions.",
"keywords": [
"multiple sclerosis",
"home-based care",
"disability",
"symptom management",
"healthcare utilization",
"value-based care"
],
"content": "Background\n\nWhile great strides have been made in the treatment of relapsing forms of multiple sclerosis (MS), many individuals have or will enter a progressive phase of this disease. This phase of disease is dynamic, highly complex and disabling, which presents extensive challenges in all aspects of care delivery1,2. The progressive phase of MS meets criteria for the description of “a consuming illness” outlined by the Patient-Centered Medical Home Model (PCMH)3. Specific problems and rate of progression vary, but many will have dysfunction in gait4,5, movement of extremities4, bladder and bowel function6,7, speech and swallowing8, and respiratory musculature9,10. Over half will have physical pain11, generally related to neuropathy and/or spasticity12. Cognitive dysfunction13 and depressive mood disorders14–17 are prevalent. Family members are also likely to suffer from caregiver burden18, mood disorders19 and strain on their own health18. As the disease progresses, a significant proportion of patients will need assistive devices, including power mobility, urinary catheters, gastric tubes, hospital beds, home modifications, and other devices1,2. Complications can be life threatening, including falls20,21, urinary tract infections (UTIs)22, respiratory conditions (pneumonia and influenza)23,24, and pressure ulcers21. Consequently, patients’ health related quality of life decreases substantially as disability ensues25. The MS Society’s White Paper captures the cry of those with progressive MS, who feel “disconnected,” “underserved,” “isolated,” “forgotten,” and “overwhelmed”26. Patients ultimately face the loss of independence in virtually all aspects of life. Still, families desire to keep their loved one at home when possible but acknowledge the overwhelming impact of the disease on the entire family27. Moreover, due to the limitations in mobility, cognition and communication, access to care is highly challenging28,29. As a result, patients living with progressive phase MS have numerous complex and dynamic healthcare needs that require a range of primary care, specialty, multidisciplinary, and community resources for a long period of time (their life expectancy)30,31. However, the current care delivery and payment systems contribute barriers and challenges in providing comprehensive coordinated care32, leading to unnecessary healthcare utilization and delayed effective treatments33. Without the comprehensive disease management by a designated provider, the quality of care received is often suboptimal or poor due to the fragmented care system in which the acute care based providers are unfamiliar with patients’ needs, have little knowledge and experience caring for patients with MS34. The combination of the current ambulatory care system utilizes a fee-for-service payment model combined with shortage of MS care specialists leaves no time to address complex chronic care issues or advance care planning35–37.\n\nTo address these problems and gaps in inadequately caring for patients living with progressive MS, we propose a home-based, patient-centered, comprehensive care management program led by a ‘MS-Comprehensivist’. The program is designed to provide a full range of medical and social services for patients and their caregivers, including a transdisciplinary team of primary care providers, specialists, care managers, rehabilitative, social home health, and personal care services. The MS-Comprehensivist who is an advance practice nurse specialized in MS care is responsible to 1) make regular house calls to address patients/family specific needs, 2) coordinate the care with the primary and specialty providers, 3) identify and mobilize other community resources, and 4) provide staff training and patient education in co-managing the complex complications and symptoms. This program, referred to as Multiple Sclerosis At Home Access (MAHA), incorporates core principles of the chronic care and patient-centered medical home models. The purpose of the proposed study is to examine the feasibility (e.g., acceptability, utility, implementation, financial sustainability, adaptation and integration)38 and effect of the MAHA model on patient-centered outcomes (i.e., complications, quality of life, satisfaction) and health care utilization outcomes (i.e., unplanned hospitalization and ED visits). The following specific aims were designed to achieve this purpose.\n\nSpecific Aim 1: To evaluate the effect of the MAHA model on 1) the numbers of complications; 2) patients’ quality of life; and 3) patients’ satisfaction over time (baseline and quarterly).\n\nSpecific Aim 2: To evaluate the effect of the MAHA model on 1) the numbers of emergency room (ER) visits, and 2) the numbers of unplanned hospitalizations.\n\nSpecific Aim 3: To evaluate the feasibility of the MAHA model.\n\n1) Acceptability by assessing patient and family experience with the program, as well as provider satisfaction\n\n2) Utility by examining the actual use of the program and number of referral received by other providers\n\n3) Implementation by evaluating the amount and type of resources needed to implement and factors affecting implementation;\n\n4) Adaptation by evaluating the selected elements of the program delivered by tele-health is as effective as face-to-face format.\n\n5) Financial sustainability and cost saving by comparing the estimated total cost of the program and financial compensation from payers.\n\n\nConceptual framework\n\nThe conceptual framework supporting the MAHA program (Figure 1) is designed based on the core elements in Wagner’s Chronic Care Model (CCM) and Donabedian’s Structure-Process-Outcome (SPO) model39. First, based on Wagner’s CCM, the MAHA model emphasizes the re-design of existing community and healthcare systems to be patient-centered40. From the patient-provider level, the unique MAHA model again is structured surrounding patients’ needs with the following key components: 1) the productive interaction between informed, activated patient/family and the MAHA team; 2) a transdisciplinary team led by an MS-Comprehensivist who is an advance practice nurse with expertise in MS care, chronic disease management and primary care; 3) care coordination and effective communication among care team members. As a result of re-designing care systems and processes, it is expected that patient outcomes (e.g, complication, quality of life and satisfaction of care) are improved, leading to reduced unplanned healthcare utilizations.\n\nBesides its function as an acronym for Multiple Sclerosis At Home Access, the term MAHA is derived from its city of origin in Omaha, Nebraska, which was settled by Native Americans of the Omaha tribe. In their language, Omaha means “against the wind or current,” which reflects American Indians survival experience from severe weather, disease and scarcity41. While caring for and supporting those MS patients with profound disabling and chronic, complex complications, the caregivers and providers often feel overwhelmed by the environmental barriers, much like the Omaha tribe must have felt centuries ago. Consistent with the core values of these original settlers (earth and sky)41, we honor holistic care which includes evidence-based medicine (earth) and equally holistic care including social, emotional, and spiritual applications (sky).\n\n\nMethods\n\nThe study is a prospective, two-group, randomized experimental design with nine data collection points (baseline and every three months). MS patients recruited from a neurology clinic will be randomized into two groups: the intervention or usual care group. The usual care group receives the current standardized MS care, while the intervention group receives usual care plus 24-months of the MAHA intervention provided by a transdisciplinary team led by a MS-Comprehensivist. The usual care group will receive the intervention at the end of the 24-month period if the intervention is found to be effective by our a priori criteria. The study is subject to review and approval by the University of Nebraska Medical Center Institutional Review Board (IRB) and informed consent will be obtained from all participants prior to the study.\n\nPotential subjects will be identified and recruited from the University of Nebraska Medical Center (UNMC) neurology clinic, where the principle investigator, who has ethical access at the clinic site, will be responsible for identifying the potential participants, screening for eligibility and referring eligible subjects for recruitment.\n\nInclusion criteria. Patients are eligible for the study if they: 1) have a diagnosis of progressive MS; 2) receive a Kurtzke Expanded Disability Status Scale (EDSS) score ≥6.5 (requires bilateral assist for ambulation and cannot walk > 120 meters; http://www.nationalmssociety.org/NationalMSSociety/media/MSNationalFiles/Brochures/10-2-3-29-EDSS_Form.pdf); and 3) have a home residence located within 60 miles of the Omaha metropolitan area.\n\nThe sample size estimation is computed based on Specific Aim 1: the increased score of quality of life (QoL) and Specific Aim 2: the reduced number of unplanned healthcare utilizations associated with complications. An estimated 50 patients per group will provide 80% power to detect a 5-point difference [Usual Care mean (SD): 45(8.6), MAHA mean (SD) 50(8.6)] between groups in the SF-36 Mental Health Component Score QoL. This sample size also provides 80% power at a 5% significance level to detect a 34% reduction in unplanned visits/admits (from 2.5 per person per year to 1.65, using the Poisson distribution, which is most appropriate for count/rate data). These are feasible and clinically important differences. Based on our preliminary analyses, 125 patients are currently expected to meet these criteria. Furthermore, based on our previous patient survey results, a high proportion of patients (~80% or n = 100) have expressed a genuine interest in participating in MS research for the benefit of others with this disabling disease. Furthermore, we estimate 50 patients will be a reasonable panel size for the MS-Comprehensivist or care team leader (CTL) in the MAHA model (unpublished report).\n\nThe MAHA model is a patient- and family-centered system in which care is tailored around the complex, chronic needs of those with progressive disabling MS. The intervention program is designed to address a fundamental question: “how will this affect the patient and/or family?” The majority of care and medical services will be provided at home, thus avoiding frequent and cumbersome clinic visits. The intervention strategies were developed based on the frequent requests and suggestions from patients with progressive MS, along with caregivers and providers experiencing daily struggles with fragmented care. Furthermore, the model is also supported by initiatives from the health policy literature developed by the National Multiple Sclerosis Society (NMSS), emphasizing the need for home- and community-based services (http://www.nationalmssociety.org/Treating-MS/Comprehensive-Care).\n\nMAHA Team Structure. The intervention will be delivered by a transdisciplinary team led by a MS-Comprehensivist, also referred to as Care Team Leader (CTL). The care provided is comprehensive and holistic, addressing patient/family physical, emotional and spiritual, and social needs. The transdisciplinary team includes the core team partners and “neighbors and best friends” (Figure 2). The CTL role will be filled by a nurse practitioner (NP) with specialty training in MS and extensive experience managing patients with chronic illness. The core team partners include the CTL, MS neurologist, primary care provider (PCP), and selected home health agencies. To address MS patients’ complex needs and reduce unplanned healthcare services, the CTL will closely collaborate with patients and family, the MS neurologist and the PCP to develop patient-centered care plans and goals, and to co-manage symptoms and complications. The selected home health agencies will provide nursing and personal care staff, physical and occupational therapists (PT/OT), and social work services. They are responsible for carrying out the care plan and conducting collaborative, on-going evaluation of the care plan with the CTL. Given MS patients’ limitations in mobility, PT/OTs will also be involved in plans of care to facilitate the restoration and maintenance of function oriented toward activities of daily living (ADLs). PTs from the home health agencies will be trained by physical therapists who have extensive experience in the education and care of MS patients. “Neighbors & Best Friends” represent subspecialty providers and community resources. The “neighbors”, or subspecialty providers (e.g., urologist, rehabilitative professionals, palliative care specialist, ophthalmologist, wound care specialist, etc.) provide consultation services and advise the CTL in preventative and treatment strategies for common complications or issues not responsive to standard strategies. The “Best Friends”, include community resources, including clergy, respite services, the MS Society, League of Human Dignity, and Office on Aging, among others.\n\nMAHA Team Processes (10 C’s). The MS-Comprehensivist or CTL will be responsible to: 1) provide regular and frequent visits to patients’ homes using a pre-planned schedule based on each individual’s care needs; 2) coordinate patients’ care by collaborating and communicating closely with the core team partners, and “neighbors and best friends”; and 3) train the home health staff in the specialty care of MS patients. In the process of care, the MAHA model stresses patient/family engagement (i.e., informed and activated) and productive interaction between patients and providers through ten fundamental elements (10 Cs; Figure 3).\n\nKey data collection instruments, assessments, and measures are summarized in Table 1. This information will be collected from three data sources: 1) patient reports to CRC personnel, 2) patient reports to the CTL or other MAHA team member at each home visit, and 3) medical record review.\n\n*Baseline questionnaires will be performed at the baseline clinic visit\n\nSpecific Aim 1 and 2. The data analysis will follow the intention-to-treat (ITT) protocol. To ensure groups are comparable, descriptive analyses, including t-tests and Chi-square tests will be conducted to compare the characteristics of each group at baseline. The occurrence of complications and other self-reported measures, such as ADLs, Multiple Sclerosis QoL Inventory (MSQLI), CSQ-8, and Caregiver Burden Inventory (CBI) will be compared between groups using a linear mixed model to account for the repeated measurements over time and adjust for baseline scores of the respective measures. To determine if differences exist between groups after adjusting for potential confounding variables, such as age, gender, comorbidity, etc. or imbalances between groups, unplanned healthcare utilization (i.e., visits/admissions) will be analyzed using Poisson regression models. The proportion of any versus no use of each health service (e.g., proportion with any inpatient admissions versus the proportion with no admissions) will be compared between groups using Chi-square tests. The time-by-group interaction will be investigated to determine if any changes over time are consistent between groups.\n\nSpecific Aim 3. To assess the acceptability, utility, implementation and adaptation of the program, we will create a toolkit that comprehensively describes our program structure, processes, patients/provider/system outcomes, as well as lessons learned. This toolkit will serve to evaluate the MAHA program and identify system-, provider-, and patient-levels of barriers and facilitators. To evaluate financial sustainability, we will specifically track the Current Procedural Terminology (CPT) codes for evaluation and management visits (clinic and home visits), care coordination, and care plan oversight. Reimbursement amounts associated with these codes will be summed for each study group at six and 12 months. T-tests and log transformed linear regression models will be used to compare total reimbursement amounts between groups. Also, staffing and associated salary estimates will be computed based on home visit time logs maintained by the MAHA team members, on-call activity logs, and number of home visits in the intervention group. We will project the necessary percent FTE and salary to execute the intervention model based on a range of panel sizes (e.g., 25, 50, 100 patients) per MS-Comprehensivist/CTL. The ratio of total salary costs-to-reimbursement amount for a given number of patients will be calculated for each group and compared using t-tests.\n\n\nDiscussion\n\nThe study is highly relevant to MS patients overwhelmed by the challenges of accessing healthcare and the myriad of complications related to such a progressive, disabling disease. The purpose is to examine the feasibility and effectiveness of a home-based, patient-centered, comprehensive care model on patient reported and healthcare utilization outcomes. Although the tools, expertise, and strategies to prevent many of these complications and to improve quality of life exist and prove to be effective, their use and implementation is hindered by a siloed system, fragmented care processes, and an inappropriate volume-based payment system. The proposed study is designed to evaluate an innovative model to meet the complex needs and overcome the self-care challenges of those MS patients in a progressive, disabling stage of disease.\n\nImplication to Practice: The study findings and all project materials will assist other institutions and providers in adapting the MAHA model to manage MS and other populations living with chronic complex conditions. In addition, the study findings may inform NMSS to develop and/or update guidelines in managing MS patients in progressive stage, leading to the improved care and prolonged lives of MS patients.\n\nImplication to Education: The toolkits and manuals we developed for patient/provider education could be integrated into the curriculum in various healthcare professional programs and modified to be continuing education packets for clinicians.\n\nIf proven successful, we will further adapt the model components and intervention strategies to be delivered via telehealth to reach rural/remote populations facing significant challenges in accessing care. Based on our preliminary analysis and the expertise of our team, we believe the MAHA intervention will be the missing ingredient for mitigating the challenges in managing populations living with chronic and complex illness. The long-term goal of this study is to conduct a larger scale study and create a template of chronic complex disease management to be expanded and disseminated in managing all population living with disabling and consuming conditions.",
"appendix": "Author contributions\n\n\n\nAll authors contributed to the development of this study protocol. Dr. Young prepared the manuscript. Dr. Healey and Dr. Charlton conceived and designed the intervention and the underlying conceptual framework. Dr. Healey, Dr Zabad, and Dr. Wester did preliminary study to gather feasibility evidence of this study protocol. Dr Schmid and Dr. Charlton assisted study design, the development of outcome measures and data management plan. All authors contributed to revising the manuscript and have agreed to the final version of this protocol.\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\nFraser R, Ehde D, Amtmann D, et al.: Self-management for people with multiple sclerosis: report from the first international consensus conference, November 15, 2010. Int J MS Care. 2013; 15(2): 99–106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchapiro RT: The symptomatic management of multiple sclerosis. Ann Indian Acad Neurol. 2009; 12(4): 291–295. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican College of Physicians. The patient centered medical home neighbor: The Interface of the patient-centered home with Specialty/Subspecialty practices. American College of Physicians Policy Paper. 2010. Reference Source\n\nMinden SL, Frankel D, Hadden L, et al.: The Sonya Slifka Longitudinal Multiple Sclerosis Study: methods and sample characteristics. Mult Scler. 2006; 12(1): 24–38. PubMed Abstract | Publisher Full Text\n\nPike J, Jones E, Rajagopalan K, et al.: Social and economic burden of walking and mobility problems in multiple sclerosis. BMC Neurol. 2012; 12: 94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChia YW, Fowler CJ, Kamm MA, et al.: Prevalence of bowel dysfunction in patients with multiple sclerosis and bladder dysfunction. J Neurol. 1995; 242(2): 105–108. PubMed Abstract | Publisher Full Text\n\nNortvedt MW, Riise T, Frugård J, et al.: Prevalence of bladder, bowel and sexual problems among multiple sclerosis patients two to five years after diagnosis. Mult Scler. 2007; 13(1): 106–112. PubMed Abstract | Publisher Full Text\n\nDe Pauw A, Dejaeger E, D'hooghe B, et al.: Dysphagia in multiple sclerosis. Clin Neurol Neurosurg. 2002; 104(4): 345–351. PubMed Abstract | Publisher Full Text\n\nGosselink R, Kovacs L, Decramer M: Respiratory muscle involvement in multiple sclerosis. Eur Respir J. 1999; 13(2): 449–454. PubMed Abstract\n\nSrour N, LeBlanc C, King J, et al.: Lung volume recruitment in multiple sclerosis. PLoS One. 2013; 8(1): e56676. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinden SL, Frankel D, Hadden LS, et al.: Disability in elderly people with multiple sclerosis: An analysis of baseline data from the Sonya Slifka Longitudinal Multiple Sclerosis Study. NeuroRehabilitation. 2004; 19(1): 55–67. PubMed Abstract\n\nFoley PL, Vesterinen HM, Laird BJ, et al.: Prevalence and natural history of pain in adults with multiple sclerosis: Systematic review and meta-analysis. Pain. 2013; 154(5): 632–642. PubMed Abstract | Publisher Full Text\n\nRao SM, Leo GJ, Bernardin L, et al.: Cognitive dysfunction in multiple sclerosis. I. Frequency, patterns, and prediction. Neurology. 1991; 41(5): 685–691. PubMed Abstract | Publisher Full Text\n\nChwastiak L, Ehde DM, Gibbons LE, et al.: Depressive symptoms and severity of illness in multiple sclerosis: epidemiologic study of a large community sample. Am J Psychiatry. 2002; 159(11): 1862–8. PubMed Abstract | Publisher Full Text\n\nWood B, van der Mei IA, Ponsonby AL, et al.: Prevalence and concurrence of anxiety, depression and fatigue over time in multiple sclerosis. Mult Scler. 2013; 19(2): 217–224. PubMed Abstract | Publisher Full Text\n\nMarrie RA, Horwitz R, Cutter G, et al.: The burden of mental comorbidity in multiple sclerosis: frequent, underdiagnosed, and undertreated. Mult Scler. 2009; 15(3): 385–392. PubMed Abstract | Publisher Full Text\n\nKorostil M, Feinstein A: Anxiety disorders and their clinical correlates in multiple sclerosis patients. Mult Scler. 2007; 13(1): 67–72. PubMed Abstract | Publisher Full Text\n\nAcaster S, Perard R, Chauhan D, et al.: A forgotten aspect of the NICE reference case: an observational study of the health related quality of life impact on caregivers of people with multiple sclerosis. BMC Health Serv Res. 2013; 13: 346. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLehan T, Arango-Lasprilla JC, Macias MÁ, et al.: Distress associated with patients' symptoms and depression in a sample of Mexican caregivers of individuals with MS. Rehabil Psychol. 2012; 57(4): 301–307. PubMed Abstract | Publisher Full Text\n\nGunn H, Creanor S, Haas B, et al.: Frequency, characteristics, and consequences of falls in multiple sclerosis: findings from a cohort study. Arch Phys Med Rehabil. 2014; 95(3): 538–45. PubMed Abstract | Publisher Full Text\n\nRedelings MD, McCoy L, Sorvillo F: Multiple sclerosis mortality and patterns of comorbidity in the united states from 1990 to 2001. Neuroepidemiology. 2006; 26(2): 102–107. PubMed Abstract | Publisher Full Text\n\nManack A, Motsko SP, Haag-Molkenteller C, et al.: Epidemiology and healthcare utilization of neurogenic bladder patients in a US claims database. Neurourol Urodyn. 2011; 30(3): 395–401. PubMed Abstract | Publisher Full Text\n\nSumelahti ML, Hakama M, Elovaara I, et al.: Causes of death among patients with multiple sclerosis. Mult Scler. 2010; 16(12): 1437–1442. PubMed Abstract | Publisher Full Text\n\nHirst C, Swingler R, Compston DA, et al.: Survival and cause of death in multiple sclerosis: a prospective population-based study. J Neurol Neurosurg Psychiatry. 2008; 79(9): 1016–1021. PubMed Abstract | Publisher Full Text\n\nNaci H, Fleurence R, Birt J, et al.: The impact of increasing neurological disability of multiple sclerosis on health utilities: a systematic review of the literature. J Med Econ. 2010; 13(1): 78–89. PubMed Abstract | Publisher Full Text\n\nMS Society: Strategic response white paper 2011-2015. Reference Source\n\nMeca J, Del Mar Mendibe Bilbao M: Burden of multiple sclerosis on caregivers and patients and degree of satisfaction with current treatment: The MS-feeling study (P3. 148). Neurology. 2014; 82(10 Supplement): P3.148. Reference Source\n\nThompson AE: JAMA patient page. The Americans with Disabilities Act. JAMA. 2015; 313(22): 2296. PubMed Abstract | Publisher Full Text\n\nIezzoni LI: Eliminating health and health care disparities among the growing population of people with disabilities. Health Aff (Millwood). 2011; 30(10): 1947–1954. PubMed Abstract | Publisher Full Text\n\nSkovgaard L, Bjerre L, Haahr N, et al.: An investigation of multidisciplinary complex health care interventions--steps towards an integrative treatment model in the rehabilitation of people with multiple sclerosis. BMC Complement Altern Med. 2012; 12: 50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNancy Holland EdD R: Comprehensive nursing care in multiple sclerosis. Springer Publishing Company; 2010. Reference Source\n\nInstitute of Medicine (US) Committee on Quality of Health Care in America. Crossing the Quality Chasm: A New Health System for the 21st Century. Washington (DC): National Academies Press (US); 2001. PubMed Abstract\n\nNusrat S, Levinthal D, Bielefeldt K: Hospitalization rates and discharge status in multiple sclerosis. Mult Scler Int. 2013; 2013: 436929. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWallin MT: Integrated multiple sclerosis care: new approaches and paradigm shifts. J Rehabil Res Dev. 2010; 47(5): ix–xiv. PubMed Abstract | Publisher Full Text\n\nDall TM, Storm MV, Chakrabarti R, et al.: Supply and demand analysis of the current and future US neurology workforce. Neurology. 2013; 81(5): 470–478. PubMed Abstract | Publisher Full Text | Free Full Text\n\nColeman K, Austin BT, Brach C, et al.: Evidence on the Chronic Care Model in the new millennium. Health Aff (Millwood). 2009; 28(1): 75–85. PubMed Abstract | Publisher Full Text\n\nBodenheimer T, Chen E, Bennett HD: Confronting the growing burden of chronic disease: can the U.S. health care workforce do the job? Health Aff (Millwood). 2009; 28(1): 64–74. PubMed Abstract | Publisher Full Text\n\nBowen DJ, Kreuter M, Spring B, et al.: How we design feasibility studies. Am J Prev Med. 2009; 36(5): 452–457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDonabedian A: The quality of care. How can it be assessed? JAMA. 1988; 260(12): 1743–1748. PubMed Abstract | Publisher Full Text\n\nWielawski IM: Improving chronic illness care. Birmingham: HSMC, University of Birmingham and NHS Institute for Innovation and Improvement. 2006. Reference Source\n\nSwanton JR: The Indian tribes of North America. Genealogical Publishing Com; 1952. Reference Source\n\nMahoney FI, Barthel DW: Functional evaluation: The barthel index. Md State Med J. 1965; 14: 61–65. PubMed Abstract\n\nCella DF, Dineen K, Arnason B, et al.: Validation of the functional assessment of multiple sclerosis quality of life instrument. Neurology. 1996; 47(1): 129–139. PubMed Abstract | Publisher Full Text\n\nNovak M, Guest C: Application of a multidimensional caregiver burden inventory. Gerontologist. 1989; 29(6): 798–803. PubMed Abstract | Publisher Full Text\n\nLarsen DL, Attkisson CC, Hargreaves WA, et al.: Assessment of client/patient satisfaction: development of a general scale. Eval Program Plann. 1979; 2(3): 197–207. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10803",
"date": "23 Oct 2015",
"name": "Anisha Doshi",
"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 aims to define a new study pre-protocol examining the efficacy of a home-based comprehensive care model, Multiple Sclerosis At Home Access (MAHA), in patients with Progressive Multiple Sclerosis (MS) versus the current healthcare delivery models. In particular the study pre-protocol describes a prospective 24 month block randomised controlled model using an estimated cohort of 50 patients in each arm with the following inclusion criteria; Progressive MS, Kurtze EDSS above or equal to 6.5,and live in a residence within 60 miles of the Omaha metropolitan area. The study is subject to review and approval by the University of Nebraska Medical Center Institutional Review Board (IRB). It would be useful to outline the currently available comprehensive care model. The proposed study measures and data analysis should be sufficient in answering the outcome measures and specific study aims. In our opinion, this study pre-protocol, if approved, will add to the already established data with regard to comprehensive care models, and in particular may be of relevance to those focussing on patients with significant disability due MS as well as to other conditions. References:PMID 23692584PMID 22247419PMID 20047364",
"responses": []
},
{
"id": "11257",
"date": "02 Dec 2015",
"name": "M. Rashad Massoud",
"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 important study and is very well designed. The study has a clear objectives and aims to overcome four challenges in care for patients with multiple sclerosis. The objective of the article is to describe the study design and methods that were used to implement and evaluate the use of the home-based comprehensive care model (MAHA) to overcome the following four challenges:Difficulty in care accessCaregiver burdenSelf-care challengesSocietal burdenThe intervention uses the MAHA and Chronic Care Model. 50 patients will be randomly assigned to receive the intervention or usual care. The authors hypothesized that there would be fewer complications, higher quality of life, greater satisfaction with care, and reduced healthcare utilization to patients who received the intervention.The authors described the theory of change well and it is plausible. We have one suggestion to the investigation. If the objective is to improve care in patients with progressive multiple sclerosis, then iterative testing of changes, and learning from these tests, may be more valuable than testing the effectiveness of a \"set intervention\".This type of inquiry might lend itself to an ongoing type of treatment and modifications of the implementation of the intervention as opposed to an endline assessment. This would allow for a deeper understanding of the elements of the intervention that did and did not work. More importantly, one could look into why and how they worked or did not.The title and the abstract are appropriate for the article. We look forward to seeing the results of the study.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-872
|
https://f1000research.com/articles/4-825/v1
|
16 Sep 15
|
{
"type": "Opinion Article",
"title": "A call to arms to help heal medicine’s greatest ailment - Publication bias and inadequate research transparency",
"authors": [
"Martin Mayer"
],
"abstract": "The paradigm of evidence-based medicine has made impressive advancements since its conception and implementation, but publication bias and issues with inadequate research transparency have remained persistent and pestilent problems. These closely-related issues have markedly detrimental effects on the evidence base from which researchers operate and medical providers make health care decisions, and this can result in involuntary violation of professional and ethical duties and supererogatory motives to serve the public; likewise, it puts patients at risk of receiving medical interventions or advice based on incomplete or ill-understood evidence. By informing readers about the scope of these issues, the failed attempts to correct these issues, and current efforts underway (including a measure in which the lay population can participate), this article serves as a call to arms to help eradicate these incredibly important problems.",
"keywords": [
"publication bias",
"research transparency",
"evidence-based medicine",
"medical ethics",
"research ethics",
"research misconduct"
],
"content": "Introduction\n\nThe problem of a certain proclivity to emphasize “positive” or “successful” findings was specifically mentioned at least as early as 1909 in The Boston Medical and Surgical Journal (Figure 1).1,2 There is even discussion of the importance objectively recording both positive and negative results as early as 1792,3 and even Diagoras of Melos of 500 BC/BCE recognized that recording only positive outcomes can be misleading.4\n\nInterested readers can access the full article through the open-access James Lind Library (http://www.jameslindlibrary.org/editorial-1909/). The excerpts are reproduced here under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).\n\nSterling appears to have been one of the first to attempt to assess publication bias in a more systematic way in his seminal 1959 article.5 Of 362 research reports in four psychology journals, 294 used tests of statistical significance. Impressively, 97.3% of these (286/294) were “positive” (i.e. rejected the null hypothesis); thus, only 2.7% (8/294) were “negative” (i.e. failed to reject the null hypothesis). He astutely noted:\n\n“Some onus appears to be attached to reporting negative results. Certainly such results occur with lesser frequency in the literature than they may reasonably be expected to happen in the laboratory – even if it is assumed that all experimenters are outstandingly clever in selecting hypotheses.”5(p34)\n\nSterling also noted this issue permeated other disciplines. Unfortunately, when Sterling looked again at the close of his career, he found essentially no difference.6\n\nDespite these early warnings, publication bias has continued unrestrained. Systematic reviews demonstrate publication bias is indisputably pervasive in the medical literature, with the two most recent systematic reviews from 2010 and 2013 finding around 50% or more of studies go unpublished.7–9 Evidence of inadequate research transparency is similarly worrisome,7,10–12 and examination of specific examples of these two closely-related issues in action adds poignant tangibility to their seriousness.13 These issues distort the evidence base from which medical providers and researchers should be operating, and therefore put patients at risk of direct or indirect harm. Unfortunately, previous efforts to rectify these issues have failed.\n\n\nWell-intended but ineffective remedial attempts\n\nIn 2004, the International Committee of Medical Journal Editors (ICMJE) proclaimed a clinical trial must register “at or before the onset of patient enrollment” in order to be considered for publication in its member journals.14(p1250) The ICMJE urged nonmember journals to adopt the same policy. At the time, ClinicalTrials.gov was the only suitable registry.14 The ICMJE penned another editorial a year later outlining what satisfactory registration entailed.15 Unfortunately, this has not worked – even for member journals – because the decree has not been upheld.16–19 The most recent appraisal of this issue looked at five psychiatry journals that have the highest impact factors in the field of psychiatry and also adhere to ICMJE guidelines: The American Journal of Psychiatry, Archives of General Psychiatry/JAMA Psychiatry, Biological Psychiatry, Journal of the American Academy of Child and Adolescent Psychiatry, and The Journal of Clinical Psychiatry.19 Using a search period of January 1, 2009, through July 31, 2013, researchers identified a total of 181 published clinical trials that required prospective registration as aforementioned. Only 60 of these 181 clinical trials (33.1%) actually prospectively registered the trial with the primary outcome(s) clearly defined; only 26 of these 181 trials (14.4%) prospectively registered with the primary outcome(s) clearly defined, had no discrepancies between the protocol and the article, and did not retrospectively modify the primary outcome(s) in the registry.\n\nTitle VIII, Section 801 of the Food and Drug Administration Amendments Act of 2007 (FDAAA of 2007) states all applicable clinical trials must register prior to initiation (technically within 21 days of the first patient being enrolled).20,21 The FDAAA of 2007 also requires trial results to be posted in summary form to ClinicalTrials.gov within 12 months of trial completion unless the trial meets certain exemption criteria and the responsible party applies for and is granted an extension.20,21 Applicable clinical trials include clinical trials of drugs or biologic agents subject to FDA regulation (other than phase I trials), clinical trials of devices subject to FDA regulation (other than small feasibility studies), and postmarketing surveillance studies of pediatric devices required by the FDA.20,21 Generally, applicable trials also meet one of the following criteria: one or more trial sites are in the U.S.; the trial is being completed as part of an investigational new drug application or investigational device exemption; or the trial involves a drug, biologic agent, or device that is manufactured in the U.S. or its territories.20,21 The FDAAA of 2007 only applies to trials starting after September 27, 2007, or considered ongoing as of December 26, 2007.20,21 These disappointingly insufficient date boundaries already set up the FDAAA of 2007 for a partial success at best, as they do not account for the vast amount of clinical trial data that accumulated before the mandate’s effective start date.\n\nRegarding extensions, permission may be granted to delay submission up to 30 days after product approval if the product has not yet been approved by the time of trial completion (termed a “certification of initial use”). Similarly, a “certification of new use” may be submitted if there are plans to seek FDA approval, clearance, or licensure for new use of an already-approved medical product; in such instances, submission may be delayed for either up to two years or 30 days after one of the following occurs: the FDA determines approval status, the FDA issues a response letter, or the application is withdrawn.20,21\n\nThe penalty for failing to post results as mandated is “not more than” $10,000 initially, which is followed by a 30-day period to correct the infraction, and then “not more than” $10,000 per day thereafter while in violation of the edict.20(p98)\n\nUnfortunately, these measures have also failed to provide an adequate remedy.22–28 Importantly, the failure to properly publish studies in general, the failure to post results to ClinicalTrials.gov even when mandated by the FDAAA of 2007, and the failure to publish even when registered with ClinicalTrials.gov are not failures unique to industry; indeed, there is evidence that studies funded by academic and government organizations also suffer considerably from publication bias, and in some cases, such studies may be worse in this regard than industry-funded studies.22–28 In spite of this, the aforementioned fines have never been imposed.\n\nThe most recent appraisal of this issue was published by Anderson and colleagues, and although it certainly adds to our knowledge about ClinicalTrials.gov, it ultimately paints the same unfortunate picture: ClinicalTrials.gov has failed.28\n\nAnderson and colleagues identified 32,656 trials that were very likely to fall under the mandated reporting requirements of the FDAAA of 2007; the authors termed these trials highly likely applicable clinical trials (HLACTs). Such verbiage was necessary because they could not be absolutely certain which trials were subject to the FDAAA of 2007 based on the publicly-available information. Such an impediment carries an inherent and saddening irony given the inadequacies of ClinicalTrials.gov thereby suggested. In order to maximize accuracy given this obstacle, they used an algorithm based on input from the National Library of Medicine via personal communication with Deborah Zarin, the Director of ClinicalTrials.gov.\n\nAfter whittling this sample via exclusion criteria (trial status, trial completion date, or data completeness in registry entry), their final sample was 13,327 HLACTs completed or terminated between January 1, 2008, and August 31, 2012. The final follow-up time for the five-year study period was September 27, 2013. Industry funded 65.6% of the HLACTs, the National Institutes of Health (NIH) funded 14.2%, and other government or academic institutions funded 20.2%.\n\nOnly 1,790 trials (13.4%) reported results within 12 months, and even when expanding the consideration to the five-year study period, only 5,110 (38.3%) reported results at any time up to September 27, 2013.\n\nAt 12 months after trial completion, only 818 trials (6.1%) had a legally-acceptable delay; furthermore, by the end of September 27, 2013, only 2,100 trials (15.8%) had requested a delay or certified their qualification for a delay in reporting to ClinicalTrials.gov.\n\nThese numbers, unacceptable as they may be, are still better than the corresponding numbers for the 25,646 non-HLACTs, where only 1,287 (5.0%) and 2,473 (9.6%) reported results within 12 months and at any time during the five-year period, respectively.\n\nTheir analysis also provides additional evidence that industry is not the only guilty party. In fact, in their analysis, industry was best at reporting, albeit still with a wholly unacceptable performance (see Table 1 for reporting rates based on trial funding).\n\nIn addition to the ICMJE proclamation and the FDAAA of 2007 proving to be, thus far, inadequate remedies, a recent survey suggested reviewers may also fall short.29 The usable survey response rate was 37.5% (1,136 respondents with usable surveys out of 3,033 potential participants completing the survey), and 676 respondents indicated they had reviewed a clinical trial in the past two years; unfortunately, only 232 out of the 676 (34.3%) reported assessing trial registry information when reviewing a manuscript.29\n\nData presented are number (percentage) in each group and come from Table 2 of Anderson and colleagues’ analysis.28\n\nIn late 2014, the U.S. Department of Health and Human Services (HHS) and the NIH proposed changes that seek to: clarify certain aspects of the current legislation; increase somewhat the elements of applicable trials that must be reported; and expand the definition of trials that are required to register and provide results, including a new NIH policy requiring all clinical trials receiving NIH funding to register and submit results in a manner similar to that required of trials falling under the FDAAA of 2007 mandate.30–32 While this expansion seems at first glance to be desirable and optimistic, one cannot help but notice the utter failure to enforce the FDAAA of 2007 since its inception; thus, it is difficult to have faith that an expansion of responsibilities will help in any material way unless there are concurrent and enforced measures to ensure adherence, and the current state of affairs – even with the recently-proposed changes – leaves much to be desired, as enforcement remains an unaddressed issue. Furthermore, these changes still do not address trial data prior to the effective start date of the FDAAA of 2007, and beyond date limitations, these changes still only guarantee access to the elements that must be reported, not all the data.\n\nRecently, the European Medicines Agency adopted a policy change requiring publication of full clinical study reports (CSRs) for all applications resulting in a new drug approval after January 1, 2015.33,34 While certainly a big step forward, it also does not account for any trial data prior to its date of enactment.\n\nThis should not be seen as a simple pessimistic accusation of an entirely lackadaisical approach on the part of those intimately involved in the above measures; however, it is an unwavering and unequivocal assertion that these measures, in their current state and execution, are ineffective or incomplete remedies. However, we must persistently pursue a remedy for these issues until true resolution occurs, and there are a number of current measures that deserve consideration.\n\n\nPotential remedies currently being tested\n\nDuring its pursuit of the clinical trial data on oseltamivir (Tamiflu®), The BMJ launched the Open Data campaign in commitment to clinical research transparency (http://www.bmj.com/open-data), and Peter Doshi (an associate editor at The BMJ) leads the Restoring Invisible and Abandoned Trials (RIAT) initiative.35 The RIAT initiative is a call to publish or be published; specifically, it calls for the original researchers involved in known-to-be abandoned or misreported trials to correctly publish (or republish, if necessary) such trials in the peer-reviewed literature. In the absence of such corrective behavior, the RIAT initiative describes the mechanism by which it will pursue publication independent of the original researchers. The first trial utilizing the RIAT initiative was published in May of 2014,36 and one hopes the RIAT initiative will remain perpetually active.\n\nFormally launched in 2013, the AllTrials initiative has gained much momentum and has played a remarkable role in combating these issues (http://www.alltrials.net/). AllTrials is a joint initiative of (in alphabetic order): Bad Science, The BMJ, the Centre for Evidence-based Medicine, the Cochrane Collaboration, the James Lind Initiative, PLoS, and Sense About Science. It is being led in the U.S. by Dartmouth’s Geisel School of Medicine and the Dartmouth Institute for Health Policy and Clinical Practice, and its official U.S. launch occurred just this year in late July. The AllTrials initiative calls for: (1) registration of all trials, including a summary of the protocol, before the trial begins (with past trials being registered retrospectively); (2) summaries of trial results being publicly available within one year of completing the trial, with the results being posted where the trial was registered (with results of past trials being made available now); and (3) full reports (e.g. CSRs) being made publicly available whenever they exist or are created (http://www.alltrials.net/find-out-more/all-trials/). The AllTrials initiative also discusses measures of monitoring and enforcement, including how current infrastructures could be better utilized. The AllTrials initiative does not call for releasing individual patient data, and it respects the potential need for redaction of identifying information (such redaction, where necessary, is not an unduly arduous task). As of the time of this writing, the AllTrials initiative has 86,030 individual supporters and 612 organizations (http://www.alltrials.net/supporters/). The AllTrials initiative remains very active in this arena, and its petition remains open for anyone to sign, even those outside the medical and scientific community.\n\nAmong the organizations to sign the petition is GlaxoSmithKline (GSK), which was a surprising and welcome addition. True transparency would be a formidable and promising step in the right direction, and Patrick Vallance, President of Pharmaceuticals Research and Development at GSK, and Sir Andrew Witty, Chief Executive Officer of GSK, have both committed to greater transparency; however, the full extent of GSK’s commitment has yet to be fully elucidated.37–40 For instance, a recent perspective piece paints a very positive picture of the raw data access mechanism for GSK-sponsored trials, a mechanism that is now also being used by several other pharmaceutical companies.38 A purportedly independent panel oversees this process, including being responsible for reviewing and approving applications for access; however, all the authors of this perspective piece (who are also members of the panel) have industry ties declared in the associated disclosures form.38 This is not simply a captious attempt to “poison the well,” nor does it imply the panel members are inherently nefarious; however, it remains an inarguably important consideration when reading the perspective piece and considering the independence of the process. The novelty of this mechanism may result in an ongoing evolution of the process, and Zarin has raised important questions and concerns.39 Likewise, the only perspectives available from individuals who have been on the other side of this process are those of Jon Jureidini and colleagues, and they suggest an unsatisfactory experience that raised uncertainties.37,40 (Jureidini and colleagues took on the RIAT project of rewriting GSK’s Study 329 on paroxetine in adolescents, which is now in press [written communication with Jon Jureidini on August 25, 2015, with permission granted to publish this information].)\n\nEarly in January of 2015, a committee from the Institute of Medicine released a report on clinical trial data sharing.41 Though the committee admits its extensive writing on the matter is not necessarily an all-encompassing solution, it nevertheless provides meaningful discussion concerning basic principles, operational guidance, and recommendations to help – as the title of the report says – maximize benefit and minimize risk of clinical trial data sharing. Four times in the report, they call for fostering “a culture in which data sharing is the expected norm.”41(p2,5,23,65)\n\nWe need that culture.\n\nThis was echoed when the World Health Organization (WHO) updated its position on public disclosure of clinical trial results.42 It reiterates the need for prospective and transparent registration and the ethical imperative of reporting results, and it also calls for: (1) submitting results for publication in a peer-reviewed journal via an open-access mechanism (unless there is a specific reason why an open-access mechanism cannot be used) within 12 months of study completion, with publication expected within 12 months after submission; (2) making results publicly available via another mechanism within 24 months of trial completion if (1) is not possible for some reason; (3) publishing key elements in an open-access clinical trial registry within 12 months of study completion; and importantly, (4) reporting as-of-yet unpublished trials in an open-access clinical trial registry at minimum, with urging for concomitant publication in a peer-reviewed journal.\n\n\nConclusion\n\nIssues with publication bias and tainted trial transparency loom large and threaten the very core of evidence-based practice. Iain Chalmers – just recipient of The BMJ’s lifetime achievement award in 201443 and someone who has devoted an enormous amount of his career to these issues – wrote a seminal article in 1990 claiming that underreporting research is scientific misconduct.44 This better-known piece actually followed his 1985 correspondence where he proposed outlawing the term “negative trial” and rightly regarded all well-done trials, regardless of their outcome, as being “positive contributions to knowledge.”45(p1002) Sadly, his words of cautionary wisdom and the aforementioned cautionary glimpses offered by others have been ignored for far too long by far too many.\n\nIn addition to being scientifically odious, these issues are in direct violation of the World Medical Association’s Declaration of Helsinki, which is supposed to serve as the authority on ethics in medical research involving human subjects (Figure 2).46 Continuing to tolerate these problems is not only an ethical blemish of the worst kind, but is also: wholly disrespectful to those who participate in clinical trials with the belief their participation will help improve care; a cause of inefficient use of research time and funding; and perhaps most importantly, a threat to providers’ ability to provide their patients with the best evidence-based care possible.\n\nReproduction of directly-applicable components was approved by the World Medical Association, but the World Medical Association implores readers to read and follow the Declaration of Helsinki in full (http://www.wma.net/en/30publications/10policies/b3/).46\n\nPrevious efforts to address these issues have fallen short. Current movements to combat these issues have gained vigor, and one hopes they will not ultimately fall short like their predecessors. The fight for transparency has had many noteworthy contributors, as recently summarized in a tribute feature.34 The contributions of Iain Chalmers and Peter Doshi have been briefly outlined, but other noteworthy contributors (in alphabetic order) are Douglas Altman, Kay Dickersin, Fiona Godlee, Ben Goldacre, and Peter Gøtzsche. This list is certainly incomplete, and importantly, all the current contributors and their efforts, while valiant and incredibly important, are still not enough. Without a unified voice from the medical and scientific communities and the general public, even the most concerted of efforts are at risk of being one day catalogued as well-intended but ultimately ineffective remedies.\n\nThe movement to end publication bias and inadequate research transparency has gained what some might consider to be unprecedented momentum, but it still remains in a critical condition requiring much additional support. Unless and until we fully eradicate these issues, medicine too remains in a perilous state. Indeed, publication bias and inadequate research transparency represent a gaping wound in the body of evidence from which researchers operate and medical providers make decisions regarding care. We need proper closure and healing of the wound – we owe it to our patients and those who participate in research to end these issues completely; anything less than that will never allow for such healing to occur.",
"appendix": "Competing interests\n\n\n\nTruly none, but in the interest of full disclosure, I am a member of the U.S. Board of BMJ Fellows. I do not receive any compensation from The BMJ or anyone else as a result of this. I disclose this here since this article cites works published in The BMJ and ultimately draws attention to their efforts in this arena.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nEditorial. The reporting of unsuccessful cases. Boston Med Surg J. 1909; 161: 263–264. Reference Source\n\nDickersin K: Publication bias: recognizing the problem, understanding its origins and scope, and preventing harm. In: Rothstein HR, Sutton AJ and Borenstein M, eds. Publication Bias in Meta-Analysis: Prevention, Assessment and Adjustments. West Sussex, England: John Wiley & Sons; 2005; 11–33. Publisher Full Text\n\nFerriar J: Medical Histories and Reflections. London: Cadell and Davies, 1792; 1. Reference Source\n\nPetticrew M: Diagoras of Melos (500 BC): An early analyst of publication bias. Lancet. 1998; 352(9139): 1558. PubMed Abstract | Publisher Full Text\n\nSterling T: Publication decisions and their possible effects on inferences drawn from tests of significance -- Or vice versa. J Am Stat Assoc. 1959; 54(285): 30–34. Publisher Full Text\n\nSterling TD, Rosenbaum WL, Weinkam JJ: Publication decisions revisited: the effect of the outcome of statistical tests on the decision to publish and vice versa. Am Stat. 1995; 49(1): 108–112. Publisher Full Text\n\nDwan K, Gamble C, Williamson PR, et al.: Systematic review of the empirical evidence of study publication bias and outcome reporting bias - an updated review. PLoS One. 2013; 8(7): e66844. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong F, Parekh S, Hooper L, et al.: Dissemination and publication of research findings: an updated review of related biases. Health Technol Assess. 2010; 14(8): iii, ix–xi, 1–193. PubMed Abstract | Publisher Full Text\n\nHopewell S, Loudon K, Clarke MJ, et al.: Publication bias in clinical trials due to statistical significance or direction of trial results. Cochrane Database Syst Rev. 2009; (1): MR000006. Accession # 00075320-100000000-05507. PubMed Abstract | Publisher Full Text\n\nWieseler B, Wolfram N, McGauran N, et al.: Completeness of reporting of patient-relevant clinical trial outcomes: comparison of unpublished clinical study reports with publicly available data. PLoS Med. 2013; 10(10): e1001526. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDwan K, Altman DG, Cresswell L, et al.: Comparison of protocols and registry entries to published reports for randomised controlled trials. Cochrane Database Syst Rev. 2011; (1): MR000031. 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\nMayer M: Peering into the “rabbit hole” of publication bias and inadequate research transparency: Adding tangibility to the abstract [v1; ref status: awaiting peer review, http://f1000r.es/5pp]. F1000Res. 2015; 4: 609. Publisher Full Text\n\nDe Angelis C, Drazen JM, Frizelle FA, et al.: Clinical trial registration: a statement from the International Committee of Medical Journal Editors. N Engl J Med. 2004; 351(12): 1250–1251. PubMed Abstract | Publisher Full Text\n\nDe Angelis CD, Drazen JM, Frizelle FA, et al.: Is this clinical trial fully registered?--A statement from the International Committee of Medical Journal Editors. N Engl J Med. 2005; 352(23): 2436–2438. 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): 977–84. PubMed Abstract | Publisher Full Text\n\nJones CW, Platts-Mills TF: Quality of registration for clinical trials published in emergency medicine journals. Ann Emerg Med. 2012; 60(4): 458–464.e1. PubMed Abstract | Publisher Full Text\n\nKilleen S, Sourallous P, Hunter IA, et al.: Registration rates, adequacy of registration, and a comparison of registered and published primary outcomes in randomized controlled trials published in surgery journals. Ann Surg. 2014; 259(1): 193–196. PubMed Abstract | Publisher Full Text\n\nScott A, Rucklidge JJ, Mulder RT: Is Mandatory Prospective Trial Registration Working to Prevent Publication of Unregistered Trials and Selective Outcome Reporting? An Observational Study of Five Psychiatry Journals That Mandate Prospective Clinical Trial Registration. PLoS One. 2015; 10(8): e0133718. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe Food and Drug Administration. Title VIII, Section 801, Food and Drug Administration Amendments Act of 2007. 2007. [Pages 82 (http://www.gpo.gov/fdsys/pkg/PLAW-110publ85/pdf/PLAW-110publ85.pdf#page=82) through 100 (http://www.gpo.gov/fdsys/pkg/PLAW-110publ85/pdf/PLAW-110publ85.pdf#page=100) of the Food and Drug Administration Amendments Act of 2007 (http://www.gpo.gov/fdsys/pkg/PLAW-110publ85/pdf/PLAW-110publ85.pdf)] Reference Source\n\nU.S. National Institutes of Health. FDAAA 801 requirements. 2014. Last reviewed December 2014. Accessed/link verified December 19, 2014. Reference Source\n\nPrayle AP, Hurley MN, Smyth AR: Compliance with mandatory reporting of clinical trial results on ClinicalTrials.gov: cross sectional study. BMJ. 2012; 344: d7373. PubMed Abstract | Publisher Full Text\n\nHuser V, Cimino JJ: Linking ClinicalTrials.gov and PubMed to track results of interventional human clinical trials. PLoS One. 2013; 8(7): e68409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones CW, Handler L, Crowell KE, et al.: Non-publication of large randomized clinical trials: cross sectional analysis. BMJ. 2013; 347: f6104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaw MR, Kawasumi Y, Morgan SG: Despite law, fewer than one in eight completed studies of drugs and biologics are reported on time on ClinicalTrials.gov. Health Aff (Millwood). 2011; 30(12): 2338–45. PubMed Abstract | Publisher Full Text\n\nKirillova O: Results and outcome reporting in ClinicalTrials.gov, what makes it happen? PLoS One. 2012; 7(6): e37847. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoss JS, Mulvey GK, Hines EM, et al.: Trial publication after registration in ClinicalTrials.Gov: a cross-sectional analysis. PLoS Med. 2009; 6(9): e1000144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnderson ML, Chiswell K, Peterson ED, et al.: Compliance with results reporting at ClinicalTrials.gov. N Engl J Med. 2015; 372(11): 1031–1039. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMathieu S, Chan AW, Ravaud P: Use of trial register information during the peer review process. PLoS One. 2013; 8(4): e59910. PubMed Abstract | Publisher Full Text | Free Full Text\n\nU.S. National Institutes of Health, Department of Health and Human Services. 42 CFR Part 11 [Docket Number NIH– 2011– 0003], RIN 0925–AA52, Clinical Trials Registration and Results Submission, Action: Notice of Proposed Rulemaking. Federal Register. 2014; 79(225): Accessed link/verified January 1, 2015. Reference Source\n\nU.S. National Institutes of Health. HHS and NIH take steps to enhance transparency of clinical trial results. Published November 19, 2014; Reviewed November 21, 2014. Accessed/link verified November 22, 2014. Reference Source\n\nU.S. National Institutes of Health. Summary of HHS/NIH proposals to enhance transparency of clinical trial results. Reviewed November 19, 2014; Accessed/link verified November 22, 2014. Reference Source\n\nEuropean Medicines Agency. European Medicines Agency policy on publication of clinical data for medicinal products for human use. 2014. Accessed/link verified January 1, 2015. Reference Source\n\nAdams B: The pioneers of transparency. BMJ. 2015; 350: g7717. PubMed Abstract | Publisher Full Text\n\nDoshi P, Dickersin K, Healy D, et al.: Restoring invisible and abandoned trials: a call for people to publish the findings. BMJ. 2013; 346: f2865. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTreasure T, Monson K, Fiorentino F, et al.: The CEA Second-Look Trial: a randomised controlled trial of carcinoembryonic antigen prompted reoperation for recurrent colorectal cancer. BMJ Open. 2014; 4(5): e004385. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoshi P: Putting GlaxoSmithKline to the test over paroxetine. BMJ. 2013; 347: f6754. PubMed Abstract | Publisher Full Text\n\nStrom BL, Buyse M, Hughes J, et al.: Data sharing, year 1--access to data from industry-sponsored clinical trials. N Engl J Med. 2014; 371(22): 2052–4. PubMed Abstract | Publisher Full Text\n\nZarin DA: Participant-level data and the new frontier in trial transparency. N Engl J Med. 2013; 369(5): 468–469. PubMed Abstract | Publisher Full Text\n\nJureidini JN, Nardo JM: Inadequacy of remote desktop interface for independent reanalysis of data from drug trials. BMJ. 2014; 349: g4353. PubMed Abstract | Publisher Full Text\n\nLo B, Coetzee T, Demets D, et al.: Committee on Strategies for Responsible Sharing of Clinical Trial Data, Board on Health Sciences Policy, Institute of Medicine. Sharing clinical trial data: Maximizing benefits, minimizing risk. National Academies Press, 2015. ISBN 978-0-309-31629-3. Accessed/link verified January 17, 2015. Reference Source\n\nMoorthy VS, Karam G, Vannice KS, et al.: Rationale for WHO's new position calling for prompt reporting and public disclosure of interventional clinical trial results. PLoS Med. 2015; 12(4): e1001819. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHawkes N: Lifetime achievement award 2014: Sir Iain Chalmers. BMJ. 2014; 348: g2921. PubMed Abstract | Publisher Full Text\n\nChalmers I: Underreporting research is scientific misconduct. JAMA. 1990; 263(10): 1405–1408. PubMed Abstract | Publisher Full Text\n\nChalmers I: Proposal to outlaw the term “negative trial”. BMJ (Clin Res Ed). 1985; 290(6473): 1002. Publisher Full Text | Free Full Text\n\nWorld Medical Association. Declaration of Helsinki: ethical principles for medical research involving human subjects. 2013. Accessed/link verified August 6, 2014. Reference Source"
}
|
[
{
"id": "10354",
"date": "02 Oct 2015",
"name": "Erin Aiello Bowles",
"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 paper outlines an important, rampant issue in public health research – publication bias. The author doesn’t present any scientific data so I can’t judge scientific quality. I appreciate the author’s historical presentation of the issue and the data provided from ClinicalTrials.gov. I have a few suggestions that might make the article even more worthwhile.The article spends much of the paper focusing on clinical trials. Nearly an entire page is devoted to ClinicalTrials.gov. While this is interesting, it is not the only cause of publication bias. I would cut down some of this information and provide information on additional causes. For example, what about biases from journals? When was the last time a top-tier journal published a non-significant result, even if the result was important. I believe this is where much of the bias lies. Perhaps breaking this into 2 sections would help – one section on bias as a result of the author failing to report results properly (or at all) and one section on bias as a result of journals failing to publish articles that aren’t highly statistically significant. The latter is a difficulty that many authors face that needs more attention if policies will ever change, and may even be part of the reason authors fail to properly report results. I would also encourage the author to expand on publication bias in observational studies. This is mentioned briefly on page 3 and may be an even bigger problem than publication bias in clinical trials. The author doesn’t mention that some journals (CEBP being one) have specific calls for “null results” to reduce publication bias. Additional calls and journal policies on actively publishing null results may be one solution to this problem. The author also doesn’t mention the impact that publication bias has on future research. When a study isn’t published, no one knows. No one knows what the results were or whether the trial worked. So the study may be funded again in a different form and repeated only to find the same null result. This is a complete waste of time and our tax payer dollars, particularly for government-funded studies. The NIH should really crack down on the lack of publications from grants and prohibit funding of future similar studies if the initial results are never published.",
"responses": [
{
"c_id": "1651",
"date": "13 Oct 2015",
"name": "Martin Mayer",
"role": "Author Response",
"response": "I am glad Ms. Bowles appreciates the gravity of these issues, and I am grateful for her overall positive review of my article. My article was not designed as a primary research article, and therefore there were no primary scientific data to report. Still, although this does mean one cannot judge scientific quality in terms of methodological appraisal for a primary research article (or a meta-analysis, etc.), I would nevertheless posit my article can still be judged as a positive contribution to the biomedical sciences, as one can ultimately think of science in two broad, interrelated senses: Firstly, science is ultimately a way of thinking about and approaching phenomena that involves asking answerable questions and then systematically and impartially pursuing and using the totality of the best-available evidence to reach an informed and reasonable stance, a stance that should remain appropriately malleable as new evidence accumulates and gets incorporated into the current body of knowledge on a subject; secondly, science is a collaborative endeavor to achieve such goals, to collegially seek out such working truths as best we can. This collaborative aspect speaks to my sincere appreciation of Ms. Bowles’ review and comments regarding my article. Below, I respond to her comments at length (with the comment[s] to which I am responding indicated in the responses). Ms. Bowles comments that much attention is paid to ClinicalTrials.gov, but that biases from journals may have been overlooked, and that she believes this is where much of the bias lies (comment 1); she is also concerned that the willingness of certain journals to publish “null results” might have been overlooked, and that this could be part of the solution (comment 3). To be sure, however, no such oversights occurred; although I originally decided to omit an in-depth discussion of such matters in my article for the sake of length and for reasons that will become clear as I respond to these comments, I will gladly expound here, and the available evidence does offer some reassurance and illumination, though it does not support Ms. Bowles’ comments. Although bias in the peer-review process has been demonstrated,1 the best-available evidence overall suggests the majority of the problem actually rests with researchers/authors failing to publish or otherwise make available the results of their research, not journals/editors preferentially publishing only studies with “positive”/significant findings.2-14 Indeed, this is discussed in the 2010 systematic review I cite in my article, which pooled the four studies to date that had investigated this matter.2 Song and colleagues found a pooled odds ratio of acceptance of 1.06 for manuscripts with “positive”/significant findings versus manuscripts with “negative”/non-significant findings (95% confidence interval [CI], 0.80 to 1.39), which they rightly conclude as corroborating the notion that acceptance of a submitted manuscript was not significantly associated with whether the paper had “positive”/significant or “negative”/non-significant findings. The four studies included in the pooled analysis included two studies investigating general medical journals (JAMA in one study,3 and The BMJ, The Lancet, and Annals of Internal Medicine in the other4) and two studies investigating the American volume of the Journal of Bone and Joint Surgery.5,6 All these studies individually arrived at the same result as well: once submitted to a journal for consideration, manuscripts with “positive”/significant findings are no more likely to be published than manuscripts with “negative”/non-significant findings. One of the studies5 – which investigated only manuscripts on hip or knee arthroplasty submitted to the American volume of the Journal of Bone and Joint Surgery – has a potentially suggestive title, and it drew attention to the significant difference in the average quality of “positive”/significant manuscripts versus “negative”/non-significant manuscripts when quality was assessed by a score derived by the authors from standard criteria used to assess the quality of a piece of evidence (termed the Sackett score by the authors). On average, “negative”/non-significant manuscripts had higher Sackett scores than “positive”/significant manuscripts. When considering the mean level of evidence scale officially required by the journal during manuscript preparation and submission, however, there was no significant difference between “positive”/significant manuscripts and “negative”/non-significant manuscripts. There was no difference in acceptance based on study outcome, and intriguingly, there was no difference in acceptance based on study quality as assessed by either the mean Sackett score or the mean level of evidence scale officially used and required by the journal. This finding causes one to ponder, as one would rightly expect higher-quality manuscripts to be published with greater frequency than lower-quality manuscripts. They discuss the finding of no difference in quality between accepted versus rejected manuscripts thusly: “Potential explanations for this finding include the possibility that the overall quality of submissions to The Journal is so high that quality differences among them are too subtle to be discerned with use of standard evidence-based-medicine scoring approaches or, more likely, that reviewers make their decisions on the basis of other criteria, such as timeliness, economic impact, or perceived relevance, in addition to those recommended in standard evidence-based-medicine texts.”5(p1016) However, these postulated explanations still leave one with at least some degree of lingering uncertainty regarding what to think about this finding and how to reconcile this finding with the other assessments and discussions of quality in this study. The authors also acknowledge that assessing manuscript quality and any relationship with acceptance versus rejection as being “outside the scope of [the] primary study end points as defined by [their] a priori hypotheses” and that “until or unless this finding is validated by others it should not be accorded great weight.” 5(p1016) (Indeed, investigating study quality was not a part of either of the two clearly-stated primary research hypotheses.) Furthermore, a large weakness of this study is its lack of attempt to control for potentially influential or confounding variables. Considering all of this, one should be cautious about making any strong conclusions about any findings of manuscript quality or relationships pertaining to manuscript quality and publication from this study, and even setting all this aside, one must also remember this study looked at submissions to one specialty journal and included only studies on hip and knee arthroplasty. Even the subsequent study investigating submissions to the American volume of the Journal of Bone and Joint Surgery noted that, due to the other study’s limitations, “it remains unclear whether publication bias exists in the orthopaedic journal editorial review process.”6(p596) The subsequent study thus set its primary research intent as seeking to determine whether manuscripts with “positive”/significant findings were more likely to be published after controlling for various quality measures, level of evidence, and subspecialty field.6 In this study and the two studies that investigated general medical journals3,4 – all of which investigated quality measures and utilized multivariate analyses to control for potentially influential or confounding variables – no difference was found in publication rates for submitted manuscripts with “positive”/significant results versus submitted manuscripts with “negative”/non-significant results. These studies also found that manuscripts of higher quality or level of evidence were significantly more likely to be published.3,4,6 Recently, a retrospective review of manuscripts from several journals (The BMJ, Annals of the Rheumatic Diseases, British Journal of Ophthalmology, Gut, Heart, Thorax, Diabetologia, and Journal of Hepatology) was published in PLoS ONE,7 and although retrospective studies are often viewed as having potential weaknesses compared to prospective studies, the retrospective design offers a unique benefit in this case: the elimination of any Hawthorne effect. Such an effect might occur in the aforementioned prospective studies on this issue, since editors and reviewers might, whether consciously or unconsciously, behave somewhat differently if they are aware their actions are being studied. This retrospective study also found no difference in acceptance for publication for submitted manuscripts with “positive”/significant findings compared to submitted manuscripts with “negative”/non-significant findings, this study considered quality indicators as well, and the finding of no difference was present in both the univariate and multivariate analyses. However, what if merely looking at publication in a binary manner misses a level of nuance that might still have an impact? What if journals/editors are less enthusiastic about publishing “negative”/non-significant results compared to “positive”/significant results, and therefore, whether unconsciously or consciously, do not pursue the steps to publication as diligently, leading to significant delays in publication? Ioannidis found that, among a cohort of 66 completed multicenter trials focused on human immunodeficiency virus, the 45 trials that had been submitted for publication were published slightly more quickly after submission if they had “positive”/significant results (median of 0.8 years versus 1.1 years; P = 0.04); however, the ultimate clinical significance of an approximate 3.6 month delay is unclear.8 However, in another study investigating a cohort of 133 trials submitted to JAMA, there was no difference in time from submission to publication for “negative”/non-significant results compared to “positive”/significant results (median of 7.6 months versus 7.8 months, respectively; P = 0.44).9 Of course, a potential limitation in these studies is the inherent inability to account for manuscripts/studies that are not even written up and submitted to journals. The theoretical question of what these results would look like if non-submitted manuscripts were submitted is certainly an interesting question, even if only for the sake of refining our understanding of this matter. In the absence of such, however, the aforementioned studies remain our best evidence on the matter, and it would be entirely unfair to hold any speculative counterfactual against the journals/editors. In summary, when considering what is known about publication bias and what this evidence shows, while the evidence may not allow us to confidently exculpate journals/editors entirely, it surely corroborates the notion that authors/researchers are more culpable than journals/editors. Further insight can be gained from asking the researchers themselves why they failed to write up and submit a study.2,10-16 Here too, we see the majority of the problem seems to rest not with the journals/editors, but with the authors/researchers, with the most common reasons for not writing up and submitting a study tending to be lack of time or lack of interest. Both reasons are wholly unacceptable. Being rejected by a journal actually tends to be reported by only a minority, but this too is an unacceptable excuse; manuscript rejection is largely to be expected at some point in one’s publishing endeavors (regardless of the manuscript’s findings/content), and allowing this to be a deterrent is a fault of the researchers/authors, not the journals/editors. Due to cognitive biases, authors/researchers may be more likely to assign fault or biased motives to the journal/editors (and thus may also view things in a more emotionally charged manner) when a manuscript with “negative”/non-significant results is rejected compared to when a manuscript with “positive”/significant results is rejected; however, this is merely a speculative suggestion, albeit one that would seem to fit within what we know about human behavior. In any case, one must remember authors/researchers are specifically called to adhere to the World Medical Association’s Declaration of Helsinki in making the results of their research on humans publicly available (see the second sentence of principle 36 in Figure 2 in my article). Furthermore, at least for clinical trials, ClinicalTrials.gov could serve as a repository for study methods and results even if the authors/researchers do not wish to seek traditional publication or get rejected and decide not to pursue publication elsewhere (although my article discusses mandatory submission, ClinicalTrials.gov also accepts voluntary submissions).17 Importantly, this trumps any excuse pertaining to journal/editorial rejection. On the note of ClinicalTrials.gov and the length to which I discuss it (comment 1), I do so because ClinicalTrials.gov was ushered in with great fanfare, and yet it has unequivocally failed and was doomed at the outset to be a partial success at best in its original (and current) form. Such assertions are admittedly bold, so the space I devote to dissecting this issue was seen as necessary, and truthfully, the space I devote to this topic is ultimately relatively limited given the evidence on the matter. However, the failure of ClinicalsTrials.gov to rectify the issues of publication bias and inadequate research transparency does not preclude it from being used in the aforementioned manner; moreover, with some important logistical changes and better oversight and enforcement (all of which I address in my article), ClinicalTrials.gov could actually be a remarkable tool in the fight to end publication bias and inadequate research transparency. Further questioning the role the journals/editors might play, Ms. Bowles also questions the willingness of a top-tier journal to publish non-significant results, asking when a top-tier journal last published a non-significant result (comment 1, the response to which will also further address comment 3). However, there are several recent examples of studies with “null,” “negative,” or “non-significant” results even within the so-called “Big 5,” including (in alphabetic order) Annals of Internal Medicine,18 The BMJ,19,20 Journal of the American Medical Association,21,22 The Lancet,23,24 and New England Journal of Medicine.25,26 Again, considering all of this, although the evidence cannot entirely rule out journal/editorial bias, the totality of the best-available evidence nevertheless suggests the majority of the problem of publication bias rests more with authors/researchers, not with journals/editors. However, as I discuss clearly in my article, journals/editors are not blameless; indeed, a large area of failure has been the disappointing failure to uphold the ICMJE proclamation. Ms. Bowles also comments on the seeming focus on clinical trials (comment 1), expresses a desire for further coverage of observational studies (comment 2), and wonders whether observational studies might suffer an even worse fate than trials (comment 2). I would like more research on observational studies as well, but as Song and colleagues note in their 2010 systematic review: “Empirical studies on publication and related biases have focused mainly on certain areas of research such as clinical trials of health-care interventions. There is only very limited evidence on publication bias in many other research fields including basic research and observational studies.”2(p36) Although the data are limited, there are some investigations into the matter, although many actually include a mixture of study types.2,11,27-30 When considering Ms. Bowles’ curiosity as to whether observational studies suffer even more from publication bias, the limitations in the available data ultimately preclude one from making any definitive statements, and the data we do have are not congruent. In a subgroup analysis, Easterbrook and colleagues found observational and laboratory-based experimental studies considered together may be more susceptible to publication bias than randomized controlled trials.29 However, Dickersin and colleagues did not detect any difference in clinical trials versus other studies,11 and Stern and Simes found the risk of publication bias was even stronger for the subgroup of clinical trials included in their study compared to all the studies considered together.30 Returning to the 2010 systematic review, Song and colleagues note the results on publication bias were materially indifferent when analyses were restricted to clinical trials versus including all studies.2 Although observational data are certainly useful, it seems at least possible that the focus on clinical trials is due to clinical trials representing a higher, more reliable level of evidence when making inferences about an intervention’s effects, a sentiment echoed by Dwan and colleagues in their 2013 systematic review.27 It also seems at least feasible that this is why efforts to improve the situation have focused on clinical trials. This certainly is not to say observational data are unimportant; indeed, we should strive to gain access to all data, not just data from trials. Perhaps the hopeful success of efforts currently underway for trials can be used as a framework for other study types; in the meantime, however, operating within the existing frameworks and efforts and within the confines of reality, it seems reasonable and important to continue with the efforts focused on clinical trials, understanding that no sensible person would consider gaining proper access to untainted clinical trial data as being the end of this plight if there is still evidence of issues with other study types. The end goal is, as Silverman opined, to be able to truly “narrow the area of uncertainty” as much as possible to build “working truths … that support our everyday actions at the bedside.”31(p165) As long as issues with publication bias and inadequate research transparency persist, this goal remains elusive, and we must therefore continue to fight until these issues are a sordid tale in our past. Lastly, Ms. Bowles comments that I do not mention the impact that publication bias has on future research, specifically that missing research may result in research being unknowingly repeated, thereby potentially leading to wasting time and money (comment 4). However, I actually do hint at this indirectly in the last sentence of my introduction (“These issues distort the evidence base from which medical providers and researchers should be operating, and therefore put patients at risk of direct or indirect harm.”) and the second-to-last sentence of my conclusion (“Indeed, publication bias and inadequate research transparency represent a gaping wound in the body of evidence from which researchers operate and medical providers make decisions regarding care.”). I also more directly state the effects on research time and funding earlier in my conclusion: “Continuing to tolerate these problems is not only an ethical blemish of the worst kind, but is also: wholly disrespectful to those who participate in clinical trials with the belief their participation will help improve care; a cause of inefficient use of research time and funding; and perhaps most importantly, a threat to providers’ ability to provide their patients with the best evidence-based care possible.” Still, I thank Ms. Bowles for her comment here, as I do think I could have been more explicit when discussing how these issues can lead to inefficient use of research time and funding, and I think being more explicit would have made for an even stronger overall statement of the multifaceted deleterious effects these issues have. I wholeheartedly agree with Ms. Bowles that the NIH should heavily penalize authors who fail to publish properly, and I honestly think this penalization should be extended beyond the NIH. Indeed, it seems monitoring and penalties (that are actually enforced) will be a necessary step in improving these issues. This speaks to part of the reason why I discuss in such detail how the government has failed to enforce Section 801 of the Food and Drug Administration Amendments Act of 2007, how the changes proposed in 2014 (including changes specific to trials receiving funding from the NIH) still fail to address enforcement and do not seem to offer much hope of true improvement, how the ICMJE proclamation has also not been upheld, and how current efforts underway seek to improve the state of affairs. In summary: The available evidence, while not able to exculpate journals/editors entirely, still suggests authors/researchers are more to blame for publication bias than journals/editors; journals/editors seem willing to publish “negative”/non-significant results; we need more investigation into how these issues affect observational studies, but it is reasonable and important to continue with efforts focused on clinical trials for the time being (understanding that the fight will continue until publication bias and inadequate research transparency are truly a thing of the past); and these issues can lead to substantial wasting of time and funding due to unawareness of previously-conducted studies that were not properly disseminated. As I state in my article, previous measures have failed to rectify the issues of publication bias and inadequate research transparency (though these measures could prove meaningful with logistical changes, better oversight, and actual enforcement), current movements have gained vigor, and one hopes they will not fall short like their predecessors. Again, we must continue to pursue these matters until true resolution occurs; anything less than that is unacceptable and disgraceful. References 1. Emerson GB, Warme WJ, Wolf FM, Heckman JD, et al.: Testing for the presence of positive-outcome bias in peer review: a randomized controlled trial. Arch Intern Med. 2010; 170 (21): 1934-1939 PubMed Abstract | Publisher Full Text 2. Song F, Parekh S, Hooper L, Loke YK, et al.: Dissemination and publication of research findings: an updated review of related biases. Health Technol Assess. 2010; 14 (8). PubMed Abstract | Publisher Full Text 3. Olson CM, Rennie D, Cook D, Dickersin K, et al.: Publication bias in editorial decision making. JAMA. 2002; 287 (21): 2825-2828 PubMed Abstract4. Lee KP, Boyd EA, Holroyd-Leduc JM, Bacchetti P, et al.: Predictors of publication: characteristics of submitted manuscripts associated with acceptance at major biomedical journals. Med J Aust. 2006; 184 (12): 621-626 PubMed Abstract5. Lynch JR, Cunningham MRA, Warme WJ, Schaad DC, et al.: Commercially funded and United States-based research is more likely to be published; good-quality studies with negative outcomes are not. J Bone Joint Surg Am. 2007; 89 (5): 1010-1018 PubMed Abstract6. Okike K, Kocher MS, Mehlman CT, Heckman JD, et al.: Publication bias in orthopaedic research: an analysis of scientific factors associated with publication in the Journal of Bone and Joint Surgery (American Volume). J Bone Joint Surg Am. 2008; 90 (3): 595-601 PubMed Abstract | Publisher Full Text 7. van Lent M, Overbeke J, Out HJ: Role of editorial and peer review processes in publication bias: analysis of drug trials submitted to eight medical journals. PLoS One. 2014; 9 (8): e104846 PubMed Abstract | Free Full Text | Publisher Full Text 8. Ioannidis JPA: Effect of the statistical significance of results on the time to completion and publication of randomized efficacy trials. JAMA. 1998; 279 (4): 281-286 PubMed Abstract9. Dickersin K, Olson CM, Rennie D, Cook D, et al.: Association between time interval to publication and statistical significance. JAMA. 2002; 287 (21): 2829-2831 PubMed Abstract10. Dickersin K, Chan S, Chalmers TC, Sacks HS, et al.: Publication bias and clinical trials. Control Clin Trials. 1987; 8 (4): 343-353 PubMed Abstract11. Dickersin K, Min Y-I, Meinert CL: Factors influencing publication of research results. Follow-up of applications submitted to two institutional review boards. JAMA. 1992; 267 (3): 374-378 PubMed Abstract12. Dickersin K, Min YI: Publication bias: the problem that won’t go away. Ann N Y Acad Sci. 1993; 703: 135-148 PubMed Abstract13. Weber EJ, Callaham ML, Wears RL, Barton C, et al.: Unpublished research from a medical specialty meeting: why investigators fail to publish. JAMA. 1998; 280 (3): 257-259 PubMed Abstract14. Timmer A, Hilsden RJ, Cole J, Hailey D, et al.: Publication bias in gastroenterological research - a retrospective cohort study based on abstracts submitted to a scientific meeting. BMC Med Res Methodol. 2002; 2: 7 PubMed Abstract | Free Full Text15. Krzyzanowska MK, Pintilie M, Tannock IF: Factors associated with failure to publish large randomized trials presented at an oncology meeting. JAMA. 2003; 290 (4): 495-501 PubMed Abstract16. Scherer RW, Langenberg P: Full publication of results initially presented in abstracts. Cochrane Database Syst Rev. 2007; 18 (2): MR000005 PubMed Abstract17. US National Insitutes of Health: Frequently Asked Questions. ClinicalTrials.gov. Last reviewed February 2015-Accessed October 11, 2015 Reference Source 18. Schleussner E, Kamin G, Seliger G, Rogenhofer N, et al.: Low-molecular-weight heparin for women with unexplained recurrent pregnancy loss: a multicenter trial with a minimization randomization scheme. Ann Intern Med. 2015; 162 (9): 601-609 PubMed Abstract | Publisher Full Text 19. Bolland MJ, Leung W, Tai V, Bastin S, et al.: Calcium intake and risk of fracture: systematic review. BMJ. 2015; 351: h4580 PubMed Abstract | Publisher Full Text 20. Machado GC, Maher CG, Ferreira PH, Pinheiro MB, et al.: Efficacy and safety of paracetamol for spinal pain and osteoarthritis: systematic review and meta-analysis of randomised placebo controlled trials. BMJ. 2015; 350 (h1225). PubMed Abstract | Free Full Text | Publisher Full Text | Reference Source 21. Chew EY, Clemons TE, Agrón E, Launer LJ, et al.: Effect of omega-3 fatty acids, lutein/zeaxanthin, or other nutrient supplementation on cognitive function: the AREDS2 randomized clinical trial. JAMA. 2015; 314 (8): 791-801 PubMed Abstract | Publisher Full Text | Reference Source 22. Rangan A, Handoll H, Brealey S, Jefferson L, et al.: Surgical vs nonsurgical treatment of adults with displaced fractures of the proximal humerus: the PROFHER randomized clinical trial. JAMA. 2015; 313 (10): 1037-1047 PubMed Abstract | Free Full Text | Publisher Full Text | Reference Source 23. Whitlock RP, Devereaux PJ, Teoh KH, Lamy A, et al.: Methylprednisolone in patients undergoing cardiopulmonary bypass (SIRS): a randomised, double-blind, placebo-controlled trial. Lancet. 2015; 386 (10000): 1243-1253 Publisher Full Text | Reference Source 24. Pickard R, Starr K, MacLennan G, Lam T, et al.: Medical expulsive therapy in adults with ureteric colic: a multicentre, randomised, placebo-controlled trial. Lancet. 2015; 386 (9991): 341-349 PubMed Abstract | Publisher Full Text | Reference Source 25. Pitt B, Pfeffer MA, Assmann SF, Boineau R, et al.: Spironolactone for heart failure with preserved ejection fraction. N Engl J Med. 2014; 370 (15): 1383-1392 PubMed Abstract | Publisher Full Text | Reference Source 26. Cung TT, Morel O, Cayla G, Rioufol G, et al.: Cyclosporine before PCI in Patients with Acute Myocardial Infarction. N Engl J Med. 2015; 373: 1021-1031 Publisher Full Text | Reference Source 27. Dwan K, Altman DG, Arnaiz JA, Bloom J, et al.: Systematic review of the empirical evidence of study publication bias and outcome reporting bias – an updated review. PLoS One. 2008; 3 (8): e3081 PubMed Abstract | Free Full Text | Publisher Full Text | Reference Source 28. Kavvoura FK, Liberopoulos G, Ioannidis JP: Selection in reported epidemiological risks: an empirical assessment. PLoS Med. 2007; 4 (3): e79 PubMed Abstract | Free Full Text29. Easterbrook PJ, Berlin JA, Gopalan R, Matthews DR: Publication bias in clinical research. Lancet. 1991; 337 (8746): 867-872 PubMed Abstract30. Stern JM, Simes RJ: Publication bias: evidence of delayed publication in a cohort study of clinical research projects. BMJ. 1997; 315 (7109): 640-645 PubMed Abstract | Reference Source 31. Silverman WA: Where's the Evidence?: Debates in Modern Medicine. 1999; Oxford University Press (Oxford). Reference Source"
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https://f1000research.com/articles/4-825
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https://f1000research.com/articles/4-824/v1
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16 Sep 15
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{
"type": "Research Article",
"title": "Macrophage migration inhibitory factor and placental malaria infection in an area characterized by unstable malaria transmission in central Sudan",
"authors": [
"Reem Eltayeb",
"Naser Bilal",
"Awad-Elkareem Abass",
"Elhassan M. Elhassan",
"Ahmed Mohammed",
"Ishag Adam",
"Reem Eltayeb",
"Naser Bilal",
"Awad-Elkareem Abass",
"Elhassan M. Elhassan",
"Ahmed Mohammed"
],
"abstract": "Background: The pathogenesis of malaria during pregnancy is not fully understood. A proinflammatory cytokine, macrophage migration inhibitory factor (MIF) is suggested as a factor involved in the pathogenesis of malaria during pregnancy.Methods: A cross-sectional study was conducted in Medani Hospital, Sudan to investigate MIF levels in placental malaria. Obstetrical and medical characteristics were gathered from each parturient woman using questionnaires. All women (151) were investigated for malaria using blood film and placental histology. MIF levels were measured using ELISA in paired maternal and cord blood samples.Results: There were no P. falciparum-positive blood films obtained from maternal peripheral blood, placenta or cord samples. Out of 151 placentae, four (2.6%), one (0.7%), 32 (21.2%) showed acute, chronic and past infection on histopathology examinations respectively, while the rest (114; 75.5%) of them showed no signs of infection.There was no significant difference in the median (interquartile) of maternal [5.0 (3.7─8.8) vs 6.2(3.5─12.0) ng/ml, P=0.643] and cord [8.1(3.3─16.9) vs 8.3(4.2─16.9), ng/ml, P= 0.601] MIF levels between women with a positive result for placental malaria infection (n=37) and women with a negative result for placental malaria infection (n=114). In regression models placental malaria was not associated with maternal MIF, hemoglobin or birth weight. MIF was not associated with hemoglobin or birth weight.Conclusion: There was no association between maternal and cord MIF levels, placental malaria, maternal hemoglobin and birth weight.",
"keywords": [
"Macrophage migration inhibitory factor (MIF)",
"malaria",
"birth weight",
"hemoglobin",
"Sudan"
],
"content": "Background\n\nMalaria is a large public health problem in endemic tropical countries where there are over 30 million pregnancies at risk of malaria occur in Africa each year1. Malaria during pregnancy can lead to adverse outcomes (both maternal and perinatal) e.g. anemia and low birth weight (LBW)2–4. Pregnant women in different regions of Sudan are susceptible to malaria, regardless of their age and parity5. Malaria is associated with adverse pregnancy outcomes such as anemia6 and LBW7, and it is the main cause of maternal mortality8.\n\nThe sequestration of Plasmodium falciparum–infected erythrocytes and accumulation of infected erythrocytes in placental intervillous spaces is responsible for the malaria-related pathologic changes in the placenta9,10. The exact mechanism by which malaria infection and placental inflammation result in fetal growth restriction and LBW is poorly understood. However, many chemokines and inflammatory cytokines are associated with malaria infection and malaria-related LBW11.\n\nMacrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine released from a variety of cells (T cells, monocytes, macrophages, blood dendritic cells, B cells, neutrophils, eosinophils, mast cells) and is implicated in the pathogenesis of sepsis, and inflammatory and autoimmune diseases12. MIF has been observed in the human endometrium, placental villi, cytotrophoblasts, and it has been implicated in implantation and other reproductive functions13,14. Several studies have demonstrated the role of MIF in modulating malaria severity and pathogenesis15,16. It has recently been reported that women with a positive result for placental malaria had significantly higher intervillous plasma MIF levels than women with a negative result for placental malaria17. There are few published data on MIF and placental malaria and none of them from Sudan. The current study was conducted in Medani Maternity Hospital, Central Sudan, to investigate MIF levels in women with placental malaria, and the effect – if any- on maternal hemoglobin and birth weight. This work is an addition to our previous research on malaria and its pathogenesis during pregnancy e.g. placental malaria infiltration18,19, hormones and cytokines20,21 complement, cytokines and malaria infections22,23.\n\n\nMaterial and methods\n\nA cross-sectional study was conducted during the rainy and post-rainy season (September–November) 2013 in Medani Maternity Hospital, Central Sudan which is a referral tertiary hospital. Central Sudan is characterized by unstable malaria transmission and P. falciparum is the sole malaria parasite24.\n\nThe sample size of 151 women was calculated to have 80% power and to detect a difference of 5% at α=0.05 and 10% of women might not respond or have incomplete data.\n\nAfter signing an informed consent form, information on history of obstetrics, medical history, antennal attendance characteristics, and bed net use was gathered from participants using questionnaires applied by a trained medical officer. Maternal weight and height were measured and body mass index was calculated and expressed as weight (kg)/height (m)2. Newborns were weighed immediately following birth using a Salter scale and the sex of each newborn was recorded.\n\n5ml of blood (maternal and cord) were taken and allowed to clot and centrifuged for 10 minutes at 3000 rpm and the serum was separated and stored at -20°C till the analyses.\n\nMaternal, placental, and cord blood films were prepared and stained using 10% Giemsa. If the slides were positive; the number of asexual parasites was counted per 200 leukocytes, assuming a leukocyte count of 8000 leukocytes/μl (for thick films) or per 1000 red blood cells (for thin films). Blood films were considered negative if no parasites were detected in 100 oil immersion fields of a thick blood film, which was double-checked in a blind manner by an expert microscopist. Maternal hemoglobin levels were measured by the HemoCue hemoglobinometer (HemoCue AB, Angelhom, Sweden) and recorded.\n\nPlacental histology. The method used for placental histology was mentioned previously7,18–20. In summary, a 3cm3 placental sample was obtained from the maternal surface at a location approximately halfway between the umbilical cord and the edge of the placenta. Each biopsy sample was immediately placed in 10% neutral buffered formalin. The buffer was used to prevent formation of formalin pigment, which has similar optical characteristics and polarized light activity as malaria pigment25. Placental biopsy samples were processed and were embedded in paraffin wax and 4mm thick slides were stained with hematoxylin-eosin and Giemsa. In these slides, placental malaria infection was characterized as follows26: uninfected (no parasites or pigment), acute (parasites in intervillous spaces), chronic (parasites in maternal erythrocytes and pigment in fibrin, or cells within fibrin and/or chorionic villous syncytiotrophoblast or strom), and previous (no parasites, and pigment confined to fibrin or cells within fibrin).\n\nMaternal and cord serum levels were measured using a human MIF ELISA kit (BIOLEGEND catalogue number 438408, Pacific Heights Blvd, San Diego, USA) by following the manufacturer’s protocol.\n\nData were entered into a computer using SPSS for windows (version 16.0). MIF data were not normally distributed and were compared between groups using Mann-Whitney U test. Multivariate analyses were performed using binary models for placental malaria infection as the dependent variable and linear models with hemoglobin, birth weight, and MIF (maternal and cord) levels as continuous dependent variables. Socio-demographic characteristics, education, antenatal care, residence, and placental malaria infections were the independent predictor of interest. Odds ratios (OR) and 95% confidence intervals (CI) were calculated and a P value of <0.05 was considered significant.\n\n\nEthics\n\nThe study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum, Sudan.\n\n\nResults\n\nThe basic characteristics of the investigated women were shown in Table 1. There were no P. falciparum-positive blood films obtained from maternal peripheral blood, placenta or cord samples. Out of 151 placentae, four (2.6%), one (0.7%), 32 (21.2%) showed acute, chronic and past infection on histopathology examinations respectively, while the rest (114; 75.5%) of them showed no signs of infection.\n\nNone of the investigated factors were associated with placental malaria infection, Table 2. There was no significant difference in the median (interquartile) of maternal [5.0 (3.7–8.8) vs 6.2(3.5–12.0) ng/ml, P=0.643] and cord [8.1(3.3–16.9) vs 8.3(4.2–16.9), ng/ml, P=0.601] MIF levels between women with a positive result for placental malaria infection (n=37) and women with a negative result for placental malaria infection (n=114; Figure 1).\n\nOR = Odds ratio, CI = confidence interval\n\nThere was no significant difference in the median (interquartile) MIF levels [5.6(3.6–11.5) vs [7.3(3.0–9.7) ng/ml, P=0.516] between the maternal and cord samples.\n\nIn linear regression placental malaria was not associated with maternal MIF, hemoglobin or birth weight. Likewise MIF levels were not associated with maternal hemoglobin or newborn birth weight (Table 3 and Table 4).\n\nMIF = macrophage inhibitory factor, SE = standard error\n\nMIF = macrophage inhibitory factor, SE = standard error\n\nThere was a significant association between maternal blood/placental and cord MIF (0.473 ng/ml, P<0.001), Table 4.\n\n\nDiscussion\n\nThe main findings of the current study were; there was no significant difference in the MIF levels in women positive for placental malaria infection and women negative placental malaria infection negative. There was no association between MIF, hemoglobin and birth weight.\n\nThis goes with previous reports where Singh et al. found no significant difference in the peripheral and cord MIF levels between women with placental malaria infections and women with placental malaria infections negative17. It is worth mentioning that in Singh’s later study the MIF levels in the intervillous space (which we did not measure) were significantly higher than the peripheral and cord levels and higher in women with placental malaria infection compared with women negative for placental malaria infection17. Furthermore, the observations of Singh et al. were based on microscopically-diagnosed placental malaria infection and in our cohort none of the women/placentae had microscopically detected malaria infections, except one that was diagnosed via histology. Yet high MIF was reported to be associated with adverse pregnancy outcome regardless of the presence of malaria infection17. Likewise, Chaisavaneeyakorn et al.27 observed high levels of MIF in the intervillous blood, compared with that in both peripheral and cord plasma and that intervillous (but not peripheral) MIF levels are associated with placental malaria among Kenyan women. Previous results obtained by Chaiyaroj and colleagues reported significantly higher MIF production by intervillous blood monocytes compared to peripheral ones and high MIF levels in placental plasma compared to paired peripheral plasma28. Increased secretion of MIF by syncytiotrophoblasts was observed previously using an in vitro system29. Generally MIF has been shown to play important roles during normal pregnancy30, as well as in preterm delivery31 and preeclampsia32 and therefore, intervillous MIF would be expected to be high.\n\nWe have previously shown that immunomodulatory hormones (cortisol), cytokines, monocytes and macrophages were implicated in the pathogenesis of malaria during pregnancy which affected pregnant women regardless of their age and parity5,7,8,18–21. Furthermore, histologic studies have shown that malaria-infected placentae have high numbers of macrophages loaded with malarial pigment and these cells could have a critical role in the clearance of the malaria parasites25. Perhaps the high levels of MIF levels observed (by the later studies) in the placenta of women positive for malaria is induced by the malaria parasites that accumulated in the placenta, with MIF helping to retain macrophages in the placenta. Interestingly, it has been shown that MIF is effective in activating macrophages to clear/remove intracellular parasites e.g. Leishmania major33.\n\nAs mentioned above, the malaria placental infections in the current study were past infections and this could explain the lack of significant difference in MIF levels. The other plausible explanation could be the submicroscopic/subpatent infections that we did not investigate in the current study. We have recently shown that in the same hospital, submicroscopic/subpatent infections that were detected by PCR rather than histology were significantly associated with low birth weight7.\n\n\nConclusion\n\nThe current study failed to show a significant association between maternal blood/placental and cord MIF levels, placental malaria, maternal hemoglobin or birth weight.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw dataset for Eltayeb et al., 2015 ‘Macrophage migration inhibitory factor and placental malaria infection in an area characterized by unstable malaria transmission in Central Sudan’, 10.5256/f1000research.7061.d10203934",
"appendix": "Author contributions\n\n\n\nRE and IA coordinated and carried out the study. NEB and AA participated in the statistical analysis. EME and AAM participated in the clinical work and conducted the laboratory work. All the authors have read and approved the final version of this manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no funding was involved in supporting this work.\n\n\nAcknowledgement\n\nAuthors would like to thank the women who were involved in the study and the midwives and the nursing staff of the Medani Hospital.\n\n\nSupplementary materials\n\nData collection questionnaire.\n\nQuestionnaire was applied by a medical professional to participants in the study. ANC: antenatal care; HB: haemoglobin; WT: weight\n\nClick here to access the data.\n\n\nReferences\n\nWHO: World Malaria Report 2012. Geneva, Switzerland: World Health Organization; 2013. Reference Source\n\nMenendez C, Ordi J, Ismail MR, et al.: The impact of placental malaria on gestational age and birth weight. J Infect Dis. 2000; 181(5): 1740–1745. PubMed Abstract | Publisher Full Text\n\nRogerson SJ, Pollina E, Getachew A, et al.: Placental monocyte infiltrates in response to Plasmodium falciparum malaria infection and their association with adverse pregnancy outcomes. Am J Trop Med Hyg. 2003; 68(1): 115–119. PubMed Abstract\n\nAhmed R, Singh N, ter Kuile FO, et al.: Placental infections with histologically confirmed Plasmodium falciparum are associated with adverse birth outcomes in India: a cross-sectional study. Malar J. 2014; 13: 232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAli AA, Elhassan EM, Magzoub MM, et al.: Hypoglycaemia and severe Plasmodium falciparum malaria among pregnant Sudanese women in an area characterized by unstable malaria transmission. Parasit Vectors. 2011; 4: 88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdam I, Khamis AH, Elbashir MI: Prevalence and risk factors for anaemia in pregnant women of eastern Sudan. Trans R Soc Trop Med Hyg. 2005; 99(10): 739–43. PubMed Abstract | Publisher Full Text\n\nMohammed AH, Salih MM, Elhassan EM, et al.: Submicroscopic Plasmodium falciparum malaria and low birth weight in an area of unstable malaria transmission in Central Sudan. Malar J. 2013; 12: 172. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdam I, Elhassan EM, Haggaz AE, et al.: A perspective of the epidemiology of malaria and anaemia and their impact on maternal and perinatal outcomes in Sudan. J Infect Dev Ctries. 2011; 5(2): 83–7, Review. PubMed Abstract | Publisher Full Text\n\nMiller LH, Baruch DI, Marsh K, et al.: The pathogenic basis of malaria. Nature. 2002; 415(6872): 673–9. PubMed Abstract | Publisher Full Text\n\nMuthusamy A, Achur RN, Bhavanandan VP, et al.: Plasmodium falciparum-infected erythrocytes adhere both in the intervillous space and on the villous surface of human placenta by binding to the low-sulfated chondroitin sulfate proteoglycan receptor. Am J Pathol. 2004; 164(6): 2013–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUmbers AJ, Aitken EH, Rogerson SJ: Malaria in pregnancy: small babies, big problem. Trends Parasitol. 2011; 27(4): 168–75. PubMed Abstract | Publisher Full Text\n\nCalandra T, Roger T: Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol. 2003; 3(10): 791–800. PubMed Abstract | Publisher Full Text\n\nArcuri F, Ricci C, Ietta F, et al.: Macrophage migration inhibitory factor in the human endometrium: expression and localization during the menstrual cycle and early pregnancy. Biol Reprod. 2001; 64(4): 1200–5. PubMed Abstract | Publisher Full Text\n\nArcuri F, Cintorino M, Vatti R, et al.: Expression of macrophage migration inhibitory factor transcript and protein by first-trimester human trophoblasts. Biol Reprod. 1999; 60(6): 1299–303. PubMed Abstract | Publisher Full Text\n\nAwandare GA, Ouma Y, Ouma C, et al.: Role of monocyte-acquired hemozoin in suppression of macrophage migration inhibitory factor in children with severe malarial anemia. Infect Immun. 2007; 75(1): 201–210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain V, McClintock S, Nagpal AC, et al.: Macrophage migration inhibitory factor is associated with mortality in cerebral malaria patients in India. BMC Res Notes. 2009; 2: 36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh PP, Lucchi NW, Blackstock A, et al.: Intervillous macrophage migration inhibitory factor is associated with adverse birth outcomes in a study population in Central India. PLoS One. 2012; 7(12): e51678. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalih MM, Mohammed AH, Mohmmed AA, et al.: Monocytes and macrophages and placental malaria infections in an area of unstable malaria transmission in eastern Sudan. Diagn Pathol. 2011; 6: 83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBatran SE, Salih MM, Elhassan EM, et al.: CD20, CD3, placental malaria infections and low birth weight in an area of unstable malaria transmission in Central Sudan. Diagn Pathol. 2013; 8: 189. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBayoumi NK, Bakhet KH, Mohmmed AA, et al.: Cytokine profiles in peripheral, placental and cord blood in an area of unstable malaria transmission in eastern Sudan. J Trop Pediatr. 2009; 55(4): 233–7. PubMed Abstract | Publisher Full Text\n\nBayoumi NK, Elhassan EM, Elbashir MI, et al.: Cortisol, prolactin, cytokines and the susceptibility of pregnant Sudanese women to Plasmodium falciparum malaria. Ann Trop Med Parasitol. 2009; 103(2): 111–7. PubMed Abstract | Publisher Full Text\n\nAlim A, E Bilal N, Abass AE, et al.: Complement activation, placental malaria infection, and birth weight in areas characterized by unstable malaria transmission in central Sudan. Diagn Pathol. 2015; 10(1): 49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChandrasiri UP, Randall LM, Saad AA, et al.: Low antibody levels to pregnancy-specific malaria antigens and heightened cytokine responses associated with severe malaria in pregnancy. J Infect Dis. 2014; 209(9): 1408–17. PubMed Abstract | Publisher Full Text\n\nMalik EM, Atta HY, Weis M, et al.: Sudan Roll Back Malaria Consultative Mission: Essential Actions to Support the Attainment of the Abuja Targets. Sudan RBM Country Consultative Mission Final Report. Geneva: Roll Back Malaria Partnership; 2004. Reference Source\n\nBulmer JN, Rasheed FN, Francis N, et al.: Placental malaria. I. Pathological classification. Histopathology. 1993; 22(3): 211–218. PubMed Abstract | Publisher Full Text\n\nBulmer JN, Rasheed FN, Morrison L, et al.: Placental malaria. II. A semi-quantitative investigation of the pathological features. Histopathology. 1993; 22(3): 219–225. PubMed Abstract | Publisher Full Text\n\nChaisavaneeyakorn S, Moore JM, Othoro C, et al.: Immunity to placental malaria. IV. Placental malaria is associated with up-regulation of macrophage migration inhibitory factor in intervillous blood. J Infect Dis. 2002; 186(9): 1371–5. PubMed Abstract | Publisher Full Text\n\nChaiyaroj SC, Rutta AS, Muenthaisong K, et al.: Reduced levels of transforming growth factor-beta1, interleukin-12 and increased migration inhibitory factor are associated with severe malaria. Acta Trop. 2004; 89(3): 319–327. PubMed Abstract | Publisher Full Text\n\nLucchi NW, Peterson DS, Moore JM: Immunologic activation of human syncytiotrophoblast by Plasmodium falciparum. Malar J. 2008; 7: 42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoung A, Thomson AJ, Ledingham M, et al.: Immunolocalization of proinflammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term. Biol Reprod. 2002; 66(2): 445–449. PubMed Abstract | Publisher Full Text\n\nIetta F, Todros T, Ticconi C, et al.: Macrophage migration inhibitory factor in human pregnancy and labor. Am J Reprod Immunol. 2002; 48(6): 404–409. PubMed Abstract | Publisher Full Text\n\nTodros T, Bontempo S, Piccoli E, et al.: Increased levels of macrophage migration inhibitory factor (MIF) in preeclampsia. Eur J Obstet Gynecol Reprod Biol. 2005; 123(2): 162–166. PubMed Abstract | Publisher Full Text\n\nJüttner S, Bernhagen J, Metz CN, et al.: Migration inhibitory factor induces killing of Leishmania major by macrophages: dependence on reactive nitrogen intermediates and endogenous TNF-alpha. J Immunol. 1998; 161(5): 2383–90. PubMed Abstract\n\nEltayeb R, Bilal NE, Abass A, et al.: Dataset 1. Raw dataset for Eltayeb et al., 2015 ‘Macrophage migration inhibitory factor and placental malaria infection in an area characterized by unstable malaria transmission in Central Sudan’. F1000Research. 2015. Data Source"
}
|
[
{
"id": "10368",
"date": "22 Sep 2015",
"name": "Bernhard Zelger",
"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\nProper study, well outlined and performed.Some minor critique regarding thickness of paraffin slides which should read 4 micromillimeter or with Greek letter μm, not \"4mm\". \"strom\" should read correctly \"stroma\". Finally, acronyms of authors in section \"Authors contributions\" are not clear to me with regard to the list of authors on title page. Please, amend, respectively.",
"responses": []
},
{
"id": "13184",
"date": "04 May 2016",
"name": "Diana Boraschi",
"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 in total favour of publishing reports of negative results, if the study is well conducted, as this one is. Malaria during pregnancy is an outstanding health issue that needs significant investigation. Biomarkers able to predict adverse outcomes are needed, and inflammation-related factors are a logical choice.This study on the correlation between MIF (either in the maternal or in the cord blood), placental infection and pregnancy outcomes (maternal hemoglobin and birth weight) goes in this direction, and quite clearly shows that there is no correlation. The number of cases is possibly low, and no cases with blood malaria were found in the cohort. Although possibly preliminary, the study is nevertheless very clear and straight forward in its design, conduction and logical conclusions.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-824
|
https://f1000research.com/articles/4-823/v1
|
16 Sep 15
|
{
"type": "Research Article",
"title": "Maternal and newborn seroprevalence of Hepatitis E virus at Medani Hospital, Sudan",
"authors": [
"Reem Eltayeb",
"Gasim I. Gasim",
"Elhassan M. Elhassan",
"Halla Abdullahi",
"Duria A. Rayis",
"Ishag Adam",
"Reem Eltayeb",
"Gasim I. Gasim",
"Elhassan M. Elhassan",
"Halla Abdullahi",
"Duria A. Rayis"
],
"abstract": "Hepatitis E virus (HEV) infection can lead to adverse maternal and perinatal outcomes and is a risk for mortality in pregnant women. There are few published data on the seroepidemiology of HEV infection in pregnant women and none are available from Sudan specifically. A cross-sectional study was conducted to investigate the seroepidemiology of anti-HEV Immunoglobulin G (IgG) antibodies in mothers and newborns in Medani Hospital, Sudan. Socio-demographic, clinical and obstetric characteristics of the women were gathered using questionnaires. Anti-HEV IgG antibodies were investigated in the paired maternal and newborn sera using an enzyme-linked immunoassay (ELISA). Two hundred and nine women were enrolled to the study. The mean (SD) of their age, parity and gestational age was 27.5 (5.5) years, 2.2(1.5) and 38.8 (1.8) weeks, respectively. Out of these 209, 25 (12.5%) women had a positive result for anti-HEV IgG and two (1.0%) newborns had a positive result for anti-HEV IgG.In logistic regression none of the investigated factors (age, parity, gestational age, residency and education) were associated with anti-HEV IgG seropositivity. There is a high seroprevalence of HEV among pregnant women in central Sudan regardless of their age, parity and gestational age. Optimal preventive measures against HEV infection should be employed.",
"keywords": [
"Pregnancy",
"vertical transmission",
"HEV",
"newborn",
"Sudan"
],
"content": "Introduction\n\nHepatitis E virus (HEV) is a single-stranded RNA virus and an emerging infectious agent where it can cause acute viral hepatitis worldwide with estimated 20 million cases of HEV infection occur globally and 70 000 deaths (Aggarwal & Gandhi, 2010; Rein et al., 2012). Due to hormonal and immunological changes, pregnant women are more prone to have severe form of HEV infections where there is increasing evidence that HEV is an important contributor to maternal and perinatal morbidity and mortality, especially in the developing countries (Ahmed et al., 2008; Bose et al., 2011; Bose et al., 2014; Kumar et al., 2001; Navaneethan et al., 2008; Ornoy & Tenebaum, 2006; Rayis et al., 2013). In previous studies, Stoszek et al. (2006) and Patra et al. (2007) reported prevalence rates of 84.3% and 60% of anti-HEV antibodies among pregnant women in Egypt and India, respectively.\n\nIn Sudan, a high mortality rate was reported among pregnant women in an outbreak of HEV in Darfur and in eastern Sudan (Boccia et al., 2006; Rayis et al., 2013). In spite of this there are no published data on the seroprevalence of anti-HEV IgG in Sudan and screening of HEV is not part of the antenatal care programme. Research on the seroprevalence of HEV is of paramount importance for health policy makers as well as for the practicing clinicians and it will yield data necessary for developing preventive measures. This study was conducted to determine the seroprevalence of anti-HEV IgG among a population of pregnant women and their newborns in Medani, Sudan.\n\n\nMaterials and methods\n\nA cross-sectional study was conducted at the delivery ward of Medani Maternity Hospital, Sudan during the period of March 2013. Medani Hospital is a tertiary care hospital in central Sudan, located in Al Gezira state which is the second largest state in Sudan. Women with a singleton pregnancy were approached to be enrolled to the study. Mothers who experienced stillbirths and those who had been diagnosed with diabetes or hypertension were excluded from the current study. After signing an informed consent form, socio-demographic, medical and obstetric characteristics were gathered using a questionnaire that was applied in the local language (Arabic; see Supplementary file 1 for sample questionnaire). A convenience sampling method was used where consecutive eligible women were recruited every day until the total desired sample size was achieved (206). The sample size of 206 women was calculated based on a 2-sided hypothesis test using Epiinfo software (Centre for Disease Control, USA; version 6) that yielded 80% power and a confidence interval of 95% with 10% of women expected to have incomplete data or samples.\n\nBody mass index (BMI) was calculated by maternal weight and height that were measured. Five millilitres of blood was collected from each woman along with the corresponding infants’ umbilical cord. The umbilical cords were stored in plain tubes and all samples were labelled and kept at room temperature for 30 minutes, centrifuged at 2000 rpm for 10 minutes to separate blood components. Serum was stored at -20 degrees until analyzed for anti-HEV IgG using specific antibody profiles using HEV IgG-specific ELISA (Euroimmun, Lübeck, Germany).\n\n\nStatistics\n\nData were entered in computer using SPSS software (IBM, UK; version 16) for Windows. Statistics were described as mean (SD) for continuous data and as frequency and percentages (%) for categorized data. T-test and X2 were used to compare continuous and categorized data, respectively between women who were seropositive for HEV IgG antibodies and women with a negative result for HEV antibodies. Binary logistic regression was conducted where seropositivity for HEV IgG antibodies was the dependent variable and socio-demographic, clinical characteristics were the independent variable. Odds ratio and 95% confidence interval were calculated, and P values of < 0.05 were considered statistically significant.\n\n\nEthics\n\nEthical approval was obtained from the Ethical committee of University of Khartoum.\n\n\nResults\n\nTwo hundred and nine women were enrolled in the study. The mean (SD) of their ages, parities and gestational ages were 27.5 (5.5) years, 2.2 (1.5) and 38.8 (1.8) weeks, respectively. Out of these 209, 25 (12.5%) women had a positive result for anti-HEV IgG and two (1.0%) newborns whose mothers also had a positive anti-HEV IgG showed a positive result for anti-HEV IgG.\n\nThere was no significant difference in the age, parity, education, gestational age, BMI or history of miscarriage between seropositive and seronegative anti-HEV IgG women (Table 1). Likewise, in logistic regression none of these investigated factors were associated with anti-HEV IgG seropositivity (Table 2).\n\n\nDiscussion and conclusions\n\nThe main findings of the current study were: the high prevalence (12.1%) of HEV IgG antibodies among pregnant women regardless of their age, parity, residence and educational levels. Recently Caron & Kazanji (2008) found that 14.1% of 840 pregnant women in Gabon had anti-HEV IgG and the prevalence was significantly higher in the urban areas than in the rural ones (13.5 vs. 6.4%). Yet a much higher (28.66%) HEV IgG seroprevalence was observed among Ghanaian pregnant women, especially pregnant women who were 21–25 years of age and women in their third trimester (Adjei et al., 2009). Furthermore, a high prevalence of HEV infection among pregnant women was reported in their neighboring countries, Egypt (84.3%) (Stoszek et al., 2006), Ethiopia (59%) (Tsega et al., 1993) and during an epidemic in Darfur, Sudan (31.1%; Boccia et al., 2006). Interestingly a low (3.6%) prevalence of anti-HEV IgG was reported in Iran and can be explained by the good hygiene and water supply in the investigated area (Rostamzadeh et al., 2013) and in Mexico (5.7%), where pregnant women of advancing age were more likely to be seropositive for HEV IgG (Alvarado-Esquivel et al., 2014).\n\nIn the current study, two of the newborns showed a positive result for anti-HEV IgG. Recently, Mesquita et al. (2013) observed that four pairs (mother and newborn) of participants of the 12 pairs tested were seropositive for anti-HEV IgG. In Egypt, anti-HEV IgG was detected in 31% of 29 neonates with clinical suspicion of congenital infections (El Sayed Zaki et al., 2013). It is worth mentioning that although vertical HEV infection is common and can lead to a high neonatal mortality, HEV is a self-limiting infection in survivors with short-lasting viremia (Khuroo et al., 2009).\n\nPreviously (2001), Kumar and colleagues reported 100% transmission of anti-HEV IgG from the mother to the infant and suggested transplacental transmission of IgG. It has been recently demonstrated for the first time that HEV replication occurs in human placenta and that placenta is a proposed site of extrahepatic replication of HEV in humans (Bose et al., 2014). HEV is mainly transmitted by the fecal-oral route, zoonotic transmission from animal reservoirs (including donkeys) to humans, blood borne, human to human, and vertical transmission from mother to child have been reported (Mirazo et al., 2014).\n\nThe high seroprevalence of HEV amongst pregnant women in central Sudan may suggest that HEV may be widespread among pregnant women in the country as well as in the general population. Furthermore, because the virus is transmitted mainly through the fecal-oral route, sanitary and hygiene conditions should be in optimum conditions to reduce the risk of infection. Perhaps the policy of not screening for HEV antibodies in pregnant women in Sudan is based on the assumed low prevalence; screening for HEV should be employed based on the results of the current study. Moreover, antenatal screening of pregnant women would ensure that treating clinicians could take further precautions to protect against perinatal HEV transmission, minimizing the risk. One of the limitations of the current study is the failure to investigate the HEV genotyping which should be considered in the future study.\n\nIn summary, the current study revealed a high seroprevalence of HEV among pregnant women in central Sudan regardless of their age, parity and gestational age. Optimal preventive measures against HEV infection should be employed.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw dataset for Eltayeb et al., 2015 ‘Maternal and newborn seroprevalence of Hepatitis E virus at Medani Hospital, Sudan’, 10.5256/f1000research.7041.d101652",
"appendix": "Author contributions\n\n\n\nRE - data collection, laboratory work, manuscript preparation. GIG - study design, data analysis, and manuscript preparation. EME - data collection, data analysis. HA - data collection, manuscript preparation. DAR - data analysis, manuscript preparation. IA - study design, data analysis, and manuscript preparation. All authors have read and approved the final content 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\nAuthors would like to thank all the nursing and midwives staff of Medani Hospital, Sudan and the women who participated in the study.\n\n\nSupplementary materials\n\nSupplementary file 1. Questionnaire administered to Mothers at the Medani Hospital, Sudan.\n\nAfter signing an informed consent form, socio-demographic, medical and obstetric characteristics were gathered using this questionnaire, translated from the local language (Arabic).\n\nClick here to access the data.\n\n\nReferences\n\nAdjei AA, Tettey Y, Aviyase JT, et al.: Hepatitis E virus infection is highly prevalent among pregnant women in Accra, Ghana. Virol J. 2009; 6: 108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAggarwal R, Gandhi S: The global prevalence of hepatitis E virus infection and susceptibility: a systematic review. Geneva, Switz: World Health Organization, 2010. Reference Source\n\nAhmed RE, Karsany MS, Adam I: Brief report: acute viral hepatitis and poor maternal and perinatal outcomes in pregnant Sudanese women. J Med Virol. 2008; 80(10): 1747–8. PubMed Abstract | Publisher Full Text\n\nAlvarado-Esquivel C, Sánchez-Anguiano LF, Hernández-Tinoco J: Hepatitis E virus exposure in pregnant women in rural Durango, Mexico. Ann Hepatol. 2014; 13(5): 510–7. PubMed Abstract\n\nBoccia D, Guthman JP, Klovstad H, et al.: High mortality associated with an outbreak of hepatitis E among displaced persons in Darfur, Sudan. Clin Infect Dis. 2006; 42(12): 1679–1684. PubMed Abstract | Publisher Full Text\n\nBose PD, Das BC, Kumar A, et al.: High viral load and deregulation of the progesterone receptor signaling pathway: association with hepatitis E-related poor pregnancy outcome. J Hepatol. 2011; 54(6): 1107–13. PubMed Abstract | Publisher Full Text\n\nBose PD, Das BC, Hazam RK, et al.: Evidence of extrahepatic replication of hepatitis E virus in human placenta. J Gen Virol. 2014; 95(Pt 6): 1266–71. PubMed Abstract | Publisher Full Text\n\nCaron M, Kazanji M: Hepatitis E virus is highly prevalent among pregnant women in Gabon, central Africa, with different patterns between rural and urban areas. Virol J. 2008; 5: 158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEl Sayed Zaki M, El Aal AA, Badawy A, et al.: Clinicolaboratory study of mother-to-neonate transmission of hepatitis E virus in Egypt. Am J Clin Pathol. 2013; 140(5): 721–6. PubMed Abstract | Publisher Full Text\n\nEltayeb R, Gasim G, Elhassan E, et al.: Dataset 1 in: Maternal and newborn seroprevalence of Hepatitis E virus at Medani Hospital, Sudan. F1000Research. 2015. Data Source\n\nKhuroo MS, Kamili S, Khuroo MS: Clinical course and duration of viremia in vertically transmitted hepatitis E virus (HEV) infection in babies born to HEV-infected mothers. J Viral Hepat. 2009; 16(7): 519–23. PubMed Abstract | Publisher Full Text\n\nKumar RM, Uduman S, Rana S, et al.: Sero-prevalence and mother-to-infant transmission of hepatitis E virus among pregnant women in the United Arab Emirates. Eur J Obstet Gynecol Reprod Biol. 2001; 100(1): 9–15. PubMed Abstract | Publisher Full Text\n\nMesquita JR, Conceição-Neto N, Valente-Gomes G, et al.: Antibodies to hepatitis E in Portuguese mothers and their newborns. J Med Virol. 2013; 85(8): 1377–8. PubMed Abstract | Publisher Full Text\n\nMirazo S, Ramos N, Mainardi V, et al.: Transmission, diagnosis, and management of hepatitis E: an update. Hepat Med. 2014; 6: 45–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNavaneethan U, Al Mohajer M, Shata MT: Hepatitis E and pregnancy: understanding the pathogenesis. Liver Int. 2008; 28(9): 1190–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrnoy A, Tenenbaum A: Pregnancy outcome following infections by coxsackie, echo, measles, mumps, hepatitis, polio and encephalitis viruses. Reprod Toxicol. 2006; 21(4): 446–57. PubMed Abstract | Publisher Full Text\n\nPatra S, Kumar A, Trivedi SS, et al.: Maternal and fetal outcomes in pregnant women with acute hepatitis E virus infection. Ann Intern Med. 2007; 147(1): 28–33. PubMed Abstract | Publisher Full Text\n\nRayis DA, Jumaa AM, Gasim GI, et al.: An outbreak of hepatitis E and high maternal mortality at Port Sudan, Eastern Sudan. Pathog Glob Health. 2013; 107(2): 66–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRein DB, Stevens GA, Theaker J, et al.: The global burden of hepatitis E virus genotypes 1 and 2 in 2005. Hepatology. 2012; 55(4): 988–97. PubMed Abstract | Publisher Full Text\n\nRostamzadeh Khameneh Z, Sepehrvand N, Khalkhali HR: Seroprevalence of hepatitis e among pregnant women in Urmia, Iran. Hepat Mon. 2013; 13(11): e10931. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStoszek SK, Abdel-Hamid M, Saleh DA, et al.: High prevalence of hepatitis E antibodies in pregnant Egyptian women. Trans Roy Soc Trop Med Hyg. 2006; 100(2): 95–101. PubMed Abstract | Publisher Full Text\n\nTsega E, Krawczynski K, Hansson BG, et al.: Hepatitis E virus infection in pregnancy in Ethiopia. Ethiopia Med J. 1993; 31(3): 173–181. PubMed Abstract"
}
|
[
{
"id": "14346",
"date": "14 Jun 2016",
"name": "Stephanie M. Borchardt",
"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\nAbstract The title is appropriate for the content of the article and the abstract is a suitable summary of the work, though the last sentence of the abstract could include examples of “optimal preventive measures”.\n\nArticle Content Introduction: Whenever describing “prevalence rates” be clear whether or not it represents seroprevalence. Also, when referring to “anti-HEV antibodies” include whether it is in reference to IgM or IgG. If there is any information on duration of detectable IgM/IgG after infection with HEV it would be helpful to include it. Lastly, if there is information on clinical significance of HEV infection it would be helpful to include it. For example, with West Nile virus infection 90% will be asymptomatic, 10% will have West Nile fever and <1% will have a more serious clinical course. And what is the clinical significance of a pregnant woman who is IgG positive for HEV and similarly her infant?\nMaterials and Methods: Please explain why mothers with diabetes or hypertension were excluded. Table 1: Please clarify whether “haemoglobin, g/dl” was significantly different (p=0.008) between the two groups as the values (i.e., 9.9 vs. 10.4) appear to be very similar.\n\nConclusions The fourth paragraph would be more appropriate for the introduction. More description is needed on “optimal preventive measures”. The author suggests that pregnant women be screened prenatally for anti-HEV antibodies. Please clarify whether this would be for IgG or IgM. Screening for IgG would simply reveal whether or not the woman has ever been infected with HEV. To my knowledge there would be no transmission risk therefore please clarify the benefit of a prenatal screening program and what should be done for women found to be anti-HEV positive and infants born to them.",
"responses": []
},
{
"id": "14088",
"date": "23 Jun 2016",
"name": "Anna Maria Spera",
"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 current manuscript, authors reviewed the seroprevalence of HEV in mothers and newborns admitted at Medani hospital (Sudan): The paper addresses a very important issue, since there are few published data on the seroepidemiology on HEV infection in pregnant women and no one available from that country. However, some concerns should be addressed before its acceptance:\n1. The English hasn’t met the standard of publication and should be polished carefully;\n2. The introduction section of this manuscript should be completed as follows:\nKey facts about HEV infection should be added such as the geographical distribution ( currently available at the discussion section), transmission, virus life cycle, risk factors and protective factors which justifies the inclusions and exclusions criteria along with clinical findings (signs and symptoms, treatment and prevention). Nevertheless this is a study on seroprevalence of HEV infection, pointing out few information on the disease may improve the quality of the text;\n\nThe “aim” of the study which is the reason why authors have been decided to write the article, should be entitled and /or underlined. Furthermore, since the fact that the improvement of the sanitary and hygiene conditions along with the antenatal screening of pregnant women for HEV infection is a specific target of the work, as specified in the discussion section we would point out this outcomes even in the aim section of the paper\n3. Moving on the Material and Methods section we suppose that it would be better to enlist the inclusion variables summarized in table 1 as long as the risks factors collected in table 2. In addition, authors should describe better the way to gather data (questionnaire and blood samples collected from women along with the corresponding infants umbilical cord). Finally, would be better to express the age interval of pregnant women eligible for the study (not only the mean).\n4. With regard to “discussion and conclusion section” we would divide it into two sub-sections. In the discussion, we would remove (or briefly summarize) the epidemiological data in order to focus on the transmission of the infection. As can be expected, we would better distinguish between the fecal-oral route and the vertical transmission of HEV. In fact the first one explain the overlap of pregnant women and general population affected by HEV infection: this may justify the necessity of improve sanitary and hygiene conditions; while the second one may explain the high prevalence of anti-HEV IgG in newborns that can lead to a high neonatal mortality and justifies the necessity of promoting antenatal screening of pregnant women in order to take further precautions to prevent perinatal HEV transmission.",
"responses": []
},
{
"id": "15796",
"date": "22 Aug 2016",
"name": "Shalini Rajaram",
"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\nHepatitis E Infection if it occurs in pregnancy can be acute, fulminant and fatal but mere presence of antibodies does not confer a risk like in Hepatitis B or Hepatitis C infection.\n\nWHO - Presence of these antibodies does not imply presence of or increased risk of disease. The usefulness of such data for epidemiological purposes may also be limited due to variable and possible sub-optimal performance of available serological assays\n\nIt cannot be recommended for routine screening as suggested by authors.\n\nAuthors to reply as to what the ‘AIM’ of the study is and why did they choose this seromarker",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-823
|
https://f1000research.com/articles/4-804/v1
|
15 Sep 15
|
{
"type": "Case Report",
"title": "Case Report: Traumatic anterior cerebral artery aneurysm in a 4-year old child",
"authors": [
"Sunil Munakomi",
"Karuna Tamrakar",
"Pramod Chaudhary",
"Binod Bhattarai",
"Iype Cherian",
"Karuna Tamrakar",
"Pramod Chaudhary",
"Binod Bhattarai",
"Iype Cherian"
],
"abstract": "Traumatic intracranial aneurysm in the proximal part of the anterior cerebral artery in the pediatric population has not been documented so far. Here we report the case of a 4 year-old child who developed a pseudo-aneurysm after minor head trauma and was managed successfully with trapping of the aneurysm. A ventriculo-peritoneal shunt was placed as the child became dependent on extraventricular drain during the post-operative period. The patient made excellent recovery in neurological status within 1 month of post-operative clinical follow up.",
"keywords": [
"trauma",
"pseudo-aneurysm",
"cerebral angiography"
],
"content": "Introduction\n\nOverall incidence of traumatic intracranial aneurysm (TICA) is 1% and is usually associated with penetrating head injury or contagious skull fracture1. The first reported pediatric TICA was in 1829; a right middle meningeal artery aneurysm in an autopsy report of 12 year-old boy with blunt head injury on the right temporal region2. Prevalence of TICA is more among males, with a ratio of 2:1 or 3:1 relative to females, most likely reflecting the greater frequency of trauma among males. The majority of cases are associated with trauma, with approximately 30% of reported cases occurring in children and adolescents before the age of 20. Petrous or cavernous part of internal carotid artery aneurysm (ICA) is associated with skull base fracture. Supraclinoid aneurysm may develop due to blunt arterial contusion by the anterior clinoid process or sudden stretching of the artery during impact to the head. Posterior circulation TICA can develop either due to direct osseous injury or stretching or compression of an artery against the tentorium. However, non-traumatic aneurysms are rare in the pediatric population; the relative frequency of trauma-induced aneurysms in children is high. Most common locations are distal vasculatures like anterior cerebral artery (ACA) (38%), petrous, cavernous, supraclinoid ICA (29%), distal branch of middle cerebral artery (MCA) (25%), and vertebrobasilar system (8%)3. Here we report the case of a 4 year-old girl who presented with delayed intracranial hemorrhage from a ruptured traumatic aneurysm involving the proximal anterior cerebral artery, managed by trapping of the aneurysm.\n\n\nCase presentation\n\nA 4 year-old girl from Sarlahi district in Nepal was brought to our emergency room with a history of sudden onset of severe headache and generalized tonic clonic seizures. She had a history of a minor fall injury while playing at preschool. At that time there was no loss of consciousness, nausea or vomiting and she remained well for the following two days. Two days later at around 3 a.m., the child screamed out in her sleep and complained of severe headache followed by an episode of generalized tonic clonic seizure. The child was then rushed to the hospital. At presentation, her Glasgow Coma Score was 14/15 (E4/M6/V5). No anisocoria was present. She was hemiparetic on the right side with a power grade of 3/5. Computed tomography (CT) scan of the head showed focal intra cerebral hemorrhage in the medial basifrontal region and subarachnoid hemorrhage (SAH) in the inter-hemispheric and in the left sylvian fissure (Figure 1). No significant past medical or surgical illnesses were elicited. She was managed conservatively in the neurosurgical intensive care unit. In repeated serial CT scans, the hematoma was found to be resolving and the child’s motor power in the right side had improved to 4+/5. The child was discharged with advice of regular follow up. The child was again brought to the emergency room one month following the initial hemorrhage with a history of headache and repeated episodes of vomiting. GCS was 15/15 with both pupils equal and reacting to light. CT scan showed re-bleed in the left medial basifrontal region with ventricular extension (Graeb score of 8/12) (Figure 2). The child developed acute hydrocephalus with sudden drop in conscious level which was managed with emergent placement of external ventricular drain (EVD) from the right Kocher’s point. Conventional cerebral angiography showed delayed filling of a 9.6 mm × 6.8 mm aneurysm arising from the proximal part of the left anterior cerebral artery without a discrete neck (Figure 3). Left ACA complex was not visualized except the aneurysmal sac. Both the distal anterior cerebral complex i.e. A2 segments were filled via the right anterior cerebral artery (Figure 4). Left pterional craniotomy and trapping of the aneurysm was performed without any intraoperative complications. H complex was redefined and A1 was found to be blind which itself was a culprit for repeated rupture. Post-operatively the child remained irritable whenever the EVD drainage was clamped off and repeated CT scans revealed persistent hydrocephalus. She was therefore managed with a right ventriculo-peritoneal shunt. On the day of discharge, 23 days after admission, she was playful with grade 4+ power of right sided limbs which became normal in the 2 week follow up period. The child made excellent recovery during 1 month of clinical follow up with no focal neurological deficit and remained asymptomatic. Angiographic follow up after 3 months showed complete obliteration of the aneurysm (Figure 5; Figure 6).\n\n\nDiscussion\n\nTICA in pediatric populations is rare and accounts for 0.5–4.6% of all aneurysms4. These aneurysms are more vulnerable to bleed than true aneurysms due to the lack of a true wall and also the neck, so that they usually present with intracranial hemorrhage (ICH). ICH has been reported in > 60% of TICA cases5. Associated mortality among TICA patient in the pediatric age group has been reported as high as 50%6. TICA is usually located in the periphery and appears to be irregular in shape without visualization of a defined neck. The most common location of TICA is the distal anterior cerebral artery and middle cerebral artery. Petrous or cavernous segment of ICA is also frequently involved. Anterior circulation is more involved than posterior circulation which accounts for less than 10% of all TICA7. Due to the lack of an endothelial layer in the aneurysm, they more likely result from penetrating injuries rather than closed head injuries. Non penetrating injuries are usually secondary to acute shearing forces as in rapid deceleration injury or due to skull fracture with underlying dural or cortical contusions. They usually develop as a consequence of sudden stretching or compression against the rigid dural structure such as the falx or the tentorium. Minor head injury without significant brain parenchymal injury or bony fractures may also cause vascular injuries which later develop into a pseudo-aneurysm.\n\nIn PubMed (www.pubmed.gov), a search of “pediatric traumatic intracranial aneurysm” returned 27 cases, and among them 10 were of patients aged 0–47–11. 7 were of the age 5–101,3,5 and 8 were 11–18 years old1,10,12. 12 were males and 7 were females whereas the remaining cases were mentioned as only infant or child8,9,11. 21 children presented with history of traumatic brain injury; 2 had sustained gunshot injury13,14, 2 cases presented with TICA following VP shunting and craniopharyngioma surgery15,16. An additional 2 cases developed aneurysm due to shaken baby syndrome10,17. The most frequently involved vessel was the distal anterior cerebral artery (11 cases)7, followed by the petrous segment internal carotid artery (5 cases)9, distal middle cerebral artery (2 cases)3, posterior cerebral artery (2 cases)5, posterior inferior cerebellar artery (1 case)18, basilar artery (2 cases), vertebral artery (2 cases)1,19, common carotid artery (1 case)19 and superficial temporal artery (1 case)20. Involvement of posterior circulation was relatively uncommon as compared with anterior circulation vessels. Traumatic aneurysm of proximal ACA in pediatric populations has not been documented in English literature so far. This is the first reported case of TICA of proximal ACA in a child under 5 years of age. Congenital aneurysms are true aneurysms that occur in the branching site of the circle of Willis. They are very rare in the pediatric population as compared to traumatic dissection. Based on the clinical-radiological aspect, the author’s reported case should be the traumatic dissection of ACA leading to pseudo-aneurysm rather than that of congenital origin.\n\nManagement has mostly been confined to surgical clipping, and few reports involve the endovascular treatment of aneurysm. Among 18 treated cases; 8 cases underwent clipping3,7,20,21, trapping was done in 4 cases10,21 and coil embolization was performed in 6 cases16,21. Parent vessel occlusion was done in 3 cases21. Spontaneous thrombosis had occurred in 2 cases1,5 and 2 children died regardless of treatment22. Although spontaneous complete occlusion of TICA is thought to be extremely rare, 15% of spontaneous thrombosis of TICA has been reported in the literature23. Pseudo-aneurysms generally do not have discrete necks and are often friable so that surgical clipping may not be an option. Trapping, in which clips are placed on the parent vessel both proximal and distal to the aneurysm, followed by aneurysmal excision, is a preferred method of treatment. Clinical outcome in a pediatric patient with TICA depends on severity of the injury, with a potentially high mortality from rupture or re-bleeding of the aneurysm22.\n\n\nConclusion\n\nTICA occurrence in the pediatric population is very low. Delayed presentation of intracranial hemorrhage with acute deterioration after minor head trauma in the pediatric age group warrants cerebral angiography for proper diagnosis and management. Trapping of traumatic aneurysm arising from the main arterial trunk is a more tenable procedure. Arterial reconstruction or bypass may prove to be acquiescent in some complex circumstances.\n\n\nConsent\n\nBoth written and verbal informed consent for publication of images and clinical data related to this case was sought and obtained from the mother of the patient.",
"appendix": "Author contributions\n\n\n\nDr Karuna, Dr Pramod and Dr Sunil reviewed the literature, designed the study and formatted the paper. Dr Binod and Dr Cherian revised and edited the final format.\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\nLoevner LA, Ting TY, Hurst RW, et al.: Spontaneous thrombosis of a basilar artery traumatic aneurysm in a child. AJNR Am J Neuroradiol. 1998; 19(2): 386–388. PubMed Abstract\n\nSmith S: On the Difficulties attending the Diagnosis of Aneurism, being a Contribution to Surgical Diagnosis and to Medical Jurisprudence. Am J Med Sci. 1873; 66(132): 401–409. Publisher Full Text\n\nVentureyra EC, Higgins MJ: Traumatic intracranial aneurysms in childhood and adolescence. Case reports and review of the literature. Childs Nerv Syst. 1994; 10(6): 361–379. PubMed Abstract | Publisher Full Text\n\nKanaan I, Lasjaunias P, Coates R: The spectrum of intracranial aneurysms in pediatrics. Minim Invasive Neurosurg. 1995; 38(1): 1–9. PubMed Abstract | Publisher Full Text\n\nMorón F, Benndorf G, Akpek S, et al.: Spontaneous thrombosis of a traumatic posterior cerebral artery aneurysm in a child. AJNR Am J Neuroradiol. 2005; 26(1): 58–60. PubMed Abstract\n\nCrowell RM, Ogilvy CS: Traumatic intracranial aneurysms. In: Ojemann RG, Ogilvy CS, Crowell RM, et al. (eds): Surgical management of neurovascular disease. Baltimore: Williams & Wilkins, 1995; 377–384.\n\nRaju BS, Purohit AK, Murthy SR, et al.: Traumatic distal anterior cerebral artery aneurysm in a child: a case report. Neurol India. 2001; 49(3): 295–298. PubMed Abstract\n\nTan TC, Chan CM, Chiu HM: Traumatic intracranial aneurysm in infancy. Br J Neurosurg. 2001; 15(2): 137–9. PubMed Abstract | Publisher Full Text\n\nChambers N, Hampson-Evans D, Patwardhan K, et al.: Traumatic aneurysm of the internal carotid artery in an infant: a surprise diagnosis. Paediatr Anaesth. 2002; 12(4): 356–61. PubMed Abstract | Publisher Full Text\n\nLevine NB, Tanaka T, Jones BV, et al.: Minimally invasive management of a traumatic artery aneurysm resulting from shaken baby syndrome. Pediatr Neurosurg. 2004; 40(3): 128–31. PubMed Abstract | Publisher Full Text\n\nKim B, Lee SK, Terbrugge KG: Endovascular treatment of traumatic intracranial aneurysm in an infant. A case report. Interv Neuroradiol. 2003; 9(2): 199–204. PubMed Abstract | Free Full Text\n\nHahn YS, Welling B, Reichman OH, et al.: Traumatic intracavernous aneurysm in children: massive epistaxis without ophthalmic signs. Childs Nerv Syst. 1990; 6(6): 360–4. PubMed Abstract | Publisher Full Text\n\nAlvarez JA, Bambakidis N, Takaoka Y: Delayed rupture of traumatic intracranial pseudoaneurysm in a child following gunshot wound to the head. J Craniomaxillofac Trauma. 1999; 5(4): 39–44. PubMed Abstract\n\nHachemi M, Jourdan C, Di Roio C, et al.: Delayed rupture of traumatic aneurysm after civilian craniocerebral gunshot injury in children. Childs Nerv Syst. 2007; 23(3): 283–7. PubMed Abstract | Publisher Full Text\n\nJenkinson MD, Basu S, Broome JC, et al.: Traumatic cerebral aneurysm formation following ventriculoperitoneal shunt insertion. Childs Nerv Syst. 2006; 22(2): 193–6. PubMed Abstract | Publisher Full Text\n\nOgilvy CS, Tawk RG, Mokin M, et al.: Stent-assisted coiling treatment of pediatric traumatic pseudoaneurysm resulting from tumor surgery. Pediatr Neurosurg. 2011; 47(6): 442–8. PubMed Abstract | Publisher Full Text\n\nLam CH, Montes J, Farmer JP, et al.: Traumatic aneurysm from shaken baby syndrome: case report. Neurosurgery. 1996; 39(6): 1252–5. PubMed Abstract | Publisher Full Text\n\nSure U, Becker R, Petermeyer M, et al.: Aneurysm of the posterior inferior cerebellar artery caused by a traumatic perforating artery tear-out mechanism in a child. Childs Nerv Syst. 1999; 15(6–7): 354–6. PubMed Abstract | Publisher Full Text\n\nMahmoud M, Roshdi E, Benderbous D: Traumatic pseudoaneurysms of the common carotid and vertebral artery in a four-year-old child. Interv Neuroradiol. 2012; 18(3): 348–52. PubMed Abstract | Free Full Text\n\nAhn HS, Cho BM, Oh SM, et al.: Traumatic pseudoaneurysm of the superficial temporal artery in a child: a case report. Childs Nerv Syst. 2010; 26(1): 117–20. PubMed Abstract | Publisher Full Text\n\nHetts SW, Narvid J, Sanai N, et al.: Intracranial aneurysms in childhood: 27-year single-institution experience. AJNR Am J Neuroradiol. 2009; 30(7): 1315–24. PubMed Abstract | Publisher Full Text\n\nBuckingham MJ, Crone KR, Ball WS, et al.: Traumatic intracranial aneurysms in childhood: two cases and a review of the literature. Neurosurgery. 1988; 22(2): 398–408. PubMed Abstract\n\nNakstad P: Spontaneous occlusion of traumatic pericallosal aneurysm and pericallosal artery. Neuroradiology. 1987; 29(3): 312. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10317",
"date": "22 Sep 2015",
"name": "Naoya Kuwayama",
"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 case report of a 4-year-old child with a delayed intracerebral hematoma resulting from the traumatic pseudoaneurysm of the left A1 segment.As authors emphasized, this is a very rare case occurring in childhood and the management of the patient, including the surgical treatment, was excellent. In the discussion, the epidemiology, diagnosis, and treatment option of the traumatic intracranial aneurysms were briefly but well documented.Finally, this article can be considered as acceptable, although the following points should be addressed:They should discuss the possibility that the pseudoaneurysm had already developed when the child first complained of a headache 2 days after head injury.They should describe the details about the surgical method focusing how they trapped the aneurysm with one clip. Did they clip only the neck or A1 segment itself?I cannot understand what “H complex was redefined” means in the operation.",
"responses": []
},
{
"id": "15779",
"date": "30 Aug 2016",
"name": "Venkatesh S. Madhugiri",
"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 of a complex aneurysm. However, a few comments on some aspects of the paper are presented here\nTrivial trauma of the kind described does not usually lead to aneurysmal rupture. Could the rupture of the aneurysm have caused the fall? Moreover, when the CT after the first ictus (the trivial fall) disclosed thick subarachnoid hemorrhage in the Sylvian and suprasellar cistern rather over the convexity of the hemispheres (as would usually be expected with trauma), did the authors not consider doing a DSA or CTA in the first instance?\n\nIt is very unusual for a major vessel to blindly end in an aneurysm. There is generally a distal take-off of the distal vessel even from giant aneurysms. This is borne out by the fact that the L A2 was being filled across the ACoA on the R ICA injection. Have the authors considered the possibility that this was an A1 aneurysm? The proximal A1 could have been partly thrombosed due to extension of thrombus from the aneurysm into the parent vessel. This could explain the poor visualization of the L A1 whereas the L A2 distal to the aneurysm was being filled by the ACoA.\n\nThe authors should describe the intra-operative findings in slightly greater detail. The L A1 was blind, per the authors. Was there no connection between the L A1 and A2? It is more likely that the aneurysm obscured this vessel. Was the aneurysm thrombosed? Did it appear to be a true aneurysm or pseudo-aneurysm? Was the sac opened after clipping and dissected away from surrounding structures to clarify the anatomy?\n\nThe preop DSAs shows vasospasm of the L M1. The authors should comment on what treatment, if any, was considered or given for this. The spasm appears to have resolved on the postop DSA. When was this obtained?\n\nPediatric traumatic intracranial aneurysms are indeed rare. In the Discussion the authors state \"a search of “pediatric traumatic intracranial aneurysm” returned 27 cases.\" However, PubMed search strategies need to built more carefully if valid results are to be obtained. A cursory search using the strings \"((aneurysm) AND intracranial) AND trauma\" and subsequently applying a filter for only children (birth-18 years) returned 584 results. Even if only 10% of these results were relevant to the search, this would still result in 58 papers dealing with this issue.\nThe full search employed was -\n\n((\"aneurysm\"[MeSH Terms] OR \"aneurysm\"[All Fields]) AND intracranial[All Fields]) AND (\"injuries\"[Subheading] OR \"injuries\"[All Fields] OR \"trauma\"[All Fields] OR \"wounds and injuries\"[MeSH Terms] OR (\"wounds\"[All Fields] AND \"injuries\"[All Fields]) OR \"wounds and injuries\"[All Fields]) AND (\"infant\"[MeSH Terms] OR \"child\"[MeSH Terms] OR \"adolescent\"[MeSH Terms])",
"responses": []
},
{
"id": "16091",
"date": "07 Sep 2016",
"name": "Guru Dutta Satyarthee",
"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\nAuthors report an extremely uncommon entity of postraumatic aneurysm in a child, who developed hydrocephalus and showed rapid neurological worsening, managed with the placement of an external ventricular drain, followed by successful surgical management of surgery of aneurysm. The index case, a four-year-old girl, presented with a history of sudden onset severe headache in association with generalized tonic clonic seizures with antecedent history of a minor fall. On examination, the Glasgow coma score was 14, right sided hemiparesis, a CT scan of the head revealed presence of subarachnoid hemorrhage in the inter-hemispheric fissure and left sylvian fissure and associated intracerebral hemorrhage in the basifrontal region, however, a vascular aetiology was not suspected and managed conservatively. Ideally digital substraction angiography ( DSA) should have been carried out and managed accordingly to the findings. She again presented after one month with re-bleed, required external ventricular drain placement for hydrocephalus. DSA revealed an aneurysm in the region of left anterior cerebral artery, although both distal anterior cerebral arteries were filling with right internal carotid artery injection, and underwent craniotomy and trapping of the aneurysm.\n\nHydrocephalus development is a common and devastating complication of aneurysmal subarachnoid hemorrhage with incidence of 20% to 30% cases. Onset of hydrocephalus can be acute as early as within 48 hours or delayed by up to weeks following ictus. Early recognition of symptoms and signs and computed tomography findings are important for planning of management. As cases showing rapid deterioration need external ventricular drain, to tide the crisis, which may be converted to ventriculo-peritoneal shunt as it aids in management of raised intracranial pressure, helps in clearance of intraventricular blood. 1,2\n\nIntracranial traumatic aneurysm is a relatively rare clinical entity caused by blunt, penetrating, or iatrogenic head trauma3,4. It can be classified histologically as false or true aneurysm. In false aneurysms or pseudoaneurysm disruption involves all layers of the arterial wall associated with the presence of hematoma in the surrounding soft tissue that prevents extravasations of blood and is considered the most common form of traumatic intracranial aneurysms5. In true aneurysms, the intimal layer, internal elastic lamina and media layers are disrupted but with an intact tunica adventitia. The intracranial space into which the hemorrhage occurs depends on the vessel and the segment of the vessel involved. Once an extracranial artery becomes intradural, the external elastic lamina disappears transforming the arterial wall into a relative weak vessel. 4,5\n\nThe reported risk of hemorrhage in posttraumatic pseudoaneurysm is 19%, with a peak incidence of rupture at 2–3 weeks after the injury, resulting in 32–54% mortality.5\n\nTreatment options include surgical repair and endovascular interventions. The treatment approach is often needed to be a tailor made approach for individual cases, and depends on factors such as atypical locations, irregular shape, relation with parent vessel and branches, age of patient, and shapes are a factor 1,3,6. Miley et al. analysed DSA of 74 patients with the diagnosis of closed head injury, vascular injury detected in 24 cases and out of which four cases had total of 4 traumatic intracranial pseudoaneurysm, were located in the supraclinoid segment of the internal carotid artery in two case, one in the cavernous segment\n\nand rest one in the paraophthalmic segment6.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-804
|
https://f1000research.com/articles/4-766/v1
|
10 Sep 15
|
{
"type": "Research Article",
"title": "Pre-surgery status determines inflammation levels post-elective surgery",
"authors": [
"Pijus Barman",
"Ratnadeep Mukherjee",
"Jatasankar Mohapatra",
"Balachandran Ravindran",
"Pijus Barman",
"Ratnadeep Mukherjee",
"Jatasankar Mohapatra"
],
"abstract": "In the present study we quantified a panel of systemic inflammation parameters in patients undergoing elective surgery with a view to evaluate pre-surgical inflammation status in relation to consequences post-surgery. The investigation revealed significantly decreased levels of plasma TNF-α, IL1-β, IL7, IL-8, MIP-1a and IL-1Ra in 79% of patients at 6 hrs post-surgery which have been designated by us a ‘hypo-responsive’ cases and the balance 21% of patients displayed significantly elevated levels of the above cytokines in plasma that have been designated a ‘hyper-responsive’ phenotype by us. Expression of HLA-DR, CD40, CD80, TLR-2, TLR-4 and CD36 on circulating monocytes as shown by multicolour flow-cytometry was significantly decreased post-surgery in hypo-responsive patients. Similarly, PBMCs of hypo-responsive cases responded very poorly in vitro when stimulated with toll-like receptor (TLR) agonists. There was an inverse association between levels of plasma inflammatory cytokines pre-surgery and hypo-responsive consequences post-surgery. Similarly, patients displaying the hyper-responsive phenotype were found to express very low levels of inflammatory cytokines pre-surgery. Taken together the current study offers two novel findings: a) a bimodal inflammatory response post-elective surgery viz., one major cohort displaying hypo-responsive state and another minor group a hyper-responsive phenotype and b) pre-surgery inflammation status determining the direction of inflammation consequence post-surgery. These findings seem to offer laboratory tools for predicting onset of inflammation post-surgery – considering that SIRS and sepsis are consequences of surgery induced inflammation this study offers predictive indicators for clinical complications post-surgery.",
"keywords": [
"DAMPs",
"PAMPs",
"Sterile inflammation",
"Sepsis",
"SIRS",
"TLRs"
],
"content": "Abbreviations\n\nTNF, tumour necrosis factor; IL, interleukin; MIP, macrophage inflammatory protein; HLA-DR, human leukocyte antigen-DR; TLR, toll-like receptor; PBMC, peripheral blood mononuclear cells; SIRS, systemic inflammatory response syndrome.\n\n\nIntroduction\n\nSepsis and multiorgan failure (MOF) are the leading causes of death in surgical patients1–3. Several studies have described surgery induced systemic inflammatory response syndrome (SIRS)4,5 and compensatory anti-inflammatory response syndrome (CARS)6–9 as prevalent causes of these complications. A causal relationship of events such as induction of cytokine storm10–12, immune cell activation and infiltration of activated immune cells13 with development of SIRS following surgery has been reported in a cohort of patients. Upregulated expression of toll-like receptors (TLRs) by activated monocytes/macrophages and hyper-reactivity of peripheral blood mononuclear cells to pathogen associated molecular patterns (PAMPs) have further been shown to be associated with this overwhelming inflammatory response post-injury14. Paradoxically there are also reports claiming downregulated expression of TLRs on circulatory monocytes as well as impaired responses to PAMPs in patients post-surgery15,16.\n\nExisting literature does not offer clarity on factors that contribute to these diagonally opposite biological outcomes in patients post-surgery. We hypothesised that the pre-surgery status of patients could determine post-surgery consequences – while previous studies have demonstrated immunodynamics and inflammation profiles post-surgery17 very little has been understood about the correlation between pre-operative inflammatory status and outcome post-injury. An association between lower pre-operative plasma IL-6 and early allograft dysfunction due to high systemic inflammation18 and higher pre-operative systemic inflammation has been demonstrated with increased risk of infection post-surgery19,20. These reports however suffer from lack of robust and comprehensive measurement of cellular and molecular parameters of inflammation and the current study was designed to fill this lacuna.\n\nHere we report the quantification of plasma cytokines, expression of monocyte surface receptors such as TLRs, scavenger receptors, HLA-DR and other co-stimulatory molecules in patients before and after elective surgery. Our results revealed downregulation of specific inflammatory mediators in 6 hrs post-surgery plasma in most of the patients while the rest displayed distinct increased levels of plasma mediators. Increased as well as decreased levels of plasma inflammatory molecules correlated with surface expression of monocyte receptors such as TLRs, scavenger receptor CD36, HLA-DR and co-stimulatory molecules. Further, our findings demonstrate an inverse association between pre-surgical status and inflammation profile post-surgery viz., patients with relatively higher basal levels of plasma cytokines and monocyte surface receptors developed a hypo-inflammatory response while patients with relatively lower basal levels of plasma cytokines and monocyte surface receptors developed a hyper-inflammatory response post-surgery.\n\n\nMaterials and methods\n\nLipopolysaccharide from Escherichia coli serotype O55:B5 (cat. No. L2880-100MG) and PAM3CSK4 (cat. No. IMG-2201) were purchased from Sigma-Aldrich and Imgenex India Pvt. Ltd. respectively. RBC lysis (cat. No. 00-4333-57), cell fixation (cat. No. 00-8222-49) and permeabilization (cat. No. 00-8333-56) buffers were purchased from eBiosciences. Bio-Plex Pro Assays-27 plex kit was purchased from Bio-Rad (cat. No. M500KCAF0Y). DNA isolation kit (QIAamp DNA Mini kit [250]; cat. No. 51306) was purchased from Qiagen. Q-PCR SYBR mix (2X Brilliant III STBR Green QPCR Master Mix, cat. No. 600882-51) was purchased from Agilent Technologies.\n\nPatients admitted for gastrointestinal and general surgery in the Department of General & Laparoscopic surgery, Neelachal Hospital Pvt. Ltd. Bhubaneswar between September 2013 and October 2014 were recruited for this study. All of the surgeries included in this study were cases of elective surgery. A total of 19 patients between the age of 18 years and 75 years were included. Inclusion criterion was surgical interventions with a minimum incision of 3 inches. Pregnant women, terminally ill patients and patients admitted for emergency surgery or accidental trauma cases were excluded from the study. Details of patients, type of anaesthesia used, duration of surgery, duration of hospital stay, pre-surgical total blood cell counts etc. are shown in Table 1. Total 23 healthy volunteers who were not on any medication since two weeks prior to blood collection participated in this study. The project protocol was approved by the human ethics committee of Institute of Life Sciences (no. 28/HEC/13) and the IRB committee. Written informed consent was obtained from each of the patients for voluntary participation in the study.\n\nData are presented as mean of 23 healthy controls and 19 surgery patients. Patients were categorized into two groups on the basis of post-operative inflammatory responses viz., ‘hypo-responsive’ and ‘hyper-responsive’. Values represent the mean ± SEM, with median and middle quartiles indicated in parentheses. RBCs, red blood cells.\n\nValues represent the mean ± SEM, with median and middle quartiles indicated in parentheses\n\nBlood was collected in ACD 15% (V/V) containing tubes. Sampling was done in two batches - initially 8 subjects were selected and sampled twice, immediately before anaesthesia (0hps) and 6 hrs after completion of surgery (6hps). After analyzing the data, 11 more patients were included and sampling was done thrice for this cohort; immediately before anaesthesia (0hps), 6 hrs and 24 hrs after completion of surgery (6hps and 24hps respectively). Whole blood was aliquoted and used immediately for complete blood count (CBC), for conducting ex vivo stimulations and analysis by flow-cytometry. Plasma was isolated from the rest of the sample by centrifugation and was stored at -80°C for conducting further assays.\n\nTwo hundred microliters of whole blood was used for complete blood count (CBC) in haematology analyzer (Sysmex XS800i).\n\nFifty microliters of freshly collected blood samples were stimulated with TLR ligands; PAM3CSK4 and LPS (10ng/ml for both) for 2 hrs at 37°C. Cells were fixed, permeabilized (following manufacturer’s instructions) and stained with antibodies to CD14-(FITC), CD66b-(APC) and TNF-α-(PE-Cy7) (antibody panel-1). Another aliquot (50ul) was used for multicolour immune staining with antibodies to CD14-(APC-Cy7), TLR2-(PE-Cy7), TLR4-(APC) and CD36-(FITC) (antibody panel-2) and CD14-(APC-Cy7), HLA-DR-(PE-Cy7), CD40-(APC) and CD80-(FITC) (antibody panel-3). After staining RBCs were lysed with RBC lysis buffer following manufacturer’s protocol. Stained cells (with antibody panels -1, 2 and 3) were acquired and analyzed by flow cytometry using BD FACS LSR Fortessa; data were analysed using BD FACS Diva software (version 7.0). To nullify day to day variations flow-cytometer settings were maintained uniform by performing bead based instrument settings following the protocol provided by BD Biosciences. To get rid of false positive or false negative events, gating was done using fluorescence minus one (FMO) controls. Monocyte expression of intracellular cytokines was shown as mean fluorescence intensity (MFI) whereas expression of surface markers was scored as percentage of positive cells.\n\nPlasma cytokines were measured by Bio-plex Pro Assays-27 plex following the manufacturer’s instructions and the final reading was taken in Bio-plex 200 system from Bio-rad.\n\nIsolation of plasma DNA. 100μl of plasma was mixed with 100μl of PBS and centrifuged at 700×g for 5 minutes at 4°C. Upper 190μl volume was collected without agitating the lower portion and centrifuged at 18000×g for 15 minutes at 4°C. From this upper 170μl was transferred into a new tube and plasma DNA was eluted using QIAamp DNA Mini kit following manufacturer’s instructions. Preparation of standard curve: mitochondrial cytochrome-b gene (mtCyt-b) was amplified from eluted DNA by end-point PCR using following primer set; forward 5’CCACCCCATCCAACATCTCC3’ and reverse 5’CTCGAGTGATGTGGGCGATT3’. Concentration of PCR product (copy number/ng of DNA) was calculated and standards (109 to 1 copy number/μl) were prepared by log10 dilution. Standards were amplified for the same gene (mtcyt-b) by real-time quantitative PCR (RT qPCR) and standard curve was prepared by plotting DNA copy number and cT value in X and Y-axis respectively. Calculation of mitochondrial DNA copy number: cT values of mtCyt-b for plasma DNA samples were obtained by RT qPCR. Real copy number of mtDNA was calculated from reference standard curve by using observed cT values. Protocol was adapted from Kiichi Nakahira et al.21.\n\nGraphPad Prism (version 5.01) software was used for statistical analysis and results of all experiments were expressed as mean±SEM. Comparisons between groups were made by nonparametric unpaired Student’s t-test (Mann-Whitney test) for Table 1, nonparametric paired Student’s t-test (Wilcoxon matched pairs test) for Supplementary Figure 2 and Supplementary Figure 3 and one way ANOVA choosing nonparametric paired test (Friedman test) for rest of the figures. P values were analysed by two-tailed test and P<0.05 was considered as significant (at 95% confidence intervals).\n\n\nResults\n\nEffect of surgery on systemic inflammatory responses was studied by estimating 27 different plasma biomarkers in patients pre- and post-surgery. Eighteen out of 27 mediators tested revealed levels detectable by the assay (Supplementary Table 1). Four of the 19 patients displayed hyper-inflammation as shown by increased plasma levels of TNF-α, IL-1β, IL-ra, IL-7, IL-8 and MIP1a in comparison to pre-surgery levels while in the remaining 15 patients all the 6 inflammatory molecules decreased consistently within 6 hrs post-surgery (Figure 1). Comparison of plasma parameters pre-surgery with post-surgery levels allowed us to differentiate bimodal inflammation consequences in patients after surgery. The dichotomy of inflammatory cytokine response between the two groups persisted at 24 hrs post-surgery also (Supplementary Figure 1). For conceptual clarity the expression ‘hypo-responsive’ and ‘hyper-response’ will be used in the manuscript to classify the former and later groups of patients. Other plasma molecules did not show persistent bimodal inflammatory response post-surgery (Supplementary Table 1). Age, type of anaesthesia used, duration of surgery or hospital stay and several blood cell parameters were comparable between the two groups (Table 1).\n\nCytokine contents (pg/ml) in plasma collected from patients at 0 and 6 hrs post-surgery were measured by bead based multiplex immunoassay. Percent changes in 6hps plasma cytokines (with respect to 0hps) were calculated and values were plotted individually. Data points above and below the X-axis indicate increased and decreased levels of cytokines respectively. Results are presented as separate data points for 19 patients among whom 15 showed decreased and 4 showed increased levels of TNF-α, IL-1β, IL-1Ra, IL-7, IL-8 and MIP-1a at 6hps plasma.\n\nThe following receptors on monocytes were scored by multicolour flow-cytometry pre- and post-surgery in all patients: toll-like receptors TLR2 and TLR4, CD-36 a scavenger receptor, co-stimulatory molecules CD40 and CD80, and HLA-DR. Expression levels of all the above listed receptors were significantly decreased (P<0.05 to P<0.01) in hypo-responsive patients at 6 hrs post-surgery, very similar to inflammatory plasma cytokine levels in this group (Figure 2). There was however no significant change in any of the receptor levels in hyper-responsive patients (Figure 2).\n\nWhole blood collected from patients at 0, 6 and 24 hrs post-surgery was stained with fluorescent conjugated anti-human antibodies for CD14, HLA-DR, CD40, CD80, TLR4, TLR2 and CD36 in two different panels as mentioned in materials and methods section. Percentage of HLA-DR+, CD40+, CD80+, TLR4+, TLR2+ and MFI of CD36 on CD14+ monocytes derived by flow-cytometric analysis is shown. Values are presented as mean±SEM of 7 hypo-responsive and 4 hyper-responsive individuals. Dotted black lines indicate mean values of respective parameters for healthy controls (n=16). Statistical comparisons were performed among all time points by one way ANOVA (*P<0.05 and **P<0.01).\n\nDecreased plasma cytokines as well as receptors on monocytes 6 hrs post-surgery in hypo-responsive patients as shown above indicated an intrinsic defect in responding to TLR agonists. This was experimentally tested by stimulating whole blood with LPS, a TLR4 agonist and PAM3CSK4, a TLR2 agonist at different time points post-surgery and scoring intracellular TNFα in circulating monocytes (Ly6G-CD14+). The results are shown in Figure 3 – circulatory monocytes of hypo-responsive patients tested 6 hrs post-surgery responded significantly less to LPS (P<0.05) as well as PAM3CSK4 (non significant) when compared with stimulation of their cells pre-surgery –the decreased activation was more prominent to LPS than to PAM3CSK4 (Figure 3). The impaired responses recovered to pre-surgery levels at 24 hrs post-surgery. There was no effect in hyper-responsive patients in terms of response to TLR ligands. The response to both TLR2 and TLR4 agonists were comparable pre- and post-surgery in these patients.\n\nWhole blood samples collected from patients at 0, 6 and 24 hrs post-surgery were stimulated with LPS (10ng/ml) (A) and PAM3CSK4 (10ng/ml) (B) for 2 hrs in presence of brefeldin-A (1X). Cells were surface stained for CD14 and CD66b followed by fixation, permeabilization and intracellular staining for TNF-α. MFI of TNF-α in CD14+CD66b- gated monocytes was measured by flow-cytometry and values were presented as mean±SEM of hypo-responsive (n=7, left panel) and hyper-responsive (n=4, right panel) individuals separately. Statistical comparisons were performed among all time points using one way ANOVA (*P<0.05).\n\nPlasma levels of 27 host molecules in normal healthy controls were compared with patients before undergoing surgery. Figure 4a reveals that levels of IL-1β, IL-8, MIP-1a and TNF-α are significantly higher (P<0.05 to P<0.001) in hypo-responsive patients when compared with healthy controls. The levels between hyper-responsive group and healthy controls were however comparable. Similarly pre-surgery expression of CD36, CD40, CD80 and HLA-DR on circulating monocytes were significantly more (P<0.05 to P<0.001) on hypo-responsive patients when compared with controls and there was no significant difference between healthy controls and hyper-responsive cases. These observations suggest that hyper- or hypo-inflammation observed post-elective surgery is determined by pre-existing plasma levels of inflammatory molecules and pathogen responsive surface receptors on monocytes.\n\nPlasma levels (pg/ml) of TNF-α, IL-1β, IL-1Ra, IL-7, IL-8 and MIP-1a in healthy controls (n=23) and both hypo (n=15) and hyper-responsive (n=4) patients pre-surgery measured by bead based multiplex immunoassay are shown (A). Surface expression of HLA-DR, CD40, CD80, TLR4, TLR2 (percent of positive cells) and CD36 (MFI) on CD14+ peripheral blood monocytes collected from healthy controls (n=16) and both hypo (n=15) and hyper-responsive (n=4) patients pre-surgery was scored by flow-cytometry (B). Values are presented as mean±SEM and statistical significance for hypo and hyper-responsive individuals with respect to healthy controls was tested by t-Test (*P<0.05, **p<0.01 and ***P<0.001).\n\nAbsolute copy number of mtDNA in 0 and 6 hrs post-operative plasma were scored to check role of endogenous danger molecules ‘DAMPs’ post-surgery. DNA was isolated from equal volume of plasma samples and copy number of mitochondrial cytochrome-b DNA was scored by real-time quantitative PCR. Results showed significant decrease (P<0.01) in copy number of mtcyt-b DNA in 6 hrs post-surgery plasma of hypo-responsive individuals. Although not statistically significant hyper-responsive individuals also followed the same trend (Supplementary Figure 3).\n\n\nDiscussion\n\nInflammation status post-surgery has been a contentious issue - some patients display features of high inflammation and signs of SIRS and others display hypo-responsive or immune paralysis phenotypes. The current study was undertaken to investigate if pre-operative inflammation status would contribute and determine post-operative inflammatory responses in patients undergoing elective surgery. The study design excluded patients undergoing surgery post trauma which could by itself contribute to induction of inflammation before surgery. The results revealed a characteristic bimodal host inflammatory response following elective surgery. The majority of patients with relatively higher pre-existing systemic inflammation displayed lower inflammation parameters post-surgery and, conversely, a small cohort of patients with decreased levels of inflammation before surgery responded vigorously with significantly elevated inflammatory molecules. A comparative analysis of 27 plasma inflammatory biomarkers revealed downregulation of cytokines such as TNF-α, IL-1β, IL-7 and IL-8, chemokines MIP-1a and the antagonist of IL-1 cytokine, IL-1Ra in 79% of subjects whereas the same mediators were upregulated in 21% of subjects at 6 hrs post-surgery. In the former category of patients decreased levels of plasma mediators persisted at 24 hrs also while in the latter the levels increased further at the same time point. On the basis of these initial findings we designated patients with downregulated plasma inflammatory biomarkers as hypo-responsive and those with upregulated plasma inflammatory biomarkers as hyper-responsive individuals. Earlier investigators in our view may have missed such bimodal distribution of inflammation due to faulty analysis of data - most studies compute mean and deviation of each of the parameters pre- and post-surgery without taking cognisance of shift in inflammation parameters in each of the patients.\n\nFactors such as age, sex, type of anaesthesia used, duration of surgery, degree of surgical injury etc. were all comparable between hypo-responsive and hyper-responsive patients. However TLRs that contribute significantly to induction of inflammation by PAMPs and DAMPs were significantly decreased on circulating monocytes in hypo-responsive patients and were either unaltered or marginally increased on monocytes of hyper-responsive individuals at different time points post-surgery. Expression of HLA-DR and co-stimulatory molecules (CD80 and CD86) on monocytes were downregulated in hypo-responsive patients, an observation similar to other reports22,23. Expression of monocyte CD36, a dominant scavenger receptor involved in phagocytosis was significantly downregulated only in hypo-responsive patients. The study of receptors on monocytes and plasma levels of cytokines are only suggestive of hypo- or hyper-inflammation status and its validation would depend on demonstration of response of immune cells to stimulation by PAMPs and DAMPs tested ex vivo – stimulation of whole blood with TLR-2 and TLR-4 agonists revealed significantly decreased induction of TNF-α by monocytes collected from hypo-responsive individuals indicating immunoparalysis.\n\nFurther, although previous studies have revealed a positive association of elevated plasma mtDNA with excessive inflammation in critically ill surgery patients21,24,25 we observed reduced copy number of plasma mtDNA in both hypo- and hyper-responsive individuals following surgery. This may be indicative of the fact that increased plasma cytokines along with high copy number of mtDNA are responsible for post-surgical complications while in uncomplicated situations although the plasma cytokines increase the host system takes control over excessive inflammation by reducing the number of plasma mtDNA.\n\nTaken together our data revealed that basal pre-surgery levels of plasma TNF-α, IL-1β, IL-7, IL-8, MIP-1a and monocyte expression of TLR-2, TLR-4 and CD80 etc. are significantly elevated in hypo-responsive patients as compared to hyper-responsive counterparts. These findings also explain several earlier reports on immuneparalysis as well as hyper-inflammation – a paradoxically opposite scenario in cohorts of patients post-surgery18–20.\n\nOur observations of bimodal response in patients post-surgery also provides us a model to categorise patients undergoing elective surgery as ‘immune paralysis’ prone vs ‘hyper-Inflammation’ prone. We propose that surgical trauma predominantly and essentially leads to hypo-responsiveness and tolerance critical for regulating innate immune activation for uneventful recovery post-surgery and that failure to do so in a small cohort of patients could result in persistent hyper-inflammation leading to susceptibility to SIRS or sepsis and that pre-existing inflammation status before surgery could play a critical role in determining the clinical outcome. It is however not clear currently from this study if patients who displayed hyper-inflammation phenotype post-surgery would have developed SIRS and/or sepsis since the patients were not followed beyond 24 hrs. It is a major limitation of this study in our view but we are currently addressing this issue and its validation could result in development of robust biomarkers for predicting outcome of surgery. The results of this study hopefully will lead to similar analysis in different surgical cohorts by other investigators. Further, the ability to predict inflammation outcome could also assist in decision making before administering immunosuppressive drugs post-surgery.\n\n\nData availability\n\nF1000Research: Dataset 1. Complete blood count raw data, 10.5256/f1000research.6991.d10160326\n\nF1000Research: Dataset 2. Mitochondrial cytochrome-b raw data, 10.5256/f1000research.6991.d10160427\n\nF1000Research: Dataset 3. Flow-cytometry .FCS files and analyzed data files, 10.5256/f1000research.6991.d10161228",
"appendix": "Author contributions\n\n\n\nPB conducted most of the laboratory assays, analysed the data and wrote the draft manuscript, RM performed analysis of multi-colour flowcytometry assays, JM conducted the surgery on patients and clinically evaluated each of the patients who participated in the study and BR conceived and designed the project, interpreted the data and finalysed the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by the core grants to Institute of Life Sciences, Bhubaneswar, India from Department of Biotechnology, Government of India. PB was supported with a fellowship from Council of Industrial and Scientific Research, New Delhi.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe Institute of Life Sciences is fully funded by Department of Biotechnology, Government of India. The authors acknowledge with thanks all the patients and volunteers who participated in the study and the staff of Neelachal Hospitals for their co-operation.\n\n\nSupplementary materials\n\nPercent changes of TNF-α, IL-1β, IL-1Ra, IL-7, IL-8 and MIP-1a in 6hps and 24hps plasma (with respect to 0hps) were plotted separately for hypo and hyper-responsive groups. Data represent mean of 7 hypo-responsive (left panels) and 4 hyper-responsive (right panels) individuals. Although there were quantitative differences none of the mediators showed statistical significance between 6hps and 24hps time points in any group.\n\nAbsolute numbers of immune cells in peripheral blood of patients at 0, 6 and 24 hrs post-surgery were scored. Percent changes of total leukocyte counts (TLCs), neutrophils, lymphocytes, monocytes, eosinophils and basophils at 6hps and 24hps are presented as mean±SEM of hypo-responsive (n=7, blue bars) and hyper-responsive (n=4, red bars) patients. Statistical analysis showed no significant difference between two groups for any parameter at any time point.\n\nA) Amplification plot shows regular increase in cT values of serially diluted (1:10) cytochrome-b DNA standards and the samples falling within the range of standards. B) The standard curve derived by plotting log10 transformed copy number of cytochrome-b DNA and cT values shows a linearity over a high range (R2=0.9949). C) Absolute copy numbers of plasma mitochondrial DNA derived from standard curve were plotted for healthy controls and patients pre-surgery (C, left panel) as well as for patients at 0 and 6 hrs post-surgery (C, right panel). Dotted black line indicates mean of healthy controls. Data showed mean±SEM of 23 healthy controls, 15 hypo-responsive and 4 hyper-responsive individuals. Statistical analysis was done by using t-test and/or one way ANOVA (*P<0.05 and **p<0.01).\n\nCytokine contents (pg/ml) in plasma collected from healthy controls (n=23) and patients at 0, 6 and 24 hrs post-surgery (n=19) measured by bead based multiplex immunoassay. Sur, surgery; hps, hours post-surgery; HC, healthy control.\n\n\nReferences\n\nCarrico CJ, Meakins JL, Marshall JC, et al.: Multiple-organ-failure syndrome. Arch Surg. 1986; 121(2): 196–208. PubMed Abstract | Publisher Full Text\n\nHaga Y, Beppu T, Doi K, et al.: Systemic inflammatory response syndrome and organ dysfunction following gastrointestinal surgery. Crit Care Med. 1997; 25(12): 1994–2000. PubMed Abstract | Publisher Full Text\n\nLobo SM, Rezende E, Knibel MF, et al.: Early determinants of death due to multiple organ failure after noncardiac surgery in high-risk patients. Anesth Analg. 2011; 112(4): 877–83. PubMed Abstract | Publisher Full Text\n\nTalmor M, Hydo L, Barie PS: Relationship of systemic inflammatory response syndrome to organ dysfunction, length of stay, and mortality in critical surgical illness: effect of intensive care unit resuscitation. Arch Surg. 1999; 134(1): 81–7. PubMed Abstract | Publisher Full Text\n\nPittet D, Rangel-Frausto S, Li N, et al.: Systemic inflammatory response syndrome, sepsis, severe sepsis and septic shock: incidence, morbidities and outcomes in surgical ICU patients. Intensive Care Med. 1995; 21(4): 302–9. PubMed Abstract | Publisher Full Text\n\nMeisel C, Schefold JC, Pschowski R, et al.: Granulocyte-macrophage colony-stimulating factor to reverse sepsis-associated immunosuppression: a double-blind, randomized, placebo-controlled multicenter trial. Am J Respir Crit Care Med. 2009; 180(7): 640–8. PubMed Abstract | Publisher Full Text\n\nHall MW, Knatz NL, Vetterly C, et al.: Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome. Intensive Care Med. 2011; 37(3): 525–32. PubMed Abstract | Publisher Full Text\n\nBoomer JS, To K, Chang KC, et al.: Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA. 2011; 306(23): 2594–605. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDöcke WD, Randow F, Syrbe U, et al.: Monocyte deactivation in septic patients: restoration by IFN-gamma treatment. Nat Med. 1997; 3(6): 678–81. PubMed Abstract | Publisher Full Text\n\nWei M, Kuukasjärvi P, Laurikka J, et al.: Cytokine Responses in Low-Risk Coronary Artery Bypass Surgery. Int J Angiol. 2001; 10(1): 27–30. PubMed Abstract | Publisher Full Text\n\nStrey CW, Marquez-Pinilla RM, Markiewski MM, et al.: Early post-operative measurement of cytokine plasma levels combined with pre-operative bilirubin levels identify high-risk patients after liver resection. Int J Mol Med. 2011; 27(3): 447–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReikeras O, Borgen P, Reseland JE, et al.: Changes in serum cytokines in response to musculoskeletal surgical trauma. BMC Res Notes. 2014; 7: 128. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJimenez MF, Watson RW, Parodo J, et al.: Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg. 1997; 132(12): 1263–9; discussion 1269–70. PubMed Abstract | Publisher Full Text\n\nLahiri R, Derwa Y, Bashir Z, et al.: Systemic Inflammatory Response Syndrome After Major Abdominal Surgery Predicted by Early Upregulation of TLR4 and TLR5. Ann Surg. 2015. PubMed Abstract | Publisher Full Text\n\nVersteeg D, Dol E, Hoefer IE, et al.: Toll-like receptor 2 and 4 response and expression on monocytes decrease rapidly in patients undergoing arterial surgery and are related to preoperative smoking. Shock. 2009; 31(1): 21–7. PubMed Abstract | Publisher Full Text\n\nIkushima H, Nishida T, Takeda K, et al.: Expression of Toll-like receptors 2 and 4 is downregulated after operation. Surgery. 2004; 135(4): 376–85. PubMed Abstract | Publisher Full Text\n\nGentile LF, Cuenca AG, Efron PA, et al.: Persistent inflammation and immunosuppression: a common syndrome and new horizon for surgical intensive care. J Trauma Acute Care Surg. 2012; 72(6): 1491–501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFriedman BH, Wolf JH, Wang L, et al.: Serum cytokine profiles associated with early allograft dysfunction in patients undergoing liver transplantation. Liver Transpl. 2012; 18(2): 166–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoyes LH, Leitch EF, McKee RF, et al.: Preoperative systemic inflammation predicts postoperative infectious complications in patients undergoing curative resection for colorectal cancer. Br J Cancer. 2009; 100(8): 1236–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohri Y, Miki C, Kobayashi M, et al.: Correlation between preoperative systemic inflammation and postoperative infection in patients with gastrointestinal cancer: a multicenter study. Surg Today. 2014; 44(5): 859–67. PubMed Abstract | Publisher Full Text\n\nNakahira K, Kyung SY, Rogers AJ, et al.: Circulating mitochondrial DNA in patients in the ICU as a marker of mortality: derivation and validation. PLoS Med. 2013; 10(12): e1001577; discussion e1001577. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMokart D, Textoris J, Chow-Chine L, et al.: HLA-DR and B7-2 (CD86) monocyte expressions after major cancer surgery: profile in sepsis. Minerva Anestesiol. 2011; 77(5): 522–7. PubMed Abstract\n\nKawasaki T, Ogata M, Kawasaki C, et al.: Surgical stress induces endotoxin hyporesponsiveness and an early decrease of monocyte mCD14 and HLA-DR expression during surgery. Anesth Analg. 2001; 92(5): 1322–6. PubMed Abstract | Publisher Full Text\n\nSimmons JD, Lee YL, Mulekar S, et al.: Elevated levels of plasma mitochondrial DNA DAMPs are linked to clinical outcome in severely injured human subjects. Ann Surg. 2013; 258(4): 591–6; discussion 596–8. PubMed Abstract | Free Full Text\n\nZhang Q, Raoof M, Chen Y, et al.: Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 2010; 464(7285): 104–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarman P, Mukherjee R, Mohapatra J, et al.: Dataset 1 in: Pre-surgery status determines inflammation levels post-elective surgery. F1000Research. 2015. Data Source\n\nBarman P, Mukherjee R, Mohapatra J, et al.: Dataset 2 in: Pre-surgery status determines inflammation levels post-elective surgery. F1000Research. 2015. Data Source\n\nBarman P, Mukherjee R, Mohapatra J, et al.: Dataset 3 in: Pre-surgery status determines inflammation levels post-elective surgery. F1000Research. 2015. Data Source"
}
|
[
{
"id": "21476",
"date": "03 Apr 2017",
"name": "Michael Bauer",
"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\nBarman et al. report data on the impact of “pre-surgery status” on post-operative surrogates of the inflammatory response to surgery. The overall concept to assess pre-existing health status as a confounder for post-operative inflammation is not completely new and the authors fail to discuss their concept in the light of current understanding of “trained immunity” as opposed to “tolerance”. Unfortunately, the report lacks critical information that is mandatory in clinical studies: For instance, the authors claim that patients undergoing elective surgery over a period of more than a year were enrolled, yet there are a total of 19 patients (a CONSORT flow diagram is missing). This low number limits interpretation with only 4 patients attributed to “HR” group. No information as to the type of surgery – except the elective nature – is provided. Statistics are sub-par and the normalization to presumably very low pre-surgery values with “1000%” increases post-op are obsolete.",
"responses": []
},
{
"id": "21176",
"date": "11 Apr 2017",
"name": "Rami A Namas",
"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 entitled \"Pre-surgery status determines inflammation levels post-elective surgery\" presents a single center prospective observational study that evaluates the impact of systemic inflammation prior to elective surgery and its correlation with outcomes post-surgery. General remarks: The research article is well written and demonstrates a clear hypothesis and sound methodology to approach the results. However, the very small patient sample size used in this study represents a major drawback making it difficult to discern any meaningful conclusions. Moreover, the study fails to deliver how these observations can change the patient clinical course for better outcomes and improve standards of care. Major Concerns: 1-\n\nAlthough this was a prospective study conducted over a one year period, surprisingly, only 19 patients undergoing elective surgery were enrolled. Since the authors did not outline a flow chart of patient recruitment in a surgery department that is expected to receive greater number of patients during a one-year period. Was there an additional exclusion criteria that has not been mentioned in the methods? Did the authors perform an a priori power analysis to justify the sample size?\n\n2-\n\nIt would have been more appropriate to show all the 19 patients’ demographics and characteristics in Table 1 (prior to dichotomizing into either hypo- or hyper-responsive) compared to the healthy controls (HC). Although the authors suggest the impact of age as a confounding factor, the statistically significant difference in age between the HC and the hypo-responsive group warrants cautious interpretation of any downstream findings. It would have been more appropriate to case control match the HC group for age with the hypo- and hyper- responsive groups to avoid the confounding impact of age. In addition, the table does not show if the 19 patient had any pre-existing comorbidities and whether these patients where on medications for a chronic disease condition which also might impact their inflammatory status. Moreover, the type of elective surgery was not mentioned in Table 1 although the operation time was comparable.\n\n3-\n\nThe authors choose to classify patients based on the absolute cytokine/chemokine values post-surgery relative to the pre-surgery values. Out of the 27 inflammatory mediators, 6 inflammatory mediators where either low or high at 6 hours post-surgery in the hypo- and hyper-responsive groups respectively. However, there is no mention of the remaining 21 inflammatory mediators in the results section. Did they have comparable levels or did they oscillate? Another concern that could have been avoided is the intention to plot the inflammatory mediators’ values in percentage (Figure 1) rather than actual values. The authors might have wanted to “show case” their point of hypo vs. hyper but by doing this they lost the opportunity to present their findings to illustrate the inflammatory mediator trajectories in a more dynamic fashion. Although they attempt to do so in Figure 4A (pre-surgery values), adding the post-surgery values would have been more comprehensive and easily comparable for the reader.\n\n4-\n\nAnother concern revolves around the sample size used to carry out the flow cytometry and in vitro experiments. The authors do not justify the downscale of the hypo-responsive group from 15 patients to only 7 patients and HC from 23 to only 16 healthy volunteers. Why the remaining 8 hypo-responsive patients excluded from either analysis? Did the 7 hypo-responsive patients’ demos and characteristic differ from their parent cohort i.e. the 15 hypo-responsive patients mentioned in Table 1? These are very critical points to the validity of the flow cytometry and in vitro results.\n\n5-\n\nBased on the critiques mentioned above, any conclusions should be interpreted very cautiously. In the discussion section, the authors conclude “Our observations of bimodal response in patients post-surgery also provides us a model to categorize patients undergoing elective surgery as ‘immune paralysis’ prone vs ‘hyper-Inflammation’ prone.” and “that pre-existing inflammation status before surgery could play a critical role in determining the clinical outcome.”\n\nOn what basis where these conclusions made? If the hypo-responsive group reflected the immune paralysis phenotype (according to the authors) would not they exhibit differential primary outcomes as end points? For example, a longer hospital stay or more incidence of post-surgery infections? Given that the total hospital length of stay in both hypo- and hyper- responsive groups was comparable (average of 4 days each) such conclusions cannot be discerned. How these inflammatory perturbations post-surgery affect long term outcomes?\n\nOverall the study is of an observational nature that characterizes a differential systemic inflammation profile in a relatively small post-surgery patient’s cohort which warrants a larger external validation cohort.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-766
|
https://f1000research.com/articles/4-764/v1
|
10 Sep 15
|
{
"type": "Review",
"title": "Dual mechanisms governing reward-driven perceptual learning",
"authors": [
"Dongho Kim",
"Sam Ling",
"Takeo Watanabe",
"Dongho Kim",
"Sam Ling"
],
"abstract": "In this review, we explore how reward signals shape perceptual learning in animals and humans. Perceptual learning is the well-established phenomenon by which extensive practice elicits selective improvement in one’s perceptual discrimination of basic visual features, such as oriented lines or moving stimuli. While perceptual learning has long been thought to rely on ‘top-down’ processes, such as attention and decision-making, a wave of recent findings suggests that these higher-level processes are, in fact, not necessary. Rather, these recent findings indicate that reward signals alone, in the absence of the contribution of higher-level cognitive processes, are sufficient to drive the benefits of perceptual learning. Here, we will review the literature tying reward signals to perceptual learning. Based on these findings, we propose dual underlying mechanisms that give rise to perceptual learning: one mechanism that operates ‘automatically’ and is tied directly to reward signals, and another mechanism that involves more ‘top-down’, goal-directed computations.",
"keywords": [
"Perceptual learning",
"Vision",
"Reward",
"goal-directed",
"contingency",
"temporal contiguity",
"automatic",
"task-irrelevant"
],
"content": "Introduction\n\nPerceptual learning is the process by which sensory systems in humans or animals improve their ability to perform a perceptual task, often after extensive experience with a particular stimulus. It had long been believed that this type of learning was tied to one’s task performance on that stimulus. Support for this came from a number of studies that found benefits of perceptual learning for features that were relevant to a task, whereas features that were merely exposed showed little-to-no learning1–3. Taken together, these studies supported the hypothesis that conscious effort directed toward a sensory feature, by means of processes such as attention, is necessary for the feature to be learned2–4.\n\nIn recent years, however, evidence for a new type of perceptual learning has emerged – one that may not necessitate higher-level, goal-directed processes, such as attention5–14. In a study by Watanabe et al. (2001), evidence was found that perceptual learning could transpire outside of the window of attention. Specifically, observers were asked to perform a demanding task at the center of a display, while they were exposed to an array of moving dots presented in the periphery. Importantly, only 5% of the dots moved coherently in a fixed direction, while the remaining dots moved randomly. Because the motion signal was task-irrelevant, it was assumed that little-to-no attention was actively deployed to that stimulus. Moreover, sensitivities to the 5% and 10% coherent motion were measured before (pre-test) and after (post-test) a training period. The strength of the 5% coherent motion was so weak that subjects were not able to discriminate or detect the coherent motion direction above chance, either at the pre-test or the post-test. Nevertheless, the result of the post-test revealed that repeated exposure improved sensitivity for the 10% coherent motion in the exposed direction. The authors interpreted these results as evidence for a new type of perceptual learning, coined ‘task-irrelevant perceptual learning’, which occurs without attention5–7.\n\nIs mere task-irrelevant exposure to a stimulus truly sufficient for perceptual learning? A follow-up study demonstrated that mere exposure is, in fact, insufficient; performance benefits of exposure only occurred when there was a temporal pairing between a task-irrelevant motion signal and task-relevant targets9. Most interestingly, task-irrelevant learning appeared to only occur in instances in which the target was successfully recognized15. Why is that? One interpretation is that successful recognition of the target letter led to a sense of accomplishment for the participant, which elicited an internal reward signal. As a consequence, task-irrelevant perceptual learning may arise as a result of repeated pairing between a stimulus and internal reward signals, which are released diffusively throughout the brain, affecting both task-relevant and task irrelevant stimuli5.\n\nAlthough the aforementioned studies did not explicitly test this reward-based hypothesis, a number of studies have since emerged, derived from work in animal models and humans, supporting the hypothesis that reward signals are sufficient in order for perceptual learning to manifest. In this article, we will review and synthesize work that has examined how reward signals play a role in shaping perceptual learning. Many models in cognition assume that goal-directed behavior plays a dominant role in governing learning. Goal-directed behavior is a class of behavior aimed towards completion of a task – a subset of self-attributed motives commonly assumed to require high-level cognitive processes, such as attention and decision-making. For instance, a classic example of a goal-directed behavior is the online computation of the probabilistic contingency between the presence of a stimulus, and receiving a reward. In phenomena such as reward-driven perceptual learning, an individual’s estimation of the ‘contingency’ between rewards and visual stimuli has been shown to impact learning rates, clearly indicating that goal-directed processes are involved. However, not all behaviors necessarily tap into these high-level processes. For instance, reward-driven perceptual learning has also been shown to occur in the absence of any task, as well as outside of an individual’s awareness. Interestingly, this suggests that reward signals can gate the emergence of learning, untainted by other higher-level cognitive processes. To explain these results, we propose dual underlying mechanisms of reward-driven perceptual learning: one mechanism that operates ‘automatically’, free from goal-directed processes, and another mechanism that involves more ‘top-down’, goal-directed computations, and requires conscious estimation of learning contingencies. Moreover, we propose that perceptual learning, in combination with paradigms used to suppress images from visual awareness, can be leveraged as tools to probe this more ‘automatic’ component of learning.\n\n\nHow do rewards shape perceptual learning, independent of goal-directed processes?\n\nThe reward-driven hypothesis for task-irrelevant perceptual learning is based on the assumption that internal reward signals are released when subjects successfully recognize a target item, with the temporal pairing between a task-irrelevant feature and the reward signals playing a crucial role in determining task-irrelevant perceptual learning. However, it is possible that the task-based component in those aforementioned studies is unnecessary, and that it truly is the reward signal itself that triggers task-irrelevant perceptual learning. How does one test this hypothesis? The lion’s share of perceptual learning studies employ a training procedure by which observers perform a task that is the same or similar to evaluating the amount of learning. However, this makes it difficult to truly understand the effects of reward on perceptual learning, because the role of rewards in such paradigms is necessarily entangled with higher-level cognitive processes, such as attention and goal-directed decision-making, when participants are consciously performing a task on a stimulus. In order to truly understand how rewards gate perceptual learning, one should empirically disentangle rewards process from other cognitive processes. Classical conditioning is a process by which learning is acquired through repeated pairings of a stimulus and a reinforcer16. Interestingly, classical conditioning does not necessitate any task during conditioning. Therefore, by leveraging classical conditioning, one can gain a true understanding of how reward signals modulate perceptual learning, untainted by goal-directed decision processes.\n\nIn a definitive test of the reward-signal hypothesis for perceptual learning, Seitz, Kim & Watanabe (2009) discovered that perceptual learning could occur even without any task involvement whatsoever. To do so, this study used a classical conditioning procedure in which human subjects, who were deprived of food and water, passively viewed visual stimuli while receiving occasional drops of water as rewards17–19. To ensure that perceptual learning was driven purely by reward signals, this study used a technique known as continuous flash suppression20 throughout the training regime, which is known to render visual stimuli imperceptible. Surprisingly, learning occurred through stimulus-reward pairing in the absence of a task and without awareness of the stimulus presentation. Since neither task nor attention was involved during the training procedure, these results study strongly implicate the continuous temporal pairing between stimulus feature and reward signals as being the necessary and sufficient elements needed for perceptual learning at least in some conditions to occur.\n\n\nHow do rewards shape perceptual learning, in the presence of goal-directed processes?\n\nWhile reward signals alone appear to be sufficient to trigger perceptual learning10, this result does not preclude goal-directed, conscious behavior from also playing a modulatory role in perceptual learning. According to the theory of motivation, implicit motives represent a more primitive motivational system derived from affective experiences21, and it is likely that the task-irrelevant perceptual learning that has been observed rides on this motivational system to yield its effects. However, behavior is also driven by self-attributed (or explicit) motives, which are based on more cognitively elaborated constructs. Such goal-directed behavior, which requires higher-level cognitive processes, likely also governs perceptual learning. Indeed, it has long been suggested that such higher-level cognitive processes, such as attention and/or decision making, also act as main factors in driving perceptual learning1,22–25.\n\nWhat roles, then, do goal-directed behaviors play in perceptual learning? Specifically, do reward signals and goal-directed decision processes elicit a similar pattern of effects on perceptual learning? To examine the similarity between these ‘automatic’ and ‘top-down’ processes in perceptual learning, a recent study developed a methodology that combines perceptual learning with a novel training procedure, which employs either classical conditioning or operant conditioning. In the classical conditioning variant of the study, human subjects, who were deprived of food and water, passively viewed visual stimuli while receiving liquid rewards during a ‘training regime’10,17–19. This experiment was similar to the aforementioned study10, with the notable exception being that there were various reward-contingencies at play, with the orientation content of a visual stimulus paired with a certain probability of receiving a liquid reward26. To vary the probability of reward-delivery, three different stimulus orientations were used for each subject: 1) the zero-contingency orientation had a reward-probability equal to the background reward-rate of 50%, 2) the positive-contingency orientation had an 80% probability of reward, and 3) the negative-contingency orientation had a 20% probability of reward. In the operant conditioning variant of the study, a goal-directed behavior component was added27. In contrast to the classical conditioning variant of the experiment, here subjects performed a ‘go/no-go task’ in response to the orientation stimuli during a training regime. Specifically, if subjects pressed a spacebar, a liquid reward was delivered at a probability contingent on the orientation of that presented stimulus (for example, 80% for a stimulus tilted 135°, 50% for a stimulus oriented 75°, and 20% for a stimulus oriented 15°).\n\nResults from the classical conditioning variant of perceptual learning showed that learning occurred for both the positive-contingency orientation stimulus as well as the zero-contingency orientation stimulus, but no significant change was found for the negative-contingency orientation stimulus. In contrast, results of the operant conditioning variant of perceptual learning revealed that learning only occurred for the positive-contingency orientation, with no learning found for either the zero-contingency orientation or the negative-contingency orientation27. These results suggest that reward-driven perceptual learning without goal-directed processing is distinct from reward-driven perceptual learning with goal-directed processes.\n\nWhen there is no goal-directed behavior, a consistent pairing between a visual stimulus and reward seems to be the underlying mechanism for perceptual learning to occur5,9,10,28. In that case, “temporal contiguity” between rewards and visual stimuli play a crucial role for perceptual learning to occur5,16. However, if goal-directed processes are involved, contingency information between rewards and visual stimuli overrides pure temporal contiguity. In other words, the top-down component can override the automatic components of reward-driven perceptual learning. The operant conditioning variant of perceptual learning demonstrated that learning of a visual stimulus occurred only when that visual stimulus informatively predicted the upcoming rewards29–32.\n\nThese results square with a study by Law and Gold (2009), where monkeys carried out a goal-directed behavior, performing a visual task to receive rewards. In that study, connections between sensory neurons and the goal-directed decision process that interprets the sensory information were first modified by reward driven reinforcement signals. Subsequently, that same mechanism acted to further refine these connections to more strongly weight inputs from the most relevant sensory neurons, thereby improving perceptual sensitivity.\n\n\nCommon mechanisms between perceptual learning and conditioning\n\nConditioning is the form of learning in which repeated pairings of arbitrary features with rewards or punishments leads to a representation of the rewards or punishment evoked by the paired features29,33. At face value, this resembles the task-irrelevant perceptual learning revealed in Seitz and Watanabe (2003), which occurred only when the visual feature was paired with the presentation of a rewarded target. A number of subsequent studies have demonstrated that task-irrelevant perceptual learning in humans can occur for visual stimuli that are consistently paired with internal or external rewards5,9,10,28, and this connection holds true for animal models as well34,35. Taken together, these studies suggest common mechanisms shared between conditioning and perceptual learning.\n\nHow generalizable are the rules governing conditioning to the domain of perceptual learning? One common theme to the perceptual learning and conditioning literatures is that of contingency29,31,36. Excitatory conditioning occurs when the probability of a reward is higher for a conditioned stimulus than at other times, which is referred to as positive contingency. Likewise, when the probability is lower (negative contingency), negative conditioning occurs29,31,32,37. Since the contingency rule is a hallmark of conditioning, along with contiguity and prediction error29, a question arises as to whether perceptual learning follows the same rules of contingency as found in conditioning. Were that the case, then one would expect to observe positive learning, negative learning, or no learning in accordance with the contingency between the predicted signal and the reward. Perceptual learning appears to be governed by both classical and operant conditions principles, depending on the situation. Under a task that promotes high-level processing of the stimulus-reward structure, perceptual learning mirrored the rules of operant conditioning, occurring only for the positive-contingency orientation, with no learning in either the zero-contingency orientation or the negative-contingency orientation27. However, under a task that prevented high-level processing of the contingency structure, the effects of perceptual learning much more closely resembled learning of the ‘temporal contiguity’ between visual features and rewards in classical conditioning, with learning transfer occurring not only for the positive-contingency stimuli, but also for zero-contingency stimuli (of note, there were 50% stimuli-rewards pairings in zero-contingency stimuli). Although there has been considerable debate in regards to whether classical conditioning depends on a contingent relation between conditioned stimulus and unconditioned stimulus38, perceptual learning under a task that prevented high-level processing of the contingency structure is more closely aligned with classical conditioning, in which learning is more influenced by contiguity than contingency39.\n\n\nConclusion\n\nPerceptual learning can occur in the absence of a task and outside the window of awareness, suggesting that reward signals gate the occurrence of perceptual learning. This may emerge through mechanisms akin to classical conditioning, impinging on very early visual sensitivity. In that case, ‘temporal contiguity’ between rewards and visual stimuli plays a crucial role5,16. However, when goal-directed processes are introduced, the contingency between rewards and visual stimuli overrides classical condition-like operations, instead influencing perceptual learning based on the stimulus-reward contingencies. This suggests that there exists two underlying mechanisms that give rise to perceptual learning: one mechanism that operates ‘automatically’ and is tied directly to reward signals, and another overriding mechanism that involves ‘top-down’, goal-directed computations.",
"appendix": "Author contributions\n\n\n\nAll authors contributed ideas, text, and critique, 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 supported by NIH grant number R01 EY015980.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAhissar M, Hochstein S: Attentional control of early perceptual learning. Proc Natl Acad Sci U S A. 1993; 90(12): 5718–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchoups A, Vogels R, Qian N, et al.: Practising orientation identification improves orientation coding in V1 neurons. Nature. 2001; 412(6846): 549–53. PubMed Abstract | Publisher Full Text\n\nShiu LP, Pashler H: Improvement in line orientation discrimination is retinally local but dependent on cognitive set. Percept Psychophys. 1992; 52(5): 582–8. PubMed Abstract | Publisher Full Text\n\nAhissar M, Hochstein S: The reverse hierarchy theory of visual perceptual learning. Trends Cogn Sci. 2004; 8(10): 457–64. PubMed Abstract | Publisher Full Text\n\nSeitz AR, Watanabe T: A unified model for perceptual learning. Trends Cogn Sci. 2005; 9(7): 329–34. PubMed Abstract | Publisher Full Text\n\nSagi D: Perceptual learning in Vision Research. Vision Res. 2011; 51(13): 1552–66. PubMed Abstract | Publisher Full Text\n\nSasaki Y, Nanez JE, Watanabe T: Advances in visual perceptual learning and plasticity. Nat Rev Neurosci. 2010; 11(1): 53–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGutnisky DA, Hansen BJ, Iliescu BF, et al.: Attention alters visual plasticity during exposure-based learning. Curr Biol. Elsevier Ltd; 2009; 19(7): 555–60. PubMed Abstract | Publisher Full Text\n\nSeitz AR, Watanabe T: Psychophysics: Is subliminal learning really passive? Nature. 2003; 422(6927): 36. PubMed Abstract | Publisher Full Text\n\nSeitz AR, Kim D, Watanabe T: Rewards evoke learning of unconsciously processed visual stimuli in adult humans. Neuron. 2009; 61(5): 700–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeclercq V, Seitz AR: Fast-TIPL occurs for salient images without a memorization requirement in men but not in women. Baker CI, editor. PLoS One. 2012; 7(4): e36228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeclercq V, Le Dantec CC, Seitz AR: Encoding of episodic information through fast task-irrelevant perceptual learning. Vision Res. 2014; 99: 5–11. PubMed Abstract | Publisher Full Text\n\nBeste C, Dinse HR: Learning without training. Curr Biol. 2013; 23(11): R489–99. PubMed Abstract | Publisher Full Text\n\nArsenault JT, Nelissen K, Jarraya B, et al.: Dopaminergic reward signals selectively decrease fMRI activity in primate visual cortex. Neuron. 2013; 77(6): 1174–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeitz AR, Lefebvre C, Watanabe T, et al.: Requirement for high-level processing in subliminal learning. Curr Biol. 2005; 15(18): R753–5. PubMed Abstract | Publisher Full Text\n\nMackintosh NJ: Conditioning and associative learning. Clarendon Press, Oxford, 1983; 1. Reference Source\n\nDorris MC, Glimcher PW: Activity in posterior parietal cortex is correlated with the relative subjective desirability of action. Neuron. 2004; 44(2): 365–78. PubMed Abstract | Publisher Full Text\n\nLauwereyns J, Watanabe K, Coe B, et al.: A neural correlate of response bias in monkey caudate nucleus. Nature. 2002; 418(6896): 413–7. PubMed Abstract | Publisher Full Text\n\nLeon MI, Shadlen MN: Effect of expected reward magnitude on the response of neurons in the dorsolateral prefrontal cortex of the macaque. Neuron. 1999; 24(2): 415–25. PubMed Abstract | Publisher Full Text\n\nTsuchiya N, Koch C: Continuous flash suppression reduces negative afterimages. Nat Neurosci. 2005; 8(8): 1096–101. PubMed Abstract | Publisher Full Text\n\nMcClelland DC, Koestner R, Weinberger J: How do self-attributed and implicit motives differ? Psychol Rev. 1989; 96(4): 690–702. Publisher Full Text\n\nKahnt T, Grueschow M, Speck O, et al.: Perceptual learning and decision-making in human medial frontal cortex. Neuron. 2011; 70(3): 549–59. PubMed Abstract | Publisher Full Text\n\nXiao LQ, Zhang JY, Wang R, et al.: Complete transfer of perceptual learning across retinal locations enabled by double training. Curr Biol. Elsevier Ltd; 2008; 18(24): 1922–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang R, Zhang JY, Klein SA, et al.: Task relevancy and demand modulate double-training enabled transfer of perceptual learning. Vision Res. 2012; 61: 33–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang JY, Zhang GL, Xiao LQ, et al.: Rule-based learning explains visual perceptual learning and its specificity and transfer. J Neurosci. 2010; 30(37): 12323–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim D, Watanabe T: Different properties between reward-driven exposure-based and reward-driven task involved perceptual learning. J Vis. 2010; 10(7): 1112. Publisher Full Text\n\nKim D, Seitz AR, Watanabe T: Visual perceptual learning by operant conditioning training follows rules of contingency. Vis cogn. 2015; 23(1–2): 147–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatanabe T, Náñez JE, Sasaki Y: Perceptual learning without perception. Nature. 2001; 413(6858): 844–8. PubMed Abstract | Publisher Full Text\n\nSchultz W: Behavioral theories and the neurophysiology of reward. Annu Rev Psychol. 2006; 57: 87–115. PubMed Abstract | Publisher Full Text\n\nShannon C: A mathematical theory of communication. Bell Syst Tech J. 1948; 27(3): 379–423 & 623–656. Publisher Full Text\n\nRescorla RA: Probability of shock in the presence and absence of CS in fear conditioning. J Comp Physiol Psychol. 1968; 66(1): 1–5. PubMed Abstract | Publisher Full Text\n\nRescorla RA: Behavioral studies of Pavlovian conditioning. Annu Rev Neurosci. 1988; 11: 329–52. PubMed Abstract | Publisher Full Text\n\nWasserman EA, Miller RR: What's elementary about associative learning? Annu Rev Psychol. 1997; 48: 573–607. PubMed Abstract | Publisher Full Text\n\nLaw CT, Gold JI: Neural correlates of perceptual learning in a sensory-motor, but not a sensory, cortical area. Nat Neurosci. 2008; 11(4): 505–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaw CT, Gold JI: Reinforcement learning can account for associative and perceptual learning on a visual-decision task. Nat Neurosci. Nature Publishing Group; 2009; 12(5): 655–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchultz W, Dayan P, Montague PR: A neural substrate of prediction and reward. Science. 1997; 275(5306): 1593–9. PubMed Abstract | Publisher Full Text\n\nRescorla RA: Pavlovian conditioning: It's not what you think it is. Am Psychol. American Psychological Association. 1988; 43(3): 151–60. PubMed Abstract | Publisher Full Text\n\nPapini MR, Bitterman ME: The role of contingency in classical conditioning. Psychol Rev. 1990; 97(3): 396–403. PubMed Abstract | Publisher Full Text\n\nMackintosh N: A theory of attention: Variations in the associability of stimuli with reinforcement. Psychol Rev. 1975; 82: 276–98. Publisher Full Text"
}
|
[
{
"id": "10747",
"date": "09 Oct 2015",
"name": "Wim Vanduffel",
"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 review aimed to provide mechanistic explanations for different variants of perceptual learning (PL). I enjoyed reading the manuscript and am inclined to follow the logic of the authors although a bit more data confirming the conjectures made by the authors would be desirable. The authors first review a number of papers from their laboratory that showed evidence for task-irrelevant perceptual learning. As opposed to more traditional forms of task-relevant PL, task-irrelevant PL occurs in the absence of attention and is supposed to be driven by external or internal rewards. Based on their data, the authors suggest the existence of two mechanisms giving rise to perceptual learning: an automatic reward-driven and a top-down goal-directed one. The studies listed in the review also suggest an interesting and common mechanism shared between conditioning and task-irrelevant perceptual learning. I only have a number of suggestions that the authors may take into account to improve the manuscript.The authors attempt to dichotomize mechanisms underlying task-relevant vs task irrelevant PL. Are these two parallel systems? Is it possible that only one mechanism exists which can be 'adjusted' based on task constraints? In other words, have they considered only one basic principle, whereby goal-directed PL is simply a variant of task-irrelevant PL? For example, one could argue that rewards are the driving force for perceptual learning dependent plasticity and that additional higher cognitive signals such as selective attention strengthen (or override) the reward-driven changes? Given the fact that the authors promote two different mechanisms, it would be interesting if they can speculate about the neuronal pathways involved. What should be done next? Are additional experimental demonstrations for task-irrelevant perceptual learning required? At this moment a rather limited set of stimulus parameters have been used in task-irrelevant PL paradigms. Does one need additional tests, for example by using other stimulus features, other sensory modalities? What about comparative experiments including other species? What about causal experiments trying to identify structures driving PL-dependent plasticity?",
"responses": []
},
{
"id": "10916",
"date": "26 Oct 2015",
"name": "Leonardo Chelazzi",
"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 have greatly enjoyed reading this engaging and inspiring review article. The authors of the article provide a concise and highly focused review of the recent literature on visual perceptual learning, and notably of the so-called task-irrelevant perceptual learning (TIPL). The main thrust of the article is to draw a distinction between an automatic form of TIPL, whereby the repeated occurrence of reward in close temporal contiguity with a visual stimulus (feature) determines perceptual learning of that stimulus (feature), and a more complex and cognitively demanding form of perceptual learning, whereby perceptual learning occurs as a result of the task-dependent contingency between a given stimulus (feature) and reward.I find this dichotomous view of perceptual learning, and of its modulation by reward, especially captivating and important, especially because we have recently elaborated a similar distinction between types of reward-dependent learning in the attentional domain that conform to classical conditioning and types that conform to operant conditioning (Chelazzi et al., 2013). Similar to what elaborated here, in prior studies (Della Libera et al., 2011) we could detect an impact of reward on attentional learning that conformed to classical conditioning using task variants in which participants were led to believe that rewards were received independently of their performance, in a lottery-like fashion. Conversely, in other work (Della Libera & Chelazzi, 2009) we could detect an impact of reward on attentional learning that conformed to operant conditioning using task variants in which participants were led to believe that rewards received in relation to specific stimuli depended on their attentional performance towards the same stimuli. I actually encourage the authors to note such remarkable consistency between notions that are being developed within the domain of perceptual learning and similar notions that are being developed within the domain of attentional learning. In this regard, it is important to eliminate any potential confusion. Perceptual learning refers to improvements in perceptual performance (increased sensitivity) in relation to specific stimuli (features) as a result of prolonged practice with/exposure to the stimuli (features). In contrast, attentional learning refers to an increase in the efficiency with which participants are able to select, or sometimes ignore, specific stimuli within multi-elements displays. It might well be the case that the two learning phenomena are intimately related, but such a possibility has never been formally tested, at least to the best of my knowledge.One aspect that the authors of the present article might want to elaborate on further is the way in which they conceive the task goal-dependency of the perceptual learning effects necessitating top-down control. In operant conditioning, the key link between a given stimulus and reward is the behavioral response directed towards the stimulus. Reward can only be obtained to the extent that an instrumental response is produced towards the stimulus. Any contingency between the stimulus and reward is not sufficient to elicit operant conditioning. In keeping with this notion, in our own work (Chelazzi et al., 2013) we have proposed that in the operant conditioning-like context, the cognitive system is shaped by reward in such a way that particular attentional operations (either selection or suppression, which, in different terms, can be named prioritization or deprioritization) are reinforced in relation to specific stimuli. In this perspective, what is being learned is a specific cognitive act in relation to a given stimulus and the learning is guided by the reward contingency.As a further development of the distinction put forward by the authors, I suggest that most likely the two forms of perceptual learning ought to differ in other important ways, including the resistance to extinction, the level of generalization across stimuli and tasks, and so forth.One other aspect that the authors might want to consider is the extent to which the task-dependency of what they describe as the second and more complex form of perceptual learning actually requires that top-down control is exerted in relation to the stimulus for which learning is measured or whether for this type of learning to occur it is sufficient that top-down control is exerted in relation to any stimulus, not necessarily the one for which learning is measured. In the latter scenario, top-down control is engaged, though it is not directed towards the stimulus for which learning is measured, performance monitoring processes are at work, and any resulting reward is linked to ongoing performance. I suppose that this level of top-down control may be sufficient for the second type of perceptual learning to materialize.Finally, Title and abstract are perfectly adequate.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-764
|
https://f1000research.com/articles/4-738/v1
|
09 Sep 15
|
{
"type": "Review",
"title": "Fatty acids from diet and microbiota regulate energy metabolism",
"authors": [
"Joe Alcock",
"Henry C. Lin",
"Henry C. Lin"
],
"abstract": "A high-fat diet and elevated levels of free fatty acids are known risk factors for metabolic syndrome, insulin resistance, and visceral obesity. Although these disease associations are well established, it is unclear how different dietary fats change the risk of insulin resistance and metabolic syndrome. Here, we review emerging evidence that insulin resistance and fat storage are linked to changes in the gut microbiota. The gut microbiota and intestinal barrier function, in turn, are highly influenced by the composition of fat in the diet. We review findings that certain fats (for example, long-chain saturated fatty acids) are associated with dysbiosis, impairment of intestinal barrier function, and metabolic endotoxemia. In contrast, other fatty acids, including short-chain and certain unsaturated fatty acids, protect against dysbiosis and impairment of barrier function caused by other dietary fats. These fats may promote insulin sensitivity by inhibiting metabolic endotoxemia and dysbiosis-driven inflammation. During dysbiosis, the modulation of metabolism by diet and microbiota may represent an adaptive process that compensates for the increased fuel demands of an activated immune system.",
"keywords": [
"microbiota",
"metabolism",
"diet"
],
"content": "Introduction\n\nIt has long been appreciated that the Western diet—high in simple carbohydrates, processed meat, and fat—is associated with adverse health outcomes, including obesity, metabolic syndrome, and type 2 diabetes1,2. In particular, consumption of saturated fatty acids and industrially produced trans fatty acids is linked with metabolic syndrome and obesity3. However, controversy continues to surround the relative importance of fat in the diet overall and which fats are healthier or more harmful4. Saturated fat in diet has received much attention for its ability to induce chronic low-grade inflammation, widely recognized as a key link to the pathologies of obesity, type 2 diabetes, and cardiovascular disease5. Dietary fat drives chronic low-grade inflammation by expanding white adipose tissue (WAT), promoting macrophage recruitment to WAT, and generating adipose inflammation (reviewed in 6). Increased release of fatty acids from expanded WAT results in decreased muscle cell surface expression of the glucose transport protein GLUT4, reducing insulin-stimulated glucose uptake and inhibiting glycogen synthesis7. Impaired glucose uptake by GLUT4 is a key feature of insulin resistance (IR), which is a precursor to the development of type 2 diabetes.\n\nImpairment of insulin action and inflammation from dietary fat have been described as resulting from the body’s limited capacity to store energy as fat8. In this view, dietary energy intake in excess of adipose storage capacity causes ectopic fat deposition in non-adipose tissues. Obesity and ectopic fat, in turn, are associated with muscle and liver accumulation of diacylglycerol (DAG)9 and ceramide, a sphingolipid derived from saturated fatty acids such as palmitate10. Toxic lipid molecules, generated through de novo synthesis from dietary fat, have pleiotropic effects on metabolism (reviewed in 11). DAG and ceramide have been shown to impair mitochondrial function, inhibit insulin signaling by acting on peroxisome proliferator-activated receptors (PPARs) and protein kinases, and cause inflammation via the nuclear transcription factor nuclear factor-kappa-B (NF-κB)9,10,12.\n\nDespite equal energy content, dietary fatty acids that differ in structure can have opposite effects on inflammation and IR. The divergent fatty acid effects on metabolism cast doubt on a simplistic view of IR as a problem of limited adipose storage capacity. For example, saturated fatty acids but not polyunsaturated fatty acids (PUFAs) caused IR in Sprague-Dawley rats, although both dietary fats resulted in increased plasma-free fatty acids13. Similarly, incubation with saturated fatty acids palmitate and stearate caused IR in human skeletal muscle, whereas unsaturated oleate had opposing effects on insulin action14. In two human trials, substituting dietary saturated fat with polyunsaturated fat or monounsaturated fat improved insulin sensitivity and reduced visceral adiposity15,16. Certain dietary fats reduce adipose inflammation and IR, even in the overfed state17,18.\n\nTo understand why fats often have opposing metabolic effects, we note that the gut microbiota, the collection of microorganisms that inhabit our bodies and outnumber human cells by an order of magnitude, is sensitive to dietary composition and is linked to changes in metabolism and obesity19,20. The composition of the diet and gut microbiota interact to modify the risk of many chronic inflammatory diseases, including obesity, diabetes, and inflammatory bowel disease21. The metabolic responses to various fats might be best understood in light of dietary fat’s ability to drive changes in the makeup and function of the intestinal microbiota.\n\n\nThe relationship of dietary fat, microbiome, and insulin resistance\n\nRecent studies have highlighted the central role of the gut microbiota in generating inflammation and regulating obesity and metabolism19. The microbiota consists of the collection of microbes living in and on our bodies, numbering as many as 100 trillion that reside mostly in the lower intestine22. Advances in sequencing technology and metagenomics have vastly increased the ability to identify intestinal microbes associated with obesity23 as well as mechanisms implicating microbiota in weight gain, such as increased energy harvest24. Compared with germ-free animals, conventionally raised mice have 60% more body fat even as the food intake was less25. This finding was explained by the suppression by gut microbes of the expression of a host intestinal protein known as fasting-induced adipocyte factor (FIAF). Because FIAF is an inhibitor of lipoprotein lipase (LPL), in the presence of gut microbes, less FIAF means reduced inhibition of LPL, resulting in more LPL, the enzyme responsible for importing and storing triglycerides. In a germ-free animal, greater FIAF increased the expression of genes responsible for fatty acid oxidation via stimulation of PPAR-γ coactivator and AMP-activated protein kinase26. Experiments that transferred microbes from obese and lean donors to germ-free mice support a causal role for microbiota in regulating fat mass and metabolism27. In a recent study, fecal microbiota from identical twins discordant for obesity were inoculated into germ-free mice, resulting in the transfer of the obese or lean phenotype of their donors28. Interestingly, when the resulting obese and lean mice were co-housed, the microbiota in lean mice appeared to have a selective advantage, transforming the microbiota of co-housed obese mice and causing weight loss. However, when obese mice were fed a high-fat diet, they could not be “rescued” by co-housing them with their lean counterparts28. In this example and others, an interaction of high-fat diet and specific microbiome appears necessary to cause systemic inflammation28,29.\n\n\nHigh-fat diet is linked to inflammation and insulin resistance\n\nA diet high in fat is sufficient to induce obesity and IR in many animal models4,30–33 and is associated with changes in gut microbiota and intestinal permeability. Two markers of inflammation, tumor necrosis factor-alpha and NF-κB activation, were induced in C57BL/6 mice fed a high-fat diet34. The essential role of the gut microbiota in this response was demonstrated by the absence of this effect in germ-free mice fed the same high-fat diet34. Because inflammatory markers increased before diet-induced obesity, inflammation that follows a high-fat diet may have a causal role in obesity34.\n\nHighlighting the potent effects of dietary fat, a single high-fat meal was sufficient to induce pro-inflammatory signaling and IR35,36. IR and inflammation following a high-fat meal resulted from increased intestinal permeability to endotoxin35,36. In addition, because lipid A, the insoluble fraction of the endotoxin lipopolysaccharide, could be carried into the lymphatic system by chylomicrons, a high-fat meal could promote postprandial entry of endotoxin into the circulation even in the absence of increased intestinal permeability. In male C57BL6/J mice, high-fat feeding resulted in weight gain and a two- to three-fold increase in circulating endotoxin, a condition termed metabolic endotoxemia4. Weight gain and IR were equivalent in the group fed a high-fat diet and mice receiving a subcutaneous infusion of endotoxin4. From these results, it was proposed that the Western diet, high in fat and low in fiber, causes a dysbiosis that results in the translocation of gut-derived bacterial endotoxin37. Supporting the role of gut microbiota in this process, IR and weight gain were blocked with antibiotic pre-treatment38. IR by this mechanism involves endotoxin detection by the Toll-like receptor TLR4 and downstream pro-inflammatory signaling39–41. Recently, Everard et al. showed that the metabolic effects of high-fat diet require MyD88 (myeloid differentiation primary response gene 88), a central adaptor molecule for many TLRs with a key role in regulating inflammation and metabolism41. Mice with the MyD88 deletion were protected against high fat-induced metabolic endotoxemia and had increased regulatory T cells, findings that were linked with decreased IR and inflammation41. Additionally, MyD88 deletion altered the composition of the gut microbiota; transfer of those microbes into germ-free mice protected the recipient mice from high fat-induced IR. These results suggest that bi-directional control involving microbiota and the MyD88 pathway regulates metabolism and inflammation.\n\n\nSaturated fats are linked to dysbiosis and metabolic endotoxemia\n\nIn this and following sections, we review how specific dietary fats alter the microbiome and change insulin sensitivity. Saturated fatty acids have been shown to have direct stimulatory effects on TLR expression42 and Jun N-terminal kinase (JNK) activity43 promoting IR via mechanisms independent of the gut microbiota. However, these direct effects may be less consequential than the influence of the gut microbiota on host metabolism, as underscored by the finding that germ-free animals are protected from high-fat diet-induced obesity and IR25,34. Saturated fatty acids have been shown to cause dysbiosis and intestinal inflammation in interleukin-10−/− mice by encouraging overgrowth of a bile-tolerant Gram-negative bacteria, Bilophila wadsworthia44. In another study of C57BL/6J mice, a diet high in saturated fat caused increased growth of three types of sulfidogenic bacteria, primarily in colonic mucosa45; these bacteria produce hydrogen sulfide gas as a metabolic by-product which can damage the intestinal barrier and cause endotoxemia. Feeding C57BL/6 mice a diet high in saturated fat decreased expression of tight junction proteins, causing increased intestinal permeability, endotoxemia46, and elevated lipopolysaccharide-binding protein47. In addition to higher fecal and plasma endotoxin levels, mice fed a diet high in saturated fat had fewer Bifidobacteria and increased Enterobacteriaceae in fecal culture46. Laugerette et al. showed an increased intestinal Escherichia coli population along with elevated plasma and adipose inflammation in animals fed saturated fat (palm oil) compared with unsaturated fats47. Taken together, these results support the hypothesis that certain diets high in saturated fatty acids may modify the structure and function of the gut microbiota, causing inflammation and IR in animal models (Figure 1).\n\nGut microbiota play a central role in the metabolic endotoxemia model of obesity and insulin resistance. Diets high in fat and low in fiber alter the function and composition of the gut microbiota. These changes can increase systemic lipopolysaccharide (LPS) exposure, thereby contributing to low-grade inflammation and impairing insulin-stimulated glucose uptake by muscle. JNK, Jun N-terminal kinase; MyD88, myeloid differentiation primary response gene 88; NF-κB, nuclear factor-kappa-B; SCFA, short-chain fatty acid; TLR, Toll-like receptor.\n\n\nOmega-6 polyunsaturated fatty acids can cause dysbiosis and inflammation\n\nGhosh et al.48,49 showed that C57BL/6 mice fed a diet rich in omega-6 (n-6) PUFAs (corn oil) resulted in bacterial overgrowth and dysbiosis. The high n-6 PUFA diet, alone among the high-fat diets studied, was also associated with bacterial invasion of the intestinal epithelial cell layer48. Corn oil supplementation caused decreased spontaneous locomotor activity, hyperinsulinemia, and IR in female C57BL/6 mice50. This animal study provides an interesting insight into the “couch potato” sedentary state in humans, suggesting that diet and microbiota can influence voluntary physical activity.\n\nDietary n-6 PUFAs were linked with changes in the composition of the gut microbiota in C57BL/6 mice49. These changes included increased abundance of Enterobacteriaceae and segmented filamentous bacteria, bacterial groups associated with inflammation49. N-6 PUFA feeding to C57BL/6 mice was shown to increase the numbers of intestinal Proteobacteria51 and change gut microbiota composition along with weight gain and fatty infiltration of the liver52. Huang et al. also reported an increase in intestinal Proteobacteria after n-6 PUFA feeding in C57BL/6 mice and greater macrophage infiltration of adipose than observed with saturated fat diets53. Excess dietary N-6 PUFAs caused higher adipose expression of resistin, a hormone linked with inflammation and IR, than was observed after consumption of saturated fat53.\n\n\nOmega-3 polyunsaturated fatty acids protect against dysbiosis and promote insulin sensitivity\n\nGhosh et al. demonstrated that altered gut microbiota caused by n-6 PUFAs in 2-year-old C57BL/6 mice was prevented when omega-3 (n-3) PUFAs (fish oil rich in DHA and EPA) were added to the diet48, suggesting that n-3 PUFAs can protect against dysbiosis. N-3 EPA and DHA reversed bacterial overgrowth and reduced fatty diet-induced inflammation by recruiting regulatory T cells to the small intestine48. However, Mujico et al. showed no similar protection from n-3 PUFA supplementation from dysbiosis caused by saturated fatty acids18. Another recent study showed that mice fed fish oil had decreased abundance of Helicobacter and Pseudomonas and Firmicutes, organisms associated with ulcers, infection, and weight gain, respectively54. One mechanism that may account for dietary n-3 PUFA’s reduction of Helicobacter and Pseudomonas is that those organisms are sensitive to the direct bactericidal effects of EPA and DHA55,56. Bacterial killing by n-3 PUFAs and other fatty acids is likely important to the overall composition of the microbiota and the function of the intestinal barrier57,58.\n\nDietary fish oil strengthened intestinal barrier function and reduced plasma endotoxin levels in swine17. Fish oil has also been linked with reduced TLR activation and MyD88 signaling in swine59. In addition to having beneficial effects on metabolic endotoxemia, n-3 PUFAs were shown to stimulate the G-protein coupled fatty acid receptor GPR120, promoting insulin sensitivity by increasing cell surface expression of GLUT460,61. N-3 PUFAs have additional anti-diabetic effects by activating GPR 40, causing increased insulin secretion from pancreatic β cells.\n\n\nMonounsaturated fatty acids antagonize dysbiosis and promote insulin sensitivity\n\nMujico et al. reported that oleic acid (a monounsaturated fatty acid) prevented high-fat diet dysbiosis in female ICR mice and increased the abundance of intestinal Bifidobacteria, a group associated with improved intestinal barrier function18. Oleic acid supplementation prevented weight gain and restored the proportion of microbial phyla altered by a high-fat diet18. Hidalgo et al. showed that butter produced changes in murine gut microbiota similar to those found in obese humans but that olive oil prevented those changes62. Interestingly, virgin olive oil had different effects on the microbiota compared with refined olive oil, suggesting that the non-lipid phenolic components of olive oil may account for some of its benefits62. Dietary supplementation with monounsaturated oleic acid in young adults improved insulin sensitivity, an effect not seen with saturated fat63. Downstream effects of microbiota may be responsible in part for improved insulin sensitivity and reduced type 2 diabetes observed with diets rich in olive oil and other monounsaturated fats64,65.\n\n\nShort-chain fatty acids and prebiotics promote insulin sensitivity via fatty acid receptors\n\nShort-chain fatty acids (SCFAs) are fully saturated but have fewer carbon atoms than long-chain saturated fatty acids, such as palmitate. SCFAs often have anti-inflammatory signaling properties (reviewed in 57). For instance, SCFAs such as butyrate tend to reduce inflammation by activating the SCFA receptor GPR4366. GPR43 activation increased energy expenditure and decreased adipose tissue insulin sensitivity while increasing insulin sensitivity in muscle and liver in C57BL/6 mice66. GPR43-deficient mice were obese on normal diet, whereas mice overexpressing GPR43 remained lean on a high-fat diet66. SCFAs are also a by-product of microbial fermentation of indigestible carbohydrates that are termed prebiotics when given therapeutically to alter the microbiota. Prebiotic treatment increased butyrate production in Wistar rats and was associated with increased Bacteroidetes, whereas high-fat diet reduced formation of butyrate and increased liver fat and inflammation39. Butyrate has anti-obesity effects by stimulating the expression of angiopoietin-like protein-4 (ANGPTL4) in human epithelial cells, leading to reduced expression of LPL and increased lipolysis67. Thus, microbes that preferentially generate butyrate through fermentation have a favorable effect on metabolism.\n\nPrebiotic treatment had additional metabolic benefits by increasing the abundance of Akkermansia muciniphila, a group of mucin-foraging bacteria that were depleted in obese and type 2 diabetic mice68. A. muciniphila separately prevented visceral adipose inflammation, increased anti-inflammatory regulatory T-cell numbers, and improved insulin sensitivity in C57BL/6 mice69. Improvement of glucose tolerance in db/db fiber-fed mice was recently shown to be transmissible with fecal transplantation, even when the recipient mice were never exposed to dietary fiber70. The insulin-sensitizing effects of dietary fiber in donor and recipient mice in that study were attributed to increased Lactobacillus and Bifidobacterium, decreased Alistipes, and changes in amino acid fermentation70. These findings underscore the importance of fiber-fermenting gut bacteria in regulating insulin sensitivity by the action of SCFAs and because of changes in gut microbiota function.\n\n\nDiscussion\n\nThe studies in this review showing a central role of the microbiota in regulating metabolism and immune function challenge traditional concepts of lipotoxicity as a primary cause of IR and metabolic syndrome8. Explanations of metabolic diseases that center on toxic lipid mediators from overfilled adipose depots are inadequate to explain the widely variable effects of equicaloric fats that have been reviewed here and elsewhere (for example, 71). A more unifying explanation is that the metabolic changes, inflammation, and changes in fat storage leading to obesity are outcomes of dietary fat acting on both the host and the gut microbiota as well as of diet-driven crosstalk between the host and the microbiota (Figure 2). Protection from obesity and IR in the germ-free state25 and with antibiotic treatment38 provides strong support for this view, which is a departure from the traditional understanding of how metabolic disorders are caused by dietary fat.\n\n(a) Dietary fats directly stimulate G protein-coupled receptor (GPR) fatty acid receptors and alter intestinal tight junction protein expression, also affecting the expression and activity of Toll-like receptors (TLRs) and the adaptor protein MyD88 (myeloid differentiation primary response gene 88), modulating nuclear transcription factor activity, and regulating inflammation and metabolism. (b) Dietary fats and prebiotics impact the composition and function of the gut microbiota and affect intestinal permeability. Significant cross-talk occurs between microbiota-derived signals, such as endotoxin and short-chain fatty acid (SCFA), and the pathways described in (a). Cues from both sources, diet and microbiota, are integrated to modulate inflammation and insulin sensitivity. PPAR, peroxisome proliferator-activated receptor.\n\nIt has been suggested that microbiota-induced changes in metabolism can be adaptive for the mammalian host (for instance, by diverting energy to fetal growth during pregnancy)72. The general concordance between insulin sensitivity/resistance and fat-driven changes in the microbiome described in this review (Table 1) suggests an alternative evolutionary explanation. Specifically, nutrients may serve a signaling function to the immune system in mammals by conveying information about diet-driven changes in the gut microbiota57. This hypothesis makes two predictions: (1) nutrients that lead to dysbiosis may generate pro-inflammatory signaling, and (2) nutrients that prevent dysbiosis may trigger anti-inflammatory signaling. A review of the effects of dietary fats on inflammation and gut microbes tended to be in line with these predictions57. As suggested by the present review, the metabolic effects of dietary fats can often be predicted by their effects on the microbiome, perhaps because metabolism and inflammation share similar regulatory pathways. We further propose that the modulation of metabolism by fats and microbiota may be adaptive in fueling the increased energy needs of immune cells activated by dysbiosis. By blocking glucose uptake, IR reduces energy utilization by tissues dependent on GLUT4 glucose uptake (predominantly skeletal muscle and fat) and diverts energy access to tissues not reliant on insulin-stimulated GLUT473–75. Phagocytes (for example, macrophages) and intestinal epithelial cells do not rely on GLUT4. As a result, glucose energy is expected to be preferentially delivered to activated innate immune cells in the gut during the IR state.\n\n*Chemical structure (number of carbon atoms:number of double bonds).\n\n**Described in animal models only.\n\nSaturated fats have been reported to cause dysbiosis and have been linked in animal studies with increased Enterobacteriaceae, increased Firmicutes/Bacteroidetes ratio, and decreased Bifidobacteria, among other changes. Dietary omega-6 (n-6) polyunsaturated fatty acid (PUFA) also is reported to cause an Enterobacteriaceae dysbiosis in mice. These microbiota alterations are associated with increased intestinal permeability, insulin resistance, and inflammation. Dysbiosis caused by saturated fat and n-6 PUFA was reversed with supplementation with monounsaturated oleic acid, omega-3 (n-3) PUFA, and short-chain fatty acid (SCFA) precursors (prebiotics). Protection from dysbiosis with oleic acid, n-3 PUFA, and prebiotic supplementation is accompanied by decreased inflammation and increased insulin sensitivity. These patterns are consistent with increased insulin-independent glucose uptake by the activated immune system during dysbiosis and opposite shifts in energy utilization when dysbiosis is absent.\n\n\nOngoing controversies\n\nDespite murine studies suggesting dysbiosis, inflammation, and metabolic disease from n-6 PUFAs, some human studies have shown no harm, and possible benefit, from consuming n-6 fats76. A recent longitudinal cohort study in Finland showed reduced risk of metabolic syndrome with increased n-6-to-n-3 PUFA ratio in serum77. Although molecular and animal studies imply a therapeutic benefit of n-3 PUFAs for metabolic syndrome and diabetes, observational studies of n-3 fat and type 2 diabetes have been mixed, indicating a possible reduction of type 2 diabetes risk with fish consumption in Asian populations78 but no benefit from fish consumption in a recent European case control study79. Elevated circulating n-3 fatty acids were recently linked to increased insulin sensitivity in overweight men80 and randomized trials have shown improved parameters related to metabolic syndrome with n-3 PUFA supplementation, including increased adiponectin and improved triglyceride levels in overweight women81,82. Self-reported diets high in n-3 alpha-linolenic acid, n-6 linoleic acid, and monounsaturated oleic acid have been associated with improved glucose metabolism76. Taken together, these findings indicate possible protection from metabolic syndrome from n-3 PUFAs and support the idea that unsaturated fats are more metabolically healthy than saturated fats. However, a recent study challenged the concept that saturated fats are harmful83. Unlike experiments in which milk fat caused dysbiosis and inflammation in mice44, experiments in humans given a diet high in dairy fat showed no increase in inflammation83. Moreover, improvement in insulin sensitivity and reduced adipose fat occurred more in human subjects assigned a diet low in carbohydrates rather than a diet low in saturated fat84. These results suggest that weight loss and improvements in metabolism can result from diets that prioritize reduction of carbohydrates rather than fats.\n\nOne explanation for the disparities between human and animal studies is that people are not mice85 and murine models may be poorly suited to understand human metabolism. Mammalian-microbiota co-evolutionary history is different for humans and other animals86, and a likely consequence is that foods modify the human microbiome differently and have distinct regulatory effects on immunity and metabolism. To date, we cannot define exactly what those species-level differences are. Without that information, it is too early to give a blanket recommendation for or against any class of dietary fat, especially when specific fatty acids in the same class vary in effects and depend also on an individual’s unique microbiota and genetic background. Molecular and human epidemiologic data strongly indicate that some fats are better for metabolic health than others. More human and comparative studies will be needed to determine whether metabolically healthy fats are those that maintain a healthy microbiota.\n\n\nAbbreviations\n\nDAG, diacylglycerol; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FIAF, fasting-induced adipose factor; GLUT-4, glucose transporter type 4; GPR, G protein-coupled receptor; IR, insulin resistance; LPL, lipoprotein lipase; MyD88, myeloid differentiation primary response gene 88; n-3, omega-3; n-6, omega-6; NF-κB, nuclear factor-kappa-B; PPAR, peroxisome proliferator-activated receptor; PUFA, polyunsaturated fatty acid; SCFA, short-chain fatty acid; TLR, Toll-like receptor; WAT, white adipose tissue.",
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Int J Antimicrob Agents. 2002; 20(4): 258–62. PubMed Abstract | Publisher Full Text\n\nShin SY, Bajpai VK, Kim HR, et al.: Antibacterial activity of bioconverted eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) against foodborne pathogenic bacteria. Int J Food Microbiol. 2007; 113(2): 233–6. PubMed Abstract | Publisher Full Text\n\nAlcock J, Franklin ML, Kuzawa CW: Nutrient signaling: evolutionary origins of the immune-modulating effects of dietary fat. Q Rev Biol. 2012; 87(3): 187–223. PubMed Abstract | Publisher Full Text\n\nDesbois AP, Smith VJ: Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol. 2010; 85(6): 1629–42. PubMed Abstract | Publisher Full Text\n\nLiu Y, Chen F, Odle J, et al.: Fish oil enhances intestinal integrity and inhibits TLR4 and NOD2 signaling pathways in weaned pigs after LPS challenge. J Nutr. 2012; 142(11): 2017–24. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nEsposito K, Maiorino MI, Ceriello A, et al.: Prevention and control of type 2 diabetes by Mediterranean diet: a systematic review. Diabetes Res Clin Pract. 2010; 89(2): 97–102. PubMed Abstract | Publisher Full Text\n\nDue A, Larsen TM, Hermansen K, et al.: Comparison of the effects on insulin resistance and glucose tolerance of 6-mo high-monounsaturated-fat, low-fat, and control diets. Am J Clin Nutr. 2008; 87(4): 855–62. PubMed Abstract | Faculty Opinions Recommendation\n\nKimura I, Ozawa K, Inoue D, et al.: The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43. Nat Commun. 2013; 4: 1829. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKorecka A, de Wouters T, Cultrone A, et al.: ANGPTL4 expression induced by butyrate and rosiglitazone in human intestinal epithelial cells utilizes independent pathways. Am J Physiol Gastrointest Liver Physiol. 2013; 304(11): G1025–37. PubMed Abstract | Publisher Full Text\n\nEverard A, Lazarevic V, Gaïa N, et al.: Microbiome of prebiotic-treated mice reveals novel targets involved in host response during obesity. ISME J. 2014; 8(10): 2116–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShin NR, Lee JC, Lee HY, et al.: An increase in the Akkermansia spp. population induced by metformin treatment improves glucose homeostasis in diet-induced obese mice. Gut. 2014; 63(5): 727–35. PubMed Abstract | Publisher Full Text\n\nHe B, Nohara K, Ajami NJ, et al.: Transmissible microbial and metabolomic remodeling by soluble dietary fiber improves metabolic homeostasis. Sci Rep. 2015; 5: 10604. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen W, Gaskins HR, McIntosh MK: Influence of dietary fat on intestinal microbes, inflammation, barrier function and metabolic outcomes. J Nutr Biochem. 2014; 25(3): 270–80. PubMed Abstract | Publisher Full Text\n\nKoren O, Goodrich JK, Cullender TC, et al.: Host remodeling of the gut microbiome and metabolic changes during pregnancy. Cell. 2012; 150(3): 470–80. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLochmiller RL, Deerenberg C: Trade-offs in evolutionary immunology: just what is the cost of immunity? Oikos. 2000; 88(1): 87–98. Publisher Full Text\n\nStraub RH, Cutolo M, Buttgereit F, et al.: Energy regulation and neuroendocrine-immune control in chronic inflammatory diseases. J Intern Med. 2010; 267(6): 543–60. PubMed Abstract | Publisher Full Text\n\nOdegaard JI, Chawla A: Pleiotropic actions of insulin resistance and inflammation in metabolic homeostasis. Science. 2013; 339(6116): 172–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKurotani K, Kochi T, Nanri A, et al.: Plant oils were associated with low prevalence of impaired glucose metabolism in Japanese workers. PloS One. 2013; 8(5): e64758. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVanhala M, Saltevo J, Soininen P, et al.: Serum omega-6 polyunsaturated fatty acids and the metabolic syndrome: a longitudinal population-based cohort study. Am J Epidemiol. 2012; 176(3): 253–60. PubMed Abstract | Publisher Full Text\n\nZheng JS, Huang T, Yang J, et al.: Marine N-3 polyunsaturated fatty acids are Inversely associated with risk of type 2 diabetes in Asians: a systematic review and meta-analysis. Atkin SL, editor. PLoS One. 2012; 7(9): e44525. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuijsse B, Boeing H, Drogan D, et al.: Consumption of fatty foods and incident type 2 diabetes in populations from eight European countries. Eur J Clin Nutr. 2015; 69(4): 455–61. PubMed Abstract | Publisher Full Text\n\nAlbert BB, Derraik JGB, Brennan CM, et al.: Higher omega-3 index is associated with increased insulin sensitivity and more favourable metabolic profile in middle-aged overweight men. Sci Rep. 2014; 4: 6697. PubMed Abstract | Publisher Full Text\n\nBrowning LM, Krebs JD, Moore CS, et al.: The impact of long chain n-3 polyunsaturated fatty acid supplementation on inflammation, insulin sensitivity and CVD risk in a group of overweight women with an inflammatory phenotype. Diabetes Obes Metab. 2007; 9(1): 70–80. PubMed Abstract | Publisher Full Text\n\nKrebs JD, Browning LM, McLean NK, et al.: Additive benefits of long-chain n-3 polyunsaturated fatty acids and weight-loss in the management of cardiovascular disease risk in overweight hyperinsulinaemic women. Int J Obes (Lond). 2006; 30(10): 1535–44. PubMed Abstract | Publisher Full Text\n\nLabonté MÈ, Cyr A, Abdullah MM, et al.: Dairy product consumption has no impact on biomarkers of inflammation among men and women with low-grade systemic inflammation. J Nutr. 2014; 144(11): 1760–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGower BA, Goss AM: A Lower-Carbohydrate, Higher-Fat Diet Reduces Abdominal and Intermuscular Fat and Increases Insulin Sensitivity in Adults at Risk of Type 2 Diabetes. J Nutr. 2015; 145(1): 177S–183S. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeok J, Warren HS, Cuenca AG, et al.: Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A. 2013; 110(9): 3507–12. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDethlefsen L, McFall-Ngai M, Relman DA: An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature. 2007; 449(7164): 811–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10241",
"date": "09 Sep 2015",
"name": "Rebecca Pedersen",
"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",
"responses": []
},
{
"id": "10242",
"date": "09 Sep 2015",
"name": "Timothy G. Dinan",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-738
|
https://f1000research.com/articles/4-691/v1
|
07 Sep 15
|
{
"type": "Opinion Article",
"title": "How to avoid pitfalls in antibody use",
"authors": [
"Diana Pauly",
"Katja Hanack"
],
"abstract": "Antibody use is ubiquitous in the biomedical sciences. However, determining best research practices has not been trivial. Many commercially available antibodies and antibody-conjugates are poorly characterized and lack proper validation. Uncritical application of such useless tools has contributed to the reproducibility crisis in biomedical research. Despite early initiatives such as MIAPAR or PSI-PAR, a best practice guideline for antibody characterization is still not in prospect. Here, we analyze 24 antibody-related databases and compare their content with regard to validation aspects and coverage. We also provide a flowchart for end-users with all necessary steps to facilitate finding and choosing specific and sensitive antibodies for their experiments. Based on a growing demand for better and standardized validation procedures and characterization guidelines for antibody molecules we have summarized our findings in a five-point plan. We intend to keep the discussion alive and hope that properly used antibodies will remain as central to biomedicine as they are today.",
"keywords": [
"antibodies",
"validation",
"characterization",
"target",
"application",
"databases",
"unique identifier"
],
"content": "\n\nAntibodies are ubiquitous tools in biomedical research to characterize and study proteins, protein-protein or protein-DNA interactions. Theoretically, they can be applied in almost every field but practically it is not as straightforward as one might naively expect. Although a lot of money has been invested and the global research antibody market is estimated to reach 2.64 billion Euros by 20191, many antibody-related products still face the problem that they are not properly validated and/or critical experimental data are not accessible. This leads - justifiably - to a growing unease among researchers, evidenced in several recent high-profile publications regarding the lack of standardization, validation and reference of those crucial research tools2–5. Antibody-related products are often expensive and fail to meet the customers’ expectations. We have now more than ten years of experience in generating monoclonal antibodies and we are well aware of the difficulties and obstacles on the way to highly effective antibodies6–11. On the one hand, we realize that end-users expect to obtain highly specific tools. On the other hand, the potential use-cases of antibody technology are too broad for a simplistic order-and-use scenario. Antibody-use and especially antibody generation are complex procedures that are liable to both false-positives as well as false-negatives, if not carried out appropriately. The following questions should be at the starting point of any such project:\n\n1. What is the target protein I want to characterize?\n\n2. What is the application in which the antibody should work?\n\n3. Which samples will be tested (serum, tissue, cells)? How is my target protein structured in these samples?\n\nWe have compiled a flow diagram for end users and manufacturers in Figure 1, which outlines the entire procedure. Already the step, finding the right antibody in multiple databases, is like looking for the proverbial needle in a haystack. Only by sheer luck or with an efficient strategy is it possible to find your antibody of choice in one of the over 24 antibody databases (Supplementary file 1). An inordinate amount of time has to be invested to extract the important characteristics of an antibody from a jumble of detailed and often unreferenced information in these databases. There are a few specialized sites which can help in some well-defined research areas, like the Antibody Validation Database, the ENCODE project or the San-Diego Epigenome Center, which focus on histone modifications or the Office of Cancer Clinical Proteomics Research Antibody Portal and the Abminer website, which concentrate on antibodies specific for cancer-associated proteins and tissues. For essentially all other antibodies, filtering information in databases with millions and millions of antibody products is a great challenge. The most useful databases are those which include independent antibody results either by citing published research reports or by including user reviews. Ten out of the 24 websites we investigated include antibody-related publications in their result screens and only 7 offer an easy to find platform for user reviews or comments (Supplementary file 1). Moreover, any of these two functionalities ideally ought to be combined with an option for comparison (available in 8 of 24 databases). There are additional unique features of individual databases which facilitate the search, including credits for user-reviews (e.g. 1DegreeBio, pAbmAbs, Biocompare), rating systems (e.g. Antibody-Adviser, 1DegreeBio, Biocompare, pAbmAbs, CiteAb, AntibodyReview), special initiatives for independent validation (e.g. Antibodies Online, Antibody Validation Database, Antibodypedia, The human protein atlas) and direct ordering from the database (e.g. Developmental Studies Hybridoma Bank, Antibodies Online). However, sometimes it seems easier to hire a detective than to order a specific antibody.\n\nIn many cases, we do not succeed in tracking down the right antibody required for our project (Figure 1). In these cases, we resort to project-specific antibody-generation based on monoclonal and polyclonal antibodies. In the planning stage, we place particular emphasis on the characterization of the target antigen and possible epitopes useful for immunization. This is necessary in order to find surface-related sequences available for antibody binding and to minimize possible cross reactivities of the antibody. Of course we agree with James Trimmer that “antibodies are not magic reagents”5, but properly designed, characterized, validated and used, some can come close.\n\nAn antibody can only bind the target used during immunization. The decision about the immunization strategy is all too often made without end-user input. Therefore, we would like to remind commercial producers of antibodies of their responsibility and support the growing demand for better validation and standardization tools12 (Figure 1). Therefore, we urge the community to revitalize the groundbreaking standardization ambitions of 2010 and revise the “Minimum information about a protein affinity reagent” (MIAPAR) and “Proteomics Standards Initiative-Protein affinity binders” (PSI-PAR) towards a simplified, common guideline for usage of affinity binders13,14.\n\nWe strongly disagree with the statements by Bradbury and Plückthun (2015) that polyclonal and hybridoma-generated monoclonal antibodies should be discarded from the biomedical research portfolio2. We also decline the exclusive value of recombinant antibodies. The disadvantages of polyclonal sera and monoclonal antibodies can be minimized by proper research practices (Table 1), such that they are far outweighed by the advantages. It is impossible to deny that sequencing antibodies is helpful in order to reliably produce them recombinantly. The main problem, however, is not the lack of sequence data but the absence of standardized assessments of antigen binding. In most common use cases, with proper research practices, sequencing antibodies becomes a matter of convenience rather than necessity.\n\nFurther we are convinced that there is an urgent need for proper identification of antibodies in order to avoid irreproducibility of research results and confusion of product similarities by rebranding of single antibodies. Sequencing of antigen-binding subunits is only one solution to add a unique, persistent identifier to each of these binders. Other initiatives, like the Encode accession number or Research Resource Identifier (RRID) will also help to identify existing antibodies in published reports15,16. In general, it should be the aim of the research community to prevent balkanization also of the persistent identifiers of antibodies and agree on a single identifier system with open standards. We are very interested in passing on our experience in antibody generation in order to create better standardization and validation workflows.\n\nAddressing all the identified problems in the antibody field, we suggest a 5-point plan:\n\n1. Combine all information about available antibodies in one comprehensive repository.\n\n2. Standardize antibody validation.\n\n3. Standardize antibody reference specifications in publications and add a unique identifier to each reagent.\n\n4. Sequence important and relevant antibodies for future reliable use.\n\n5. Generate specific, reliable and consistent binders for missing antigens using all techniques available.",
"appendix": "Author contributions\n\n\n\nDP conducted survey; DP, KH analyzed and interpreted data; DP, KH draft manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed for D. Pauly. K. Hanack is co-founder of new/era/mabs GmbH (Potsdam, Germany), who are generating antibodies. This activity does not interfere with her scientific interests and use of antibodies.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary materials\n\nSupplementary file 1: Antibody search websites.\n\nabbreviations: Ab antibody, WB Western Blots, ELISA enzyme-linked immunosorbent assay, IHC immunohistochemistry, FACS fluorescence-activated cell sorting, IF immunofluorescent, IP immunoprecipitation, DB dot blot, ChIP chromatin immunoprecipitation, siRNA small interfering RNA, CRISPR clustered regularly interspaced short palindromic repeats, shRNA small hairpin RNA, Immuno-MS immunoprecipitation with mass spectrometry analysis, SPR surface plasmon resonance spectroscopy, NAPPA nucleic acid programmable protein array, EM electron microscopy15,17–20.\n\nClick here to access the data.\n\n\nReferences\n\nPivotalscientificltd: The Research Antibody Market - 3rd Edition. [Internet], 2015. Reference Source\n\nBradbury A, Plückthun A: Reproducibility: Standardize antibodies used in research. Nature. 2015; 518(7537): 27–9. PubMed Abstract | Publisher Full Text\n\nVasilevsky NA, Brush MH, Paddock H, et al.: On the reproducibility of science: unique identification of research resources in the biomedical literature. PeerJ. 2013; 1: e148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReiss PD, Min D, Leung MY: Working towards a consensus for antibody validation [v1; ref status: not peer reviewed, http://f1000r.es/4o9]. F1000Res. 2014; 3: 266. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaker M: Reproducibility crisis: Blame it on the antibodies. Nature. 2015; 521(7552): 274–6. PubMed Abstract | Publisher Full Text\n\nPauly D, Kirchner S, Stoermann B, et al.: Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay. Analyst. 2009; 134(10): 2028–39. PubMed Abstract | Publisher Full Text\n\nPauly D, Chacana PA, Calzado EG, et al.: IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. J Vis Exp. 2011; (51): pii: 3084. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPauly D, Nagel BM, Reinders J, et al.: A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples. PLoS One. 2014; 9(5): e96371. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWand I, Holzlöhner P, Neupert S, et al.: Cooperation of dendritic cells with naïve lymphocyte populations to induce the generation of antigen-specific antibodies in vitro. J Biotechnol. 2011; 156(3): 173–81. PubMed Abstract | Publisher Full Text\n\nMesserschmidt K, Heilmann K: Toxin-antigen conjugates as selection tools for antibody producing cells. J Immunol Methods. 2013; 387(1–2): 167–72. PubMed Abstract | Publisher Full Text\n\nNeumann-Schaal M, Messerschmidt K, Grenz N, et al.: Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates. Immunol Lett. 2013; 151(1–2): 39–43. PubMed Abstract | Publisher Full Text\n\nBordeaux J, Welsh AW, Agarwal S, et al.: Antibody validation. Biotechniques. 2010; 48(3): 197–209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBourbeillon J, Orchard S, Benhar I, et al.: Minimum information about a protein affinity reagent (MIAPAR). Nat Biotechnol. 2010; 28(7): 650–3. PubMed Abstract | Publisher Full Text\n\nGloriam DE, Orchard S, Bertinetti D, et al.: A community standard format for the representation of protein affinity reagents. Mol Cell Proteomics. 2010; 9(1): 1–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandt SG, Marinov GK, Kundaje A, et al.: ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 2012; 22(9): 1813–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBandrowski A, Brush M, Grethe JS, et al.: The Resource Identification Initiative: A cultural shift in publishing [v1; ref status: indexed, http://f1000r.es/5fj]. F1000Res. 2015; 4: 134. Publisher Full Text\n\nMajor SM, Nishizuka S, Morita D, et al.: AbMiner: a bioinformatic resource on available monoclonal antibodies and corresponding gene identifiers for genomic, proteomic, and immunologic studies. BMC Bioinformatics. 2006; 7: 192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEgelhofer TA, Minoda A, Klugman S, et al.: An assessment of histone-modification antibody quality. Nat Struct Mol Biol. 2011; 18(1): 91–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBjörling E, Uhlén M: Antibodypedia, a portal for sharing antibody and antigen validation data. Mol Cell Proteomics. 2008; 7(10): 2028–37. PubMed Abstract | Publisher Full Text\n\nSoll DR: The Developmental Studies Hybridoma Bank, a national resource created by the National Institutes of Health. How does it work? Mater Methods. 2014; 4: 876. Reference Source"
}
|
[
{
"id": "11019",
"date": "23 Nov 2015",
"name": "Simon Glerup",
"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\nPaula and Hanack touch upon a very timely and important topic. Namely, the use of antibodies in research and how this affects scientific reproducibility. The authors intend to provide a guideline to how antibodies should be selected (or generated) before the onset of a scientific project.This is an opinion article and therefore reflects the personal view of the authors. Although, I do not fully agree with all of the statements in the paper, I still consider this an interesting contribution to the discussion of how we can achieve a transparent use of research antibodies.My main concern is their view on standardization of antibody validation. Antibodies may require very different protocol and buffer conditions in order to reach their optimal performance. One example is described by Ghatak et al. http://www.sciencedirect.com/science/article/pii/S2215016114000211. A standardized antibody validation system would completely miss such information. Instead, I consider a system for effective sharing of antibody performance details from scientists around the World. This would be much more effective for achieving a reproducible use of antibodies in research.",
"responses": []
},
{
"id": "11018",
"date": "20 Jan 2016",
"name": "Anita Bandrowski",
"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 report entitled \"How to avoid pitfalls in antibody use\" by Pauly and Hanack is a reasonable opinion piece with useful information and results from their survey. As such, it deserves indexing. I have already used the supplementary figure a couple of times as reference.However, in my opinion, the piece does require a thorough tightening up in writing style. The authors should improve the writing to make the piece more formal to align with standard journal practices, removing most uses of \"I\", \"we\" and \"my\", strewn throughout the article.For example:current text \"What is the target protein I want to characterize?\"may read better as \"What is the target protein to be characterized?\"current text \"How is my target protein structured in these samples?\"may read better as \"What is the configuration (concentrated protein, within complex tissue, membrane bound) of the target protein in the samples?\"current text \"Of course we agree with James Trimmer that “antibodies are not magic reagents”5, but properly designed, characterized, validated and used, some can come close.\"may read better as \"Indeed, James Trimmer the founder of NeuroMab states that “antibodies are not magic reagents”5, but properly designed, characterized, validated and used, some can come close.\" The informal style detracts from the message and should be updated to increase impact.For an example of masterful text, in this style please see:http://www.sciencedirect.com/science/article/pii/S2212968516300022",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-691
|
https://f1000research.com/articles/4-179/v1
|
01 Jul 15
|
{
"type": "Review",
"title": "Individuality, phenotypic differentiation, dormancy and ‘persistence’ in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology",
"authors": [
"Douglas Kell",
"Marnie Potgieter",
"Etheresia Pretorius",
"Marnie Potgieter"
],
"abstract": "For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically ‘nonculturable’ on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as ‘persisters’. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one’s bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit.",
"keywords": [
"Dormancy",
"persisters",
"sepsis",
"microbiome",
"inflammation",
"culturability",
"iron dysregulation"
],
"content": "Introduction\n\n“It is now well established that some micro-organisms can, under certain conditions, be deprived of all visible signs of life and yet these organisms are not dead, for, when their original conditions are restored, they can return to normal life and activity.”1\n\n“Bacterial populations in both batch and continuous culture are much more heterogeneous than is normally assumed, and such cultures may consist of several types of subpopulations simultaneously differing in viability, activity and integrity of the cells.”2\n\nConsider a typical axenic flask or broth culture of bacteria (Figure 1), arguably the staple of modern laboratory microbiology. We seed a suitable growth medium with an appropriate inoculum of cells known to be capable of replicating in that growth medium. After a lag phase the number of culturable cells (the ‘viable count’3,4, as judged by plate counts of the number of colony-forming units observable on the same medium solidified by agar or a similar material) is observed to increase, typically exponentially, for a number of generations (the growth phase or exponential phase). Apart from the changes in nutrient concentration, and for non-synchronised cultures, it is generally taken that cells pass smoothly through their cell cycles en route to doubling their numbers by binary fission. The population distribution of organisms in different parts of their cell cycle during the exponential phase is thereby unchanged and thus in a steady state (from which the cell cycle parameters can even be inferred5). In time this increase in cell numbers ceases, usually because of the exhaustion of a nutrient in a closed system, or sometimes in part or whole because of the build-up of toxins. Again, after a further period, the viable or colony count decreases (often to quite low levels if such starvation is carried out for extended periods). Inoculation of a new broth culture with a similar number of viable cells from this culture usually provides a simple repeat of the previous culture6, and in the absence of mutation may reasonably be anticipated, for organisms proliferating asexually, to be played out indefinitely.\n\nAfter the end of stationary phase the viable count decreases over time, but very rarely to precisely zero. Some authors recognise an extended “period of prolonged decrease”825 during which some of the survivors undergo significant dynamics, and in which mutants are selected. Our interest here is largely in cells that have not mutated.\n\nThe development of continuous7, nutrient-limited (‘chemostat’8) or feedback-controlled (‘turbidostat’9–11) cultures was and is entirely consistent with this view of steady-state microbial doubling via homogeneous cell cycles that are common, within statistical fluctuations, to each cell. The same is true for cultures undergoing serial transfer (where there is slightly more of a focus on selection for genotypic variants that grow faster – see e.g. 12–14).\n\nThere should be nothing controversial in the above passage, but in fact it hides a variety of assumptions that themselves conceal a considerable feast of very interesting physiology. The chief one here is that – given that all cells in the culture are genetically homogeneous and see the same ‘environment’, and modulo where they are in their cell cycles – all such cells are indeed supposed to represent a single population (as per Figure 2). If they do not, and as we shall see they never do15–18, we are dealing with differentiated systems. It turns out that a particular subset of typical cell cultures – a phenotypically dormant or non-growing sub-population, occurring even in non-sporulating bacteria2 – is widespread to the point of ubiquity. This leads to an exceptionally important biology with significant consequences both for our understanding of microorganisms and our ability to harness and domesticate them. Although the relevant literatures rarely cite each other or overlap, it is clear that similar phenomena are common to bacterial behaviour in the natural environment, the laboratory, and in a variety of samples of clinical interest. This theory or hypothesis that we develop here comes about from the synthesis19 of a large amount of data, and is summarised in Figure 3 and Figure 4.\n\nOne main population is shown. A second, smaller population is also shown; these might represent dormant cells.\n\n(1) A bacterial system contains distinct subpopulations, that we classify as culturable, dormant and ‘non-culturable’ (2). Specific attention is given to persister cells (3), and the inter-relationship (4) between the subpopulations. Subpopulations within environmental biology are discussed (5), followed by subpopulations within laboratory cultures (6). Particular emphasis is placed on phenotypic switching between the culturable and dormant subpopulation of laboratory cultures (7). Generalized detection techniques typically fail to detect dormant cells, and we review the various reasons for this failure and discuss alternatives (8). Resuscitation of and endotoxin production by such dormant cells underpins many diseases not normally seen as having a microbial component.\n\n\nPhenotypic differentiation to dormancy – some early indications\n\nWhile dormancy and resuscitation of rotifers had been observed by Leeuwenhoek himself in 17021, some of the earliest modern indications for a physiologically significant phenotypic differentiation of microbial cultures came in the 1940s. In a conceptually simple experiment (illustrated in Figure 5), Bigger20 exposed staphylococcal cultures to concentrations of penicillin that would normally be sufficient to kill them completely (and they did kill all but 1 in a million). However, these (10-6) survivors, that Bigger20 and McDermott21 (and many modern commentators have) referred to as ‘persisters’, were not genetic mutations selected for resistance to penicillin, since when they were inoculated into fresh broth they were just as susceptible as were those in the first culture. Bigger recognised (correctly) that the only explanation that made any kind of sense was that despite being exposed to nominally the same conditions, these cells were operationally dormant (even if metabolically active22,23) and thus phenotypically resistant to the penicillin (that anyway kills only dividing cells24,25). Similarly, Luria and Latarjet26 noted that approximately 1% of the cells in a culture of Escherichia coli displayed a phenotypic resistance to normally sterilising doses of ultraviolet irradiation. Many similar experiments since (e.g. 27–29), discussed in more detail below, have recapitulated this basic phenomenon. (We note here that high-frequency antigenic ‘phase’ variation can occur due e.g. to changes in microsatellite DNA30; detailed discussions of such genotypic changes31, including those that can affect the extent of dormancy in persistent bacteria32, are outwith the scope of the present, purely phenotypic analyses.)\n\nThis kind of protocol can be used to determine if the resistant subpopulation has accumulated genetic mutations that encoded resistance or whether, as focused on here, the resistance is purely phenotypic. A detailed analysis of the shape of the time-survivor curves may also be informative827.\n\n\nDormancy as an operational property\n\nFor the avoidance of doubt, and in accordance with Keilin’s description with which we opened, we shall define dormancy as:\n\n“a reversible state of {often} low metabolic activity, in which cells can persist for extended periods without division; we shall see that this often corresponds to a state in which cells are not 'alive' in the sense of being able to form a colony when plated on a suitable solid medium, but one in which they are not 'dead' in that when conditions are more favourable they can revert to a state of 'aliveness' as so defined”2.\n\nWe thus stress33 the recognition that dormancy is not solely an innate property of a bacterial cell; it is a property assessed by one or more experiments, so whether a cell appears to be dormant depends on both the cell and the experiment used to assess that dormancy. (This principle shares a similar philosophical foundation to the independence from any specific experiment, or otherwise, of the perceived state of objects within the quantum theory33–35.) As do Postgate3,4,36 and Barer37–41, we take the hallmark of a viable or living bacterial cell to be its ability to replicate or its ‘culturability’. This means that we cannot tell via culturability that a cell is alive, only (after a cell division) that it was alive33,42. Dormant cells – even if ‘not immediately culturable’ – must by definition be resuscitable to form culturable cells. Although the term ‘nonculturable’ is quite commonly used to describe not-immediately-culturable cells it is best avoided, as we cannot try every possible combination43 of incubation conditions that might serve to resuscitate a cell in a sample. ‘Non-cultured’, ‘as-yet-uncultured’ or ‘operationally nonculturable’ are better terms. Culturable, (operationally) non-culturable and (operationally) dormant bacteria in the differentiated bacterial (cellular) system can therefore be seen as distinct subpopulations of the system, and culturable and dormant bacteria as reversible states of the same population. The relationships between such subpopulations of the bacteria within a differentiated cellular system are shown in Figure 6.\n\nGiven our operational definition of dormancy as including reversible culturability, we note that different kinds of assays for the presence or activity of bacteria necessarily reflect cells in different kinds of physiological states (and can thereby be used to discriminate them). Thus direct counts with stains such as acridine orange (a list of these and other methods is given in Table 1 of 33) do not determine culturability, only presence or activity. Similarly, macromolecular sequencing methods such as those based on rDNA and its amplification (e.g. 44–49) almost certainly reflect mainly dormant cells plus any actively dividing ones (in that ‘naked’ DNA is usually degraded fairly rapidly in serum or the environment). The difference between culturable counts and total sequence-based counts probably provides one of the best methods for detecting and enumerating dormant cells when they cannot yet be brought back into culture. It is particularly noteworthy (and see also 50 and below) that the amount of prokaryotic DNA in whole blood exceeds by 10–100-fold that detectable in serum51, implying adsorption onto or sequestration within blood cells.\n\nWe shall return to clinical and laboratory microbiology later, but it is to environmental microbiology that we now turn to discuss the culturability of typical microbes. While the same general truths undoubtedly pertain in viruses (e.g. 52,53), and in yeasts, fungi, archaea, mycoplasmas and other unicellular organisms, our focus will be on prokaryotes.\n\n\nBacterial culturability and dormancy in environmental microbiology\n\nIt has long been known that the number of bacteria observable microscopically exceeds, typically 100-fold, those that can readily be grown axenically in standard isolation media (i.e. to proliferate in liquid culture or to form colonies on solid media). The latter has been referred to as ‘the great plate count anomaly’54, and has been amply confirmed by more modern, culture-independent sequencing methods. A selection of papers and reviews serve to document both the numerical anomaly and the much greater biodiversity detectable by sequencing (e.g. 55–73). It is thus useful to discriminate (1) bacteria that have been cultured, that are typically available in culture collections, and whose growth requirements are known, from (2) bacteria that may be recognised as novel via macromolecular sequencing (typically of ribosomal DNA68,74–77) but that have not yet been cultured and whose growth requirements may not yet even be known. Much (sequencing) evidence indicates that the bulk of the ‘missing microbes’ or ‘dark matter’78,79 in natural ecosystems falls into this second category80, and that ‘single cell’ methods may be required to culture them81.\n\nThere are at least four general reasons of principle why these organisms have not yet been cultured. We consider each in turn (although more than one may contribute in individual cases).\n\nIt is an elementary observation in microbiology, and the basis for selective isolation media, that not all bacteria grow on all media and in all conditions. Leaving aside truly syntrophic bacteria (that for thermodynamic or unknown nutritional reasons require another organism for growth (e.g. 82–88)), some organisms may have quite fastidious growth requirements. A number of bacteria determined as causative of disease, whose role had originally been inferred only through microscopic observation, were later cultured and could be shown to fulfil Koch’s postulates. These include Helicobacter pylori89,90 (with an unusually high requirement for urea to fuel its alkalinogenic urease activity91) and Legionella pneumophila92–95 (with an unusually high requirement for cysteine). Note that even the supposedly rich LB medium96 (Lysogeny Broth, often erroneously called Luria-Bertani medium, see http://schaechter.asmblog.org/schaechter/2009/11/the-limitations-of-lb-medium.html) is not in fact a particularly rich medium97–99. An especially nice example100,101 is provided by Tropheryma whipplei, the causative organism of Whipple’s disease102,103. It resisted attempts (over many decades) to bring it into axenic culture until systematic genome sequencing104,105 showed its requirements for a variety of common amino acids that it was unable to synthesise itself, the provision of which permitted its growth. The MetaGrowth database106 is now available for similar purposes. Another good example is Coxiella burnetii, the causative agent of Q fever, for which a genome-derived growth medium (‘acidified citrate cysteine medium’) permitting axenic culture has now been developed107,108. Other examples are given by Stewart109 and by Singh and colleagues100, and include marine bacteria of the highly common SAR11 clade71,110,111. Of course these kinds of phenomena are not absolute; much evidence indicates that host stress hormones may act as growth or virulence factors for a variety of Gram-negative organisms, representing a kind of ‘microbial endocrinology’ (e.g. 112–114).\n\nOrganisms in nature are often living in low-nutrient conditions115–119. It is thus reasonable (and unsurprising) that the isolation of microbes from starved, oligotrophic environments benefits from the use of low-nutrient conditions63,109,120–122; some manifest this ‘starvation’ through their size, as ‘ultramicrobacteria’ (see e.g. 123–129). In a similar vein, taking cells from low-nutrient natural environments directly onto, say, a highly aerobic agar plate may produce stresses that effectively kill them, so that afterwards they would not even grow on the kinds of media (as in the previous section) that would support their growth. Thus, Tanaka and colleagues130 showed interactions between phosphate and agar when autoclaved together that led to the production of compounds inimical to bacterial growth. Gellan may be a better solidifying agent here82. However, we recognise that it may be hard to discriminate cells that we kill in the act of trying to isolate and grow them from ‘already dead’ bacteria.\n\nWhile this possibility certainly exists, and is included for completeness, it is actually the least likely for a number of conceptual and empirical reasons. The first is that if an organism is present in a particular environment it must have been able to grow and divide in it at some point in the more or less recent past, even if the result of such growth was its utilisation of a finite amount of necessary nutrients or growth factors whose exhaustion caused replication to cease. (Interestingly, in soil it seems that sequestration, rather than complete exhaustion, of nutrients is the more significant phenomenon131–133.) Secondly, it is highly unlikely that evolution could select for unicellular organisms that cannot replicate. Thirdly, environmental organisms can be shown to metabolise even when they cannot be shown to divide (e.g. in the ‘Direct Viable Count’ method134 and in any number of other tests that detect metabolic activity33,135). And finally, as we shall see in the next section, careful methods of resuscitation/cultivation do indeed allow a very significant fraction of organisms that can be isolated from a variety of environments (e.g. the gut136–139) to be resuscitated and to grow very effectively.\n\nAs indicated in the introduction, it is now well established that even laboratory cultures, that from a macroscopic point of view are growing exponentially, contain subpopulations of non-growing cells. These cells are dormant by definition, because they may later be resuscitated and grow. It is easy to ascribe an evolutionary advantage of this culture differentiation from the perspective of the benefits of having a sub-population that by not growing is more resistant to environmental stresses (e.g. 140–142). Indeed, this general kind of phenotypic differentiation strategy, in which the variance in reproductive rate is traded off at the expense of the mean, has been referred to as bet hedging66,142–152 and is actually adaptive153,154. An important point here153 is that in many natural environments, asexually reproducing organisms such as bacteria are likely to be (spatially) close to their ancestors and descendants, such that inclusive fitness theory155,156 implies that it is entirely reasonable for them to behave altruistically, e.g. by ‘bet hedging’. This is also discussed further below.\n\nIt is also reasonable that in isolated (closed) natural environments, nutrients and thus sources of energy must be exhausted at some point, and thus for simple energetic reasons multiplication becomes impossible and a dormant state likely (if later resuscitation proves it to be so). Similarly, it is likely that in the absence of energy, nutrients and/or signalling molecules, and based on more ecological or community considerations (e.g. 157–159), it is necessary to add any or each of them to ‘prime’ bacteria to resuscitate. This has indeed been shown58,159–163, including for sources of energy164,165, iron-acquiring compounds166 (siderophores167–169), cell wall muropeptides170, and various signalling molecules171,172 (especially pheromones153,154,173,174) that exist in natural environments58,159,175. We note too that ‘kick starting’ dormant cells may require the synthesis of transporters necessary for the uptake of all kinds of molecules176–179. Overall, the idea that most bacteria that may be observed in the natural environment are ‘unculturable’ is incorrect.\n\nFinally here, and though this is obvious it is well worth rehearsing, the simple fact that we can store non-growing microbes under desiccated or frozen conditions or as agar ‘stabs’ in culture collections for extended periods means that most microbes are certainly well adapted to entering and leaving dormancy.\n\n\nPheromonal proteins\n\nA related and unexpected discovery came from analyses of starved laboratory cultures of the actinobacterium Micrococcus luteus, in which almost all cells lost culturability2,180–182. However, they were not dead but dormant, as they could be resuscitated by using a combination of weak nutrient media and a signalling molecule found in spent culture supernatants183–188. The original studies used flow cytometry to discriminate the physiological state of individual cells189–193 (see also 194,195). By using another ‘single cell’ assay based on dilution to extinction (that avoids artefacts connected with the regrowth of ‘initially viable’ bacteria33), we were able to purify the signalling molecule. It turned out to be a protein, named Rpf (for ‘resuscitation-promoting factor’)196. In M. luteus there is only one homologue197, and the gene (product) is essential for both resuscitation and multiplication196,198. Rpf contains a highly conserved 70 amino acid ‘Rpf domain’ and is widely (and probably ubiquitously) distributed throughout the actinobacteria199–202, but with examples elsewhere203,204. Most organisms that have a homologue have more than one. Thus M. tuberculosis has five homologues205–207. Rpfs can have peptidoglycanase and muralytic activity208–213 and known crystal structures are consistent with this214–219. These activities can certainly account for at least some220 of the resuscitation-promoting properties. As an extracellular protein that may be required for growth, and with a high level of immunogenicity221, it is obviously an excellent candidate target for inclusion in appropriate vaccines against pathogenic actinobacteria196,208,222–229. It is also more directly of potential utility in stimulating bacterial communication and resuscitation in a variety of cultures in both samples taken from Nature230–240 and in the laboratory241–254.\n\n\nCulturability, dormancy and persistence in laboratory cultures of non-fastidious bacteria\n\nHaving established the frequency of occurrence of microbial dormancy in the natural environment, it is of interest to understand better the mechanisms by which microbes might effect this dormancy and potential resuscitation. Unsurprisingly, microbiologists have turned to E. coli, and considerable progress has been made23,255–262.\n\nThe starting position is as in Figure 1 and Figure 6, to the effect that at any given moment in a typical culture a small fraction of the population is dormant. Since clearly the same fraction cannot (or is wise not to) remain in dormancy indefinitely in the presence of suitable nutrients that permit the growth of its siblings, we must invoke at least one mechanism that can cause the bacteria to ‘oscillate’ between growing and dormant states. Many simple gene expression network topologies admit this behaviour145,263–267, including a simple feedback loop with delay268,269, and we note that even whole cultures can exhibit oscillations and deterministic chaos270. While flow cytometric observations (e.g. 192,271) show that even ‘homogeneous’ laboratory cultures show highly heterogeneous distributions in cellular volume (not just between X and 2X) and expression profiles (and see 272), our particular focus will be on ‘binary’ or ‘bistable’ systems in which individual cells either are or are not operationally culturable.\n\nExperimentally, it is also common to assess the phenotypic ability of subpopulations of cells to tolerate normally inhibitory concentrations of bactericidal drugs273,274, this being a marker for that fraction of cells that is dormant at the stage in question. Note that the persistence phenotype is not induced by the drugs258. Changes or transitions in the state of a particular cell in a population between the various phenotypic states is a phenomenon that may be (and is commonly) referred to as ‘phenotypic switching’.\n\nA particularly well-developed example of this ‘bet hedging’ or phenotypic switching between physiologically dormant and growing states may be observed in laboratory cultures of organisms such as E. coli demonstrating ‘persistence’147,150,151,275–281. In general, any scheme in which both a first gene product inhibits cellular proliferation and in which this first gene product may be titrated out potently282 by a second gene product that thereby undoes the inhibition of proliferation, can have the effect of phenotypically switching cells between growth and dormancy. This seems to be precisely what is going on, and such pairs of gene products have been referred to (somewhat misleadingly283) as toxin-antitoxin (TA) pairs283–290. One such involves the well-known pp(p)Gpp metabolic system that can serve to inhibit DNA gyrase23,291–294, and points to the fact that in these circumstances, persisters may be quite metabolically active22,23,292,295, even if transiently incapable of reproduction. Another phenotype switching mechanism, underlying colony phenotype switching, comes from metabolic bifurcations driven by the levels of a particular metabolite296.\n\nAny mechanisms that permit cells to communicate with each other can amplify switching effects by cell synchronisation, and by definition such ‘social’ signals act as pheromones, whose apparent ‘altruism’ can be explained on the basis of kin selection theory153. There is considerable interest, largely outwith our scope here, in these evolutionary aspects (e.g. 297–304). Such systems are commonly, but far too broadly relative to the term’s origin305, referred to as ‘quorum-sensing’. However, they do offer opportunities for limiting bacterial virulence (e.g. 306–313).\n\n\nClassical clinical microbiology of culturable organisms\n\nUntil relatively recently, almost all of clinical microbiology314,315 was based on rather classical methods of plate counting316, coupled to assessment of antibiotic sensitivity. Various means of automated blood culture that assess metabolism exist (although they require typically 48–72h to show a ‘positive’)317. Positive tests, often implicitly involving culture (and not just metabolism) within the assay, would be followed by other tests seeking to identify the organisms detected, nowadays typically by nucleic acid sequence-based methods49,318–321. However, these and other tests for the presence of antigens or even antibodies322 cannot speak to the question of culturability (and of course antigens such as lipopolysaccharide (LPS) are shed by dying cells).\n\nThe existence of bacterial DNA in even ‘healthy’ blood has long been known323, and since naked DNA would be degraded and living cells would soon kill the host, the (seemingly) obvious conclusion that the prokaryotic DNA must reflect dormant cells seems neither to have been drawn nor acted upon.\n\n\nSome well-established cases of dormancy in clinical microbiology\n\nThe idea that (typically intracellular) dormancy is a major component in some infectious diseases (including in the absence of antibiotics that may serve to light up ‘persisters’) is of course well-established, and the main purpose of this brief section is simply to remind readers of this. Such a reminder serves as a prelude to a longer discussion of the very many clinical circumstances where we consider that the role of dormant microbes is not widely appreciated, and where they are not really considered to involve a communicable or microbial component at all. Thus Table 1 shows a few organisms (and references) for which we consider that most readers would regard the idea of and evidence for dormancy as more or less uncontroversial. We do not include disease-causing infectious agents where they are better known for their ability to persist in the natural environment. Organisms such as Legionella pneumophila that represent significant public health issues, fall into this category, and Legionella and other persisters (in environments such as water system biofilms) are indeed well known (e.g. 324–328), although they too have special adaptations to an intracellular lifestyle (e.g. 329).\n\nExamples are given for both low- and high-GC Gram positives, as well as a number of Gram-negative organisms.\n\n\nGeneralised failure of classical techniques to detect dormant bacteria in clinical microbiology\n\nAs noted above for environmental microbiology, dormant bacteria can represent as much as 99% of the organisms that may be observed microscopically or by macromolecular sequencing, but classically (and by definition) they are not enumerated by culture-based methods that determine ‘immediate culturability’33. Such culture-based methods are also widely used in clinical microbiology. However, if we were to plate out 100 μL of a culture containing 200 bacteria.mL-1, of which 99% were dormant at any instant, we would expect (based on a Poisson distribution) to see fewer than 1 propagule or colony-forming unit per sample. We have noted above that it can be determined by sequencing that many of the non-cultured environmental organisms largely differ from those in standard culture collections. Certainly the examples given above in clinical microbiology, such as Tropheryma whipplei, were both observed microscopically and were sequenced prior to being brought into axenic culture.\n\nThe PCR method is exquisitely sensitive (down to one cell or propagule per sample), and we note that contamination artefacts from the PCR reagents represent a real issue that must always be checked (e.g. 357–361), albeit this is no less true of blood cultures362. We have rehearsed elsewhere50 five classes of argument that collectively make it implausible that these are all contamination artefacts; probably the most persuasive is simply the sheer number of prokaryotic DNA molecules that can be measured in blood and serum (e.g. 363–365). While some of the most recent nucleic acid sequencing methods (e.g. 366–371) do operate on single molecules, the analysis of prokaryotes usually used a broad-range PCR step to amplify small-subunit rDNA to assess their presence, whether in environmental62,68,75,372 or clinical370,373–385 samples. Using this, and while these methods alone cannot tell whether they were operationally dormant or dead, a very considerable number of studies have been performed in which ‘culture-negative’ clinical samples showed the presence of prokaryotes (at least as judged by sequence-based methods). This has some profound consequences.\n\n\nBroad-range PCR methods indicate the widespread presence of prokaryotic DNA in culture-negative clinical samples\n\nWhile PCR-based methods have long been used to assess the species involved in culture-positive samples386, e.g. from blood, our interest here is in samples that are culture-negative387 that may yet (and indeed likely do) contain dormant cells. Among the first such indications of this was the study by Relman’s group323, who showed that the blood of even healthy controls contained significant amounts of prokaryotic DNA. Table 2 lists some studies in which broad-range PCR has been used to amplify and detect prokaryotic rDNA in culture-negative samples.\n\nNote that we have sought to exclude examples where anaerobic bacteria could be detected by PCR but not cultured simply because cultures were not anaerobic, and also cases (e.g. 388,389) where high antibiotic concentrations might have prevented culture.\n\nIn environmental microbiology, as mentioned above, there were many early indications (as observed microscopically or flow cytometrically) for the presence of bacteria that did not (or not easily) prove resuscitable or culturable. In a similar vein, many studies have shown microscopically observable organisms in culture-negative but disease-positive samples. This is true both for diseases considered to be due to microbial pathogens and, in fact, for many others normally considered non-communicable50.\n\n\nMicroscopically observable and potentially dormant bacteria in clinical disease\n\nMicroscopic observations in tissues have been a major part of the discovery process by which certain bacteria were indeed identified as the cause of various diseases. Billings411, Price412, Domingue393,413–415, Mattman416, Ewald417 and Onwuamaegbu and colleagues418 review the extensive and largely forgotten early literature. Domingue and Schlegel419 also mentioned that they could recover culturable bacteria, probably mainly from L forms (see 50,416,420), from lysates of normal and diseased blood. It was to be assumed that these cells were not replicating at significant rates in the blood itself. However, we can find no evidence that this was ever followed up. Our own work421,422, summarised in 50, showed that both bacillary and coccoid cells could be found attached to and within the erythrocytes of patients with Parkinson’s disease and Alzheimer’s disease, at rather greater concentrations than in samples taken from nominally healthy controls.\n\nIn a similar way, our preliminary data show that bacteria are visible in plasma, as well as in whole blood smears in various inflammatory conditions. Here we show bacteria in platelet-rich plasma (PRP) taken from a patient with systemic lupus erythematosus and smeared onto a glass cover slip (Figure 7A and Figure 7B). We also show the same from patients with hereditary hemochromatosis (Figure 7C) and type 2 diabetes (Figure 7D). We also noted microbiota associated with erythrocytes in thromboembolic ischemic stroke (Figure 8A and Figure 8B). (Our microscopy methods are as published previously (e.g. 422–431), but fuller publications will appear elsewhere.) The ultramicroscopic evidence that these are indeed small bacteria and not say, cellular debris or microparticles (see 432) is presently mainly morphological, though we note the considerable evidence for the presence of bacterial DNA in blood (see previous sections and e.g. 51,323,433).\n\nA and B) Platelet rich plasma (PRP) from a patient with systemic lupus erythematosus (SLE). A) Platelet with bacteria visible in the surrounding smear (pink arrows); B) areas in smear with bacteria (pink arrows); C) Erythrocyte with associated bacteria from patient with confirmed hereditary hemochromatosis; D) Erythrocytes with bacteria from patients with diagnosed type II diabetes. A–C Scale bar: 1 μm and D 400 nm.\n\nBacteria in whole blood from a patient with thromboembolic ischemic stroke A) Microbiota in whole blood; scale bar: 200 nm. B) Erythrocyte with bacteria; scale bar: 1 μm.\n\nIt is worth rehearsing the very great significance of this. With erythrocytes being present at some 5×109.mL-1 in human blood, even if only one erythrocyte in a thousand harboured just a single dormant bacterium (that would be hard to detect microscopically, but see 433–437), the dormant bacterial load would still be 5,106.mL-1. This is both far from negligible, and serves to exclude the (always potentially worrisome) claim that ‘it is all contaminants’.\n\n\nA culturable blood microbiome\n\nA recent and highly significant paper by Damgaard and colleagues438 bears discussion. These workers note438 that while bacterial growth can normally be elicited during sterility testing in vitro from fewer than 1 in a 1000 blood units439–441, transfusion-transmitted infections occur with a very much higher frequency (more like 10–12%442,443, or even more444), and are responsible for a high fraction of transfusion-associated deaths445–447. Although it was acknowledged that venepuncture-associated contamination or an effect of transfusion in suppressing the immune system might contribute, it was also recognised438 that one means by which to account for this would be that ‘normal blood’, and in particular its erythrocyte components, might also contain infectious agents that might be able to grow post-transfusion. Indeed, these authors found438 that under anaerobic conditions a small number of colony-forming units (ca 4–5.mL-1) could be recovered by direct plating from fully 62% of blood units, with ‘controls’ producing an average of just 1 cfu.mL-1. More of the bacteria were associated with red blood cells than with plasma, and rDNA was used to identify them. These data are entirely consistent with the idea that dormant bacteria are present in the blood of even ‘normal’ individuals (note that periodontitis was not a criterion for donor exclusion here438), that they are probably lurking in or on erythrocytes448,449, and that they can be resuscitated and grow under the correct conditions.\n\n\nEvidence for a microbial component in a very large variety of ‘non-communicable’ diseases\n\nWe have surveyed the literature for evidence in which a microbial component has indeed been observed to be an accompaniment of, and probably a major contributory factor to, a variety of (typically inflammatory) diseases that are normally considered ‘non-communicable’. Rarely has the physiological state of these microbes been considered, but since it would be obvious if they were growing, it is most likely that they are indeed dormant. Table 3 summarises these highly extensive associations. While some are just associations, and we could have extended this table considerably, some studies (e.g. 450) contain very detailed aetiological arguments that leave little room for doubt. Overall, the sheer size of the Table does strongly indicate the commonality of many of the microbially based mechanisms underpinning or accompanying various autoimmune and inflammatory diseases. In conditions such as atherosclerosis, transient ischemic attacks (TIAs), and stroke, it is very easy to conceive how resuscitating bacteria might serve to block the flow of blood, for instance. At all events, our main point here is that the evidence for a microbial contribution to many diseases supposedly lacking a microbial component is both multi-factorial and very considerable. Indeed, the purpose of a synthetic review such as this is to provide such pointers for more detailed studies in individual cases. Our specific interest is with the chief mechanisms by which these supposedly dormant bacteria might resuscitate and act as triggers of disease.\n\nWe purposely largely confine ourselves to bacteria here, but include the occasional parasite, fungus, mycoplasma and virus. While obesity is usually seen as a cause of other diseases, rather than a disease itself, we note the influence of endotoxaemia on obesity451–456. We note too the extensive evidence for the role of LPS in inflammation457–459, and the experimental models (e.g. for Parkinson’s460) where it can induce disease directly. We do not much discuss diseases such as Crohn’s disease where the extensive uncertainty over the extent of involvement of mycobacteria (e.g. 461–463) needs no extra rehearsal (albeit it serves to illustrate the difficulties of identifying the role of hard-to-cultivate bacteria in chronic diseases). Further, while similar phenomena may be observed in a variety of cancers (e.g. 464–469), for reasons of space we have determined that this must be the subject of a separate work.\n\n\nRelation between iron dysregulation, sepsis and other comorbidities\n\nMany of the diseases in Table 3 are precisely those inflammatory diseases that we have listed before as coupled to iron dysregulation167,168,429,432,713. A consequence of our analysis is that iron dysregulation and sepsis (as judged either by genuine infection by culturable bacteria or their inflammatory products such as LPS) should be associated causally with these various other diseases.\n\nThis leads to a variety of predictions and postdictions that we rehearse. A purposely simple (and simplistic) indication of a plausible chain of events (for which each step is underpinned by substantial evidence) is given in Figure 9, both in general terms (for unspecified diseases) and for a couple of steps to type 2 diabetes. Figure 9 aims specifically to highlight the relationship between the ability of available iron to stimulate bacterial growth and the potential disease sequelae thereof.\n\nWhile it is recognised that this simple diagram is very far from capturing the richness of these phenomena, there is abundant evidence for each of these steps, but sample references for the numbered interactions are (1)828–831 (especially including the release of free iron from ferritin432), (2)832–834, (3)268,453,455,835–842, (4)456,713,843–846, (5)167,168,432 (6)847,848, (7)849–855 (8)856 (9)857–859 (10)860,861.\n\nFirst of all, it is well established that free iron may be raised in sepsis and related conditions714–722, as may serum ferritin723–727 (that has mainly dumped its iron432). We have here argued that this is likely to be a significant contributor to the relationship between overt or cryptic infection and the many iron-related inflammatory diseases discussed here and elsewhere167,168,432,713. Note that patients suffering from iron overload diseases such as hereditary haemochromatosis are especially susceptible to infection (see e.g. 728–730 and Table 3). Certainly the idea that iron-related metabolism and siderophores are virulence factors (e.g. 731–743) is established unequivocally. In many diseases (e.g. lupus744,745 or type 1 diabetes746) it is considered that patients with the disease are more prone to sepsis, but we suggest here that (as with stroke561,565,566,568–570,747–755) it may more likely be the converse that is truer: patients suffering from latent infections are in fact more prone to acquiring, having, or exacerbating the state of these other conditions, in a vicious cycle (see Figure 9).\n\nThis was discussed at considerable length previously168, and that discussion is not repeated here (though a few more recent and pertinent references include756–759). However, while (shockingly, given the evidence) it does not even appear in the guidelines760, there is considerable evidence168 that iron chelation slows, inhibits or overcomes sepsis. On this basis, iron chelation may be a suitable alternative to antibiotics in preventing multiple inflammatory diseases (and such chelation may be nutritional rather than pharmacological in nature, e.g. 167). However, it is clear that we also need to learn to kill ‘dormant’ bacteria, and this usually requires that they are growing.\n\nIt is well established that the re-use of protein motifs in natural (and directed761) evolution means that most drugs, especially the more lipophilic ones, are promiscuous in the sense that they bind to multiple targets177,762 (on average six known ones for marketed drugs763). This said (and while we are very far from wishing to encourage the unnecessary use of antibiotics), the prediction here is that appropriate antibiotics will prove to have clinical benefit in diseases commonly seen as non-communicable. This is certainly known to be the case for a number of autoimmune diseases764 such as rheumatoid arthritis765–770, multiple sclerosis771–777 and psoriasis778–780. Vaccination may prove equally effective781,782.\n\n\nConcluding comments: on the systems properties of dormancy and virulence\n\nWe have here brought together some of the relevant elements of environmental, laboratory, and clinical microbiology. We have argued that while their languages may differ (e.g. ‘dormancy’ vs ‘persistence’), very similar phenomena have been observed in each of these spheres (plausibly underlying a commonality of mechanism). Certainly the ability to culture microbes, and not merely to observe them (whether microscopically or via their macromolecular sequences or chemical products), remains an important goal of basic microbiology. This is likely to have significant payoffs in bioprospecting (e.g. 163,783). However, we are sure that improved methods of detecting and identifying these dormant bacteria, whether this is done via chemical imaging, through macromolecular amplification and/or sequencing, or through resuscitation and culturing, will have a major role to play in increasing the awareness of their existence and importance.\n\nClearly dormant, persistent bacteria are likely to be relatively avirulent when they are in such dormant states, and able to bypass the attentions of the innate immune system (albeit the production of superantigens by at least some microorganisms784,785 may be what triggers autoimmune diseases). This ‘stealth’ antigenicity is probably why they have been largely unnoticed by us too786, and their routine estimation via molecular methods787 seems highly desirable. Indeed, virulence varies widely between individual strains (e.g. 788,789). Modern molecular microbiology places much emphasis on the virulence of the pathogen, with concepts such as ‘pathogenicity islands’790–795, ‘virulence genes’796,797, and the ‘virulome’798 being commonplace. However, if dormant microbes resuscitate (or are to be resuscitated) in vivo we shall need to pay much more attention to the environmental triggers that cause this to happen than we probably have so far799 (given that the pathogen genotype is fixed800,801). In other words, virulence, like dormancy, is a phenotypic as well as a genotypic property. We remain largely ignorant of the means by which an optimal immune system has been selected for (or against) by longer-term evolution on the basis of microbial exposures in early life, and how this may have changed with more recent changes in human lifestyle802–805. Nor do we understand how such microbes might enter and exit blood cells (and see 50,330,806–810) (albeit the known endosymbiotic origins811,812 of eukaryotic organelles must have presaged such mechanisms). Similarly, we do not yet know what may cause these dormant microbes to resuscitate (and/or to exit their intracellular niches). However, the potential for iron-associated replication and (e.g.) LPS production and shedding does provide a very straightforward explanation for the continuing low- or medium-grade inflammation characteristic of the many inflammatory diseases we have considered here and elsewhere167,168,429,432,713 (Figure 9).\n\nOne approach to Science is based on varying independently something considered a cause and observing its predicted effects (e.g. 178,813,814). To assess causality in microbiology it is usual (e.g. 792,815–817) to invoke what are (variously818) referred to the Henle-Koch or Koch’s postulates. These are based on the nature and presence, but not the physiological state, of an agent that might be believed to ‘cause’ (or at least contribute to) an infectious disease. Consequently, dormancy poses something of a challenge to the full completion of the required tests. Indeed a number of authors417,792,818–821 have recognised that these tests may need revision in the light of the ability to identify disease-causing microbes by sequence alone. We suspect that a key element here will be the ability to resuscitate dormant organisms in vivo and to see the effects of that on clinical disease.\n\nAs phrased by Silvers822, “Several of our contributors showed how discoveries and insights could emerge with what seemed great promise, and yet be pushed aside, discarded, and forgotten – only to re-emerge once again, sometimes many years later, and become, in their new formulation, accepted as important”. In this sense, and as presaged in the opening quotation1, it seems that ideas, as well as bacteria, can remain dormant for extended periods823,824.",
"appendix": "Author contributions\n\n\n\nThis review originated as part of a discussion between the corresponding authors, who have a funded collaboration as outlined under ‘grant information’, and was partly written during a visit of EP and MP to Manchester. All authors contributed to the writing of the manuscript and have agreed to its final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe thank the Biotechnology and Biological Sciences Research Council (grant BB/L025752/1) as well as the National Research Foundation (NRF) of South Africa for supporting this collaboration. This is also a contribution from the Manchester Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM) (BBSRC grant BB/M017702/1).\n\n\nReferences\n\nKeilin D: The problem of anabiosis or latent life: history and current concept. Proc R Soc Lond B Biol Sci. 1959; 150(939): 149–91. 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[
{
"id": "9602",
"date": "23 Jul 2015",
"name": "Michael R Barer",
"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\nKell, Potgieter and Pretorius present a stimulating and argumentative review ranging from the interrelationships between the culturablilty of bacteria and their viability and any links these descriptions may have to defined physiological states, through a discussion of environmental bacteria and ultimately focusing on the human-associated microbiota, particularly those found in blood (without associated symptoms of sepsis) and their proposed roles in disease. Two central themes are developed beyond those that have been discussed extensively elsewhere: 1) the proposal that failure to culture bacteria from many samples often reflects dormancy and 2) that such dormant bacteria interact with host iron regulation to contribute to or directly cause a panoply of chronic diseases largely labelled as non-communicable.At a general level I support the provocative stance taken by the authors. With 861 cited references, at the very least they provide a valuable resource for anyone wishing to consider the potential microbial contribution to diseases traditionally considered free of this aetiological component. Of course Helicobacter infection stands as a monument to the stupidity of dismissing this possibility in the face of carefully assembled evidence. Indeed this reviewer, who many years ago, was presented with a case of duodenal ulcer in his final medical exams, would probably have experienced quite a different career had he claimed a role for infection in causing his patient’s pathology.In considering the specific points presented I have multiple concerns, the most significant of which I will indulge in outlining below.Semantics present a central problem in considering bacterial viability and physiology and I broadly support the approach taken here. The authors do try to define their terms but some problems remain. In particular I take issue with the very broad application of term “Persisters” which should be reserved for cells that survive (have the potential to replicate) after exposure to an antimicrobial stress to which kills most cells in an actively growing culture of the organism concerned. Conflation of this term with “Dormancy” implies on the one hand that the persisting cells must have been dormant and on the other that dormancy and persistence represent the same physiological state in bacteria. This difficulty resurfaces later when they define dormancy but other problems emerge before then.I was next concerned by the extensive use of the term “Differentiation”. I completely agree that what we used to think of as uniform bacterial populations are probably never so but the degree to which subpopulations may be considered differentiated rather than reflecting a range of adaptive responses or indeed, some degree of injury, is not considered here and again I think this leads to problems in considering their hypotheses under a unitary banner downstream. I consider differentiation to require phenotypic changes that are not directly reversible, as in the case of sporulation, whereas adaptation can involve expression of a single gene that can be reversed by its subsequent repression. I do agree that cell cycle contributes to the range of phenotypes in a pure bacterial culture and that this is not the only reason for their diversity (but was not enlightened by use of the term “modulo” in this regard).The operational definition of dormancy given deliberately leaves open the possibility of metabolic activity and seems only to require that the cell so defined should not divide; this did not allow me to recognise which operational tests might be applied to enumerate or detect dormant cells. Subsequently the detection of molecular signals indicative of bacterial presence in samples from which they were not isolated in culture is taken as evidence of dormancy. In the first case do we accept any non-dividing cell as dormant and in the second I can (and will) offer multiple alternate explanations other than dormancy. Moreover, returning briefly to the conflation between dormancy and persisters, the recent work of John McKinney and colleagues shows that antibiotic exposed persisting cells are not necessarily non-dividing cells in the mycobacterial system he studied.Alternative interpretations of the presence of bacterial 16SrDNA sequences in blood when culture fails to detect the organisms from which they derive, include the presence of dead, injured or moribund cells. If they are shown to be repeatedly present then they must either be able to persist in the face of clearance mechanisms or be supplied at a rate equal to their clearance; both seem equally plausible to the dormancy explanation to me. Moreover, why the first three explanations offered for “Not-yet-cultured” should apply to environmental bacteriology but not to clinical samples escapes me.I am led to the conclusion that the authors have chosen to label evidence for discrepancies between culture and nucleic acid detection of bacteria in blood to give their hypotheses a simple headline. I have no problem with the proposal that human blood and tissues classically considered sterile in the absence of overt symptoms of infection are frequently exposed to bacteria and bacterial products that in many cases contribute to serious chronic disease. However, I consider the burden of available evidence currently provides many potential explanations within the field of microbiomics/metagenomics in contrast to the dormancy hypothesis offered here. Further, I feel this broad application of dormancy to bacterial phenotypes which, even in the case of Rpf dependency, have not been shown to result from a programme of gene expression that could be considered as differentiation, diminishes the value of the term. Indeed there remains no direct proof that dormancy of Mycobacterium tuberculosis underpins what we call latent tuberculosis infection and it is not essential to the observed clinical or pathological pattern, notwithstanding the widespread acceptance of this view by most researches, including me.I am not fundamentally opposed to the ideas presented by Kell and colleagues but I do not think they are assisted by lack of attention to the contradictions I have identified above.Finally I come to the iron dysregulation hypothesis and its pro-inflammatory consequences. It is beyond my expertise to comment on the plausibility of the inorganic chemistry deployed here or to review the evidence relating to more than a fraction of the conditions listed. The importance of the struggle between pathogens and host for access to iron is beyond question. When I entered the medical field of infectious disease it was fully recognised that depriving bacteria from iron was a potential therapeutic angle and indeed iron chelation was studied. Desferioxamine, a widely used agent in iron overload, was investigated and found to effectively deliver iron to the pathogen and the approach was set aside. More recently this agent has been identified as a major risk factor in serious fungal infection and guidance specifically recommends its avoidance.\n\nNewer agents seem not to suffer from this problem and the approach deserves renewed attention. However, I would not underestimate the ability of pathogens to outwit our pharmaceutical industry in the battle to sequester iron. While there are reasons beyond the host –pathogen tug-of war for iron to consider chelation as a therapeutic option, the potential for adverse effects is significant and I think the suggestion that omission of iron chelation from recent guidance on sepsis management is “shocking” is not justified.Focussing briefly on the specific diseases cited and their relation to bacterial exposure in one form or another, I find that evidence cited frequently rests on what can be considered “fringe” hypotheses that have little currency in their respective fields. This is not to discourage their continued pursuit but it does weaken the strength of the authors’ argument when investigation of the supporting literature frequently leads to papers that are given little credence in the specialist field. Of course “cave Helicobacter” must remain on the table. But there, an accidental technical breakthrough led to an avalanche of convincing laboratory and clinical data. In summary Kell, Potgieter and Pretorius have produced an interesting read which bring many important ideas to our attention. I am not convinced of the breadth of conditions to which they argue their ideas are applicable and I await with interest, demonstration of of how they may be practically pursued and some selected definitive proofs that iron-driven inflammatory disease is as important as they claim.",
"responses": [
{
"c_id": "1527",
"date": "07 Sep 2015",
"name": "Douglas Kell",
"role": "Author Response",
"response": "\"Kell, Potgieter and Pretorius present a stimulating and argumentative review ranging from the interrelationships between the culturablilty of bacteria and their viability and any links these descriptions may have to defined physiological states, through a discussion of environmental bacteria and ultimately focusing on the human-associated microbiota, particularly those found in blood (without associated symptoms of sepsis) and their proposed roles in disease. Two central themes are developed beyond those that have been discussed extensively elsewhere: 1) the proposal that failure to culture bacteria from many samples often reflects dormancy and 2) that such dormant bacteria interact with host iron regulation to contribute to or directly cause a panoply of chronic diseases largely labelled as non-communicable.At a general level I support the provocative stance taken by the authors. With 861 cited references, at the very least they provide a valuable resource for anyone wishing to consider the potential microbial contribution to diseases traditionally considered free of this aetiological component. Of course Helicobacter infection stands as a monument to the stupidity of dismissing this possibility in the face of carefully assembled evidence. Indeed this reviewer, who many years ago, was presented with a case of duodenal ulcer in his final medical exams, would probably have experienced quite a different career had he claimed a role for infection in causing his patient’s pathology.In considering the specific points presented I have multiple concerns, the most significant of which I will indulge in outlining below.\"Many thanks for the above; it is perfectly accurate and we have nothing to add here. \"Semantics present a central problem in considering bacterial viability and physiology and I broadly support the approach taken here. The authors do try to define their terms but some problems remain. In particular I take issue with the very broad application of term “Persisters” which should be reserved for cells that survive (have the potential to replicate) after exposure to an antimicrobial stress to which kills most cells in an actively growing culture of the organism concerned. Conflation of this term with “Dormancy” implies on the one hand that the persisting cells must have been dormant and on the other that dormancy and persistence represent the same physiological state in bacteria. This difficulty resurfaces later when they define dormancy but other problems emerge before then.\"This is entirely fair; we see that we occasionally elided the terms ‘dormancy’ and ‘persistence’ to imply synonymy, when either there is none or at least there is no evidence for it. We think the best solution is to add a little section pointing out the semantic difficulties, repeating the operational nature of the definitions, and specifying that in very few cases do we actually know the true physiological state of individual cells – which is what matters with regard to replicatory potential. This material mainly appears in the section defining dormancy, and its title has been extended to note the semantic issues. \"I was next concerned by the extensive use of the term “Differentiation”. I completely agree that what we used to think of as uniform bacterial populations are probably never so but the degree to which subpopulations may be considered differentiated rather than reflecting a range of adaptive responses or indeed, some degree of injury, is not considered here and again I think this leads to problems in considering their hypotheses under a unitary banner downstream. I consider differentiation to require phenotypic changes that are not directly reversible, as in the case of sporulation, whereas adaptation can involve expression of a single gene that can be reversed by its subsequent repression. I do agree that cell cycle contributes to the range of phenotypes in a pure bacterial culture and that this is not the only reason for their diversity (but was not enlightened by use of the term “modulo” in this regard).\"We mainly agree, and suggest what we think is a useful clarification or extension. We note again that “reversibility” is established post hoc, but there are at least two meanings involved. At one level we are discussing a reversibility of states. Let us take a spore and a vegetative cell, which obviously, for sporulating bacteria, can indeed interconvert (“reversibly”). However, another level or meaning implies a mechanistic reversibility, i.e. the path from A to B is simply traversed in the opposite direction when B reverts or interconverts to A. Not only is this not what we mean but (also for thermodynamic reasons) it is certainly not what is done (sporulation and germination in B. subtilis are definitely quite separate processes, as indicated by the referee, and one is not at all the reverse of the other). We have added clarificatory comments accordingly. (One might also have added, but we have not in the ms as it would distract, that similar issues apply to the ‘reversibility’ of enzymes and of biochemical pathways (gluconeogenesis is not mechanistically a reversal of glycolysis, even if the “start” and “end” states are the same molecules.) \"The operational definition of dormancy given deliberately leaves open the possibility of metabolic activity and seems only to require that the cell so defined should not divide; this did not allow me to recognise which operational tests might be applied to enumerate or detect dormant cells. Subsequently the detection of molecular signals indicative of bacterial presence in samples from which they were not isolated in culture is taken as evidence of dormancy. In the first case do we accept any non-dividing cell as dormant and in the second I can (and will) offer multiple alternate explanations other than dormancy. Moreover, returning briefly to the conflation between dormancy and persisters, the recent work of John McKinney and colleagues shows that antibiotic exposed persisting cells are not necessarily non-dividing cells in the mycobacterial system he studied.\"The hallmark of the dormant macrostate, stated in quotation marks in the second paragraph of the ‘dormancy’ section, is indeed that the cells in question do not immediately grow when attempts to culture them under “suitable” conditions (that normally admit their growth), are often (but not necessarily) of low metabolic activity, but are not operationally dead since they can be resuscitated. On this basis we think that this should allow the referee or anyone else to determine the operational tests. It follows that we do not accept ‘any’ non-diving cell as dormant since only resuscitable cells can – post hoc – be considered dormant, and certainly a non-dividing cell it may be irreversibly injured or operationally dead. However, the presence of molecular signals (e.g. 16S) in samples from which nothing (or many fewer colonies or OTUs) may be recovered by culture is certainly an indication of the possibility of resuscitation, and hence dormancy.The referee is entirely correct that we had missed John McKinney’s recent and very relevant work, and we mention it accordingly. \"Alternative interpretations of the presence of bacterial 16SrDNA sequences in blood when culture fails to detect the organisms from which they derive, include the presence of dead, injured or moribund cells. If they are shown to be repeatedly present then they must either be able to persist in the face of clearance mechanisms or be supplied at a rate equal to their clearance; both seem equally plausible to the dormancy explanation to me. Moreover, why the first three explanations offered for “Not-yet-cultured” should apply to environmental bacteriology but not to clinical samples escapes me.\"The referee is entirely correct with regard to the last sentence, and the whole point (or at least a major theme) of our review is precisely that what is well established in environmental microbiology has had much less impact in clinical microbiology (referee 2 makes this exact point, even more explicitly). We agree that in a steady state such cells must be supplied at a rate equal to that of their clearance, and that the fact that clearance is lower than probably expected implies a significant ability to evade the innate and adaptive immune systems. We also take it that for common organisms (not very slow growers such as certain mycobacteria) the former rates must be much lower than those typically attainable in laboratory cultures, else we would have classical sepsis. We have added a few comments on these issues accordingly, in the section entitled ‘Generalised failure of classical techniques to detect dormant bacteria in clinical microbiology’. \"I am led to the conclusion that the authors have chosen to label evidence for discrepancies between culture and nucleic acid detection of bacteria in blood to give their hypotheses a simple headline. I have no problem with the proposal that human blood and tissues classically considered sterile in the absence of overt symptoms of infection are frequently exposed to bacteria and bacterial products that in many cases contribute to serious chronic disease. However, I consider the burden of available evidence currently provides many potential explanations within the field of microbiomics/metagenomics in contrast to the dormancy hypothesis offered here. Further, I feel this broad application of dormancy to bacterial phenotypes which, even in the case of Rpf dependency, have not been shown to result from a programme of gene expression that could be considered as differentiation, diminishes the value of the term. Indeed there remains no direct proof that dormancy of Mycobacterium tuberculosis underpins what we call latent tuberculosis infection and it is not essential to the observed clinical or pathological pattern, notwithstanding the widespread acceptance of this view by most researches, including me.I am not fundamentally opposed to the ideas presented by Kell and colleagues but I do not think they are assisted by lack of attention to the contradictions I have identified above.\"All of the above is entirely fair, and we do not disagree. We hope that the changes we have now made to the ms to weaken the ostensible claims (and misplaced synonymies) now meet the referee’s approval. For instance we have stressed that while the presence of suitable molecular sequences (e.g. 16S) implies that it is worth seeking to resuscitate the organisms from which it came, an absence would imply that it is not. A success in resuscitating organisms from a sample that initially appeared sterile would from our operational definition imply that those ones were indeed dormant, and we’d like to think that this had now been clarified. \"Finally I come to the iron dysregulation hypothesis and its pro-inflammatory consequences. It is beyond my expertise to comment on the plausibility of the inorganic chemistry deployed here or to review the evidence relating to more than a fraction of the conditions listed. The importance of the struggle between pathogens and host for access to iron is beyond question. When I entered the medical field of infectious disease it was fully recognised that depriving bacteria from iron was a potential therapeutic angle and indeed iron chelation was studied. Desferioxamine, a widely used agent in iron overload, was investigated and found to effectively deliver iron to the pathogen and the approach was set aside. More recently this agent has been identified as a major risk factor in serious fungal infection and guidance specifically recommends its avoidance. Newer agents seem not to suffer from this problem and the approach deserves renewed attention. However, I would not underestimate the ability of pathogens to outwit our pharmaceutical industry in the battle to sequester iron. While there are reasons beyond the host –pathogen tug-of war for iron to consider chelation as a therapeutic option, the potential for adverse effects is significant and I think the suggestion that omission of iron chelation from recent guidance on sepsis management is “shocking” is not justified.\"The point about desferrioxamine is well made (and we mention it, with citations), but the molecule is of course in fact a natural prokaryotic siderophore, from Streptomyces pilosus. We have replaced the term ‘shocking’ with something more suitable. \"Focussing briefly on the specific diseases cited and their relation to bacterial exposure in one form or another, I find that evidence cited frequently rests on what can be considered “fringe” hypotheses that have little currency in their respective fields. This is not to discourage their continued pursuit but it does weaken the strength of the authors’ argument when investigation of the supporting literature frequently leads to papers that are given little credence in the specialist field. Of course “cave Helicobacter” must remain on the table. But there, an accidental technical breakthrough led to an avalanche of convincing laboratory and clinical data.\"It is probably a philosophical distraction to rehearse how often in science something outside the mainstream is blocked for many years by ‘vested interests’. However, we may as well mention Peyton Rous, whose discovery of a viral cause of certain cancers was sidelined for decades (he received a Nobel prize when he was 87, 40 years after first being nominated https://en.wikipedia.org/wiki/Francis_Peyton_Rous!). Closer to (prokaryotic) home, Barry Marshall has edited a book (Marshall BJ (ed.): Helicobacter pioneers: firsthand accounts from the scientists who discovered helicobacters. Melbourne: Blackwell, 2002.) whose invited contributors had all long recognised a bacterial cause of ulcers and treated their patients accordingly, on the simple grounds that the antibiotics worked! Of course Marshall and Warren (and the wider world) knew nothing of this at the time of their discovery of H. pylori. Under these circumstances (as here) we rely on the overall weight of evidence (as much as its place of publication) to support our views. In Philosophy of Science circles this bolstering of a view via overlapping circles of self-consistent reasoning and data is referred to as ‘coherence’. Accordingly, in this sense, we have tried to make this a coherent story, and rehearse this point in the concluding section. \"In summary Kell, Potgieter and Pretorius have produced an interesting read which bring many important ideas to our attention. I am not convinced of the breadth of conditions to which they argue their ideas are applicable and I await with interest, demonstration of of how they may be practically pursued and some selected definitive proofs that iron-driven inflammatory disease is as important as they claim.\"We have no further comments at this stage. Many thanks again for a very thoughtful review."
}
]
},
{
"id": "9285",
"date": "11 Aug 2015",
"name": "Vanya Gant",
"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 review Kell et al’s review relating to individuality, phenotypic differentiation, dormancy and “persistence” as a clinical microbiologist, infectious diseases doctor, with an interest in developing and assessing the impact of rapid sequence-based molecular blood and lung diagnostics in the critically ill. This review reminded me of Mussorsky’s Pictures at an Exhibition, a collection of hastily composed pieces whose theme was to take an interested individual through an art gallery, and to tarry awhile in front of 10 Tableaux, interspersed with musical elements referring to the “Promenade” through the gallery.And so it is with Kell et al’s review. After an introductory Promenade relating to matters of bacterial dormancy and its relationship with just about any other conceivable physical state between life and death, exhaustively referenced together with the thought provoking Postgate-ian concept of the difficulties inherent in differentiating bacterial life from death if you only have an instant in time to measure it – we are then presented with several pictures, garlanded for us in extensively referenced detail by the authors. Were mindmaps not enough to capture the reader’s curiosity as to this magnum opus of a kind, we are invited to walk through Kell et al’s gallery of mental pictures depicting scenes of the Yet to be Cultured, Those bacteria that aren’t culturable yet but are certainly not dead, the biological importance of bacterial pheromones, the evils of Iron - thence to the Clinical Microbiology Room of Pictures with a liberal helping of systems biology throughout.I am a proponent of, and believer in, the present and future potential of Nucleic Acid Technology (NAT) for pathogen detection in Clinical microbiology and I use such techniques on a daily basis. When appropriately deployed, it allows me to find those “unculturable” pathogens as drivers for individual clinical cases of infection. Perhaps strangely, this is a relatively new paradigm for most practising clinicians, and one which likely will generate fundamental discoveries highly relevant to human disease, and for all we know as equally important as Helicobacter. That such sequences should be found in blood is hardly surprising, given that human beings have between 10 and 100 times more bacterial cells than their own, living (or persisting, or dormant) on and in them. This groups’ demonstration of bacteria adhering to red cells (also in red cells) is certainly very intriguing, and such suggested “atopobiosis” is more expansively dealt with in another publication and prompts far more questions than it answers – in a good way. Another obvious question relates to how these adherent bacteria may remain undetected and intact in the presence of numerous moieties central to both innate and acquired immunity (complement and antibody to name but two) as well as escaping phagocytosis in the liver and spleen. It would certainly be interesting to look at red cells in the grave condition of erythrophagocytosis, a condition whose mechanism is in most cases obscure –it might even be that adherent bacteria “opsonize” the red cells in these cases. This reader, however, does baulk at the very serious work to be done as regards untangling the mechanistic nature of an “association” with several diseases, and certainly at this stage it would be very unwise to suggest it’s anything more than that. Further work of this nature should be approached and undertaken with extreme caution and rigor in view of the myriad possible explanations other than causative ones; the Measles vaccine/autism saga comes to mind here. It is likely therefore that such technologies will perforce “lift the lid” on what might lie beyond the Culturable, and its relationship to human disease. This is explored in Table 3, which represents a tour de force as concerns the sheer volume of references relating to all that appears to associate human Disease and organisms, mostly bacteria.Unfortunately, this Table doesn’t work for me. Whilst it will serve me as a unique and accessible resource of information in this space, it is anarchic. Correctly described as “Evidence for agents in non-communicable diseases”, it lists, in no particular order, and with no apparently critical eye, references 470 to 712 as relevant to the Table subject stated above. This list’s breadth as concerns both organisms and clinical diseases is extraordinary; and the literature quoted in a table described as “effect of bacterial involvement” ranges from unusual cases, to mechanistic assumptions of what LPS might do, to the concept of “dysbiosis” amongst many others. I was left rather dizzy from the mental exercise needed to constantly adjust to the sheer scale and variation of why a particular organism, or something it produces, might either directly causally relate to a particular disease, or perhaps through the individuals’ immune response to it; especially now we know how outbred we are as concerns immune responsiveness. This review finishes with an impressive and lyrical chiding for Scientists, whereby those who research this field should wake up from their intellectual slumber, as might and indeed do bacteria. This review is additionally peppered with tantalizing if perhaps sometimes unfounded assumptions, some arguable and some bordering on plain unreasonable. Certainly my eyebrow raising went into overdrive when considering Kell’s conviction as concerns a Catholic Grand Unifying Theory based around the Evils of Iron, the subject of a previous equally grand Magnum Opus.This review has to be one of the most undisciplined I have read in a long time, on occasions associating seemingly disparate observations and conflating “scientifically” determined facts with clinical issues. Having said this, I should finish by applauding Kell et al’s review as a thumping good read. It’s fast paced, edgy, a real treasure trove of papers for me to read at leisure, and goes way outside the usual, expected and conventional boundaries of style of prose and rigor we “normally expect” of such scientific publications. And (warts and all, and there are many) it left this reader thinking that there indeed is Life beyond dormancy within the review’s style itself, beyond the doubtless very important but less imaginative run-of-the-mill, tightly written yet dreary “Scientific Publication”. It is almost as if this review in all its unconventionality were particularly well aligned to the current state of the Art for the Uncultured in Clinical Medicine (bacteria, not Doctors) and its potential to release significant Paradigm shifts. No doubt this reviews’ readers are made up of those who have the capacity to appreciate Kells’ latest brand of emergent, imaginative systems biology style of thinking underneath what some might consider a publication of inadequate scientific rigor.",
"responses": [
{
"c_id": "1528",
"date": "07 Sep 2015",
"name": "Douglas Kell",
"role": "Author Response",
"response": "\"I review Kell et al’s review relating to individuality, phenotypic differentiation, dormancy and “persistence” as a clinical microbiologist, infectious diseases doctor, with an interest in developing and assessing the impact of rapid sequence-based molecular blood and lung diagnostics in the critically ill.This review reminded me of Mussorsky’s Pictures at an Exhibition, a collection of hastily composed pieces whose theme was to take an interested individual through an art gallery, and to tarry awhile in front of 10 Tableaux, interspersed with musical elements referring to the “Promenade” through the gallery.And so it is with Kell et al’s review. After an introductory Promenade relating to matters of bacterial dormancy and its relationship with just about any other conceivable physical state between life and death, exhaustively referenced together with the thought provoking Postgate-ian concept of the difficulties inherent in differentiating bacterial life from death if you only have an instant in time to measure it – we are then presented with several pictures, garlanded for us in extensively referenced detail by the authors. Were mindmaps not enough to capture the reader’s curiosity as to this magnum opus of a kind, we are invited to walk through Kell et al’s gallery of mental pictures depicting scenes of the Yet to be Cultured, Those bacteria that aren’t culturable yet but are certainly not dead, the biological importance of bacterial pheromones, the evils of Iron - thence to the Clinical Microbiology Room of Pictures with a liberal helping of systems biology throughout.\"This is a lovely analogy, which we shall let readers enjoy in the open referee’s report; we are probably not capable of recasting the review in Mussorgskian style anyway! In this regard, readers might also enjoy a little known and whimsical piece on bioinformatics that takes just such an approach: Goble C, Wroe C: The Montagues and the Capulets. Comp Func Genomics 2004; 5:623-632. \"I am a proponent of, and believer in, the present and future potential of Nucleic Acid Technology (NAT) for pathogen detection in Clinical microbiology and I use such techniques on a daily basis. When appropriately deployed, it allows me to find those “unculturable” pathogens as drivers for individual clinical cases of infection. Perhaps strangely, this is a relatively new paradigm for most practising clinicians, and one which likely will generate fundamental discoveries highly relevant to human disease, and for all we know as equally important as Helicobacter. That such sequences should be found in blood is hardly surprising, given that human beings have between 10 and 100 times more bacterial cells than their own, living (or persisting, or dormant) on and in them. This groups’ demonstration of bacteria adhering to red cells (also in red cells) is certainly very intriguing, and such suggested “atopobiosis” is more expansively dealt with in another publication and prompts far more questions than it answers – in a good way. Another obvious question relates to how these adherent bacteria may remain undetected and intact in the presence of numerous moieties central to both innate and acquired immunity (complement and antibody to name but two) as well as escaping phagocytosis in the liver and spleen. It would certainly be interesting to look at red cells in the grave condition of erythrophagocytosis, a condition whose mechanism is in most cases obscure –it might even be that adherent bacteria “opsonize” the red cells in these cases. This reader, however, does baulk at the very serious work to be done as regards untangling the mechanistic nature of an “association” with several diseases, and certainly at this stage it would be very unwise to suggest it’s anything more than that. Further work of this nature should be approached and undertaken with extreme caution and rigor in view of the myriad possible explanations other than causative ones; the Measles vaccine/autism saga comes to mind here.\"These are excellent points, and we have covered some of them in the forward-looking concluding section. While they might be seen as ‘premature’ (in the sense that it requires acceptance of the basic ‘dormancy’ hypothesis in the first place) they do point to important areas where we would seek a mechanistic understanding of what is going on. \"It is likely therefore that such technologies will perforce “lift the lid” on what might lie beyond the Culturable, and its relationship to human disease. This is explored in Table 3, which represents a tour de force as concerns the sheer volume of references relating to all that appears to associate human Disease and organisms, mostly bacteria.Unfortunately, this Table doesn’t work for me. Whilst it will serve me as a unique and accessible resource of information in this space, it is anarchic. Correctly described as “Evidence for agents in non-communicable diseases”, it lists, in no particular order, and with no apparently critical eye, references 470 to 712 as relevant to the Table subject stated above. This list’s breadth as concerns both organisms and clinical diseases is extraordinary; and the literature quoted in a table described as “effect of bacterial involvement” ranges from unusual cases, to mechanistic assumptions of what LPS might do, to the concept of “dysbiosis” amongst many others. I was left rather dizzy from the mental exercise needed to constantly adjust to the sheer scale and variation of why a particular organism, or something it produces, might either directly causally relate to a particular disease, or perhaps through the individuals’ immune response to it; especially now we know how outbred we are as concerns immune responsiveness.\"We very much accept the point that the table could be improved with regard to ordering, and we have done so accordingly. However, we think that readers will recognise it for what it is (as does the referee), viz. as a useful resource and/or pointer to a large literature in which specialists in disease X may wish to read at least those papers we suggest as relevant to ‘their’ disease, while others will simply see it as a recognition of the widespread evidence for our more general claims. \"This review finishes with an impressive and lyrical chiding for Scientists, whereby those who research this field should wake up from their intellectual slumber, as might and indeed do bacteria.This review is additionally peppered with tantalizing if perhaps sometimes unfounded assumptions, some arguable and some bordering on plain unreasonable. Certainly my eyebrow raising went into overdrive when considering Kell’s conviction as concerns a Catholic Grand Unifying Theory based around the Evils of Iron, the subject of a previous equally grand Magnum Opus.\"As mentioned in the comments on the review of referee 1, the basis for this is the desire to produce a coherent story (in the sense used by Philosophers of Science), and (as referee 1 also states) it is well known that microbial growth in vivo is normally limited by iron availability. That iron dysregulation is also a hallmark of just those chronic inflammatory diseases that we highlight here is consistent with this view, and indeed serves to provide a simple explanation for this. Of course, as the referee indicates (and referee 1 does too), further demonstrations will benefit from varying iron levels as an independent variable. \"This review has to be one of the most undisciplined I have read in a long time, on occasions associating seemingly disparate observations and conflating “scientifically” determined facts with clinical issues.Having said this, I should finish by applauding Kell et al’s review as a thumping good read. It’s fast paced, edgy, a real treasure trove of papers for me to read at leisure, and goes way outside the usual, expected and conventional boundaries of style of prose and rigor we “normally expect” of such scientific publications. And (warts and all, and there are many) it left this reader thinking that there indeed is Life beyond dormancy within the review’s style itself, beyond the doubtless very important but less imaginative run-of-the-mill, tightly written yet dreary “Scientific Publication”. It is almost as if this review in all its unconventionality were particularly well aligned to the current state of the Art for the Uncultured in Clinical Medicine (bacteria, not Doctors) and its potential to release significant Paradigm shifts. No doubt this reviews’ readers are made up of those who have the capacity to appreciate Kells’ latest brand of emergent, imaginative systems biology style of thinking underneath what some might consider a publication of inadequate scientific rigor.\"Many thanks for these last comments; we have nothing further to add here."
}
]
}
] | 1
|
https://f1000research.com/articles/4-179
|
https://f1000research.com/articles/4-681/v1
|
07 Sep 15
|
{
"type": "Review",
"title": "Recent developments in osteogenesis imperfecta",
"authors": [
"Joseph L. Shaker",
"Carolyne Albert",
"Jessica Fritz",
"Gerald Harris",
"Carolyne Albert",
"Jessica Fritz",
"Gerald Harris"
],
"abstract": "Osteogenesis imperfecta (OI) is an uncommon genetic bone disease associated with brittle bones and fractures in children and adults. Although OI is most commonly associated with mutations of the genes for type I collagen, many other genes (some associated with type I collagen processing) have now been identified. The genetics of OI and advances in our understanding of the biomechanical properties of OI bone are reviewed in this article. Treatment includes physiotherapy, fall prevention, and sometimes orthopedic procedures. In this brief review, we will also discuss current understanding of pharmacologic therapies for treatment of OI.",
"keywords": [
"Osteogenesis imperfecta",
"mutations",
"recessive"
],
"content": "Introduction\n\nOsteogenesis imperfecta (OI) is an unusual heritable disease that occurs in about 1 in 10,000 to 20,000 live births1. The major clinical manifestation is skeletal fragility. Skeletal deformity, joint laxity, and scoliosis may be present2. Other extraskeletal manifestations include hearing loss, dentinogenesis imperfecta, blue/gray sclerae, hypercalciuria, aortic root dilatation, and neurologic conditions such as macrocephaly, hydrocephalus, and basilar invagination1–5. The phenotype is variable, ranging from osteoporosis presenting in adulthood to lethality in children3. Even adults with “mild” OI may have significant musculoskeletal symptoms, including arthritis, fractures, back pain, scoliosis, and tendon ruptures6.\n\nAbout 90% of patients have mutations in type I collagen genes (COL1A1 and COL1A2)3; however, many other genes have now been described. Some of the genes encode proteins related to type I collagen (for example, enzymes that modify type I collagen, chaperone proteins, and signaling proteins). In 1979, Sillence et al. proposed a classification system for OI with four types based on severity: type I mild non-deforming, type II perinatal lethal, type III severely deforming, and type IV moderately deforming7. This classification has been expanded as new genes were discovered. Phenotypic classification (types I to V with multiple genes included in some of the types) has been proposed5. Alternatively, classification by genetics has been proposed (see Table 1), which was created through modifications of references8–10.\n\nAD, autosomal dominant; AR, autosomal recessive; XL, x-linked.\n\nThere have been recent advances in the understanding of the structure and mechanical properties of bone in children with OI. These advances may lead to improved finite element (FE) models that help predict fracture risk of specific activities and help plan physiotherapy.\n\nIn addition to physiotherapy and orthopedic surgery when needed, intravenous bisphosphonates have been used extensively in moderate to severe OI in childhood. Less is known about pharmacologic treatment in adults. Anabolic therapy with PTH 1-34 has been studied in adults with OI. Future therapies may include antibodies to sclerostin, transforming growth factor beta (TGFβ) antagonism, gene therapy, and cell-based therapies.\n\n\nGenes and classification\n\nOI is most commonly caused by mutations in type I collagen. Type I collagen is a rod-like structure formed from a trimer of 2 COL1A1 and 1 COL1A2 subunits3, which requires post-translational modification. Many of the other rare forms of OI are due to defects in proteins involved in cross-linking, hydroxylation, and mineralization of type I collagen.\n\nMutations of CRTAP, which encodes cartilage-associated protein, have been shown to cause recessive OI11–14. Mutations of LEPRE1, which encodes prolyl 3 hydroxylase14–16, and PPIB (protein cyclophylin B)17–19 also cause recessive OI. The proteins described above form a complex that modifies specific prolines in the collagen and these mutations result in moderate to lethal OI.\n\nSERPINH1 mutations cause severe recessive OI20. The protein affected in SERPINH1 mutations, HSP47, is a collagen chaperone protein8. FKBP10 mutations cause recessive OI (progressively deforming)21. This gene encodes the protein FKBP65, which appears to be needed for hydroxylation of collagen telopetide lysine22. Both HSP47 and FKBP65 are needed for the proper folding of the collagen triple helix. Furthermore, Bruck syndrome (OI and congenital contractures) can be caused by homozygous mutations on FKPB1023, and Kuskokwim syndrome (congenital contractures with mild skeletal problems seen in Yup’ik people in Alaska) is caused by FKBP10 mutations24. PLOD2 mutations also cause recessive OI25. PLOD-2 encodes lysyl hydroxylase 2, which hydroxylates collagen telopeptide lysine. Bruck syndrome can also be caused by homozygous mutations of PLOD225.\n\nBMP1 (bone morphogenetic protein 1) mutations also cause recessive OI26,27. The protein, BMP1, is a protease that cleaves the c-propeptide of type I collagen26,27 but also has other substrates. SP7 mutations cause recessive OI28. SP7 encodes the protein osterix, which may be needed for osteoblast differentiation10. WNT1 mutations29–31 have been reported in early-onset osteoporosis (dominant) and OI (recessive). The protein, WNT1, may be important in the beta catenin system, which stimulates bone formation29–31.\n\nTMEM38B mutations have been reported in recessive OI32. This gene encodes TRIC-B, which may be important in intracellular calcium signaling. Defective TRIC-B may cause bone disease through defective calcium signaling in bone cells10. CREB3L1 mutations cause recessive OI33. CREB3L1 encodes the protein OASIS, which may activate transcription of COL1A134. PLS3 (plastin 3) mutations have been reported in x-linked osteoporosis35–37. Plastin 3 is expressed in osteocyte dendrites and may be important in mechanosensing35. Bone biopsies from patients with PLS3 mutations have shown cortical and trabecular osteoporosis with normal to low bone formation rates36,37. There is no mineralization defect36,37.\n\nMutations in IFITM5, a bone-restricted IFITM-like protein (BRIL) (dominant) cause type V OI38–42. These patients have prominent callus formation and ossification of the forearm interosseous membrane38–42. They also have mesh-like lamellation on bone biopsy as well as a mineralization defect38–42. There appear to be substantial differences in phenotypic presentation even with similar mutations40–42. Type VI OI is caused by mutations in SERPINF1 (protein PEDF)43,44. Children with type VI OI have elevated alkaline phosphatase, and bone biopsy reveals fish-scale pattern under polarized light as well as broad bands of unmineralized osteoid43,44. Interestingly, some patients with BRIL mutations have phenotypic type VI OI (rather than type V)45. BRIL and PEDF are related, and it appears that mutations causing gain-of-function of BRIL cause OI type V and that those causing loss-of-function of BRIL look phenotypically like OI type VI46.\n\n\nStructure and mechanical properties of bones in osteogenesis imperfecta\n\nFrom a mechanical perspective, increased fracture risk in individuals with OI could stem from a combination of reduced bone mass, decreased bone material quality, and, in some individuals, the presence of bone deformity.\n\nLow bone mass is a clinical characteristic of OI, and individuals with this disorder tend to have markedly reduced areal bone mineral density (BMD)47–49. This reduced bone mass can be the consequence of decreased bone size or decreased volumetric BMD or both49,50. Studies of iliac crest biopsies have revealed lower bone tissue quantity in children with moderate and severe OI, including reduced bone volume fraction, and decreased trabecular and cortical thicknesses51–53. Decreased bone volume, though less marked, was also noted in some children with mild OI51,52.\n\nIn cortical bone specimens from the long bone shafts of children with OI, “atypical, flattened, and large resorption lacunae”54 and abnormally elevated porosity have been observed54–57. For example, an average intracortical vascular porosity of 21% was found in bone shaft osteotomies from children with OI by synchrotron radiation micro-computed tomography55,57; the corresponding value in normal pediatric bones was 3%57. From a structural perspective, reduced bone mass can lead to increased stresses within the bone as a result of a smaller area of bone tissue present to support physiological loads. For this reason, low bone mass is likely a considerable contributor to bone fragility in OI.\n\nIn addition to the structural deficiency (low bone mass), mechanical quality of the bone material in OI is reduced. The genetic defects causing OI affect type I collagen, the main organic component of bone. As discussed earlier, most forms of OI (types I to IV) are attributed to insufficient collagen production or amino acid substitution defects within the collagen molecules or both58–63, and less common recessive forms have been associated with abnormalities in other proteins that interact with type I collagen9,64. Since type I collagen is an integral component of bone tissues, it should be no surprise that abnormalities affecting this protein would impact bone material quality. At the ultrastructural level, irregularities in collagen and mineral geometry as well as abnormalities in mineral composition have been reported65–70. Studies in mice indicated that the material abnormalities in OI have a negative impact on bone material properties71–76. A few studies have also used biopsy and osteotomy specimens to measure bone material properties in humans with this disorder. Some of these studies used nanoindentation, a technique in which a diamond-tip indenter is pressed into the polished surface of a material (in this case, bone), creating an indent a few microns in size. With this test, elastic modulus and hardness—that is, properties representing the material’s resistance to elastic (recoverable) and plastic (non-recoverable) deformation, respectively—are determined at the submicrostructural level. Based on nanoindentation, slightly higher elastic modulus and hardness were found in children with mild (type I) versus severe (type III) OI77, whereas these properties were not found to differ between children with severe (type III) versus moderately severe (type IV) phenotypes78. However, exactly how these properties compare with normal tissues remains unclear; one study reported higher elastic modulus and hardness in children with severe OI versus controls79, whereas another reported the opposite80. Furthermore, bone tissues have a complex hierarchical structure, which results in properties that differ between length scales, and nanoindentation provides only limited insight regarding bone tissue properties at the submicrostructural scale. Another limitation with this technique is that it does not measure strength, a property representing the ability of a material to carry stress without breaking or sustaining damage.\n\nRecent studies have measured cortical bone material properties, including strength, at a larger scale by using surgical bone specimens from long bone diaphyses of children with OI55,56,81. In these studies, small osteotomy specimens were machined into parallelepiped-shaped specimens and loaded to failure in either bending55,81 or compression56. Bone material strength was confirmed to be lower than normal in these children, and this property was found to be negatively related to an abnormally elevated intracortical porosity. These findings suggest that increased cortical porosity contributes to increased risk of long bone fractures in OI.\n\nIn addition to decreased bone mass and reduced bone material quality (low bone material strength), deformities of the spine and long bones are common in OI. For example, children with severe OI often exhibit anterolateral bowing of the femur and anterior bowing of the tibia7,47. Increased curvature in long bones leads to an increase in maximum stresses within the bone shaft82. The increased stresses attributed to bone deformities in OI can further contribute to the risk of bone fracture.\n\nMechanical modeling through the use of FE analysis is a well-established technique that allows detailed analysis of composite structures under a variety of load conditions. In the field of orthopedic biomechanics, FE modeling is frequently used to examine the responses of bone to loading83–86. Patient-specific FE models have been effective for bone strain and fracture strength assessment, and as recently as 2009 Fritz et al. applied these models to predict fractures in OI87,88. A femoral model including muscle forces was analyzed during all seven phases of the gait cycle and geometrically matched to bone anatomy with x-rays. The most current work includes advanced meshing techniques for improved geometric biofidelity and updated mechanical property data55. Other FE models for assessing OI bones have also been reported. Orwoll et al. used FE modeling to estimate vertebral strength in a study of the effects of teriparatide treatment in adults with OI89. Caouette et al. developed an FE model to assess fracture risk at the tibia in children with OI90. This tibia model examined fracture risk during two-legged hopping, lateral loading, and torsional loading. Future applications of FE modeling may prove invaluable for better quantification of fracture risk in OI. These models could help identify activities that pose greater risk of fracture and, through appropriate controls, may enable persons with OI to participate safely and more fully in a greater spectrum of daily and recreational activities.\n\n\nManagement\n\nThe goals of the treatment in OI are to decrease pain and fractures and to maximize mobility. Physical therapy/rehabilitation91 is particularly important in children to improve weight bearing and prevent fractures as well as to increase strength and mobility during fracture recovery. Some children may require wheelchairs or walking aids. Occupational therapy may be needed to help with daily living activities.\n\n\nPharmacologic therapy\n\nBisphosphonates (BPs) are non-hydrolysable synthetic analogs of pyrophosphate92. BPs adhere to mineralized surfaces, inhibit osteoclastic bone resorption, and have very long skeletal half-lives92. Intravenous BPs are currently the primary treatment of children with moderate to severe OI. BPs increase BMD and size in children with OI49. BPs do not appear to impair bone formation that increases cortical width in children with OI93. Observational studies suggest decreased fractures94,95, decreased bone pain, and improved vertebral shape94,95. Ability to perform activities of daily living may also be improved. However, it has been difficult to confirm all of these benefits in randomized trials, and the optimal duration of BP treatment is unknown.\n\nIn a study of children with predominantly mild OI, oral risedronate increased BMD and appeared to decrease clinical fractures96. Atypical fractures have been reported in children with OI treated with bisphosphonates97,98; however, osteneocrosis of the jaw does not appear to be a major problem in children with OI treated with BPs99–101. Several studies have been done on the use of intravenous or oral BPs in adults with OI. Although BMD increases have been reported during these treatments, fracture data are equivocal102–106. A Cochrane review found increased BMD in patients with OI treated with BPs but did not find definitive evidence of fracture reduction107. Furthermore, a recent meta-analysis of placebo-controlled trials suggested that the effects of BPs for fracture prevention in OI were inconclusive108.\n\nGrowth hormone has anabolic effects on bone. A 1-year randomized trial of the BP, neridronate, with or without growth hormone showed greater increase in BMD and growth velocity with growth hormone, but there was no fracture benefit of growth hormone109.\n\nTeriparatide (PTH1-34) is an anabolic agent that stimulates bone formation (and ultimately bone resorption). This drug decreases vertebral and non-vertebral fractures in post-menopausal women with osteoporosis110. Observational data in adults with OI suggest increased BMD with teriparatide107,111. Recently, a randomized trial of teriparatide in adults with OI showed increased BMD as well as increased vertebral strength estimated by FE analysis91. The benefits appeared to occur in mild (type I) OI but not in more severe OI (types III and IV).\n\nDenosumab is a monoclonal antibody to receptor activator of nuclear factor kappa B ligand that decreases bone resorption, increases bone density, and reduces fractures in women with post-menopausal osteoporosis112. This drug may represent a future therapy in OI. In a study of four children with type VI OI, increased BMD and mobility and improved vertebral shape were reported after denosumab treatment, and the outcomes of this study indicated that this treatment appears to be safe113. There is also a report of denosumab use in two children with OI caused by COL1A1/A2 mutations114. As with BPs, “zebra lines” were present, suggesting continued longitudinal growth114. Denosumab has been reported to cause hypophosphatemia, hypocalcemia, and secondary hyperparathyroidism in a child with fibrous dysplasia of bone115. There was rebound hypercalcemia after stopping denosumab115.\n\nSclerostin is an inhibitor of the LRP5/WnT system that decreases bone formation. Antibodies to sclerostin are in clinical trials for treatment of osteoporosis with the goal to increase bone density116. Sclerostin antibody appeared to be effective in a mouse model of moderately severe OI117,118 but less so in a mouse model of more severe OI119. TGFβ is secreted by osteoblasts and increases osteoclastic bone resorption120. Excessive TGFβ signaling may be important in some forms of OI, and anti-TGFβ therapy represents an interesting prospect for the future treatment of OI120.\n\nCell-based therapy, such as bone marrow121 or mesenchymal stem cell122–124 transplantation, has also been investigated and may have promise; but these could also have significant risks. Gene therapy with allele-specific silencing may represent a future therapy125.\n\n\nSummary\n\nAlthough most cases of OI are caused by COL1A1/A2 mutations, many new genetic causes have been identified in recent years. Some of these genes are related to the processing of type I collagen. Furthermore, we have greater understanding of the biomechanics of OI bone, including material properties, muscle and gait load effects, and fracture strength assessment. Biomechanical models could help identify activities that pose greater risk of fracture and, through appropriate controls, may enable persons with OI to participate safely and more fully in a greater spectrum of activities. Physical therapy is an important part of the management of these patients. Intravenous BPs are commonly used in children with moderate to severe OI. Some of the benefits seen in observational studies have been hard to prove in controlled studies. Treatment of adults with OI is less well studied. BPs and teriparatide appear to increase BMD, but fracture data are lacking. Teriparatide appears to increase bone strength as estimated by FE analysis in adults with mild OI. Other promising treatments for OI are under investigation.",
"appendix": "Competing interests\n\n\n\nJS is a consultant for Alexion Pharmaceuticals. The other authors declare that they have 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\nMonti E, Mottes M, Fraschini P, et al.: Current and emerging treatments for the management of osteogenesis imperfecta. Ther Clin Risk Manag. 2010; 6: 367–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArponen H, Mäkitie O, Waltimo-Sirén J: Association between joint hypermobility, scoliosis, and cranial base anomalies in paediatric Osteogenesis imperfecta patients: a retrospective cross-sectional study. BMC Musculoskelet Disord. 2014; 15: 428. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLindahl K, Langdahl B, Ljunggren Ö, et al.: Treatment of osteogenesis imperfecta in adults. Eur J Endocrinol. 2014; 171(2): R79–90. 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PubMed Abstract | Publisher Full Text\n\nWilling MC, Deschenes SP, Scott DA, et al.: Osteogenesis imperfecta type I: molecular heterogeneity for COL1A1 null alleles of type I collagen. Am J Hum Genet. 1994; 55(4): 638–47. PubMed Abstract | Free Full Text\n\nShapiro JR: Clinical and genetic classification of osteogenesis imperfecta and epidemiology. In Osteogenesis imperfecta - a translational approach to brittle bone disease. JR Shapiro, et al., Editors. Academic Press: San Diego, CA. 2014: 15–22. Publisher Full Text\n\nCassella JP, Ali SY: Abnormal collagen and mineral formation in osteogenesis imperfecta. Bone Miner. 1992; 17(2): 123–8. PubMed Abstract | Publisher Full Text\n\nCassella JP, Barber P, Catterall AC, et al.: A morphometric analysis of osteoid collagen fibril diameter in osteogenesis imperfecta. Bone. 1994; 15(3): 329–34. PubMed Abstract | Publisher Full Text\n\nStöss H, Freisinger P: Collagen fibrils of osteoid in osteogenesis imperfecta: morphometrical analysis of the fibril diameter. Am J Med Genet. 1993; 45(2): 257. PubMed Abstract | Publisher Full Text\n\nVetter U, Eanes ED, Kopp JB, et al.: Changes in apatite crystal size in bones of patients with osteogenesis imperfecta. Calcif Tissue Int. 1991; 49(4): 248–50. PubMed Abstract | Publisher Full Text\n\nTraub W, Arad T, Vetter U, et al.: Ultrastructural studies of bones from patients with osteogenesis imperfecta. Matrix Biol. 1994; 14(4): 337–45. PubMed Abstract | Publisher Full Text\n\nFratzl-Zelman N, Schmidt I, Roschger P, et al.: Unique micro- and nano-scale mineralization pattern of human osteogenesis imperfecta type VI bone. Bone. 2015; 73: 233–41. PubMed Abstract | Publisher Full Text\n\nKozloff KM, Carden A, Bergwitz C, et al.: Brittle IV mouse model for osteogenesis imperfecta IV demonstrates postpubertal adaptations to improve whole bone strength. J Bone Miner Res. 2004; 19(4): 614–22. PubMed Abstract | Publisher Full Text\n\nJepsen KJ, Schaffler MB, Kuhn JL, et al.: Type I collagen mutation alters the strength and fatigue behavior of Mov13 cortical tissue. J Biomech. 1997; 30(11–12): 1141–7. PubMed Abstract | Publisher Full Text\n\nMcCarthy EA, Raggio CL, Hossack MD, et al.: Alendronate treatment for infants with osteogenesis imperfecta: demonstration of efficacy in a mouse model. Pediatr Res. 2002; 52(5): 660–70. PubMed Abstract | Publisher Full Text\n\nMisof BM, Roschger P, Baldini T, et al.: Differential effects of alendronate treatment on bone from growing osteogenesis imperfecta and wild-type mouse. Bone. 2005; 36(1): 150–8. PubMed Abstract | Publisher Full Text\n\nMiller E, Delos D, Baldini T, et al.: Abnormal mineral-matrix interactions are a significant contributor to fragility in oim/oim bone. Calcif Tissue Int. 2007; 81(3): 206–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRao SH, Evans KD, Oberbauer AM, et al.: Bisphosphonate treatment in the oim mouse model alters bone modeling during growth. J Biomech. 2008; 41(16): 3371–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlbert C, Jameson J, Toth JM, et al.: Bone properties by nanoindentation in mild and severe osteogenesis imperfecta. Clin Biomech (Bristol, Avon). 2013; 28(1): 110–6. PubMed Abstract | Publisher Full Text\n\nFan Z, Smith PA, Harris GF, et al.: Comparison of nanoindentation measurements between osteogenesis imperfecta Type III and Type IV and between different anatomic locations (femur/tibia versus iliac crest). Connect Tissue Res. 2007; 48(2): 70–5. PubMed Abstract | Publisher Full Text\n\nWeber M, Roschger P, Fratzl-Zelman N, et al.: Pamidronate does not adversely affect bone intrinsic material properties in children with osteogenesis imperfecta. Bone. 2006; 39(3): 616–22. PubMed Abstract | Publisher Full Text\n\nImbert L, Aurégan J, Pernelle K, et al.: Mechanical and mineral properties of osteogenesis imperfecta human bones at the tissue level. Bone. 2014; 65: 18–24. PubMed Abstract | Publisher Full Text\n\nAlbert CI, Jameson J, Harris G: Design and validation of bending test method for characterization of miniature pediatric cortical bone specimens. Proc Inst Mech Eng H. 2013; 227(2): 105–13. PubMed Abstract | Publisher Full Text\n\nFritz JM, Grosland NM, Smith PA, et al.: Brittle bone fracture risk with transverse isotropy. In Proceedings of the 37th Annual Meeting of the American Society of Biomechanics. September 4–7. Omaha, NE, 2013. Reference Source\n\nBoyd SK, Müller R: Smooth surface meshing for automated finite element model generation from 3D image data. J Biomech. 2006; 39(7): 1287–95. PubMed Abstract | Publisher Full Text\n\nShim VB, Pitto RP, Streicher RM, et al.: The use of sparse CT datasets for auto-generating accurate FE models of the femur and pelvis. J Biomech. 2007; 40(1): 26–35. PubMed Abstract | Publisher Full Text\n\nEdwards WB, Troy KL: Simulating distal radius fracture strength using biomechanical tests: a modeling study examining the influence of boundary conditions. J Biomech Eng. 2011; 133(11): 114501. PubMed Abstract | Publisher Full Text\n\nEdwards WB, Troy KL: Finite element prediction of surface strain and fracture strength at the distal radius. Med Eng Phys. 2012; 34(3): 290–8. PubMed Abstract | Publisher Full Text\n\nFritz JM, Guan Y, Wang M, et al.: A fracture risk assessment model of the femur in children with osteogenesis imperfecta (OI) during gait. Med Eng Phys. 2009; 31(9): 1043–8. PubMed Abstract | Publisher Full Text\n\nFritz JM, Guan Y, Wang M, et al.: Muscle force sensitivity of a finite element fracture risk assessment model in osteogenesis imperfecta - biomed 2009. Biomed Sci Instrum. 2009; 45: 316–21. PubMed Abstract\n\nOrwoll ES, Shapiro J, Veith S, et al.: Evaluation of teriparatide treatment in adults with osteogenesis imperfecta. J Clin Invest. 2014; 124(2): 491–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nCaouette C, Rauch F, Villemure I, et al.: Biomechanical analysis of fracture risk associated with tibia deformity in children with osteogenesis imperfecta: a finite element analysis. J Musculoskelet Neuronal Interact. 2014; 14(2): 205–12. PubMed Abstract | F1000 Recommendation\n\nHoyer-Kuhn H, Semler O, Stark C, et al.: A specialized rehabilitation approach improves mobility in children with osteogenesis imperfecta. J Musculoskelet Neuronal Interact. 2014; 14(4): 445–53. PubMed Abstract | F1000 Recommendation\n\nLicata AA: Discovery, clinical development, and therapeutic uses of bisphosphonates. Ann Pharmacother. 2005; 39(4): 668–77. PubMed Abstract | Publisher Full Text\n\nRauch F, Travers R, Plotkin H, et al.: The effects of intravenous pamidronate on the bone tissue of children and adolescents with osteogenesis imperfecta. J Clin Invest. 2002; 110(9): 1293–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPlotkin H, Rauch F, Bishop NJ, et al.: Pamidronate treatment of severe osteogenesis imperfecta in children under 3 years of age. J Clin Endocrinol Metab. 2000; 85(5): 1846–50. PubMed Abstract | Publisher Full Text\n\nLand C, Rauch F, Munns CF, et al.: Vertebral morphometry in children and adolescents with osteogenesis imperfecta: effect of intravenous pamidronate treatment. Bone. 2006; 39(4): 901–6. PubMed Abstract | Publisher Full Text\n\nBishop N, Adami S, Ahmed SF, et al.: Risedronate in children with osteogenesis imperfecta: a randomised, double-blind, placebo-controlled trial. Lancet. 2013; 382(9902): 1424–32. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNicolaou N, Agrawal Y, Padman M, et al.: Changing pattern of femoral fractures in osteogenesis imperfecta with prolonged use of bisphosphonates. J Child Orthop. 2012; 6(1): 21–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarpintero P, Del Fresno JA, Ruiz-Sanz J, et al.: Atypical fracture in a child with osteogenesis imperfecta. Joint Bone Spine. 2015; 82(4): 287–8. PubMed Abstract | Publisher Full Text\n\nMalmgren B, Aström E, Söderhäll S: No osteonecrosis in jaws of young patients with osteogenesis imperfecta treated with bisphosphonates. J Oral Pathol Med. 2008; 37(4): 196–200. PubMed Abstract | Publisher Full Text\n\nChahine C, Cheung MS, Head TW, et al.: Tooth extraction socket healing in pediatric patients treated with intravenous pamidronate. J Pediatr. 2008; 153(5): 719–20. PubMed Abstract | Publisher Full Text\n\nHennedige AA, Jayasinghe J, Khajeh J, et al.: Systematic review on the incidence of bisphosphonate related osteonecrosis of the jaw in children diagnosed with osteogenesis imperfecta. J Oral Maxillofac Res. 2014; 4(4): e1. PubMed Abstract | Free Full Text\n\nAdami S, Gatti D, Colapietro F, et al.: Intravenous neridronate in adults with osteogenesis imperfecta. J Bone Miner Res. 2003; 18(1): 126–30. PubMed Abstract | Publisher Full Text\n\nChevrel G, Schott AM, Fontanges E, et al.: Effects of oral alendronate on BMD in adult patients with osteogenesis imperfecta: a 3-year randomized placebo-controlled trial. J Bone Miner Res. 2006; 21(2): 300–6. PubMed Abstract | Publisher Full Text\n\nShapiro JR, Thompson CB, Wu Y, et al.: Bone mineral density and fracture rate in response to intravenous and oral bisphosphonates in adult osteogenesis imperfecta. Calcif Tissue Int. 2010; 87(2): 120–9. PubMed Abstract | Publisher Full Text\n\nBradbury LA, Barlow S, Geoghegan F, et al.: Risedronate in adults with osteogenesis imperfecta type I: increased bone mineral density and decreased bone turnover, but high fracture rate persists. Osteoporos Int. 2012; 23(1): 285–94. PubMed Abstract | Publisher Full Text\n\nO'Sullivan ES, van der Kamp S, Kilbane M, et al.: Osteogenesis imperfecta in adults: phenotypic characteristics and response to treatment in an Irish cohort. Ir J Med Sci. 2014; 183(2): 225–30. PubMed Abstract | Publisher Full Text\n\nPhillipi CA, Remmington T, Steiner RD: Bisphosphonate therapy for osteogenesis imperfecta. Cochrane Database Syst Rev. 2008; (4): CD005088. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHald JD, Evangelou E, Langdahl BL, et al.: Bisphosphonates for the prevention of fractures in osteogenesis imperfecta: meta-analysis of placebo-controlled trials. J Bone Miner Res. 2015; 30(5): 929–33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAntoniazzi F, Monti E, Venturi G, et al.: GH in combination with bisphosphonate treatment in osteogenesis imperfecta. Eur J Endocrinol. 2010; 163(3): 479–87. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNeer RM, Arnaud CD, Zanchetta JR, et al.: Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med. 2001; 344(19): 1434–41. PubMed Abstract | Publisher Full Text\n\nGatti D, Rossini M, Viapiana O, et al.: Teriparatide treatment in adult patients with osteogenesis imperfecta type I. Calcif Tissue Int. 2013; 93(5): 448–52. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCummings SR, San Martin J, McClung MR, et al.: Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med. 2009; 361(8): 756–65. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHoyer-Kuhn H, Netzer C, Koerber F, et al.: Two years' experience with denosumab for children with osteogenesis imperfecta type VI. Orphanet J Rare Dis. 2014; 9: 145. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHoyer-Kuhn H, Semler O, Schoenau E: Effect of denosumab on the growing skeleton in osteogenesis imperfecta. J Clin Endocrinol Metab. 2014; 99(11): 3954–5. PubMed Abstract | Publisher Full Text\n\nBoyce AM, Chong WH, Yao J, et al.: Denosumab treatment for fibrous dysplasia. J Bone Miner Res. 2012; 27(7): 1462–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcClung MR, Grauer A, Boonen S, et al.: Romosozumab in postmenopausal women with low bone mineral density. N Engl J Med. 2014; 370(5): 412–20. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSinder BP, Eddy MM, Ominsky MS, et al.: Sclerostin antibody improves skeletal parameters in a Brtl/+ mouse model of osteogenesis imperfecta. J Bone Miner Res. 2013; 28(1): 73–80. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSinder BP, White LE, Salemi JD, et al.: Adult Brtl/+ mouse model of osteogenesis imperfecta demonstrates anabolic response to sclerostin antibody treatment with increased bone mass and strength. Osteoporos Int. 2014; 25(8): 2097–107. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRoschger A, Roschger P, Keplingter P, et al.: Effect of sclerostin antibody treatment in a mouse model of severe osteogenesis imperfecta. Bone. 2014; 66: 182–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGrafe I, Yang T, Alexander S, et al.: Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta. Nat Med. 2014; 20(6): 670–5. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHorwitz EM, Prockop DJ, Gordon PL, et al.: Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood. 2001; 97(5): 1227–31. PubMed Abstract | Publisher Full Text\n\nHorwitz EM, Gordon PL, Koo WK, et al.: Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A. 2002; 99(13): 8932–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLe Blanc K, Götherström C, Ringdén O, et al.: Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta. Transplantation. 2005; 79(11): 1607–14. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAmin MT, Shazly SA: In utero stem cell transplantation for radical treatment of osteogenesis imperfecta: perspectives and controversies. Am J Perinatol. 2014; 31(10): 829–36. PubMed Abstract | Publisher Full Text\n\nLindahl K, Kindmark A, Laxman N, et al.: Allele dependent silencing of collagen type I using small interfering RNAs targeting 3'UTR Indels - a novel therapeutic approach in osteogenesis imperfecta. Int J Med Sci. 2013; 10(10): 1333–43. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "10217",
"date": "07 Sep 2015",
"name": "Malachi J. McKenna",
"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",
"responses": []
},
{
"id": "10218",
"date": "07 Sep 2015",
"name": "Sudhaker Dhanwada Rao",
"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",
"responses": []
},
{
"id": "10219",
"date": "07 Sep 2015",
"name": "Bart Clarke",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-681
|
https://f1000research.com/articles/4-334/v1
|
23 Jul 15
|
{
"type": "Research Article",
"title": "Cerebrospinal Fluid Biomarkers of Japanese Encephalitis",
"authors": [
"Nabonita Sengupta",
"Sriparna Mukherjee",
"Piyush Tripathi",
"Rashmi Kumar",
"Amol Ratnakar Suryawanshi",
"Anirban Basu",
"Nabonita Sengupta",
"Sriparna Mukherjee",
"Piyush Tripathi",
"Rashmi Kumar",
"Amol Ratnakar Suryawanshi"
],
"abstract": "Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia. Acute encephalitis syndrome (AES) is a group of central nervous system (CNS) disorders caused by a wide range of viruses, bacteria, fungi, chemicals and toxins. It is important to distinguish between various forms of infectious encephalitis with similar clinical manifestations in order to ensure specific and accurate diagnosis and development of subsequent therapeutic strategies. Cerebrospinal fluid (CSF) is in direct contact with the CNS and hence it is considered to be an excellent source for identifying biomarkers for various neurological disorders. With the recent advancement in proteomic methodologies, the field of biomarker research has received a remarkable boost. The present study identifies potential biomarkers for JE using a proteomics based approach. The CSF proteomes from ten patients each with JE and Non-JE acute encephalitis were analyzed by 2D gel electrophoresis followed by mass spectrometry. Vitamin D-binding protein (DBP), fibrinogen gamma chain, fibrinogen beta chain, complement C4-B, complement C3 and cytoplasmic actin were found to be significantly elevated in case of JE indicating severe disruption of the blood brain barrier and DBP can be suggested to be an important diagnostic marker.",
"keywords": [
"Biomarkers",
"Japanese Encephalitis",
"Cerebrospinal fluid"
],
"content": "Introduction\n\nNon-infectious central nervous system (CNS) diseases can also have clinical presentations similar to those of infectious causes of encephalitis and should also be considered in the differential diagnosis1. Out of the viral forms of encephalitis, Japanese encephalitis (JE), is one of the most prevalent forms throughout the world, affecting mostly children and causes 15,000 deaths annually. Due to lack of suitable diagnostic strategies for Japanese encephalitis virus (JEV) and hence delayed treatment, the mortality rate of this disease is very high. Till date, JE can be diagnosed only with clinical symptoms, and serological examination of JE patients. Therefore, there is an urgent need for the development of effective treatment strategies which may be effective before the viral invasion in the brain and spinal cord. For this, an earlier diagnosis based on reliable biomarkers is essential to identify the status and intensity of JE infection. The extracellular fluid of the brain and spinal cord constitutes around 30 to 40% of the cerebrospinal fluid (CSF). Cerebrospinal fluid thus provides an accessible insight into the brain and hence is an ideal body fluid to examine for signature protein profiles for diagnosis or etiology of CNS-related disorders2.\n\n\nMethods\n\nThe study samples were obtained from the pediatric and adult medicine wards of King George’s Medical University (KGMU) in Lucknow, Uttar Pradesh. KGMU Hospital is a teaching hospital which caters mostly to the poor and severely ill from the city and surrounding districts extending upto Nepal. Ethical approval was obtained from the Institutional Ethics Committee of King George’s Medical University (KGMU), Lucknow. Written consent for CSF collection was obtained from the patient’s guardian. The experiments were carried out in accordance with the institutional approved guidelines.\n\nA total of 20 CSF samples were obtained from King George’s Medical University, Lucknow for this study, These included 10 subjects each with JE and other forms of acute encephalitis respectively (Table 1). The lumbar puncture was done within 3 hours of admission of the patients and the CSF volume collected was 2–3 ml from each individual. Inclusion criteria for CSF collection in both AE and JE patients were: i) age >3 years but excluding pregnant women, ii) presence of fever with altered sensorium of 7 days or less. Exclusion criteria were: i) a firm alternative etiological diagnosis, ii) some contraindication to drug administration.\n\nJE was confirmed by the presence of JE IgM in CSF using IgM Capture ELISA (MAC ELISA) kit developed by the National Institute of Virology (Pune) as per the manufacturer’s instructions.\n\nDue to the limited volume of the CSF samples, CSF samples from each experimental group were pooled by mixing equal volumes of each sample. The protein concentration of the samples was then determined by Bradford’s method (Bradford Protein Assay Kit, Bio-Rad) according to the manufacturer’s instructions. The respective CSF samples were then supplemented with 0.1% Triton X100 and protease inhibitors (Sigma Aldrich) and then filtered using a 0.45µm syringe filter to remove particulate matter, concentrated until the desired volume for protein enrichment was reached using a Freeze dryer (Martin Christ, Germany) and subjected to protein enrichment using Bio-Rad ProteoMiner™ Protein Enrichment Kit following the manufacturer’s instructions to normalize the levels of high abundance proteins in the CSF.\n\nThe enriched samples were then cleaned using 2D-clean up kit (Bio-Rad Laboratories, CA, US) following the manufacturer’s instruction to remove impurities such as nucleic acids, lipids, and salts. 2-DE was performed as described earlier3. Briefly, the cleaned up protein pellet was resuspended in sample rehydration buffer and for the first dimension, isoelectric focusing was performed using immobilized pH gradient (IPG) strips (Bio-Rad, USA) of 7 cm size with a pH range from 3.0–10.0 on a Protean i12TM IEF Cell (Bio-Rad, USA). For the second dimension, the proteins were separated by Tris glycine SDS-PAGE on 12% poly acrylamide gel by the method of Blackshear4. The protein spots were visualized by overnight staining with Brilliant Blue R-250, destained with H2O, methanol, and acetic acid in a ratio of 50/40/10 (v/v/v) and scanned using Licor Odyssey Infra-red Imaging System. The protein spots visualized exclusively in the JE CSF proteome were excised and stored at -80°C and processed later for in gel trypsin digestion as previously described5. The peptides extracted were analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF) mass spectrometry using AB SCIEX TOF/TOFTM 5800 System. Acquired combined MS and MS/MS spectra were analyzed with ProteinPilot 4.0 Software using MASCOT v 2.3.02 search engine from matrix sciences against the taxonomy Homo sapiens. The peak list was searched against the taxonomy Homo sapiens at protein sequence Database: UniProtKB-SwissProt sprot_2014-04-16 (544996 sequences; 193815432 residues) Search parameters were as follows: Digestion: trypsin with one missed cleavage; Fixed modification: carbamidomethyl (c); variable modification: oxidation (m); peptide mass tolerance: 100ppm for precursor ion and 0.8 Da for fragment ion with +1 charge state; instrument: MALDI-TOF-TOF.\n\nIL-8, IL-1β, IL-6, IL-10, TNFα, and IL-12 concentrations were measured by flow cytometry using a Human Inflammatory Cytokine CBA Kit, BD Cytometric Bead Array (a kind gift from Dr. Pankaj Seth, NBRC) (BD Biosciences, San Diego, CA, USA) as per the manufacturer’s instructions (n=3 for each experimental group).\n\nThe associations of Vitamin D binding protein were explored using the STRING v10 clustering tool (http://string-db.org/). The confidence score was set at the highest level (0.900) and additional 20 nodes which were indirectly interacting with DBP were asked to show by the software.\n\n\nResults\n\nThe specific JE associated proteins were identified by proteomic comparison of CSF from JEV-infected patients and patients with other forms of encephalitis. Around 16 proteins were found to be exclusively present in the JEV CSF proteome out of which 10 spots could be successfully identified (Figure 1). The observed MW and pI values of the protein spots on the 2- DE gels were compared with the theoretical MW and pI values of corresponding proteins (Table 2). The proteins identified were predominantly DBP, fibrinogen gamma chain, fibrinogen beta chain, complement C4-B, complement C3 and cytoplasmic actin. Most of the identified proteins were found to be members of the albumin multigene family.\n\nCerebrospinal fluid samples were pooled and proteins were extracted and separated on immobilized linear pH gradient IPG strips (pH 3.0–10.0) and then in the second dimension on 12% SDS-PAGE. Spots exclusively visualized in the JE- CSF were marked and excised, and identified by MALDITOF/MS and database searches. The spots are labeled on the gel according to the numbers presented in Table 2. Images are representative of 4 replicate experiments.\n\naNCBI accession number of identified proteins is mentioned.\n\nbMS/MS data of 3 peptides for each spot was searched against NCBI database in the taxonomy group of Homo sapiens using Mascot tool.\n\nThe levels of two pro-inflammatory cytokines IL-1β and TNFα were found to be significantly elevated in JE patients as compared to AES patients while there was no remarkable change in the rest (Figure 2). The data were analyzed by Student’s t-test and a statistical p value<0.05 were considered significant.\n\nIL-8, IL-1β, IL-6, IL-10, TNFα, and IL-12 concentrations were measured by flow cytometry using Human Inflammatory Cytokine Kit (BD Biosciences, San Diego, CA, USA) as per manufacturer’s instructions. Elevated levels of IL-1β and TNFα were observed in the JE samples (2A) whereas no significant changes were observed in the rest (2B). The image is a representative of 3 replicate experiments.\n\nThe STRING v10 clustering tool was used to explore currently known associations of DBP (GC in Figure 3) and out of all the predicted functional partners, low density lipoprotein related protein-2 (LRP2) which is also known as megalin was found to have the highest interaction score (Figure 3).\n\nThe colored nodes signify a direct interaction with DBP whereas the white nodes denote a distant interaction. 4 proteins, LRP2 (yellow node), Cubilin (CUBN, green node), alpha 1 actin (ACTA1, blue node) and legumain (LGMN, violet node) are directly interacting with DBP and LRP2 is being shown the highest interacting score (0.983).\n\n\nDiscussion\n\nJE is one of the most dreaded forms of epidemic and sporadic encephalitis in the tropical regions of Asia with a very high mortality rate especially among children and young adults. The CSF proteome has been explored extensively for the identification of biomarkers for diseases like Alzheimer’s disease, brain tumors, multiple sclerosis6 but until now, few studies have investigated the association between imbalance of CSF elements and severity of JE infection. Recent advancements in proteomic approaches by mass spectrometry have aided the identification of useful predictive biomarkers. The present study provides the first insight into the potential CSF biomarkers associated with JEV neuroinvasion using proteomic methodologies. The CSF proteome from JEV infected individuals was compared to that of AES patients by 2DE-MS based approach. These experiments successfully identified a set of proteins with abnormal expression patterns in the CSF of JE patients which included 6 major proteins belonging to the albumin multigene family. One of the most interesting findings was the DBP which is an abundant multifunctional protein with roles in vitamin D metabolite transport, actin sequestration, and regulation of immune responses7. In a recent study, Yang et al.8 reported DBP to be a potential diagnostic biomarker for the progression of multiple sclerosis where this protein has been shown to interact with actin and play an important role in removal of excess actin. In addition elevated levels of DBP were also shown to be adverse to recovery. Interestingly, we have also observed an increase in the levels of cytoplasmic actin in the CSF of JE patients as opposed to that of patients with AES. Elevated levels of cytoplasmic actin in the CSF have been previously related to severe axonal degeneration associated with progressive multiple sclerosis9. Hence the abnormal levels of the aforementioned interacting proteins in case of JE may be attributed to the extensive neurodegeneration taking place in the CNS. Another important finding was the abnormal increase in the levels of complement proteins C3 and C4b. It has been previously suggested that the activation of the complement system is involved in the pathogenesis of several neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis10. A recent study has also shown that complement C3 is causally involved in the inflammatory neurodegeneration11. Some studies have also shown that DBP can enhance complement component 5a-mediated macrophage chemotaxis via binding to C5a12. We also detected elevated levels of fibrinogen beta and gamma chains as well as serum albumin in the CSF of JE patients. Fibrinogen and serum albumin are normally excluded from the brain by the blood brain barrier (BBB) and their presence in the CSF is an obvious sign of the disruption of the BBB during JE infection. A recent study has shown that fibrinogen leakage upon BBB disruption triggers perivascular microglial clustering in turn causing neuroinflammation and subsequent axonal damage13. Hence the accumulation of fibrinogen in the cerebral parenchyma may play a critical role in neuroinflammation and JE pathogenesis.\n\nIn our study, DBP, complement proteins C3 and C4b and fibrinogen beta and gamma chain were found to be increased in expression in JE patients when compared to AES patients. Elevated levels of proinflammatory cytokines like IL-1β and TNFα were indicative of peripheral immune activation subsequently leading to an up-regulation of CNS cytokine production14. Since DBP has been an important finding of this study, an attempt was made to explore the interacting partners of this protein in order to decipher its possible role in JEV neuropathogenesis using STRINGv10 cluster analysis tool15. LRP-2 or megalin was found to have the highest interaction score. LRP-2 belongs to a group of surface endocytic receptors, which bind and internalize extracellular ligands which also include DBP and is known to play a key role in the clearance and entrance of many proteins from the brain or CSF16. These findings highlight a severe disruption of the BBB during JE infection ultimately leading to subsequent pathogenesis and neurodegeneration and DBP may have an important role as a potential JEV biomarker which can be established with further studies.\n\n\nConclusion\n\nWe can suggest that DBP may be an important marker for JE. Further studies need to be carried out to further validate this putative biomarker.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data of Cytokine Bead Analysis (CBA) of AE and JE infected CSF samples. The datasheet contains the cytokine profile of three patients of AE and JE CSF samples. 10.5256/f1000research.6801.d8953317\n\nF1000Research: Dataset 2. PDF version of the MALDI-TOF raw Data of the collected spots of JE CSF.\n\nEach folder consists of two PDF files, one file contains the Mascot Search Result and the other file explains the identification of the protein. 10.5256/f1000research.6801.d8960518",
"appendix": "Author contributions\n\n\n\nAB, NS and SM designed the experiments. NS and SM performed the experiments. AB and NS wrote the main manuscript and prepared the figures. RK and PT collected the samples. Mass Spectrometry was performed by AS. All authors reviewed the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nAB is awarded the Tata Innovation Fellowship (ABS/DBT/0515/062) from the Department of Biotechnology. The project is also funded by National Brain Research Centre core funding. RK is funded by a grant from NBRC.\n\nI confirm that the 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 would like to acknowledge the kind help provided by R. Rajendra Kumar Reddy, Central Proteomics Facility at Institute of Life Sciences, Bhubaneswar, India.\n\n\nReferences\n\nTunkel AR, Glaser CA, Bloch KC, et al.: The management of encephalitis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2008; 47(3): 303–327. PubMed Abstract | Publisher Full Text\n\nRansohoff RM: Immunology: Barrier to electrical storms. Nature. 2009; 457(7226): 155–156. PubMed Abstract | Publisher Full Text\n\nSengupta N, Ghosh S, Vasaikar SV, et al.: Modulation of neuronal proteome profile in response to Japanese encephalitis virus infection. PloS One. 2014; 9(3): e90211. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlackshear PJ: Systems for polyacrylamide gel electrophoresis. Methods Enzymol. 1984; 104: 237–255. PubMed Abstract | Publisher Full Text\n\nKhan SA, Suryawanshi AR, Ranpura SA, et al.: Identification of novel immunodominant epididymal sperm proteins using combinatorial approach. Reproduction. 2009; 138(1): 81–93. PubMed Abstract | Publisher Full Text\n\nBlennow K, Zetterberg H: Cerebrospinal fluid biomarkers for Alzheimer’s disease. J Alzheimers Dis. 2009; 18(2): 413–417. PubMed Abstract | Publisher Full Text\n\nGomme PT, Bertolini J: Therapeutic potential of vitamin D-binding protein. Trends Biotechnol. 2004; 22(7): 340–345. PubMed Abstract | Publisher Full Text\n\nYang M, Qin Z, Zhu Y, et al.: Vitamin D-binding protein in cerebrospinal fluid is associated with multiple sclerosis progression. Mol Neurobiol. 2013; 47(3): 946–956. PubMed Abstract | Publisher Full Text\n\nTeunissen CE, Dijkstra C, Polman C: Biological markers in CSF and blood for axonal degeneration in multiple sclerosis. Lancet Neurol. 2005; 4(1): 32–41. PubMed Abstract | Publisher Full Text\n\nFinehout EJ, Franck Z, Lee KH: Complement protein isoforms in CSF as possible biomarkers for neurodegenerative disease. Dis Markers. 2005; 21(2): 93–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBodea LG, Wang Y, Linnartz-Gerlach B, et al.: Neurodegeneration by activation of the microglial complement-phagosome pathway. J Neurosci. 2014; 34(25): 8546–8556. PubMed Abstract | Publisher Full Text\n\nKew RR, Webster RO: Gc-globulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg. J Clin Invest. 1988; 82(1): 364–369. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavalos D, Ryu JK, Merlini M, et al.: Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation. Nat Commun. 2012; 3: 1227. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLampa J, Westman M, Kadetoff D, et al.: Peripheral inflammatory disease associated with centrally activated IL-1 system in humans and mice. Proc Natl Acad Sci U S A. 2012; 109(31): 12728–12733. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzklarczyk D, Franceschini A, Wyder S, et al.: STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015; 43(Database issue): D447–452. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpuch C, Ortolano S, Navarro C: LRP-1 and LRP-2 receptors function in the membrane neuron. Trafficking mechanisms and proteolytic processing in Alzheimer's disease. Front Physiol. 2012; 3: 269. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSengupta N, Mukherjee S, Tripathi P, et al.: Dataset 1 in: Cerebrospinal Fluid Biomarkers of Japanese Encephalitis. F1000Research. 2015. Data Source\n\nSengupta N, Mukherjee S, Tripathi P, et al.: Dataset 2 in: Cerebrospinal Fluid Biomarkers of Japanese Encephalitis. F1000Research. 2015. Data Source"
}
|
[
{
"id": "9697",
"date": "29 Jul 2015",
"name": "Akhil C. Banerjea",
"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 a significant addition to our knowledge in JE encephalitis and have identified markers using proteomic approach that would also suggest damage to the blood brain barrier. The experiments carried out support the conclusions. I recommend indexation. The DBP as marker will need more JE and non-JE cases to confirm - so they must indicate that possibility.",
"responses": []
},
{
"id": "9714",
"date": "30 Jul 2015",
"name": "Anil 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 entitled “Cerebrospinal Fluid Biomarkers of Japanese Encephalitis” by Sengupta and colleagues reports their novel findings unique biomarkers for an important disease wherein encephalitis caused by Japanese Encephalitis Virus causes almost 15,000 deaths every year. Currently the JEV infection can be diagnosed only by using serology and clinical symptoms. Therefore there is an urgent need to identify biomarker(s) for this disease. They used cerebrospinal fluid from 10 JE and 10 AE patients and performed 2D and mass spectrometry on these samples. They observed 6 different proteins that were significantly increased in JE patients. They also found that IL-1β and TNF-α were also significantly elevated in JE patients. Their further analysis indicated DBP as potential biomarker in JE patients. Both the Tables and 3 figures are necessary and results in these items support the conclusions very well stated in the manuscript. This manuscript would be extremely useful to the people involved in the general area of JE research and particularly to those involved in diagnostics. The manuscript is well written and this study provides a very important and significant advancement in the field.",
"responses": []
},
{
"id": "9609",
"date": "31 Jul 2015",
"name": "Daniel Růžek",
"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\nSengupta et al. submitted a research article reporting novel CSF biomarkers of Japanese encephalitis (JE). JE is a leading cause of viral encephalitis in Asia. However, there are still many gaps in our understanding of the pathogenesis of JE in humans. Early diagnosis based on reliable biomarkers is essential to identify the status and intensity of JE infection. Here, the authors compared CSF proteomes from ten patients with JE and ten patients with another form of acute encephalitis by 2D gel electrophoresis followed by mass spectrometry. The analysis identified elevated levels of vitamin D-binding protein (DBP), fibrinogen gamma chain, fibrinogen beta chain, complement C4-B, complement C3 and cytoplasmic actin in CSF from JE patients. This indicates blood-brain barrier disruption, and the DBP also represents a novel and mechanistically important CSF biomarker of JE. The exact role of DBP in the development of JE will be investigated is further studies. The manuscript is well written, easy to follow, and provides very important data to all interested in the mechanisms of pathogenesis of neurotropic flaviviral infections. I fully recommend accepting the manuscript for indexation.\n\nMinor comments:I would be interested to know more details on the other forms of the acute encephalitis involved in the study. In the next studies, it would be interesting to compare different forms of encephalitis more specifically, like JE vs. TBE, JE vs. herpetic encephalitis, etc. In the next studies, it would be useful to investigate the biomarker levels in individual patients or to make more pools for each group. In case of one pool investigated, it is not fully clear if the levels of biomarkers are increased in all or most of the patients or if there is just only a strong production in one patient.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-334
|
https://f1000research.com/articles/4-651/v1
|
02 Sep 15
|
{
"type": "Review",
"title": "Engineering food crops to grow in harsh environments",
"authors": [
"Damar López-Arredondo",
"Sandra Isabel González-Morales",
"Elohim Bello-Bello",
"Gerardo Alejo-Jacuinde",
"Luis Herrera",
"Damar López-Arredondo",
"Sandra Isabel González-Morales",
"Elohim Bello-Bello",
"Gerardo Alejo-Jacuinde"
],
"abstract": "Achieving sustainable agriculture and producing enough food for the increasing global population will require effective strategies to cope with harsh environments such as water and nutrient stress, high temperatures and compacted soils with high impedance that drastically reduce crop yield. Recent advances in the understanding of the molecular, cellular and epigenetic mechanisms that orchestrate plant responses to abiotic stress will serve as the platform to engineer improved crop plants with better designed root system architecture and optimized metabolism to enhance water and nutrients uptake and use efficiency and/or soil penetration. In this review we discuss such advances and how the generated knowledge could be used to integrate effective strategies to engineer crops by gene transfer or genome editing technologies.",
"keywords": [
"plant nutrition",
"abiotic stress",
"plant development",
"grain yield",
"gene overexpression",
"bacterial genes",
"biotechnology"
],
"content": "Introduction\n\nCurrent agricultural systems use numerous crop varieties that have been improved through traditional breeding, which has produced a substantial increase in the yields of many crops, particularly cereals1. However, high input agriculture has been conducted with the excessive use of agrochemicals, including phosphorus (P)- and nitrogen (N)-fertilizers, herbicides, and insecticides. Moreover, the harsh conditions that crops face, such as drought, soil elemental toxicities, extreme temperatures, and high soil impedance, merit special attention because they drastically limit crop yields worldwide2,3. The predicted increase in the global population to over 9 billion by 20504 poses a critical challenge: how to develop effective stress-resistant/tolerant crops that are more competitive and can grow in marginal soils to ensure food production.\n\nThe development of efficient gene transfer systems, together with combined “omics” platforms (e.g. genomics, transcriptomics, proteomics and metabolomics), has facilitated the understanding of the physiology and biochemistry of plant adaptive responses to unfavorable environmental conditions and the identification of the key molecular players that control these responses. Several genes encoding transcription factors (TFs), transporters, and metabolic enzymes with a clear potential to improve crops have been identified. Agricultural schemes that use transgenic crops have proven to be effective and complementary alternatives because they provide multiple benefits for farmers (e.g. 37% less pesticide used, 22% higher yields, and 68% more profits); such crops are cultivated today on more than 180 million hectares globally5. The transgenic crops that are currently on the market address the crop yield per unit area by controlling insect attacks and weed competition; however, new transgenic crops that overcome the limitations caused by harsh environments are also starting to be deregulated for commercial use5. In this review, we highlight some relevant transgenic approaches regarding nutrient use efficiency, abiotic stresses and soil physical degradation. These approaches have the potential to increase crop yields in marginal lands with poor soil fertility or low water availability and to expand cropping land into places in which the agro-climatic conditions are favourable but abiotic stress reduces yields and thereby discourages agricultural production.\n\n\nThe two most limiting nutrients for crop productivity: phosphorus and nitrogen\n\nAmong all of the nutrients required by plants, P and N are the most limiting factors for agricultural production in most soils; thus, large amounts of fertilizers are commonly applied to ensure high yields. Although plants are able to use different organic compounds as sources of nutrients, P can be assimilated only in the form of orthophosphate (H2PO4-/HPO4-2, Pi), whereas N is predominantly taken up as nitrate (NO3-, Ni) or ammonia (NH4+)6,7. Moreover, the availability of Pi in the soil solution is drastically affected by the biogeochemical properties of the soil, making P-fertilization efficiency highly variable and more dependent on external inputs. To ensure high yields, farmers usually apply excessive amounts of both P- and N-fertilizers. This practice is unsustainable because crops use only 20–40% of the applied nutrients; the remainder contributes to environmental pollution, toxic algal blooms, and global warming8. Whereas N-fertilizers are synthesized from atmospheric N through a process that consumes at least 1% of global energy usage, P-fertilizers are produced from phosphate rock, a finite, non-renewable mineral resource. Consequently, both fertilizer and food prices will increase continuously. Therefore, searching for integrated strategies to increase P, N, and water use efficiency is an issue of food security and sustainability for all nations. The following paragraphs discuss the most relevant advances in engineering improved P and N uptake and use efficiency.\n\nHow can we improve P and N uptake and/or use efficiency in crops? There is no simple answer. This issue is being addressed by attempting to identify the key genes that control the global adaptive responses that plants display to low availability of N and P and to investigate the possible contributions of these genes to enhancing nutrient uptake and use efficiencies. This set of responses includes profound morphological, physiological and metabolic changes, which rely on the induction and repression of numerous genes and allow plants to survive and reproduce under nutrient-deprived conditions9–12. For instance, under limited-P regimens, plants optimize P use by activating metabolic pathways that require smaller amounts of P-containing compounds, reducing shoot growth and promoting root branching to enhance soil exploration10–12.\n\nThe uptake of N and Pi from the soil is critical and requires specialized transporter proteins6,13–18; therefore, overexpression of these transporters has been considered as a potential approach for plant improvement. However, overexpressing Pi transporters has either had little effect on Pi uptake or, in some cases, resulted in toxicity symptoms due to an excessive accumulation of Pi in the shoots19,20. Interestingly, overexpression of the Phosphate Transporter Traffic Facilitator 1 (PHF1) in rice, responsible for regulating the localization of low- and high-affinity Pi transporters to the plasma membrane21, results in enhanced low-Pi tolerance. Field data demonstrate that grain yield of PHF1-overexpressing plants in a low-Pi soil is higher than that of wild-type (WT) plants, suggesting that post-transcriptional regulation of Pi transporters could also be considered to improve crop performance in soils with low-Pi availability22.\n\nThe generation of transgenic plants to improve the N use efficiency has also been attempted in a variety of crop plants by manipulating the flux-limiting enzymes involved in N assimilation23–25. However, except in the case of alanine aminotransferase (AlaAT)26, as described below, the overexpression of enzymes has not provided reproducible or robust results to indicate that it could be an effective strategy for improving the efficiency of N use.\n\nIn addition to transporters and key enzymes, some TFs that play crucial roles as master regulators of P and N metabolism have been identified. PHR1 is a member of the MYB transcription family that activates the expression of a large set of the Pi-responsive genes that participate in the low-Pi rescue responses in Arabidopsis, and it is evolutionarily conserved from algae to vascular plants27. Overexpressing Phosphate Starvation Response 1 (PHR1) and other TFs, such as Phosphate Starvation-Induced Transcription Factor 1 (PTF1) and OsMYB2P-1, in a variety of crops, such as wheat28, rice29, and maize30, appear to confer low-Pi tolerance and improved grain yield in greenhouse or field trials. Field-testing in different geographical locations and different soil types is required to confirm that the overexpression of these TFs is a robust strategy for improving plant performance under Pi-limiting conditions without affecting performance under optimal Pi availability. Recently, it was reported that in Arabidopsis and rice, SPX1 and SPX2 repress the activity of PHR1 as a transcriptional activator in a Pi-dependent manner31,32. The data published in these reports strongly suggest that the PHR1-SPX1/SPX2 complex is one of the main sensors that regulate the plant response to low-Pi availability. Although no structure of the PHR1-Pi-SPX complex is available, regulation of the interaction between these proteins could become an important target for engineering plants with modulated responses to low-Pi availability. It could be possible to alter PHR1 or SPX1/SPX2 in such a way that the low-Pi response could be modulated to activate processes that enhance Pi uptake and assimilation while preventing the drastic reduction in shoot growth that is generally observed in Pi-starved plants (Figure 1).\n\nEngineering DRO1, AlaAT, PSTOL1, PTXD/Phi and the PHR1-Pi-SPX complex represent interesting approaches with the potential to improve crops for harsh environments. In addition, the identification and manipulation of genes involved in cell-wall components synthesis and stress-responsive epigenetic modifiers has great potential for developing optimal root systems and the improvement of plant responses to diverse stimuli. The simultaneous manipulation of some of these elements could bring robust effects to develop crops with high-yield performance, with a consequent decrease in P- and N-fertilizers input. C, cortex; E, endodermis; E´, epidermis; P, pericycle; VT, vascular tissue.\n\nIn the case of N, overexpression of the TaNFYA-B1 TF in wheat improved the yield under different regimes of P and N inputs under field conditions33. These results were attributed to enhanced root growth and the up-regulation of N- and Pi-transporters. In addition, the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) TF was recently identified as a key element in the systemic signaling pathway that directs N foraging in Arabidopsis roots, thus opening up the possibility of controlling root plasticity to improve soil exploration capacity in crops34.\n\nOverexpression of TF, transporters or enzymes generally used the CaMV35S promoter, which confers constitutive high expression levels, independently of nutrient availability in the soil, as well as in cells that normally do not express the overexpressed gene and lack the expression of other genes required for efficient nutrient assimilation. Therefore, these approaches must consider cell-specific expression and/or modulation of inter-connected biochemical or regulatory pathways to ensure an appropriate phenotype. Recently, enhancer elements that regulate the transcriptional activation of Pi-starvation responsive genes were reported35. These enhancer elements could be used to design synthetic promoters that could direct high levels of expression while maintaining cell specificity and responsiveness to Pi- or N-deprivation. An additional phase for the improvement of nutrient uptake and use efficiency will be the understanding of the regulatory networks that orchestrate plant responses to nutrient deficiency. The integration of this knowledge will serve to design strategies to direct the enhanced expression of two or more TFs simultaneously, leading to more robust improvements in the key traits to achieve a more sustainable agriculture (Figure 1). Altering expression of several TFs to have a higher level of induction upon the stress stimuli, or higher cell-specific constitutive expression, could be feasible by introducing enhancer elements by genome editing using the CRISPR/Cas9 system that allows the simultaneous modification of several genes at the same time36.\n\nInterestingly, TF-overexpressing plants that showed an increased yield under N- or Pi-limiting conditions generally developed a more robust root system. This finding corroborates the importance of the root system architecture in soil exploration and nutrient uptake. Therefore, the identification and molecular characterization of quantitative trait loci (QTL) and marker-assisted backcrossing of genes that regulate root traits that improve nutrient uptake and use efficiencies into modern varieties is of the highest importance37–40. Genes that are responsible for these QTL as well as superior allelic variations in candidate genes, identified in GeneBank collections for instance, could provide powerful potential tools for engineering crops for higher nutrient uptake efficiency in the same or other species by gene transfer or genome editing technologies.\n\nImprovements in P and N metabolism should come from enhanced nutrient uptake and assimilation and/or their subsequent remobilization to support seed or fruit production. Among reported efforts to improve Pi and N use efficiency, we identify the following three promising strategies to develop improved crops, which could make a real contribution to sustainable agriculture: the use of AlaAT to enhance N assimilation26, the use of PHOSPHORUS-STARVATION TOLERANCE 1 (PSTOL1) to enhance P assimilation41, and the development of a novel fertilization system based on the production of transgenic plants that are able to use phosphite (Phi) instead of Pi as a P source42 (Figure 1). Interestingly, each of these approaches is based on the manipulation of a single gene, but they have enormous potential to not only reduce Pi or N applications but also have a profound environmental impact. In the following paragraphs, these approaches and their implications are discussed.\n\nAlanine aminotransferase. Alanine aminotransferase (AlaAT) plays an important role in carbon fixation and N metabolism because it catalyzes the reversible reaction between pyruvate and glutamate to produce alanine and oxoglutarate43. The potential effectiveness of this approach relies on the facts that amino acids act as signals controlling N uptake and that alanine is the only amino acid whose biosynthesis is not inhibited by N deficiency43. The development of the AlaAT technology started with the expression of barley AlaAT in canola using the btg26 root-specific promoter, which resulted in the production of increased biomass under low-N conditions26. Field evaluations showed that btg26:AlaAT canola plants exhibited a 42% increase in seed yield under suboptimal N fertilization (56 kg ha-1). This yield increase correlated with lower levels of glutamine and glutamate in the shoot, increased N influx and higher levels of alanine in roots, and up-regulation of high-affinity N-transporters. The overexpression of barley AlaAT in rice, which was also driven using a root-specific promoter (OsANT1), also resulted in increased biomass and grain yield44. Recently, similar results have been reported for sugarcane45.\n\nInterestingly, the btg26 promoter is expressed mainly in the cortex and lateral roots of transgenic plants26, which are fundamental for the uptake and loading of nutrients into the vascular system. It will be interesting to determine how robust the AlaAT-overexpressing phenotype is under different stress conditions and soil types.\n\nPhosphorus-starvation tolerance 1. Because of the low mobility of Pi in the soil7, a well-developed and highly branched root system is a determinant for soil exploration and Pi uptake in soils with a low availability of this nutrient46. However, modern breeding programs have focused on developing high-yield crops by primarily selecting the above-ground phenotype and applying full fertilization during breeding processes, which probably selects against root traits that are important for nutrient uptake efficiency. The QTL Pup1 (Phosphorus Uptake1), which is responsible for low-Pi tolerance, was identified in a cross between a low-Pi-tolerant rice landrace with a low-Pi-intolerant modern rice variety41. Pup1 was found to contribute to enhanced Pi uptake and grain yield by 170% and 250%, respectively, in low-Pi soils in the modern variety47. Recently, it was found that the PSTOL1 gene, which encodes a protein kinase, is responsible for the effect of the Pup1 QTL on Pi uptake and assimilation41. The overexpression of PSTOL1 under a constitutive promoter (CaMV35S) in two types of modern rice varieties (one indica and one japonica) that naturally lack the gene resulted in an increase of over 60% in grain yield in low-Pi soils41.\n\nInterestingly, the low-P tolerance phenotype conferred by PSTOL1 correlated with a more robust root system, as transgenic plants produced almost five times more root biomass than did the non-transgenic plants. A global expression analysis of the PSTOL1-overexpressing plants revealed a set of up-regulated genes that are related to root growth and stress responses, including a putative peptide transporter41. Because peptide transporters are included in the set of N-transporters (PTR/NRT1) in plants48, it would be interesting to determine whether PSTOL1-overexpression could also improve N uptake efficiency. Optimizing the cell-specific and regulated expression of PSTOL1 will probably have an even higher impact on grain yield.\n\nThe phosphite oxidoreductase/phosphite system. The high reactivity of Pi with soil components and the constant competition of microorganisms and weeds with cultivated plants make agriculture highly dependent on P-fertilizers and herbicides. Recently, a phosphite oxidoreductase (PTXD) from Pseudomonas stutzeri was used to propose a re-design of the currently used agricultural systems42. PTXD oxidizes Phi using NAD+ as a cofactor and yields Pi and NADH as products49. The expression of PTXD in Arabidopsis and tobacco produced transgenic plants that are capable of using Phi as a sole P source.\n\nThe importance of this approach relies on the fact that Phi has distinct chemical and biochemical properties compared with Pi, including higher solubility and lower reactivity with soil components50; in addition, plants and most microorganisms are unable to metabolize Phi as a P source51–53. Therefore, the system makes the PTXD-transgenic plants more competitive over other plants, including weeds, in low-Pi soils that are fertilized with Phi. PTXD-transgenic lines required 30–50% less P to achieve optimal productivity when they were fertilized with Phi instead of Pi42 and reduced the requirement of herbicides because of the poor growth of weeds in soils fertilized with Phi.\n\nThis technology is still in its infancy, and several questions need to be addressed before its real potential is uncovered. The promising results obtained under greenhouse and field conditions suggest that a potential reduction in production costs and energy consumption could be achieved by replacing the independent application of fertilizer and herbicides with a single treatment and by reducing the cost of additional herbicides. It is important to consider that the PTXD/Phi system has the highest potential for use in acidic and alkaline soils that have very low-Pi availability and that, in spite of having appropriate climatic conditions and water availability to sustain high crop productivity, it has been used only as grassland for cattle. These areas are predominant in Brazil, China, Australia, India, and Russia, comprising over 350 million hectares that could be converted into highly productive cropping areas if the PTXD/Phi technology was incorporated into genotypes adapted to acidic and alkaline soils.\n\nThis technology provides sustainable management of P, and thus has the potential to prolong the lifetime of phosphate rock reserves and to reduce the environmental impact of eutrophication of lakes, seas, and oceans. Additionally, a paper published recently suggested that on the moon or other planets that lack oxygen in the atmosphere, on which P accumulates primarily as schreibersite mineral and that are rich in Phi, the PTXD/Phi technology could be an interesting alternative for establishing agriculture54.\n\n\nElement toxicities that limit crop productivity\n\nThere are other nutritionally related stresses that have an important impact on plant yield that deserve special attention, which we only briefly mention because they are outside the main scope of this review, namely aluminum (Al) and boron (B) toxicity. Al toxicity is a major constraint for plant yield on acidic soils, which comprise between 40 and 50% of the world’s potentially arable lands. At pH values below 5, Al3+ ions are dissolved from soil minerals and are highly toxic to plants, impairing root growth and function. Two main classes of Al resistance mechanisms have been reported: Al exclusion mechanisms, which prevent Al from entering the root apex, and Al tolerance mechanisms, in which Al enters the plant but is sequestered into the vacuole and detoxified. Since the root apex is the main site of Al toxicity, the most well-characterized exclusion mechanism involves the regulated release of organic acids (OAs) by the root tip, which chelate Al3+ ions forming non-toxic compounds that do not enter the root tip cells. Members of the Al-activated Malate Transporter (ALMT) family of anion channel transporters and the Multidrug and Toxic compound Extrusion (MATE) family of OA/H+ antiport transporters are responsible for plasma membrane malate and citrate efflux, respectively, from root cells into the rhizosphere in response to the presence of toxic concentrations of Al3+ ions (for a review see 55). Several attempts have been made to show that overexpression of MATE and ALMT genes leads to enhanced Al tolerance56. However, the effectiveness of OA efflux transporters to confer an enhanced Al3+ tolerance remains to be demonstrated under field conditions and also to be agronomically relevant.\n\nB is an essential micronutrient required for several physiological and developmental processes in plants, including meristem development, but that can also be present in toxic levels in the soil. Typical B toxicity symptoms include necrosis of marginal leaves and the inhibition of root growth (for a review see 57). It has been revealed over the last 10 years that plants have B transporters that maintain B homeostasis. B tolerance loci have been identified in high B-tolerant barley and wheat genotypes, which encode B exporters to reduce B concentrations in roots and to alter cellular distribution of B in shoots that are absent in susceptible lines. In barley, tolerance to toxic levels of B is associated with four tandem copies of Bot1 (encoding a B efflux transporter), which is highly expressed in the tolerant landraces58, whereas B tolerance in wheat is associated with a B transporter-like gene (Bot-B5b) that has high root expression levels in tolerant genotypes as compared to susceptible lines59. The finding that high expression of B exporters reduces B concentration in the plant, or that a decreased expression of the transporters that facilitate B uptake could lead to tolerance to toxic B levels, opens up the possibility of using transgenic approaches or genome editing technologies to improve the yield of different crops in soils containing toxic levels of B.\n\n\nEngineering tolerance to drought, salinity, and high temperatures\n\nDrought, saline soils, and extreme temperature are abiotic stresses that adversely affect the growth and productivity of most crops. Drought is the most aggressive form of osmotic stress and limits crop yield in approximately 50% of the total cultivated area worldwide60. Table 1 shows the numerous efforts to engineer crops for drought tolerance. Plants have evolved adaptive mechanisms to cope with abiotic stresses by remodeling morphological and physiological processes, mainly by altering their metabolism to reduce transpiration and promote osmotic adjustment through the interaction of multiple signaling pathways. Adaptive mechanisms that allow plants to cope with drought, salinity, and high temperatures include the production and accumulation of osmoprotectants, molecular chaperones, and antioxidants. Osmoprotectants are metabolites that protect cells by maintaining their water potential and by stabilizing membranes and scavenging reactive oxygen species (ROS)61. Heat shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins also play crucial roles during seed desiccation and water stress by preventing protein denaturation and aggregation62. Additionally, several genes that encode TFs have been identified as key elements that possess the potential to improve crop performance under different abiotic stresses (Table 1). This section discusses the most promising approaches to engineering crops with enhanced tolerance to drought stress, extreme temperatures, and soil salinity.\n\nNumerous genes have been shown to improve drought-tolerance in transgenic crops. In addition, some of these approaches have improved productivity and tolerance to other abiotic stresses, such as cold, heat, and high salinity. The gene source and the type of expression system—constitutive (C), inducible (I) or tissue specific (TS)—are indicated in each case. Positive and negative phenotypic alterations are also specified when data are available (GR, growth retardation; IB, increase biomass; PE, pleiotropic effect; SA, sensitivity to ABA; SOx, increased sensitivity to oxidative stress). Gene sources: Arabidopsis thaliana (At), Arthrobacter globiformis (Ag), Bacillus subtilis (Bs), Cynodon dactylon x C. transvaalensis (Cdt), Escherichia coli (Ec), Glycine max (Gm), Gossypium arboreum (Ga), Hordeum vulgare (Hv), Macrotyloma uniflorum (Mu), Malus domestica (Md), Medicago truncatula (Mt), Nicotiana tabacum (Nt), Oryza sativa (Os), Pisum sativum (Ps), Solanum habrochaites (Sh), Solanum lycopersicum (Sl), Solanum tuberosum (St), Thellungiella halophile (Th), Triticum aestivum (Ta), Vigna aconitifolia (Va). ND, not data.\n\nHSPs and LEA proteins from plants have been clearly shown to be involved in abiotic stress responses; however, as shown in Table 1, only limited attempts have been made to use the genes that encode these proteins to engineer abiotic stress tolerance in crops. Nevertheless, there are some examples that show the potential of overexpressing LEA proteins in vegetative tissues. For instance, the constitutive expression of OsLEA3-1 in rice63 and HvLEA1 in wheat64 and rice65 resulted in improved yields under drought stress without impairing yield under control conditions. Similarly, overexpressing GHSP26 resulted in improved drought and osmotic stress tolerance in cotton plants66. However, although transgenic plants that constitutively express LEA- and HSP-encoding genes have shown improved abiotic stress tolerance under both in vitro and greenhouse conditions, their efficacy under field conditions remains to be demonstrated (Table 1). Interestingly, the best results were obtained in transgenic plants expressing the cold shock protein A (CspA) and CspB genes from Escherichia coli and Bacillus subtilis, respectively. These genes encode RNA-binding proteins with chaperone activity that confer drought tolerance in maize and rice under field conditions67. In fact, CspB-expressing maize is the first genetically modified (GM) crop with enhanced water use efficiency that has been deregulated for commercial use in the USA5.\n\nThe multiple pathways involved in plant adaptations to osmotic and water stress and the complexity of their interactions can explain, to some extent, the limited success under field conditions of manipulating individual genes encoding chaperones or enzymes involved in the synthesis of osmoprotectants68,69. To develop crops with higher yields under drought, it will most likely be necessary to engineer metabolic pathways through the simultaneous manipulation of multiple critical genes. In addition, it would be interesting to explore the mechanisms that regulate desiccation tolerance in seeds to obtain new insights into the adaptive stress response pathways and to identify new candidate genes for crop improvement.\n\nManipulating proteins that regulate gene expression or the signal transduction of multiple metabolic pathways involved in abiotic stresses has proven to be useful for improving the stress tolerance of crops (Table 1). TFs that belong to the Dehydration-Responsive Element-Binding/C-repeat Binding Factor (DREBs/CBF)70, NAM-ATAF and CUC (NAC)71, and Nuclear Factor Y (NF-Y)72 families have been used to develop transgenic plants and study their performance under stress conditions. The expression of some of these TFs under drought-inducible or root-specific promoters has resulted in improved tolerance to drought, salinity, and temperature stress and a higher yield under water-limited conditions in rice73–76, wheat77, canola78, and maize72.\n\n\nGenomic resources for breeding crops with enhanced abiotic stress tolerance\n\nAs observed for N and P improvement, hundreds of QTLs related to drought and heat tolerance traits have been identified. However, only a few of them have been implemented in appropriate breeding programs for improving crop abiotic stress tolerance. Efforts have been made to improve drought tolerance in rice by using marker-assisted (MAS) breeding79 to identify and characterize the Deeper Rooting 1 (DRO1) QTL that controls the root growth angle80. Higher expression of DRO1 causes a more vertical root growth. Breeding DRO1 into a shallow-rooting rice line enables these plants to avoid drought by increasing the depth of their roots, resulting in a higher grain yield80. The DRO1 gene is the first drought tolerance QTL that was cloned, and its beneficial effects on plant growth further confirmed that the root system architecture plays a crucial role in abiotic stress tolerance. Interestingly, DRO1 has no homology to known proteins, which suggests that cloning genes associated with QTLs could provide completely novel genes for plant breeding. This example shows that a considerable improvement in drought tolerance can be achieved by altering root growth patterns and opens up the possibility of introducing DRO1 in shallow-rooting crops other than rice through the use of genetic engineering (Figure 1).\n\nThe phytohormone abscisic acid (ABA) regulates numerous processes in plants including seed dormancy and the plant responses to low water availability. ABA is perceived by soluble PYR/PYL/RCAR (pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor) receptors that belong to the START superfamily of ligand-binding proteins (for a review see81). It has been shown that constitutive overexpression of ABA receptors improves drought tolerance; however, it negatively affects yield under non-stress conditions82. This suggests that the precise regulation of the activity of individual or multiple receptors will be required to achieve enhanced drought tolerance without a yield penalty. A novel alternative to actively control tolerance to abiotic stress is the use of chemicals that can activate or repress the receptors that sense the stress or the signaling pathways activated by hormones that mediate the corresponding stress responses. Recently it was shown that it is feasible for the case of drought tolerance. Drought tolerance in Arabidopsis was achieved using an engineered PYR1 ABA-receptor that can be activated by an existing non-herbicidal agrochemical that is not a natural inducer of ABA responses. This example opens up a new avenue of crop improvement to regulate abiotic or biotic stress responses at the beginning of, or prior to, the presence of the stress in a timely and quantitative manner by the application of a non-toxic compound, reducing potential yield reductions83.\n\n\nThe hidden enemy in the soil: mechanical impedance\n\nAmong the different types of soil physical degradation, soil compaction is considered one of the most serious problems in agricultural fields because it directly alters the soil structure and modifies intrinsic soil properties, such as porosity, aeration, water potential, and soil strength84. Soil compaction increases soil impedance and thereby affects crop yield by decreasing the capacity of the root system to explore new soil horizons and absorb water and essential nutrients to sustain active growth and development. Several studies have shown that continuous mechanical impedance affects root system architecture by altering root diameter, total root length, and lateral root initiation85–87. Despite the increasing importance of soil compaction resulting from the mechanization of agriculture, this abiotic stress is the least studied to date.\n\nStudying the genetic diversity of root penetration ability could permit the identification and characterization of genes that allow roots to penetrate soils with high impedance. Genotypic variation in root penetration ability has been found in soybean88, rice89, and wheat90. Our group has found that, among Arabidopsis ecotypes, there is wide variation in the capacity of the root system to penetrate substrates with high mechanical impedance (Figure 2). At the molecular level, some studies have attempted to elucidate the detailed mechanosensing and mechanotransduction processes in roots by studying early signaling events during physical stimuli and the role of putative mechanoreceptors91–93. Although important advances have been made in this field, the precise mechanisms and specific root traits that enable roots to penetrate into hard soils remain largely unknown. Several interesting questions still need to be answered with regard to root penetration. Why can some plant species more efficiently penetrate compact soil layers? Which genes are involved in the adaptive root traits that permit some plant species or genotypes to effectively cope with soil compaction problems? And what hormonal changes occur when a plant encounters a below-ground obstacle?\n\nA) Col-0, Kz-9 and Ler Arabidopsis ecotypes show a wide variation in penetrating hard agar layers. Screening test was carried out using a double-phase agar system, which mimics soil compaction condition. B) Quantitative analysis of the root penetration ability expressed as the root penetration percentage (%) in reference to that of Col-0, showed by nine different Arabidopsis ecotypes. (*) indicates statistically significant differences: *P<0.05, **P<0.01, and ***P<0.001 level; n=120 seedlings per ecotype (one-way ANOVA).\n\nThe use of image analysis techniques based on transparent substrates and 3D imaging using X-ray and neutron tomography technologies or fluorescent and luminescent proteins in conjunction with specifically designed devices should improve our understanding of how roots respond to high mechanical impedance with much better resolution, compared with that previously possible at the macroscopic level94–96.\n\nThe plant cell wall consists primarily of polysaccharides that can be broadly classified as cellulose, cellulose-binding hemicelluloses, pectins, and lignins, which confer mechanical stability and allow adequate cell expansion through the regulation of turgor pressure generated inside plant cells97,98. Enzymes, such as endoglucanases, xyloglucan-endotransglycoxylases and expansins, play crucial roles in mediating the rearrangement of the cell wall structure. Modulating the expression of the genes involved in the synthesis and remodeling of cell wall components could allow the modification of root mechanical properties to produce stronger root systems that have a better capacity to penetrate compacted soils (Figure 1). In Arabidopsis, specific TFs, such as MYB58 and MYB63, have been found to activate lignin biosynthetic genes during secondary wall formation99. Therefore, the overexpression of these TFs under root-specific or stress-inducible promoters could result in plant roots that have strengthened cell walls with enhanced tolerance of mechanical restriction100 (Figure 1).\n\nIt is essential to consider root responses to soil compaction in current and future breeding programs. In conjunction with genetic engineering and genome editing technologies, this approach will accelerate the development of crop varieties with enhanced performance in soils degraded by compaction.\n\n\nConcluding remarks\n\nAs discussed above, engineering for tolerance to abiotic stress by manipulating key genes and using multiple tools has allowed the generation of crop plants that are tolerant to drought, extreme temperatures, and salinity, or that have a higher nutrient uptake and use efficiency. A remarkable contribution has resulted from studies with tolerant crop varieties to certain stresses instead of using model genotypes, such as the case of the PSTOL1, suggesting that we must encourage the use of tolerant genotypes in our research.\n\nThe pursuit of master regulators that control abiotic stress and determination of the best way to modulate their expression has been the most important challenge in engineering plant genetics to enhance abiotic stress tolerance. However, rapid advances in genomic technologies for the characterization of QTLs and performing genome-wide association studies101 should facilitate the identification of novel genes for engineering abiotic stress tolerance in crops. The use of systems biology that integrates “omics” data102 and generates mathematical models to achieve a more complete view of the interactions between plant responses to abiotic stress should also facilitate the design of effective strategies to engineer plants with enhanced performance under harsh conditions.\n\nEpigenetic processes, such as DNA methylation, histone modifications, generation of small RNAs (sRNA), and transposable element activity, play essential roles in modulating gene activity in response to environmental stimuli103,104. Indeed, it has been shown that drought adaptive-responses in plants can be transgenerationally transmitted through the action of these processes on specific genes105. Moreover, epigenetic processes are also involved in the switch from C3 to CAM photosynthesis and contribute to adaptation to salt stress in the halophyte Mesembryanthemum crystallinum106. In wheat, the use of the methylation inhibitor 5-azacytidine resulted in increased tolerance to salt stress at the seedling stage107. Therefore, understanding the epigenetic mechanisms that control gene expression in response to environmental cues could also become an important avenue for developing improved crops (Figure 1). However, more information is needed to clarify the complex interaction between abiotic stress responses and epigenetic changes and to identify potential stress-responsive epigenetic modifiers.\n\nWe believe that the most exciting transgenic approaches for producing plant varieties and hybrids that are much less dependent on the application of agrochemicals, including fertilizers and pesticides, have yet to be discovered. The engineering of crops for harsh environments is evolving and will rapidly incorporate new breeding technologies, including genome editing, which has already produced its first commercial product (herbicide-resistant canola). The development of effective approaches for specifically and visibly monitoring certain environmental stresses, such as P deficiency, and timely indicating the degree of the stress is also emerging and providing additional tools for improving crops108. Furthermore, the possibility of activating or repressing the expression of specific genes by introducing site-specific epigenetic changes, such as DNA methylation or histone modifications using a modified version of the CRISPR/Cas9 system99, will drastically modify how agriculture is developed by creating an integral, effective, and sustainable global agriculture. However, translating these approaches from the laboratory or the greenhouse to the field remains challenging. In our opinion, more interdisciplinary research and the active involvement of breeders and agronomists in project planning is necessary to better define project goals and align the interests of researchers with that of crop producers.",
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{
"id": "10204",
"date": "02 Sep 2015",
"name": "Guohua Xu",
"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",
"responses": []
},
{
"id": "10205",
"date": "02 Sep 2015",
"name": "Sigrid Heuer",
"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",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-651
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https://f1000research.com/articles/4-218/v1
|
10 Jul 15
|
{
"type": "Research Article",
"title": "An unexpected effect of TNF-α on F508del-CFTR maturation and function",
"authors": [
"Sara Bitam",
"Iwona Pranke",
"Monika Hollenhorst",
"Nathalie Servel",
"Christelle Moquereau",
"Danielle Tondelier",
"Aurélie Hatton",
"Valérie Urbach",
"Isabelle Sermet-Gaudelus",
"Alexandre Hinzpeter",
"Aleksander Edelman",
"Sara Bitam",
"Iwona Pranke",
"Monika Hollenhorst",
"Nathalie Servel",
"Christelle Moquereau",
"Danielle Tondelier",
"Aurélie Hatton",
"Valérie Urbach",
"Isabelle Sermet-Gaudelus",
"Alexandre Hinzpeter"
],
"abstract": "Cystic fibrosis (CF) is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT) CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml) of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE) leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC) signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular.",
"keywords": [
"Cystic fibrosis",
"epithelium",
"F508del-CFTR",
"inflammation",
"tumor necrosis factor-alpha",
"chloride channel",
"CFTR",
"correctors"
],
"content": "Introduction\n\nCystic fibrosis (CF) is a genetic disease attributable to mutations in the cystic fibrosis transmembrane regulator gene (CFTR). CFTR’s main function is encoding a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, but the most important consequences of mutated CFTR are in the airways, ascribed to both abnormal fluid transportation and excessive inflammatory responses. These abnormalities lead to the bacterial colonization of the lung, causing lung obstruction and resulting ultimately in respiratory insufficiency and death. The primary origin of this inflammatory scenario has been controversial for a long time. Dealing with this question in 2009, we wrote “…many authors consider it secondary to recurrent infections and airway colonization by opportunistic pathogens”1. Today, a growing body of evidence indicates that inflammation and infection in CF can be dissociated, and that a basal inflammatory status preexists pathogen infections2. Pezzulo and colleagues2, studying the relationship between ion transport in trachea and inflammation/infection, showed that inflammation results from bacterial infection and is independent from CFTR function. Nevertheless, reports from 2015 show that inflammation precedes infection in the CF ferret model3.\n\nDifferent studies have established a direct link between ion transport regulation and inflammation1,4. However, there is still insufficient knowledge about how the mediators of inflammation modulate CFTR expression, and consequently, if they modulate ion transport. Furthermore, most of the previous works in this area were performed in cell models over-expressing wild-type (WT) CFTR1,5–8. These studies showed that cytokines could either reduce6, or increase1 CFTR expression and function depending on the cell type and treatment duration. In Calu-3 cells derived from a pulmonary adenocarcinoma, treatment of cells for more than 24h (corresponding to chronic inflammation conditions) with a pro-inflammatory cytokine (TNFα) activated CFTR gene expression at the transcriptional level7, whereas the same treatment reduced CFTR expression in a colon adenocarcinoma-derived cell line (T84)6. The impact of cytokine treatment on epithelial ion permeability was addressed by another study, showing the involvement of complex transduction signaling pathways concerning different mitogen-activated protein (MAP) kinases8.\n\nEven less information exists about the effects of cytokines on CFTR during the acute phase of inflammation. We have previously observed that short-term (10min) treatment of Calu-3 cells by TNFα induces CFTR-dependent eicosanoid production, and CFTR-independent IL-1β secretion1. Additionally, these observations may be extended to the context of F508del/F508del patients, as we have reported that residual activity of CFTR in the nasal epithelium exists in patients with a mild phenotype, suggesting that inflammatory status may be correlated with residual CFTR function9. We hypothesize now that cytokines could affect the expression and function of mutated CFTR during the acute phase of inflammation, being in part responsible for this residual activity. The aim of this study was to evaluate the effects of acute and chronic stimulation by TNFα or IL-1β on F508delCFTR in two cell types: HeLa cells stably expressing F508delCFTR, and primary human bronchial epithelial cells (HBE) derived from F508del homozygous patients.\n\n\nMaterials and methods\n\nHuman recombinant cell culture grade TNFα was purchased from Jena Bioscience GmbH (Jena, Germany); Brefeldin A (BFA; Sigma-Aldrich, St Quentin Falavier, France, B7651); a protein kinase C inhibitor10, GF109203X, (Selleckchem, USA, S7208); Forskolin (from Coleus forskohlii) (Sigma-Aldrich, F6886), Amiloride hydrochloride hydrate (Sigma-Aldrich, A7410-5G); inh-172 (Sigma-Aldrich, C2992-5MG); genistein (Sigma-Aldrich, G6649-5MG). Anti-CFTR antibodies (abs): MM13-4 mouse monoclonal ab against N-terminus of CFTR, (Millipore, France, 05-581); 24-1 mouse monoclonal ab against C-terminus of CFTR (R&D Systems, MAB, 25031). Anti-tubulin abs: rabbit polyclonal anti-tubulin ab (Ab4074) and anti-NaKATPAse mouse monoclonal ab (Ab7671) were from Abcam (Paris, France). For western blot analysis, the secondary polyclonal goat anti-mouse abs were IRDye® 800CW (Li-Cor, Bad Homburg, Germany, 926-32210), anti-keratin 8 mouse monoclonal abs (Progen, Biotechnik GmbH, Heildelberg, Germany, 61038), and anti-NHERF1 (rabbit polyclonal Santa-Cruz Biotechnology, B2107). For immunocytochemistry, anti–Zona occludens 1 (ZO-1) rabbit polyclonal abs were purchased from Santa Cruz Biotechnology, sc-10804; secondary anti-mouse (A-11001) and anti-rabbit (A-24923) Alexa-fluor (488 and 594) IgGs were from Life technologies (Fontenay s/Bois, France). For proximity ligation, the secondary abs were provided by OLINK in the kit (DUO92004 for Duolink® in Situ PLA® Probe anti-mouse MINUS, and DUO92002 for Duolink® in Situ PLA® Probe anti-rabbit PLUS, Bioscience, Uppsala, Sweden). In situ kits for proximity ligation assay were purchased from OLINK. Human primary bronchial epithelial (HBE) cells in air-liquid interface are cultivated on microporous filters purchased from Corning Incorporated (Transwell® polyester membrane cell culture inserts, 6.5mm diameter, New York, USA). IL1β was obtained from ENZO life sciences (ALX-520-001-C010, Villeurbanne, France).\n\n\nCell culture\n\nHeLa cell culture. HeLa cells stably transfected with the wild type CFTR plasmid construct and the mutated F508del-CFTR were kindly provided by Pascale Fanen (INSERM U955, Créteil, France). Cells were cultured in Dulbecco’s modified Eagle medium, supplemented with 10% FCS, 2mM glutamine, 100g/ml streptomycin, 100 units/ml penicillin and 250g/ml Zeocin (all reagents were from Invitrogen) in an incubator at 37°C and 5% CO2.\n\nPrimary Human bronchial epithelial (HBE) cell culture. Primary HBE cells were isolated from bronchial explants of CF and non-CF patients after lung transplantation, with consent approved by Comité de protection des personnes Ile de France II, 2010-05-03 A3. Cells were isolated from bronchial tissue by enzyme digestion and were cultured in differentiation medium (DMEM/F12, supplemented with 15% of fetal calf serum) on type I collagen-coated filters. Briefly, bronchial explants were washed twice with washing medium (Eagle’s minimum essential medium + antibiotics (2.5µg/ml amphotericin B, 150µg/ml tazocillin, 25µg/ml ciprofloxacin) + dithiotheitol (DTT) + DNAse) and at least twice with (Eagle’s minimum essential medium MEM) containing only antibiotics (as above) to remove DTT. Bronchial explants were then incubated for 24h in a differentiation medium containing antibiotics (150µg/ml piperacillin plus tazobactam, 25µg/ml ciprofloxacin), Amphotericin B and protease, at 4°C with constant rotation (1500rpm for 5min). Next day, 15% fetal calf serum was added to neutralize proteases, and bronchia with medium were placed on a Petri dish. Epithelial cells were scraped with a curved scalpel from the inner surface of bronchia, centrifuged (1500rpm, 7min, 4°C) and re-suspended in trypsin (incubation 10min). After that, differentiation medium with serum (FCS) was added and cells were re-centrifuged. Cells were resuspended in an appropriate volume of FCS medium (DMEM/F12, 5% FCS, non-essential amino acids, appropriate antibiotics depending on the patient’s clinical status) and counted. The cells were plated with 106 cells/cm2 to cover apical surface of each filter coated as described above. UG2% medium (DMEM/F12, supplemented with 2% Ultroser G, appropriate antibiotics (amphotericin B, tazocillin, ciprofloxacin, concentration as above) was added to the basal side of filters. The next day, apical medium (FCS) was aspirated and cells were gently washed (to remove cells other than epithelial) with PBS-antibiotics. Starting from the second day of culture, the basal medium was changed daily. Basal medium which passed to the apical compartment was removed daily. Cells were cultured at an air-liquid interface for at least 21 days and were differentiated to form polarized epithelium, after 2 weeks of growth11. Cell differentiation was verified with immunofluorescent staining of markers: α-tubulin for ciliated cells, Zona-occludens (ZO-1) for tight junctions, keratin 8 (K8) for simple epithelia marker, mucin 5 AC (MUC5AC) for goblet cells and CFTR. The transepithelial resistance (RT) of cultures was measured and short circuit current (Isc) experiments were performed on cultures with at least 800Ω/cm2.\n\nTNFα was added to the culture medium for periods of time indicated in the Results section without fetal calf serum except for path-clamp experiments.\n\nProtein sample preparation for CFTR immunoblotting. Sample preparation protocols are described in detail elsewhere12,13. Briefly, cells were washed on ice twice with phosphate-buffered saline (PBS) solution containing 0.1mM CaCl2 and 1mM MgCl2. PBS (Mg2+- and Ca2+-free) was added to scraped cells. A first centrifugation was done at 1500g at 4°C. Cells were resuspended in a hypotonic solution containing 10mM KCl, 10mM TRIS at pH7.4, 1.5mM MgCl2 and homogenized with a mini Potter-Elvehjem tissue grinder. Cells were centrifuged at 15000g at 4°C for 15min. The resulting supernatant was re-centrifuged for 1h at 100000g at 4°C. The pellet was resuspended in a hypotonic solution. The total amount of protein was quantified by Lowry-Folin assay14.\n\nWestern blot analysis. Western blot analyses were performed as described elsewhere with slight modifications15. Briefly, equal amounts of proteins/lane were electrophoresed on an 8% SDS-PAGE and electrotransferred onto nitrocellulose membranes over 2h at 4°C in Tris-glycine buffer (Biorad) at 200 mA. Next, nitrocellulose membranes were incubated in PBS + 0.1% tween20 (Sigma, 9005-64-5) containing 5% milk (Regilait Bio, Supermarket Simply, Paris) saturation solution for 1h. Proteins were immunoblotted for 2h with the MM-13-4 ab (1/1000). After extensive washing, the nitrocellulose membranes were incubated with anti-tubulin (1/5000) or anti-NaKATPase (1/5000) abs. Next, the last washes (three times for 30min in washing buffer: PBS plus 0.1% Tween20) were done. CFTR, tubulin or NaK-ATPase were detected using Odyssey detection system (Li-Cor, Bad Homburg, Germany). The relative protein expression was assessed using the ImageJ 1.47v software (http://imagej.nih.gov/ij/index.html).\n\nELISA analysis. Analysis of interleukin-8 (IL-8) secretion was performed on basolateral culture media (UG2% medium) of primary HBE cells grown at an air-liquid interface after 24h incubation time at 37°C. Basal, non-induced, levels of IL-8 secretion were measured in the regular basolateral culture media after 24h incubation at 37°C. The effect of TNF-α on IL-8 secretion was determined on the same culture filters after 10min, 3h, and 24h incubation with 50ng/ml TNF-α. Stimulation after 10min and 3h of incubation at 37°C with TNF-α were followed by washings and incubation with regular media for 24h, which was used to measure IL-8 secretion. IL-8 secretion levels were determined using ELISA immunoassay (Human CXCL8/IL-8 Quantikine ELISA Kit from R&D Systems) following the manufacturer's instructions.\n\nWhole-cell patch-clamp recordings. The technique for patch-clamp recordings in the whole-cell configuration has been described elsewhere16,17. Stably transfected cells were plated in 35-mm glass bottom plates that were mounted on the stage of an inverted microscope. Patch experiments were performed at room temperature with an Axopatch 200A amplifier controlled by a computer via a digidata 1440 interface (Axon Instruments, USA). Pipettes were pulled from hard glass (Kimax 51) using a Sutter micropipette puller, and the tips were fire-polished. Current recordings were performed using the nystatin-perforated patch-clamp configuration16. The nystatin stock solution (50 mg/ml) was prepared daily in DMSO. The stock solution was diluted (1:250) with the internal solution, which was sonicated for 1min. The internal solution contained the following (in mM): 131 NaCl, 2 MgCl2 and 10 Hepes, pH 7.3 adjusted with NaOH. The bath solution contained (in mM): 150 NaCl, 1 CaCl2, 1 MgCl2, 35 sucrose and 10 Hepes-Na+, pH 7.3, adjusted with NaOH.\n\nCurrents were recorded by application of regular pulses of -60 mV for 1s, with a holding potential of 0 mV and an interval of 3 s.\n\nTo establish the I-V curves, regular voltage pulses were interrupted by a series of 9 voltage jumps (1-s duration each) toward membrane potentials between -100 and +80 mV. CFTR Cl- currents, ICFTR, were activated using 400 µM 8-(4-chlorophenylthio)-cAMP sodium salt (CPT-cAMP) and 100 µM 3-isobutyl-1-methylxanthine (IBMX).\n\nWhen maximal stimulation was reached, cells were bathed with 5 to 50ng/ml of TNFα in the presence of CPT-cAMP and IBMX TNFα solution, and steady-state was achieved after 7 to 10 minutes.\n\nThen 5 µM of the CFTR inhibitor, CFTRinh172, was added to the CPT-cAMP containing perfusion solution (solution +/- TNFα). ICFTR, defined CFTR currents as a difference in current amplitude recorded during maximum stimulation with solution +/- TNFα and maximum inhibition with CFTRinh172. Data were analyzed using the Student’s t-test (Origin Pro 9.1 software, RITME, France); results were considered to be statistically significant if the p value was less than 0.05 (for non-parametric tests, the Mann-Whitney U test was used).\n\nShort-circuit current experiments. For short-circuit current measurements, primary human bronchial epithelial cells (HBE) were grown on permeable filters (0.33-cm2 surface area) at an air-liquid interface for differentiation and then inserts were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA). For all measurements, a Cl- gradient was applied by differential composition of basal and apical Ringer solutions. The basal Ringer solution contains: 145mM NaCl, 3.3mM K2HPO4, 10mM HEPES, 10mM D-Glucose, 1.2mM MgCl2, and 1.2mM CaCl2; and apical solution contains: 145mM Na-Gluconate, 3.3mM K2HPO4, 10mM HEPES, 10mM D-Glucose, 1.2mM MgCl2, 1.2mM CaCl2. Cells were washed for a 30-min stabilization period in Ringer solutions and aerated with 95% O2/5% CO2 at 37°C. Transepithelial resistance (RT) was measured by applying a 15mV pulse and calculating RT by Ohm’s Law. Isc was measured with an EVC4000 Precision V/I Clamp (World Precision Instruments) and registered using a PowerLab 4/30 workstation (AD Instruments, Castle Hill, Australia). During continuous recording of Isc (in voltage-clamp mode) various inhibitors and activators were added. After stabilization of baseline Isc, amiloride (100µM) was added to the apical side of inserts to inhibit the apical epithelial sodium channel (ENaC). Then Forskolin (10µM) and IBMX (100µM) were added to apical and basolateral compartments, followed by Genistein (50µM) and then CFTR inhibitor Inh-172, added apically at a 5µM concentration.\n\nImmunocytochemistry. HeLa cells and polarized epithelial monolayers of HBE cells were fixed with ice-cold acetone for 5min, then rinsed twice with PBS. Permeabilization was done with PBS containing 0.1% Triton X100 for 15min (PBS-T). Cells were then incubated in blocking solution (3% BSA in PBS-T) for 20min. CFTR immuno-detection by confocal microscopy (see below) was performed with p.24-1 antibody diluted 1/300 (for HeLa cells) or 1/100 (for primary HBE cells) in blocking solution, during overnight incubation at 4°C. Accompanying K8 or ZO-1 staining were done simultaneously. Following this, cells were washed four times for 5min each in PBS-T 0.1% and blocked in 10% goat serum (in PBS-T). Goat secondary IgGs conjugated to Alexa 488 and 594 were added for 30min at 1/1000 dilution in 10% goat serum. After a final four washes for 5min each, Vectashield mounting medium containing DAPI (Vector Laboratories, H-1200) was used to mount cells on microscope slides.\n\nConfocal microscopy. Cells were visualized and images captured using Leica TCS SP5 AOBS confocal microscope (Heidelberg, Germany), equipped with 63x/1.4 oil differential interference contrast λ blue PL APO objective. Typically we performed multiple optical xy sections over the cell culture to reconstitute using the ImageJ software v.147, and the 3D reconstitution of polarized epithelia of HBE cells was performed with 3D Viewer plugin in ImageJ.\n\nDNA proximity ligation assay. Cells were grown on round microscopy cover slips and fixed with ice-cold acetone for 5min, then rinsed twice with PBS. In the first step of the proximity ligation assay (PLA) procedure, cells were incubated in bovine serum albumin solution (blocking solution provided by O-link) for 30min at 37°C and then with either two primary anti-keratin-8 mouse monoclonal abs, or mouse monoclonal anti-NHERF1 ab and rabbit polyclonal anti-CFTR ab for 1h at 37°C. After three washes with PBS-T 0.1%, cells were incubated for 1h at 37°C with the PLA probes (secondary abs provided in the kit) specific to mouse and rabbit IgGs, coupled to the oligonucleotides. Cells were then washed three times and incubated with a mixture of ligase and oligonucleotide-connectors (sequences homologous to the oligonucleotides conjugated to PLA probes). Connectors hybridize with PLA probes only when the distance is <40 nm and form a circle which is enzymatically ligated. Following this, polymerase and nucleotides coupled to fluorochromes were added for amplification of circular oligonucleotides as a template, using the PLA probe sequences as primers. Each step of this protocol is separated by washing with PBS-0.1% tween20 solution to remove non-specific interactions. At the end of this procedure, cells were mounted on microscope slides with Vectashield mounting medium containing DAPI and signal was detected as fluorescent orange spots. PLA results are quantitative and presented as number of spots per cell.\n\nStatistical analysis. Experiments were repeated at least three times and analyzed using the unpaired non-parametric Student’s t-test (Mann-Whitney U test) using Graphpad Prism 5 or Origin (see in patch-clamp section).\n\n\nResults\n\nWe first investigated the effect of acute TNFα treatment on HeLa cells stably transfected with F508del-CFTR as a function of time. A representative immunoblot is shown in (Figure 1A) and the relative quantification in Figure 1B. The analysis of microsomal proteins showed that the fully glycosylated mature CFTR (band C) could be detected after a 10–30min treatment with 50ng/ml of TNFα. The effect persisted for 3–6h and decreased after 24h of treatment, suggesting that TNFα might have a very rapid correcting effect on misfolded F508del-CFTR. The same treatment performed on HeLa stably expressing WT-CFTR was without effect (Figure 1C). We then tested if the effect of TNFα was concentration-dependent. Figure 1D shows the quantification of immunoblot analysis of proteins derived from F508del-CFTR HeLa cells treated with different concentrations of TNFα, ranging from to 50ng/ml for 3–6 h. The relative quantification of mature (band C) vs. core-glycosylated F508del-CFTR (band B) showed a maximal effect at 0.5ng/ml TNFα, which did not increased significantly at higher concentrations (Figure 1D).\n\n* indicates significant results. A. F508delCFTR expression after stimulation of cells with 50ng/ml TNFα for 10min, 3–6h and 24h. Band C refers to fully glycosylated F508del-CFTR, band B refers to core glycosylated F508del-CFTR. B. Relative quantification of C/B+C indicating changes in maturation of F508delCFTR after stimulation of cells with 50ng/ml TNFα for 10min, 3-6 and 24 h, p=0,0046, p=0,0234, p=0,69 (NS) for 10mins, 3–6h and 24 h, respectively. C. WT-CFTR expression after stimulation of cells with 50ng/ml TNFα for 10min, 3–6h and 24h. D. Concentration dependence of expression and the changes in maturation of F508delCFTR in response to treatment of cells with 0.5, 10 and 50ng/ml TNFα, p=0.12 (NS), p=0.01, p=0.03, respectively.\n\nImmunoblot analysis data were supported by immunocytochemistry experiments. The treatment of F508del-CFTR expressing HeLA cells with 50ng/ml of TNFα for 3–6h resulted in a marked increase of CFTR, staining suggesting an increase in F508del-CFTR expression and a possible relocalization of F508del-CFTR to the plasma membrane (Figure 2, white arrows).\n\nHeLa cells stably transfected with F508delCFTR were subjected to CFTR immunodetection and analyzed by confocal microscopy (scale bar = 10 µm). Untreated cells (left panels). Cells treated with 50ng/ml TNFα for 30 min (middle panels). Cells treated with 50ng/ml TNFα for 3–6h (right panels). White arrows show a possible membrane localization of F508delCFTR.\n\nIn the next series of experiments, we tested whether TNFα-induced delivery of F508del-CFTR to the plasma membrane was associated with CFTR-Cl- channel function. Using the nystatin-perforated patch-clamp configuration, we observed the activation of a cAMP-dependent Cl- current, which was sensitive to a CFTR inhibitor (inh172, 5μM) attesting to the presence of a CFTR current (ICFTR; Figure 3A, B and C) within 10–30min after addition of 5 or 50ng/ml TNFα to the solution (Figure 3A, B and D). Non-treated control cells did not display ICFTR (Figure 3A). These experiments are in concordance with the biochemical data showing that acute TNFα translocates functional F508del-CFTR to the plasma membrane and therefore behaves like a corrector. Application of the same protocol to HeLa cells expressing WT-CFTR did not change the amplitude of ICFTR (data not shown).\n\n* indicates significant results. A. Representative current traces recorded by holding the membrane potential at 0 mV and by pulsing the voltages in the range -100 mV to +80 mV at 20 mV steps. Current traces recorded: at the basal level (a); in the presence of CPT-cAMP/IBMX (b); in the presence of 50 TNFα+CPT-cAMP/IBMX (c); in the presence of 5 µM CFTRInh172, 50ng/ml TNFα and 400 µM CPT-cAMP/IBMX (d). B. Mean CFTR-related current amplitudes recorded at -60 mV and normalized to cell capacitance in the presence of CPT-cAMP/IBMX (O); in the presence of 50 TNFα+CPT-cAMP/IBMX (X). C. Mean current amplitudes recorded at -60 mV and normalized to cell capacitance (means + SEM, N=8): at the basal level; in the presence of CPT-cAMP/IBMX; in the presence of 50 TNFα+CPT-cAMP/IBMX; in the presence of 5 µM CFTRInh172, 50ng/ml TNFα and 400 µM CPT-cAMP/IBMX. Wilcoxon signed rank test (paired samples): basal vs TNFα p=0.014, TNFα vs CFTRinh 172 p=0.014. D. Dose-response of 0 to 50ng/ml TNFα after 10min: mean CFTR current amplitudes recorded at -60 mV and normalized to cell capacitance (means + SEM; ns for 5ng/ml N=4; p<0.05 for 50ng/ml n=8).\n\nTo test whether other pro-inflammatory cytokines induce F508del-CFTR function, we tested the effects of different concentrations of IL-1β on F508del-CFTR-expressing HeLa cells. Treatment of cells with 10 ng IL-1β for 10–30min did not induce ICFTR (Figure 4A). Treatment of the same cells with 1 or 10 ng of IL-1β for 24h did not change the maturation pattern of F508del-CFTR (Figure 4B).\n\n* indicates significant results. A. ICFTR recorded in F508delCFTR-expressing HeLa cells by patch clamp. (a) Mean CFTR related current-voltage relationships recorded in the presence of 400 µM CPT-cAMP/100 µM IBMX (O) and the presence of 10ng/ml IL1β + CPT-cAMP/IBMX (X). The current was normalized to capacity (pA/pF). B. The normalized CFTR currents are depicted as mean + SEM, in absence (control, white column) or presence of 10ng/ml IL1β for 10min (IL1β, black column) or presence of 50ng/ml TNFα for 10min (TNFα, grey column). IL1β did not significantly increase ICFTR compared to control (NS), but TNFα significantly increased ICFTR compared to IL1β (p=0.03) and control (p=0.03; unpaired Student’s t-test).\n\nTo investigate whether the acute effects of TNFα on F508del-CFTR maturation may have physiological consequences, we performed experiments on primary human bronchial epithelial cells from CF patients homozygous for the F508del mutation, cultured at an air-liquid interface. Confocal microscopy analysis of F508del-CFTR distribution in reconstituted epithelium was performed. Figure 5 shows representative images obtained in HBE cell cultures from three different patients bearing F508del/F508del mutations. Green fluorescence, corresponding to the presence of CFTR protein, increased in cell preparations treated with TNFα (50ng/ml) compared to control, suggesting an increase in F508del-CFTR expression. Furthermore, in TNFα treated cells, F508del-CFTR appeared in the same plane as ZO-1, indicating its apical localization, in contrast to lighter and diffuse cytoplasmic staining in control conditions. The redistribution of F508del-CFTR to the apical side of epithelium occurred within 10min of TNFα 50ng/ml treatment and was sustained over 24h of treatment.\n\nDifferentiated primary HBE cell cultures grown at an air-liquid interface were incubated with 50ng/ml TNFα for 10–30min, 3–6h and 24h. CFTR immunodetection was performed with 24.1 anti-CFTR antibody and analyzed with confocal microscopy. Green staining represents CFTR (Alexa Fluor 488), red color staining represents ZO-1 protein of tight junctions (Alexa Fluor 594) and blue DAPI staining visualizes nuclei. Independent TNFα treatments and CFTR immunodetection were performed on HBE cells from three different F508del/F508del CF patients. Representative images of one experiment are demonstrated (Scale bars = 20µm).\n\nWe investigated the functional consequences of F508del-CFTR insertion in the plasma membrane upon TNFα exposure using Isc measurements. Representative Isc recordings are shown in Figure 6: TNFα treatments of CF HBE cells enhanced the cAMP-sensitive Isc, which is consistent with increased activity of CFTR, as compared to non-treated control cells. Increased responses to Forskolin (Figure 6A and B) as well as potentiation by genistein or inhibition by Inh-172 (Figure 6B) were observed. In these cells, the effect was still visible after 24h of incubation with TNFα (Figure 6). Altogether, these experiments suggest that TNFα exerts a correcting effect during the acute and resolving phases of inflammation, by promoting rapid insertion of F508del-CFTR into the apical membrane of primary HBE cells derived from CF patients.\n\nShort-circuit current experiments on air-liquid cultures of HBE cells from a F508del/F508del CF patient. Amiloride, an inhibitor of Na+ current (ENaC), was present throughout the experiment. Forskolin (10µM) + IBMX (100µM) were added apically and basolaterally to increase the intracellular cAMP to activate the cAMP-dependent currents, followed by addition of genistein (50µM), a phytoestrogen known to potentiate ICFTR. The inhibitor of ICFTR, inh-172 (10µM), was added to the apical Ussing chamber to attest to the presence of ICFTR. A. Treatment of cells for 6h with 50ng/ml of TNFα induced cAMP-dependent, inh172 sensitive chloride current of higher magnitude than control non-treated cells. B. Treatment of cells for 24h with 50ng/ml of TNFα induced cAMP-dependent chloride current, potentiated by genistein and abolished by inh172 of higher magnitude than control cells. Black tracings represent Isc measurements in control untreated cell cultures whereas red tracings visualize Isc measurements in epithelia treated with TNFα.\n\nTo understand how TNFα enables the exit of F508del-CFTR from the ER, we investigated the role of several possible TNFα targets and signaling pathways, including ER to Golgi vesicular transport, keratin 8 and PKC-related signaling.\n\nIn a first set of experiments, cells were pre-treated with BFA, (35µM for 3–4h), an inhibitor of protein transport from the ER to the Golgi apparatus18. Pretreatment with BFA abolished ICFTR induced by exposure of cells to 50ng/ml of TNFα (Figure 7) indicating that vesicular trafficking is involved in TNFα action.\n\nWhole cell Cl- currents recorded in HeLa cells expressing F508delCFTR by patch-clamp experiments. * indicates significant results. A. Representative current traces recorded by holding the membrane potential at 0 mV and by pulsing the voltages in the range -100 mV to +80 mV at 20 mV steps for cells treated by 35 µM brefeldin A (BFA) for 2h. Current traces recorded: at the basal level (a); in the presence of CPT-cAMP/IBMX (b); in the presence of 50 TNFα+CPT-cAMP/IBMX (c); in the presence of CFTRInh172+50 TNFα+CPT-cAMP/IBMX (d). B. Mean CFTR-related current-voltage relationships. Current densities normalized to cell capacitance (pA/pF) were measured in the presence of TNFα (O); on cell treated for 2h by 35µM brefeldin A in the presence of TNFα (d). C. Mean CFTR current amplitudes recorded at -60 mV and normalized to cell capacitance in untreated cells (control; mean + SEM; n=8), in cells treated for 10 minutes by TNFα (50; mean + SEM, n=8) and in cells treated by 35 µM brefeldin A for 2h and for 10 minutes by TNFα (50+BFA; means + SEM, n=6). Statistics: unpaired Student’s t test between (50) and (50 + BFA), p=0.025.\n\nIn a second set of experiments, the keratin-8 proximity to F508del-CFTR in HeLa cells +/- TNFα was evaluated. Using the PLA assay, we tested whether the number of K8-F508del-CFTR pairs that are closer than 40nm was changed by TNFα treatment. The results indicate that incubation with 50ng/ml TNFα for 30min or 3 h had no effect on the number K8-F508del-CFTR pairs (Figure 8A and B) suggesting that the interaction between keratin 8 and F508del-CFTR was not a target of TNFα.\n\nA. Differential interaction between K8 and CFTR in cells treated with TNFα (50ng/ml) for varied durations. DNA proximity ligation assay of K8 and CFTR in HeLa cells transfected with F508delCFTR, scale bar = 10 µm. B. Number of dots corresponding for proteins pairs, Keratin 8 –F508delCFTR, per cell. NS, not significant.\n\nA third series of experiments were designed to investigate the possible role of protein kinase C (PKC) in TNFα action. TNFα has been reported to induce within 30min the insertion of the leptin B receptor into the plasma membrane in a PKC-dependent manner10. To test if this is also the case for F508delCFTR, cells were pre-treated for 2–4h with a PKC inhibitor, GF109203X. GF109203X prevented the TNFα-induced changes on ICFTR, suggesting that a PKC-dependent signaling pathway is involved in this process (Figure 9A and B).\n\n* indicates significant results. A. Mean CFTR-related current-voltage relationships. Current densities normalized to cell capacitance (pA/pF) were measured in the presence of 50 ng/ml TNFα (O); on cell treated for 30 min by 5µM GF109203X in the presence of TNFα (X). B. Mean CFTR current amplitudes recorded at -60 mV and normalized to cell capacitance in untreated cells (control; mean + SEM; n=8), in cells treated for 10 minutes by TNFα (50; means + SEM, n=8) and in cells treated by 5 µM GF109203X for 30min and for 10 minutes by TNFα (50+GF; means + SEM, n=6). Statistics: non-parametric unpaired Student’s t test between (50) and (50 + GF), p=0.047.\n\nAs rescued F508del-CFTR still carries a misfolding mutation, it will be recognized by the peripheral quality control19. Therefore, we wanted to evaluate the effect of TNFα on the stability of rescued F508delCFTR. Because it was shown that the adaptor protein NHERF1 stabilizes CFTR at the plasma membrane20, we investigated whether the number of NHERF1–F508delCFTR protein pairs was modified by TNFα treatment. Using the PLA assay, we observed that the former did not significantly change under TNFα treatment. These results indicate that, while TNFα enables the exit of F508delCFTR from the ER, it does not alter the peripheral quality control (Supplementary Figure S1).\n\n\nDiscussion\n\nIn this study we demonstrate a novel, rapid and unexpected effect of TNFα on F508delCFTR trafficking, maturation and function as a chloride channel. This effect was observed in HeLa cells stably transfected with F508delCFTR, and in HBE cells derived from homozygous F508del CF patients in primary culture, giving credence to the physiological relevance of this effect. Our data suggest that TNFα – induced ICFTR is due to the release of misfolded F508delCFTR from the ER to the Golgi apparatus and the subsequent insertion of late Golgi vesicles into the plasma membrane. This TNFα action was found to be dependent on PKC activity. In HeLa cells expressing F508delCFTR, the TNFα – induced ICFTR activity is transient, but in CF patients’ cells it lasted for 24h, suggesting that it may occur during chronic inflammation.\n\nTNFα has been extensively described to play a major role in the inflammatory process by inducing cytokine release from inflammatory cells as well as bronchial epithelial cells21,22. TNFα induces IL-8 and IL-1β synthesis and secretion from adenocarcinoma lung cancer cells, Calu-3 cells1. IL-1β has recently been reported to stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signalling23. For these reasons, possible involvement of other inflammatory mediators in the F508delCFTR response to TNFα could not be excluded. However, using a similar protocol as for TNFα, we report here that IL-1β, another pro-inflammatory cytokine, did not affect ICFTR. Therefore, the effect of TNFα on F508delCFTR, described here, seems to be specific to this cytokine, and is most likely distinctive of its stimulatory effect on pro-inflammatory cytokines.\n\nThe role of TNFα in enhancing chloride transport through F508delCFTR is consistent with its function in immunity. Indeed, host defense and efficient mucociliary clearance is achieved by the stimulation of chloride transport and subsequent regulation of airway surface hydration. Other mediators have been reported to play a role in epithelial transport, including pro-inflammatory mediators, such as prostaglandins, leukotrienes and interferon gamma24–26 as well as pro-resolution mediators27. In other models, such as the colon adenocarcinoma-derived cell line T84, the same treatment reduced CFTR expression and function6. In the present study, the TNFα-induced F508delCFTR activity was transient in HeLa cells, but in HBE cells from CF patients this effect was sustained over 24h. Taken together our data provide evidence for a novel effect of TNFα in stimulating F508delCFTR maturation and activation during both the acute and chronic phases of inflammation.\n\nThe rapid insertion of membrane proteins into the plasma membrane following short-term treatment by TNFα has been previously described for other membrane proteins. For example, it was observed for the leptin receptor, a primary regulator of leptin signaling believed to regulate energy homeostasis, reproduction and immunity10, and for an injury-promoting receptor in motor neurons, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptor, involved in amyotrophic lateral sclerosis28. Both proteins are inserted into plasma membrane in a PKC-dependent manner, by a mechanism that may be common, at least in part, to the one uncovered by our study on F508delCFTR. However, there is a marked difference between these studies and our observations. In the case of leptin receptor and AMPA, it is the correctly-folded proteins that are inserted into plasma membrane in response to TNFα treatments. On the contrary, TNFα has no effect on WT-CFTR, whereas it promotes insertion of an abnormally folded and prematurely degraded protein, F508delCFTR. Therefore, our results suggest that the underlying mechanisms of action between the effect of TNFα on wild-type proteins and abnormally folded proteins must differ. One possible explanation is a differential regulation of kinases. Our preliminary results indicate that ERK2 phosphorylation is diminished by TNFα within 10min and remains low for 24h. It is therefore possible that the PKC pathway, involved in F508delCFTR translocation to the plasma membrane leads to the decreased phosphorylation of ERK2. Indeed, the fact that one of PKC isoforms, PKCδ, activates ERK supports this hypothesis29. Nevertheless, the phosphorylation status of ERK1/2 in the context of transepithelial ion transport in the presence of TNFα has not been investigated. Conversely, other authors8 have reported that ERK1/2 are not involved in the TNFα-induced decrease in transepithelial resistance of human epithelial cells, and in the prevention of these effects by probiotics, although they did not determine the phosphorylation status of the kinases8. In any case, these observations would concern WT-CFTR, i.e. the properly folded protein, which under our experimental conditions is not regulated by TNFα. Of note, the chronic treatment of intestinal cells by TNFα leads (>24h) to decreased expression of WT-CFTR6,30. Thus, our study opens a new field of investigation into those signaling pathways activated by TNFα and/or other cytokines during the maturation of wild-type and misfolded proteins.\n\nThe translocation of F508delCFTR to the plasma-membrane upon exposure to TNFα and the inhibitory effect of BFA on TNFα-activated ICFTR suggest that TNFα-induced insertion of vesicles containing F508delCFTR proteins from Golgi into plasma membrane enhances cAMP-dependent chloride currents (ICFTR). Conversely, the keratin 8–F508delCFTR protein complex recently shown by us as an unwanted interaction preventing the escape of F508delCFTR from the degradation pathways17,31,32 seems not to be involved in this process.\n\nTNFα acts on the trafficking of F508delCFTR through the Golgi apparatus since blocking of vesicular exit from ER by BFA prevents the development of ICFTR (Figure 6). At later times, F508delCFTR may be stabilized at the plasma membrane by favoring the formation of a protein macrocomplex through interaction with NHERF1. This is supported by two observations: first, it has previously been reported that a multiprotein complex (NHERF1-CFTR-ezrin-actin) plays a significant role in maintaining tight junction organization and function in cystic fibrosis epithelial cells33. Second, it is known that NHERF1 itself prevents F508delCFTR from degradation20. Even if the number of F508delCFTR-NHERF1 protein complexes was not changed by short term treatment with TNFα, NHERF1 and/or other proteins that bind to the PDZ domain of CFTR might play a stabilizing role. Within this line of investigation, we and others have demonstrated that CFTR forms a protein complex with TNFα receptor, p11, Annexin 1 and cPLA21,34. Within 10 min TNFα treatment is sufficient to relocate CFTR, together with these four proteins, to lipid raft-like detergent-resistant microdomains. How this mechanism relates to the observations described in the present study and to the potential implication of NHERF1 remains to be investigated.\n\nIn agreement with our observations, all studies related to the rapid effects of TNFα on membrane proteins mentioned in this manuscript1,10,28,34 suggest that this pro-inflammatory cytokine very rapidly modifies the composition of the plasma membrane, which may (in the case of F508delCFTR) lead to profound changes in ion transport. It has also been reported that VX-809, a corrector for F508delCFTR, stabilizes NHERF1-F508delCFTR at the plasma membrane35. We propose that TNFα is one of the players in the stabilization of F508delCFTR at the plasma membrane, at least for 24h after the onset of inflammation. It is tempting to hypothesize that either TNFα behaves as VX-809 or TNFα and VX-809 actions could act in parallel.\n\nOur observations are important as they highlight a novel perspective on airway inflammation in the context of CF that could open unexpected avenues in the understanding of correcting mechanisms. Indeed, it signifies that TNFα action is, at least, not opposed to the treatment. It has to be remembered, however, that during chronic inflammation, other mediators may have different behaviors. We propose that systematic studies on acute and chronic effects of inflammation mediators on F508delCFTR trafficking and ICFTR should be undertaken in the context of correcting treatments.\n\nFinally, the effect of TNFα on F508delCFTR maturation may provide a partial explanation for the residual activity of F508delCFTR in patients with a mild CF phenotype9. We propose systematic testing in CF patients of TNFα levels and, when possible, association of these tests with nasal potential measurements, systematic immunocytochemistry of F508delCFTR in nasal cells, and determination of TNFα blood concentration. A potential correlation between these parameters and CF phenotype could be useful as a prognostic marker of disease evolution.\n\nIn summary, a corrector-like effect of TNFα on F508delCFTR raises the question of the role played by acute inflammation in CF patients during treatment with correcting compounds.\n\n\nData availability\n\nFigshare: Raw data for Bitam et al., 2015 ‘An unexpected effect of TNFα on F508del-CFTR maturation and function.’. doi: 10.6084/m9.figshare.147615636",
"appendix": "Author contributions\n\n\n\nSB designed and performed experiments, analyzed, presented all results, IP made experiments on CFHBEO cells, MH performed IL1ß experiments, NS made patch-clamp experiments on TNFα, CM made WB analysis, DT made duolink experiments and analysed the results, AH helped with short-circuit current measurements, VU, IS, AH co-wrote the manuscript, AE supervised the whole work and wrote the manuscript. All authors have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThis study was supported by the French Agency of Research grant CORCF ANR-13-BSV1-0019-01 (A. Edelman), French foundations ‘Vaincre la Mucoviscidose’ (grants obtained by A. Edelman, A. Hinzpeter and S. Bitam) and ‘Mucoviscidose-ABCF2’ grants (A. Edelman and I. Sermet-Gaudelus).\n\nI confirm that the 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 Mario Ollero for English editing.\n\n\nSupplementary material\n\nNumber of pairs F508delCFTR-NHEFR1 in controls in TNFα-treated (as indicated) cells Number of cells for control NHERF1-WTCFTR: 39 ± 13 n=90, NHERF1-F508delCFTR: 42 ± 6, n= 80 cells; cells treated for 30min: NHERF1-WTCFTR 33 ± 7.7 n= 114 cells, NHERF1-F508delCFTR 35 ± 7.1 n= 106; treated for 3 h: NHERF1-WTCFTR: 51 ± 9.1 n= 80 cells, NHERF1-F508delCFTR: 40 ± 8.2 n=104 cells, p= NS in all conditions.\n\n\nReferences\n\nBorot F, Vieu DL, Faure G, et al.: Eicosanoid release is increased by membrane destabilization and CFTR inhibition in Calu-3 cells. PloS One. 2009; 4(10): e7116. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPezzulo AA, Tang XX, Hoegger MJ, et al.: Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature. 2012; 487(7405): 109–113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKeiser NW, Birket SE, Evans IA, et al.: Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs. Am J Respir Cell Mol Biol 2015; 52(6): 683–94. PubMed Abstract | Publisher Full Text\n\nVeit G, Bossard F, Goepp J, et al.: Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia. Mol Biol Cell. 2012; 23(21): 4188–4202. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaudouin-Legros M, Colas J, Moriceau S, et al.: Long-term CFTR inhibition modulates 15d-prostaglandin J2 in human pulmonary cells. Int J Biochem Cell Biol. 2012; 44(6): 1009–1018. PubMed Abstract | Publisher Full Text\n\nBesançon F, Przewlocki G, Baró I, et al.: Interferon-gamma downregulates CFTR gene expression in epithelial cells. Am J Physiol. 1994; 267(5 Pt 1): C1398–1404. PubMed Abstract\n\nBrouillard F, Bouthier M, Leclerc T, et al.: NF-kappa B mediates up-regulation of CFTR gene expression in Calu-3 cells by interleukin-1beta. J Biol Chem. 2001; 276(12): 9486–9491. PubMed Abstract | Publisher Full Text\n\nResta-Lenert S, Barrett KE: Probiotics and commensals reverse TNF-alpha- and IFN-gamma-induced dysfunction in human intestinal epithelial cells. Gastroenterology. 2006; 130(3): 731–746. PubMed Abstract | Publisher Full Text\n\nSermet-Gaudelus I, Vallée B, Urbin I, et al.: Normal function of the cystic fibrosis conductance regulator protein can be associated with homozygous (Delta)F508 mutation. Pediatr Res. 2002; 52(5): 628–635. PubMed Abstract | Publisher Full Text\n\nGan L, Guo K, Cremona ML, et al.: TNF-α up-regulates protein level and cell surface expression of the leptin receptor by stimulating its export via a PKC-dependent mechanism. Endocrinology. 2012; 153(12): 5821–5833. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJeanson L, Kelly M, Coste A, et al.: Oxidative stress induces unfolding protein response and inflammation in nasal polyposis. Allergy. 2012; 67(3): 403–412. PubMed Abstract | Publisher Full Text\n\nBaudouin-Legros M, Hamdaoui N, Borot F, et al.: Control of basal CFTR gene expression by bicarbonate-sensitive adenylyl cyclase in human pulmonary cells. Cell Physiol Biochem. 2008; 21(1–3): 75–86. PubMed Abstract | Publisher Full Text\n\nLipecka J, Bali M, Thomas A, et al.: Distribution of ClC-2 chloride channel in rat and human epithelial tissues. Am J Physiol Cell Physiol. 2002; 282(4): C805–816. PubMed Abstract | Publisher Full Text\n\nLowry OH, Rosebrough NJ, Farr AL, et al.: Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1): 265–275. PubMed Abstract\n\nBensalem N, Ventura AP, Vallée B, et al.: Down-regulation of the anti-inflammatory protein annexin A1 in cystic fibrosis knock-out mice and patients. Mol Cell Proteomics. 2005; 4(10): 1591–1601. PubMed Abstract | Publisher Full Text\n\nHinzpeter A, Lipecka J, Brouillard F, et al.: Association between Hsp90 and the ClC-2 chloride channel upregulates channel function. Am J Physiol Cell Physiol. 2006; 290(1): C45–C56. PubMed Abstract | Publisher Full Text\n\nOdolczyk N, Fritsch J, Norez C, et al.: Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain. EMBO Mol Med. 2013; 5(10): 1484–1501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarie M, Sannerud R, Avsnes Dale H, et al.: Take the ‘A’ train: on fast tracks to the cell surface. Cell Mol Life Sci. 2008; 65(18): 2859–2874. PubMed Abstract | Publisher Full Text\n\nOkiyoneda T, Barrière H, Bagdány M, et al.: Peripheral Protein Quality Control Removes Unfolded CFTR from the Plasma Membrane. Science. 2010; 329(5993): 805–810. PubMed Abstract | Publisher Full Text\n\nKwon SH, Pollard H, Guggino WB: Knockdown of NHERF1 enhances degradation of temperature rescued DeltaF508 CFTR from the cell surface of human airway cells. Cell Physiol Biochem. 2007; 20(6): 763–772. PubMed Abstract | Publisher Full Text\n\nMartin LD, Rochelle LG, Fischer BM, et al.: Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators. Eur Respir J. 1997; 10(9): 2139–2146. PubMed Abstract | Publisher Full Text\n\nBerger M: Inflammatory mediators in cystic fibrosis lung disease. Allergy Asthma Proc. 2002; 23(1): 19–25. PubMed Abstract\n\nClauzure M, Valdivieso AG, Massip Copiz MM, et al.: Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function. PloS One. 2014; 9(6): e99257. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScheckenbach KEL, Losa D, Dudez T, et al.: Prostaglandin E2 regulation of cystic fibrosis transmembrane conductance regulator activity and airway surface liquid volume requires gap junctional communication. Am J Respir Cell Mol Biol. 2011; 44(1): 74–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchaeffer BE, Zadunaisky JA: Leukotriene modulation of chloride transport in frog cornea. Invest Ophthalmol Vis Sci. 1986; 27(6): 898–904. PubMed Abstract\n\nLi R, Maminishkis A, Banzon T, et al.: IFN{gamma} regulates retinal pigment epithelial fluid transport. Am J Physiol Cell Physiol. 2009; 297(6): C1452–1465. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVerrière V, Higgins G, Al-Alawi M, et al.: Lipoxin A4 stimulates calcium-activated chloride currents and increases airway surface liquid height in normal and cystic fibrosis airway epithelia. PloS One. 2012; 7(5): e37746. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYin HZ, Hsu CI, Yu S, et al.: TNF-α triggers rapid membrane insertion of Ca2+ permeable AMPA receptors into adult motor neurons and enhances their susceptibility to slow excitotoxic injury. Exp Neurol. 2012; 238(2): 93–102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUeda Y, Hirai Si, Osada Si, et al.: Protein kinase C activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf. J Biol Chem. 1996; 271(38): 23512–23519. PubMed Abstract | Publisher Full Text\n\nNakamura H, Yoshimura K, Bajocchi G, et al.: Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene. FEBS Lett. 1992; 314(3): 366–370. PubMed Abstract | Publisher Full Text\n\nColas J, Faure G, Saussereau E, et al.: Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect. Hum Mol Genet. 2012; 21(3): 623–634. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdelman A: Cytoskeleton and CFTR. Int J Biochem Cell Biol. 2014; 52: 68–72. PubMed Abstract | Publisher Full Text\n\nCastellani S, Guerra L, Favia M, et al.: NHERF1 and CFTR restore tight junction organisation and function in cystic fibrosis airway epithelial cells: role of ezrin and the RhoA/ROCK pathway. Lab Invest. 2012; 92(11): 1527–1540. PubMed Abstract | Publisher Full Text\n\nDudez T, Borot F, Huang S, et al.: CFTR in a lipid raft-TNFR1 complex modulates gap junctional intercellular communication and IL-8 secretion. Biochim Biophys Acta. 2008; 1783(5): 779–788. PubMed Abstract | Publisher Full Text\n\nArora K, Moon C, Zhang W, et al.: Stabilizing rescued surface-localized δf508 CFTR by potentiation of its interaction with Na+/H+ exchanger regulatory factor 1. Biochemistry. 2014; 53(25): 4169–4179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBitam S, Pranke I, Hollenhorst M, et al.: Raw data for Bitam et al., 2015 ‘An unexpected effect of TNFα on F508del-CFTR maturation and function’. Figshare. 2015. Data Source"
}
|
[
{
"id": "9762",
"date": "03 Aug 2015",
"name": "Renaud Beauwens",
"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\nCystic Fibrosis is one of the most common genetic diseases attributable to mutations in the cystic fibrosis transmembrane regulator (cftr) gene which encodes a cAMP-dependent Cl- channel, the CFTR protein present in the apical membrane of numerous epithelia. Inflammation is a hallmark of the disease contributing to its severity and has been suggested to arise independently of bacterial infection. The study by Bitam et al. questions whether mediators of inflammation affect CFTR expression and ion transport function. The most frequent mutation DF508 leads to a functional protein that is prematurely degraded. The authors investigated the effect of tumor necrosis factor a (TNFa), a proinflammatory cytokine in DF508-CFTR-transfected HeLa cells as well as in human bronchial cells expressing DF508-CFTR in primary culture. In both cases, exposure to TNFa (0.5 – 50 ng/ml) for 10 min enhanced the maturation of DF508-CFTR with appearance of its glycosylated form at the plasma membrane and CFTR-mediated chloride current inhibitable by the specific CFTR inhibitor (CFTRinh 172). The effect was already observed within 10 min and lasted at least 24 h. It involves binding to TNFa receptor, activation of protein kinase C and vesicular trafficking to the plasma membrane. TNFa induced no change in expression of WT CFTR in control cells.In summary this study shows that TNFa behaves as a corrector molecule. If sustained treatment also lead to similar increased maturation at low dose of TNFa, it could even be considered as a possible additive in the therapeutic armamentarium.The study is very interesting, well done and well-illustrated by confocal microscopy and it certainly deserves indexing.",
"responses": []
},
{
"id": "9637",
"date": "25 Aug 2015",
"name": "Bruno Miroux",
"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 report, Bitam et al. investigated the putative correcting function of TNFα in the targeting of DF508-CFTR protein to the plasma membrane. Experiments were performed on two different biological model systems: Hela stable cell lines expressing either wt or DF508-CFTR protein and a more physiologically relevant system, i.e. primary Human Bronchial Epithelial cells (HBE), isolated from bronchial explants of CF and non-CF patients after lung transplantation. In the first part of the study, they not only convincingly showed that TNFα increases cell surface expression of F508del-CFTR in HeLa cells but also that it restores the chloride conductance function of CFTR. To prove the physiological relevance of these initial findings the authors performed the same acute treatment on HBE cells and showed that it increases the expression of F508del-CFTR at the plasma membrane in a functional state. More importantly, in this model, acute stimulation of cells with TNFα had a long lasting effect, up to 24H, suggesting that TNFα could be useful for the treatment of the disease. The second part of the paper is devoted to the search of TNFα mechanism of action. Using inhibitors of various signaling pathways they showed that TNFα acts at least at two different levels: vesicular trafficking and PKC dependent signaling pathway. Altogether these set of data revealed a very interesting and intriguing correcting function of TNFα in the targeting of F508del-CFTR to the plasma membrane. Experimental evidences are statistically robust and well described. The paper is overall well written and discussed and deserves indexation.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-218
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https://f1000research.com/articles/4-642/v1
|
28 Aug 15
|
{
"type": "Review",
"title": "Advances in understanding cartilage remodeling",
"authors": [
"Yefu Li",
"Lin Xu",
"Lin Xu"
],
"abstract": "Cartilage remodeling is currently among the most popular topics in osteoarthritis research. Remodeling includes removal of the existing cartilage and replacement by neo-cartilage. As a loss of balance between removal and replacement of articular cartilage develops (particularly, the rate of removal surpasses the rate of replacement), joints will begin to degrade. In the last few years, significant progress in molecular understanding of the cartilage remodeling process has been made. In this brief review, we focus on the discussion of some current “controversial” observations in articular cartilage degeneration: (1) the biological effect of transforming growth factor-beta 1 on developing and mature articular cartilages, (2) the question of whether aggrecanase 1 (ADAMTS4) and aggrecanase 2 (ADAMTS5) are key enzymes in articular cartilage destruction, and (3) chondrocytes versus chondron in the development of osteoarthritis. It is hoped that continued discussion and investigation will follow to better clarify these topics. Clarification will be critical for those in search of novel therapeutic targets for the treatment of osteoarthritis.",
"keywords": [
"Cartilage remodeling",
"osteoarthritis",
"transforming growth factor-beta 1",
"aggrecanase",
"chondrocytes",
"chondron"
],
"content": "\n\nCartilage remodeling is a continuous process in which the existing cartilage is removed (or degraded) and replaced by new cartilage (regenerated). The balance between degradation and regeneration has been considered so critical that tipping the scale toward degradation results in osteoarthritis (OA). Therefore, an understanding of the molecular basis of the degradation and regeneration processes will undoubtedly provide valuable information for those in search of novel therapeutic protocols for the treatment of OA. In this review article, we focus on several recent discoveries on the topic of articular cartilage degeneration.\n\nWith regard to the remodeling of the extracellular matrix, mature articular cartilages are, in general, considered relatively quiescent tissues. For example, a study by Verzijl et al. indicates that the half-life of collagens in human mature cartilages is 117 years1. The long half-life of the collagens indicates a slow turnover of the collagens in the cartilages. However, the half-life of the aggrecan (the large monomer) turnover is about 3.4 years2. This suggests that the rate of the turnover may be different in the different parts of the extracellular matrix. For example, the rate of the turnover is high in the aggrecan-rich pericellular matrix of chondrocytes and the rate of the turnover is low in the collagen-rich interterritorial and territorial matrices in articular cartilages3.\n\nAlthough this review focuses on articular cartilages, we have to point out that OA is currently considered the consequence of the whole joint failure. In addition to articular cartilages, the subchondral bone, peri-articular cartilage, synovial membrane, ligaments, and menisci contribute to the development of OA4.\n\n\nBiological effect of transforming growth factor-beta 1 β on developing and mature articular cartilages\n\nIs transforming growth factor-beta 1 (TGF-β1) a culprit or protector in the development of OA? Currently, numerous pharmaceutical companies are considering the use of TGF-β1 as a stimulant to repair damaged articular cartilage for the treatment of OA. Is this the correct choice?\n\nTGF-β1 has been considered an anabolic factor to articular chondrocytes, based largely on results from in vitro and ex vivo experiments in which TGF-β1 can stimulate chondrocytes to synthesize and release proteoglycans and type II collagens5,6. In addition, three independent mouse genetic studies7–9 demonstrate that Tgf-β1 is required for the formation of articular cartilage at early stages of development in mice. Without Tgf-β1, articular cartilage is not formed properly, eventually an immature joint becomes an OA-like joint in mice. Moreover, a human genetic study reports that a two-nucleotide deletion, 741-742del AT (nonsense mutation), in SMAD-3 causes early-onset OA in a human family10. All of the aforementioned results support the argument that TGF-β1 is a protector against the development of OA. Unfortunately, the situation is not that simple. Numerous other independent investigations suggest that TGF-β1 may, in fact, be a factor in joint destruction. First, studies with animal models, by Itayem et al., suggest that intra-articular injections of TGF-β1 into adult rat knee joints cause early onset of OA11,12. Second, a human genetic study reports that a nucleotide change, 859C>T or 782C>T in SMAD-3, increases the level of TGF-β1 and activity of the TGF-β1 signaling pathway in human families is associated with early-onset OA10. This is in agreement with the observation from two other studies indicating that the level of TGF-β1 is significantly higher in human OA tissues than in healthy articular cartilages13,14. Third, we found increases in the expression of Tgf-β1 and of p-Smad2/3 in articular chondrocytes of knee joints in mouse models of OA15. The increased expression of p-Smad2/3 was associated with elevated expression of a serine protease, high-temperature requirement A1 (HtrA1), in the chondrocyte. HTRA1 is capable of degrading extracellular matrix molecules, particularly most pericellular components of articular chondrocytes16. Another independent research group also demonstrates that TGF-β1 induces HTRA1 in human primary chondrocytes17. Fourth, we determined whether the removal of Tgf-β type II receptor (Tgfbr2) from the articular cartilage of adult knee joints could attenuate the OA progression. We deleted Tgfbr2 in the articular cartilage of adult mouse knee joints and then subjected the mice to destabilization of the medial meniscus (DMM). We found a significant disparity in the progression of articular cartilage degeneration in knee joints between mice with or without Tgfbr2 at 8 and 16 weeks following the surgery. The progression toward OA was significantly (P <0.05) delayed in Tgfbr2−/− mice.\n\nSeveral studies also indicate that the increase in the amount of TGF-β1 in other joint tissues has detrimental effects on adult joints. A study by Maeda et al. suggests that a high level of TGF-β1 does more harm than good to the tendon18. One study by Bakker et al. reports that the constitutive overexpression of active TGF-β1 in adult mouse knee joints results in OA associated with an increase in the production of proteoglycans in articular cartilage, hyperplasia of synovium, and chondro-osteophyte formation19. A study by Zhen et al. demonstrates that inhibition of TGF-β1 signaling in mesenchymal stem cells of subchondral bone delays the development of OA in adult mice20.\n\nHow can this “conflicting” role of TGF-β1 in the pathogenesis of OA be explained? One plausible explanation is that effective TGF-β1 signaling acts in a dose-dependent manner. In this scenario, an appropriate level of TGF-β1 is required for the development and maintenance of articular cartilages. Therefore, TGF-β1 below or above this level results in articular cartilage degeneration. However, results from our study with mice without Tgfbr2 suggest that the TGF-β1 dose-dependent manner may not be the case. Another plausible explanation is that effective TGF-β1 signaling acts in a developmental stage-dependent manner. In this scenario, TGF-β1 is required for the development of articular cartilage; however, once a joint is formed, TGF-β1 is no longer needed. In any case, induction of TGF-β1 in an adult joint causes articular cartilage degeneration. Therefore, inhibition activity of TGF-β1, not application of TGF-β1, may be considered in treatment of OA in mature joints.\n\n\nAre ADAMTS4 and ADAMTS5 key enzymes in articular cartilage destruction?\n\nProteoglycans are the basic elements of articular cartilage and are indispensable in the ability of articular cartilage to resist compressive pressure. Thus, much of the effort in the OA research field is focused on the search for an enzyme, or enzymes, that degrades proteoglycan. In 1999, two enzymes, ADAMTS-4 and ADAMTS-5, were cloned21,22. Both of these aggrecanases degrade aggrecans (proteoglycans). This indicates that both aggrecanases may be ideal therapeutic targets in the development of disease-modifying OA drugs. In fact, two independent research groups used mouse gene-targeting techniques to delete ADAMTS-4 or ADAMTS-5. One group found that the deletion of ADAMTS-5 could protect aggrecan from being degraded in a mouse model of inflammatory arthritis. With regard to the development of OA, it is not clear whether both aggrecanases are involved in cartilage destruction23. The results from another group demonstrated that the removal of ADAMTS-5 in mice could significantly delay the progressive process of articular cartilage degeneration at 4–8 weeks following the DMM surgery24. This suggests that ADAMTS-5 may play a role in early stages of OA development. However, lack of evidence indicating elevated expression of ADAMTS-5 at early stages of articular cartilage degeneration in any one of the existing mouse models of OA raises a question as to how important a role this enzyme has in the development of OA.\n\nMore importantly, a recent study indicates that the expression of ADAMTS-5 is increased in the articular cartilage of knee joints in adult mice because of inactivation of Sox925. The elevated expression of ADAMTS-5 is associated with the disappearance of aggrecans. Surprisingly, there is no progression of articular cartilage degeneration in this model. This is contrary to our current understanding that aggrecans are indispensable for articular cartilage health. Consistent with this observation, another independent investigation indicates that an increase in the expression of bone morphogenetic protein 2 (Bmp2) elevates levels of the neo-epitope, VDIPEN341, of aggrecan in articular cartilage without inducing an acceleration of cartilage degeneration in mice. Furthermore, Davidson et al. find that the increased expression of Bmp2 does not exacerbate the degenerative condition of articular cartilage that has been induced by the DMM in mouse knee joints26.\n\nOne plausible explanation for the aforementioned observation is that the loss of proteoglycans alone may not be sufficient to initiate or accelerate articular cartilage degeneration. Instead, the degradation of both proteoglycans and type II collagen may be required in the development of OA. Interestingly, a study by Karsdal et al. demonstrates that articular cartilage degradation is completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation but irreversible after induction of metalloprotenase (MMP)-mediated aggrecan and collagen type II degradation27. This study suggests that the aggrecanases may be involved with the reversible processes of cartilage degradation (or extracellular matrix turnover) but MMPs cause the irreversible degeneration of articular cartilages.\n\nThere is evidence that the removal of ADAMTS-5 may protect joints against OA by stabilizing subchondral bone28. Thus, it will be important to understand whether aggrecanases play roles in the development of OA through other joint tissues.\n\nWe discuss the aggrecanases in this brief review. However, other enzymes, such as MMPs, elastase, and cathepsins, also play important roles in the pathogenesis of OA.\n\n\nChondrocytes versus chondron in the development of osteoarthritis\n\nPrimary chondrocytes and chondrocyte cell lines are the primary tools for in vitro experiments in cartilage research and repair. They have allowed a wealth of information to be obtained about the genetic regulation of chondrocyte function, activation of signaling pathways, and gene expression profiles in chondrocyte response to chemical or mechanical stimulation. Many investigators use chondrocytes in vitro to study physiological and pathophysiological events while mimicking in vivo biological conditions. In particular, researchers almost exclusively use primary chondrocytes as a resource to regenerate functional articular cartilage. Regarding this method, however, a question remains: are primary chondrocytes alone adequate for investigating the role of chondrocytes in OA development and articular cartilage repair?\n\nChondrocytes and their pericellular matrix are considered to be the primary structural and functional units, termed chondrons, of articular cartilage29–35. This concept was proposed by Benninghoff in 1925. About 40 years later, Szirmai further evaluated the structure of the chondron by a more systematic analysis. At that time, however, the chondron was not widely recognized as a functional unit. Some 20 years later, C.A. Poole’s research group completed additional experiments to physically isolate chondrons from cartilage and showed that chondrons are true anatomic and functional entities. Chondrons consist of chondrocytes, the pericellular matrix, and a capsule surrounding the pericellular matrix. The pericellular matrix contains laminin, fibronectin, biglycan, decrin, fibromodulin, matrilin 3, and cartilage oligo matrix protein (COMP). The pericellular capsule is composed mostly of type VI and IX collagen and proteoglycans. The capsule and the pericellular matrix separate chondrocytes from the adjacent interterritorial or territorial matrices containing type II collagen. Clearly, under normal conditions, type II collagen is not exposed to chondrocytes. It is conceivable that disruption of the pericellular matrix exposes chondrocytes to type II collagen and can alter the metabolic events in chondrocytes, eventually leading to cartilage destruction. In fact, results from human and mouse genetic studies indicate the significant role of the pericellular matrix in protecting articular cartilage against the development of OA. For example, the deficiency of one or a combination of two components of the pericellular matrix, such as type VI collagen, type IX collagen, matrilin 3, decrin, biglycan, and fibromodulin, results in early onset of OA in mice36–40. In human genetic studies, mutations in type IX collagen and COMP are associated with OA41–46.\n\nIn 1998, a study by Lee and Loeser provided evidence that the pericellular matrix of chondrocytes could play critical roles in the maintenance of normal metabolic activities of chondrocytes in articular cartilage and that the disruption of the pericellular matrix is associated with articular cartilage degeneration47. Very interestingly, the significant role of the pericellular matrix of a cell is also demonstrated in neural tissue. A study by Gogolla et al. points out that the perineuronal net (pericellular matrix of neurons) in the amygdala plays a significant role in protecting the neurons from the loss of “fear memory”48. The authors also find that functional and structural changes of sensory systems are caused by the absence of a perineuronal net in the visual cortex. The significant role of the pericellular matrix of a neural cell in the brain coincides with its perceived role within the chondron. If that is the case, maintaining the integrity of the pericellular matrix will be one of the key issues in protection against OA. We believe that more attention should be directed toward the pericellular matrix of chondrocytes for the identification of novel biomarkers and therapeutic targets for OA.\n\nData from a very recent investigation provide more evidence that chondrons are not only the functional unit in the maintenance of articular cartilage homeostasis but also the basic elements in the regeneration of articular cartilage. Chondrons derived from adult articular cartilage are more efficient than chondrocytes in the regeneration of articular cartilage. This information is particularly critical for the articular cartilage repair field49.\n\nCollagen type VI is one of the major components of the capsule of the pericellular matrix. Results from one very recent study indicate that soluble collagen type VI can be a stimulant for chondrocyte proliferation. The soluble collagen type VI may also prevent proliferating chondrocytes from being dedifferentiated in vitro50. It is well known that chondrocyte dedifferentiation is one of the major obstacles in cartilage tissue repair. A study by Zelenski et al. shows that the deletion of type VI collagen alters the mechanical properties of the pericellular matrix of chondrocytes51. This, in turn, increases the extent of cell swelling and osmotically induced transient receptor potential cation channel subfamily V member 4 (TRPV4) signaling in an age-dependent manner. These findings suggest that alterations in pericellular matrix properties can influence mechanotransduction via TRPV4 or other ion channels, which eventually leads to articular cartilage destruction.\n\nIn summary, chondrons, instead of primary chondrocytes or chondrocyte cell lines, may be a more appropriate choice for investigating the biological functions and effects of chondrocytes in the development of OA and cartilage repair.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have 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\nVerzijl N, DeGroot J, Thorpe SR, et al.: Effect of collagen turnover on the accumulation of advanced glycation end products. J Biol Chem. 2000; 275(50): 39027–31. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMaroudas A, Bayliss MT, Uchitel-Kaushansky N, et al.: Aggrecan turnover in human articular cartilage: use of aspartic acid racemization as a marker of molecular age. Arch Biochem Biophys. 1998; 350(1): 61–71. PubMed Abstract | Publisher Full Text\n\nWilusz RE, Sanchez-Adams J, Guilak F: The structure and function of the pericellular matrix of articular cartilage. Matrix Biol. 2014; 39: 25–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoeser RF, Goldring SR, Scanzello CR, et al.: Osteoarthritis: a disease of the joint as an organ. 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PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBakker AC, van de Loo FA, van Beuningen HM, et al.: Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthritis Cartilage. 2001; 9(2): 128–36. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZhen G, Wen C, Jia X, et al.: Inhibition of TGF-β signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat Med. 2013; 19(6): 704–12. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTortorella MD, Burn TC, Pratta MA, et al.: Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. Science. 1999; 284(5420): 1664–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAbbaszade I, Liu RQ, Yang F, et al.: Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol Chem. 1999; 274(33): 23443–50. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nStanton H, Rogerson FM, East CJ, et al.: ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature. 2005; 434(7033): 648–52. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGlasson SS, Askew R, Sheppard B, et al.: Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 2005; 434(7033): 644–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHenry SP, Liang S, Akdemir KC, et al.: The postnatal role of Sox9 in cartilage. J Bone Miner Res. 2012; 27(12): 2511–25. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDavidson ENB, Vitters EL, Bennink MB, et al.: Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage. Ann Rheum Dis. 2015; 74(6): 1257–64. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKarsdal MA, Madsen SH, Christiansen C, et al.: Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity. Arthritis Res Ther. 2008; 10(3): R63. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBotter SM, Glasson SS, Hopkins B, et al.: ADAMTS5-/- mice have less subchondral bone changes after induction of osteoarthritis through surgical instability: implications for a link between cartilage and subchondral bone changes. Osteoarthritis Cartilage. 2009; 17(5): 636–45. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPoole CA, Flint MH, Beaumont BW: Chondrons extracted from canine tibial cartilage: preliminary report on their isolation and structure. J Orthop Res. 1988; 6(3): 408–19. PubMed Abstract | Publisher Full Text\n\nPoole CA, Ayad S, Schofield JR: Chondrons from articular cartilage: I. Immunolocalization of type VI collagen in the pericellular capsule of isolated canine tibial chondrons. J Cell Sci. 1988; 90(Pt 4): 635–43. PubMed Abstract\n\nPoole CA, Wotton SF, Duance VC: Localization of type IX collagen in chondrons isolated from porcine articular cartilage and rat chondrosarcoma. Histochem J. 1988; 20(10): 567–74. PubMed Abstract | Publisher Full Text\n\nPoole CA, Honda T, Skinner SJ, et al.: Chondrons from articular cartilage (II): Analysis of the glycosaminoglycans in the cellular microenvironment of isolated canine chondrons. Connect Tissue Res. 1990; 24(3–4): 319–30. PubMed Abstract | Publisher Full Text\n\nPoole CA, Matsuoka A, Schofield JR: Chondrons from articular cartilage. III. Morphologic changes in the cellular microenvironment of chondrons isolated from osteoarthritic cartilage. Arthritis Rheum. 1991; 34(1): 22–35. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPoole CA: Articular cartilage chondrons: form, function and failure. J Anat. 1997; 191(Pt 1): 1–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunziker EB, Michel M, Studer D: Ultrastructure of adult human articular cartilage matrix after cryotechnical processing. Microsc Res Tech. 1997; 37(4): 271–84. PubMed Abstract | Publisher Full Text\n\nHu K, Xu L, Cao L, et al.: Pathogenesis of osteoarthritis-like changes in the joints of mice deficient in type IX collagen. Arthritis Rheum. 2006; 54(9): 2891–900. PubMed Abstract | Publisher Full Text\n\nAlexopoulos LG, Youn I, Bonaldo P, et al.: Developmental and osteoarthritic changes in Col6a1-knockout mice: biomechanics of type VI collagen in the cartilage pericellular matrix. Arthritis Rheum. 2009; 60(3): 771–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWadhwa S, Embree M, Ameye L, et al.: Mice deficient in biglycan and fibromodulin as a model for temporomandibular joint osteoarthritis. Cells Tissues Organs. 2005; 181(3–4): 136–43. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWadhwa S, Embree MC, Kilts T, et al.: Accelerated osteoarthritis in the temporomandibular joint of biglycan/fibromodulin double-deficient mice. Osteoarthritis Cartilage. 2005; 13(9): 817–27. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nvan der Weyden L, Wei L, Luo J, et al.: Functional knockout of the matrilin-3 gene causes premature chondrocyte maturation to hypertrophy and increases bone mineral density and osteoarthritis. Am J Pathol. 2006; 169(2): 515–27. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBorochowitz ZU, Scheffer D, Adir V, et al.: Spondylo-epi-metaphyseal dysplasia (SEMD) matrilin 3 type: homozygote matrilin 3 mutation in a novel form of SEMD. J Med Genet. 2004; 41(5): 366–72. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHecht JT, Nelson LD, Crowder E, et al.: Mutations in exon 17B of cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia. Nat Genet. 1995; 10(3): 325–9. PubMed Abstract | Publisher Full Text\n\nMuragaki Y, Mariman EC, van Beersum SE, et al.: A mutation in the gene encoding the alpha 2 chain of the fibril-associated collagen IX, COL9A2, causes multiple epiphyseal dysplasia (EDM2). Nat Genet. 1996; 12(1): 103–5. PubMed Abstract | Publisher Full Text\n\nMustafa Z, Chapman K, Irven C, et al.: Linkage analysis of candidate genes as susceptibility loci for osteoarthritis-suggestive linkage of COL9A1 to female hip osteoarthritis. Rheumatology (Oxford). 2000; 39(3): 299–306. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBönnemann CG, Cox GF, Shapiro F, et al.: A mutation in the alpha 3 chain of type IX collagen causes autosomal dominant multiple epiphyseal dysplasia with mild myopathy. Proc Natl Acad Sci U S A. 2000; 97(3): 1212–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nCzarny-Ratajczak M, Lohiniva J, Rogala P, et al.: A mutation in COL9A1 causes multiple epiphyseal dysplasia: further evidence for locus heterogeneity. Am J Hum Genet. 2001; 69(5): 969–80. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLee GM, Loeser RF: Interactions of the chondrocyte with its pericellular matrix. Cell Mater. 1998; 8: 135–149. Reference Source\n\nGogolla N, Caroni P, Lüthi A, et al.: Perineuronal nets protect fear memories from erasure. Science. 2009; 325(5945): 1258–61. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVonk LA, de Windt TS, Kragten AHM, et al.: Enhanced cell-induced articular cartilage regeneration by chondrons; the influence of joint damage and harvest site. Osteoarthritis Cartilage. 2014; 22(11): 1910–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSmeriglio P, Dhulipala L, Lai JH, et al.: Collagen VI enhances cartilage tissue generation by stimulating chondrocyte proliferation. Tissue Eng Part A. 2015; 21(3–4): 840–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZelenski NA, Leddy HA, Sanchez-Adams J, et al.: Type VI Collagen Regulates Pericellular Matrix Properties, Chondrocyte Swelling, and Mechanotransduction in Mouse Articular Cartilage. Arthritis Rheumatol. 2015; 67(5): 1286–94. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "10169",
"date": "28 Aug 2015",
"name": "Richard Loeser",
"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",
"responses": []
},
{
"id": "10170",
"date": "28 Aug 2015",
"name": "Farshid Guilak",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-642
|
https://f1000research.com/articles/4-639/v1
|
27 Aug 15
|
{
"type": "Correspondence",
"title": "Double blinding requirement for validity claims in cognitive-behavioral therapy intervention trials for major depressive disorder. Analysis of Hollon S, et al., Effect of cognitive therapy with antidepressant medications vs antidepressants alone on the rate of recovery in major depressive disorder: a randomized clinical trial",
"authors": [
"Douglas Berger"
],
"abstract": "This paper will focus on problems in the inability to double-blind cognitive-behavioral therapy (CBT) studies for major depressive disorder (MDD), and provides an analysis of a recently published study to show how this problem can lead to faulty conclusions.A study by Hollon et al. published in JAMA Psychiatry that compared an antidepressant medication-only arm with a combined CBT/antidepressant arm concluded that the cognitive therapy/antidepressant combination enhanced the recovery rates compared with antidepressant alone, and that the magnitude of this increment nearly doubled for patients with more severe depression.We propose that for subjects with greater severity, there could have been both antidepressant efficacy as well as more hope and expectation in the group who knew they had received combined cognitive therapy/medication, leading to an erroneous conclusion of greater efficacy for the combined group. The large subject number in this study could easily lead to an erroneous finding on statistical testing as a small amount of bias in the subjects adds-up.We opine that the conclusions of unblind CBT outcome research in conditions with subjective endpoints such as MDD need to be given with great caution. The validity of CBT (and its derivatives such as dialectical behavioral therapy) for indications other than MDD is also part of a larger problem in the inability to blind outcome studies for these interventions.",
"keywords": [
"psychotherapy",
"cognitive-behavioral therapy (CBT)",
"outcome studies",
"blinding",
"clinical trials"
],
"content": "\n\nThe validity of cognitive-behavioral therapy (CBT) efficacy for major depressive disorder (MDD) is widely accepted and is based largely on clinical intervention studies of CBT in MDD. However, clinical trials for CBT cannot be carried out under double-blind conditions as would be required of pharmacotherapy (or other somatic therapies), thus the rigor of CBT interventional studies is quite different from those modalities that can be studied under double-blinded conditions1,2.\n\nTreatment allocation cannot be blinded in CBT studies because the subjects have to actively participate in cognitive restructuring tasks. More than just saying a study was “blinded”, absolute concealment of what treatment was allocated is crucial in order to avoid bias3.\n\nCBT trials are sometimes stated to be, “single-blind” because the persons who rate the symptoms that subjects report are blind to the treatment allocation of the subject. The term “single-blind”, however, should be used with caution as single-blind is defined as the condition when subjects are blind, not the raters4. Blind (or “masked”) raters only record whatever bias may be in the subjective reports of the subjects that can be swayed by the unblinded conditions. Emphasizing that raters are blind in a CBT study can distract from the issue that subjects and treaters are not blind.\n\nAllocation concealment is crucial for indications with subjective outcomes as in MDD3. During a clinical trial, subjects with MDD report changes in the severity of subjective depressive symptoms that may be influenced by an expectation or hope for improvement3. Only interventional studies for indications with objective endpoints can ignore potential bias from lack of blinding. For example, mortality rates, MI incidence, stroke, etc. where random error is small1. In this line, a meta analysis of CBT trials that controlled for blinding found treatment effects to be small in MDD5.\n\nHowever, studies continue to report positive results of unblinded trials without voicing strong caution on the validity of the results. Hollon et al. in the October 2014 issue of JAMA Psychiatry compared an antidepressant medication only arm with a combined cognitive therapy/antidepressant arm6. All the subjects who received antidepressants did so under unblinded conditions. The cognitive therapy subjects and their treaters were also unblind to the treatment given. The study concluded that the cognitive therapy/antidepressant combination enhanced the rate of recovery compared with antidepressant alone, and that the magnitude of this increment nearly doubled for patients with more severe depression with little evidence of benefit for patients with less severe MDD. Only one line at the end of the discussion noted that the unblinded conditions could be a limitation.\n\nAn alternative conclusion could just as easily be that patients with greater severity MDD may have included more patients with a medication-responsive depression7. For those subjects with greater severity, there could have been both antidepressant efficacy as well as more hope and expectation in the group who knew they had received combined cognitive therapy/medication leading to an erroneous conclusion of greater efficacy for the combined group. A large sample size (N) as in this study is not necessarily a sign of robust results. A large N can create a significant finding on statistical testing as a small amount of bias in the subjects adds-up1. Our alternative conclusion may also be incorrect, the important issue is that the lack of allocation concealment in the study design does not allow any valid conclusion to be made either way. The antidepressant in each arm of the study provides the same amount of hope and expectation; the CBT arm has the added potential for bias from hope and expectation.\n\nIn addition, combining and comparing antidepressants that have market approval based on double-blinded placebo controlled outcome research with CBT, heretofore never studied under double-, or single-blinded conditions, in the same unblinded study is a serious problem. Handicapping one intervention group (antidepressants without the double-blinded placebo control needed for proof of efficacy), while providing advantage to another intervention group (unblinded CBT with no psychotherapy placebo which allows bias in one arm) which is then mixed with the handicapped group, confounds the study conditions and invalidates the design logic of a clinical trial.\n\nTo be sure, interventional studies for somatic therapies such as medications may also have elements of allocation non-concealment requiring caution in their interpretation. While medications can feasibly be blinded, side-effects may expose a subject to the fact that they are in the active-drug arm of a study. An exit analysis on the proportion of subjects in a study that correctly guessed the treatment arm they were in should be done, and the results of any study in an indication with subjective endpoints such as MDD that has evidence of unblinding should be suspect to have bias. Psychotherapy treatment, on the other hand, is virtually impossible to hide from the subject who is openly given the treatment. Whether medication, psychotherapy, or other intervention, no valid scientific assessment of efficacy can be made if a hurdle such as double-blinding in the study design of an indication with subjective endpoints is not rigorously implemented.\n\nAuthors must state clearly when an intervention cannot be studied with rigor, and conclusions need to be given with great caution when studies with subjective endpoints are unblinded. There is no regulatory authority like the FDA to review and approve a psychotherapeutic intervention for MDD, so that both professionals and society at large alike are dependent on the sound-bite conclusions made by authors and commentators on the results reported.\n\nThe critical problem of the inability to double-blind CBT clinical trials for MDD requires further evaluation by research groups who do not have a vested interest in CBT or related therapies. The validity of CBT (and its derivatives such as dialectical behavioral therapy) for indications other than MDD is part of a larger problem in the inability to blind outcome for these interventions.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed. The author has no financial interests, activities, relationships, and affiliations other than those affiliations listed in the title page of the manuscript. There was no data collected or analyzed for this paper.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPiantadosi S: Clinical Trials: A Methodologic Perspective, Second Edition. New York: Wiley-Interscience, 2005. Publisher Full Text\n\nHanrahan C, New JP: Antidepressant Medications: The FDA-Approval Process and the Need for Updates. Ment Health Clin. 2014; 4(1): 11–16. Publisher Full Text\n\nSchulz KF, Grimes DA: Blinding in randomised trials: hiding who got what. Lancet. 2002; 359(9307): 696–700. PubMed Abstract | Publisher Full Text\n\nFriedman LM, Furgerg CD, DeMets DL: Fundamentals of Clinical Trials, Third Edition. Springer; 1998. Reference Source\n\nLynch D, Laws KR, McKenna PJ: Cognitive behavioural therapy for major psychiatric disorder: does it really work? A meta-analytical review of well-controlled trials. Psychol Med. 2010; 40(1): 9–24. PubMed Abstract | Publisher Full Text\n\nHollon SD, DeRubeis RJ, Fawcett J, et al.: Effect of cognitive therapy with antidepressant medications vs antidepressants alone on the rate of recovery in major depressive disorder: a randomized clinical trial. JAMA Psychiatry. 2014; 71(10): 1157–1164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhan A, Leventhal RM, Khan SR, et al.: Severity of depression and response to antidepressants and placebo: an analysis of the Food and Drug Administration database. J Clin Psychopharmacol. 2002; 22(1): 40–45. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10880",
"date": "26 Oct 2015",
"name": "Rebecca Graham",
"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, a reasonably well-written summary of the complexities evident in CBT intervention trials for MDD. The only suggestion I would make is that the grammar should be checked carefully prior to publication - especially the correct use of commas. Other than that - would agree with indexation.",
"responses": []
},
{
"id": "13511",
"date": "09 May 2016",
"name": "Arif Khan",
"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. Berger has taken to task a sacred cow in our field. Psychotherapy works and any challenges to it amount to sacrilegious position! Unfortunately for the dogmatic, Dr. Berger is right. It is not possible to truly 'blind' a psychotherapy study. Simply put, the model of blinded raters is limited as both the patient and therapist are not blinded. Whether one likes it or not, the enthusiasm and conviction of a therapist is more than porous. The Freudian model is that a cigar smoking therapist begets a cigar smoking patient. In the same note the sham therapist's conviction and enthusiasm (or a lack of it) will also be picked up by the patients. We have shown that the level of blinding does change expectations and expectations for the psychotherapy + pharmacotherapy results in the expected increased benefit.1Having said this, it would be foolish of me to suggest that psychotherapy has no value. After all, practice of medicine, psychiatry and psychology is intrinsically and intricately based on human interactions. So, training and practicing of psychotherapy is essential, just like saying you have to know anatomy before you start surgery. It is an essential skill in humanistic sciences. However, just like proving you need to know anatomy before you cut somebody up surgically cannot really be tested using current models and the same applies to psychotherapy. In this context, it is important to note that different types of psychotherapy are better than other ones. This is like testing a surgical knife for different surgeries. All the psychotherapies (if practiced well) are effective just like all knives are effective. It is only a poor clinician who tries to treat a patient without the understanding and application of psychotherapy principles to distressed patients. Here, I would like to note that Dr. Berger is a known psychotherapist. Thus, his position is courageous and to be lauded.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-639
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https://f1000research.com/articles/4-635/v1
|
27 Aug 15
|
{
"type": "Review",
"title": "The role of connective tissue in the embryology of the musculoskeletal system: towards a paradigm shift",
"authors": [
"Carolina Marchuk",
"Carla Stecco",
"Carla Stecco"
],
"abstract": "This paper presents a review of literature regarding the role of embryological connective tissue in the formation of muscles and the organization of the musculoskeletal system. The intention is to introduce a potential paradigm shift with regards to understanding peripheral coordination of movement and movement patterns. This new perspective could improve comprehension of the normal physiological function of connective tissue and, whenever it is pathological, resultant symptoms. Furthermore, this paper briefly discusses some implications of this paradigm shift in the interpretation of movement patterns, posing further questions for future research.",
"keywords": [
"connective tissue",
"embryology",
"anatomy and physiology",
"movement patterns",
"fascial manipulation."
],
"content": "Introduction\n\nBefore being ejected from the ovarian follicle, the ovum is covered by a double sac: theca interna and theca externa (Moore & Persaud, 1999). Once released, the ovarian follicle is covered — like every other cell — by a phospholipid bilaminar membrane, which acts as a double sac surrounding the content of the cell. During ovulation, the ovum released from the follicle is surrounded by another membrane, a lining of mucopolysaccharides’ translucent gel called zona pellucida. After fertilization, the ovum divides itself an indefinite number of times within the zona pellucida. This shell of ‘fundamental substance’ covering the zygote constitutes the first meta-membrane of the organism. This is the first connective tissues product to fulfill this function, followed by the reticulum and collagen fibroid components. This ooze is the primordial organismic membrane as well as the original organismic environment. A little amount of cytoplasm leaves the two daughter cells in the process of the first division and it creates a thin layer of fluid which surrounds the two cells and between the cells and the zona pellucida. (Moore & Persaud, 1999). This is the interstitial or lymphatic fluid, the principal means of exchange in the community of cells within the organism. The cells keep dividing and five days later they form a hollow sphere of cells called the blastocyst. During the following two weeks the blastocyst invaginates and undergoes gastrulation, a process in which pseudopods form in each corner of the blastocyst that join with other cells to make a whole, then a crater, and finally a tunnel, differentiating into two layers: an internal and an external one (Moore & Persaud, 1999). Thus, three spaces are created: the inside of the internal sac, the environment of the external sac and a space in between these two. When invagination is complete, it gives rise to two double sacs: the amniotic sac and the yolk sac surrounded by three germinal disc layers consisting of Ectoderm, Mesoderm and Endoderm (Figure 1).\n\nThese layers are intertwined between the two sacs in the following way:\n\nThe Ectoderm will form the nervous system, neural networks and skin in contact with the amniotic sac and fluid.\n\nThe Mesoderm will form the muscles, the connective tissues (so being the precursor of the fibrous net), the blood, lymph, kidneys, part of the genital organs and the adrenal cortex glands. It is located in the middle of the ectoderm and the endoderm.\n\nThe endoderm will form the tracts of our blood vessels, the organs of the digestive system and the glands. It is in contact with the yolk sac.\n\nWe will now focus on the formation of the fascial network within the mesoderm or the middle layer. During the second week of embryological development, there is hardly any cellular differentiation; almost all cells are exact copies of each other. However, as the embryo develops, growing at an exponential rate, there is also further specialization which means that spatial reorganization becomes essential (Figure 2). At the center of the mesoderm there is a thickening called the notochord, which gives rise to the spinal column, vertebrate bodies and intervertebral discs. To the sides of the paraxial mesoderm there is a special section known as mesenchyme (literally ‘tissue in the middle’) (Snyder, 1975). Mesenchymal stem cells are progenitors of fibroblasts and other connective tissue cells. Mesenchymal stem cells migrate among cells to settle in the three linings and they secrete reticular fibers (a primitive form of collagen) into the interstitial space. These fibers are chemically connected, much like velcro, to form a network that covers all the body (Schultz & Freitis, 1996). The mesenchymal stem cells are scattered throughout the body in order to differentiate themselves into other cell types where needed. They are a perfect example of the remarkable ability of connective tissue to adapt and respond to the continuous changes of the organism. Mesenchymal cells gradually replace the reticular fibers with the collagen fibers forming an only one fibrous net in the organism. That is the reason why favor the singular “fascia” over the plural “fasciae” (Myers, 2009). Once the three layers and the fascial cohesive network are established, it begins to fold onto and over itself repeatedly. The mesoderm covers the endoderm reaching to the ventral zone, where, from its in-between position, it gives rise to the ribs, the abdominal muscles and the pelvis. At the same time, it lodges and supports the endodermic digestive tract within (Cinnamon, 1999). The mesoderm moves to the dorsal zone where it forms the neural arch of the spinal cord as well as the cranial cavity that covers and protects the Central Nervous System (CNS) (Duprez, 2002). The palate is one of the last folds to connect together (when this process is not completed, a cleft lip results) (Moore & Persaud, 1999). In the musculoskeletal system, the internal sac fixed to bone is referred to as the periosteum, but when it is a fibrous cuff surrounding an articulation it is known as an articular capsule. These connective tissue structures are always connected and they are continuous with one another throughout the body. The external sac forms, deforms, reforms and stabilizes different tensional lines in the intermuscular septum or investing fascia according to the growth and movement to which they are subjected. The tissue’s tensional lines and the direction of the muscular fibers start from this process (Myers, 2009). The pattern of Tcf4 expression will determine how the muscles of the limbs within the mesoderm will be formed. From the beginning, the limbs will be organized according to these characteristics: from proximal to distal, from dorsal to ventral and from anterior to posterior. The signal that Tcf4-expressing cells give at a local level is through the secretion of inductive factors, or also through the deposition of an instructive extracellular matrix to induce myogenic precursors to distinguish into myotubes, which are in specific areas in the limb, serving as the nucleus for the anatomical formation of a future muscle. Following these primary signals, secondary signals join and are integrated to produce an upregulation of Tcf4 in specific areas of the limb mesoderm (Blasi et al., 2015; Kardon et al., 2003). Connective tissue controls the patterns of muscle formation by guiding the migration of myoblasts through the mesoderm (Brand-Seberi & Christ, 1999). Towards the end of the fifth week, future muscular cells are grouped together into two portions: a smaller epimere portion, organized in myotomes, and a larger hypomere portion in the ventral region. These portions subsequently form the epaxial and hypaxial muscles, respectively. Nerves that traverse segmental muscles, are also divided into a primary dorsal ramus for the epimere and a ventral ramus for the hypomere. They accompany the muscles throughout their migratory path (Figure 3). In particular:\n\nPectoralis Major PM; pectoralis minor pm; intramuscular septa ims; intermuscular septa IMS. Department of Anatomy at the University of Salvador, School of Medicine, Buenos Aires, Argentina.\n\nVoluntary muscles of the head: from the neural crest cells derive connective tissue elements that guide the patterns of muscle formation in the head; they derive from paraxial mesoderm (somitomeres and somites) and include the muscles of the tongue, the eye (some of them) and are associated with the pharyngeal arches (visceral) (Langman´s Medical Embryology, 2001).\n\nVoluntary muscles of extremities: the mesenchyme is derived from dorsolateral cells of the somites, which move from each limb bud to form the muscles; this occurs near the seventh week of development and can be seen as a condensation (Chevallier et al., 1977; Cristo et al., 1977; Duprez, 2002; Zhi et al., 1996). From somatic mesoderm derives the connective tissue that makes the muscle pattern formation of the limb bones (Langman´s Medical Embryology, 2001; Sadler, 2001).\n\nSpinal nerves play an important role in the differentiation and motor innervation of the limb musculature, and they, as well, provide sensory innervation to dermatomes (Langman´s Medical Embryology, 2001).\n\nSobolevskii explains in his article that \"Comparative anatomy of the spinal cord of the terrestrial, aquatic and semi-aquatic mammals of animals\" to the nerve fascicle takes its origin from the spinal cord and elongation of nerve bands, going from the spinal cord to spinal ganglia.\n\nClassically, more attention is given to the connections between the muscles and the skeleton via the tendons of origin and insertion. The recruitment of muscle fibers generates mechanical tension that, via these tendon connections, produces either musculoskeletal movement or maintenance of a static position, thus stabilizing the body (Turrina et al., 2012). Different authors (Hujing, 2003; Huijing & Baan, 2001a; Huijing & Baan, 2001b; Stecco, 2009a; Stecco, 2009b) describe myotendinous expansions that extend from the deep fascia, or extramuscular connective tissue, of muscles to attach to the deep fascia in adjacent segments, periarticular soft tissues, intermuscular septa, interosseous membranes and neurovascular sheaths (Platzer, 1978; Standring et al., 2005; Tidball & col., 1991). Due to these connections, muscles acquire additional surfaces for leverage and the generation of movement (Huijing & Jaspers, 2005; Stecco, et al., 2010a; Stecco, et al., 2010b; Yucesoy et al., 2010). Via these myotendinous expansions, which are a type of extension of the tendons, muscles can directly stretch deep fascia in a number of body segments in a longitudinal sense (Huijing, 2009; Stecco et al., 2007a). They can also stretch it transversally through intramuscular connective tissue (endomysium, perimysium and epimysium) (Huijing & Jaspers, 2005; Monti et al., 1999; Purslow, 2010), as well as through other parts of the musculoskeletal connective tissue (such as the intermuscular septum and neurovascular bundles).\n\nIt is also well established that, during a muscular contraction, not all motor units within recruited muscles are activated simultaneously (Finni et al., 2003; Pappas et al., 2002). The presence of intramuscular connective tissue plays a fundamental role in the synchronization of the many variables involved in force production. (Rowe, 1981), as does the retinacula of deep fascia (Sanchis-Alfonso & Rosello-Sastre, 2000).\n\nIntramuscular connective tissue, from the macroscopic to the microscopic aspect, can be defined as:\n\nEpimysium: thicker than the other elements of the intramuscular tissue, it is formed by large diameter collagen fibers (Sakamoto, 1996). In the extreme of the muscle the epimysium thickens before it merges with the tendons of origin and insertion (Benjamin, 2009) and converges in the paratenon, while the muscle belly, which it covers, forms a thin layer that determines the volume of the muscle. Wherever epimysium of two muscles connects, it forms a connective tissue sheath that envelops vessels and nerves destined to reach these muscles. Between the collagen fibers, there is the ground substance, which is rich in hyaluronic acid and other glycosaminoglycans (McCombe et al., 2001). In particular, the hyaluronic acid component allows collagen fibers to slide freely, allowing relative mobility of intrafascial layers (Stecco et al., 2011).\n\nPerimysium: surrounding the muscle and the tendon of origin and insertion of the same muscle, this perimysium does not present a solution of continuity with the epimysium. The amount of perimysium within each muscle and each region of the body varies. The perimysium divides the muscle belly into fascicles of different sizes. Histologically, perimysium has a smaller percentage of elastic fibers and especially collagen fibers type I, III, IV, V, VI and XII, immersed in a matrix of proteoglycans (Kurose et al., 2006; Petibois et al., 2006). The collagen type I fibers provide the perimysium with great resistance to traction. It is probable that, in the transmission of force generated by the muscle towards bone levers, intramuscular connective tissue play an important role (Turrina et al., 2012). In addition, in between two adjacent muscle bundles is placed the same perimysium that contacts with both fascicles by its opposite surface.\n\nEndomysium: is the thinner portion of intramuscular connective tissue. It is directly in contact with the sarcolemma and therefore with every single muscle fiber. Covering the muscular fiber in its entire surface, it takes on a structural role, similar to the parenchyma within organs (Moore, 1983). Endomysium creates a network that unites adjacent fibers. It is the only intramuscular element that, within each muscular fascicle, has contact with the individual muscular fibers innervated by the same motor unit. Other muscular fibers may be interposed but they may remain inactive during the activation of a given motor unit. Thanks to the endomysium, the muscle fibers that are not recruited represent a ‘true tendon’ for the transmission of force laterally, without having to change length (Trotter, 1990). To analyze the properties and structure of a muscle must take into account the arrangement of fibers in relation to the aponeurosis and tendons. (Finni et al., 2003; Turrina et al., 2012) Figure 4.\n\nDepartment of Anatomy at the University of Salvador, School of Medicine, Buenos Aires, Argentina.\n\nIt is evident that right from embryological development, the fascia determines the structural and functional characteristics of the muscle.\n\nDuring evolution: the independence of the various segments meant that the myomeres and myosepta were required to align themselves according to new lines of force, thus abandoning metamerism altogether. Previously the entire musculature of a body intervened every time it was moved in any direction, or plane, but at this stage each part of the body required its own musculature (Chiarugi, 1975; Stecco, 2004). In this way, the method of Fascial Manipulation organized into six myofascial units, formed by mono and biarticular fibres as well as muscle spindles, were created for each segment.\n\nThe evolutionary process proceeded in the following manner:\n\n- at first metameres lengthened according to the lines of tension;\n\n- then the myosepta, or metameric septa, in part joined with the unidirectional muscle fibres to form the muscle spindles and, in part, surrounded the entire muscular mass to form the epimysium (Chiarugi, 1975; Stecco, 2004).\n\nFrom the observation of this process in bony fishes we find that lateral flexion of the trunk stimulated the myomeres to elongate in a cephalo-caudal sense which in turn induced the myosepta-fasciae to align parallel to the traction. Hence, these fibres lengthened and they connected to a number of segments (Figure 5).\n\nDepartment of Anatomy at the University of Salvador, School of Medicine, Buenos Aires, Argentina.\n\nAs a consequence, the myosepta-fasciae, which are no longer metameric, lengthened between the muscle fibres to form the perimysium. The deep muscles maintained parallel fibres between one vertebra and the next, somewhat similar to the first metameric stage. The more superficial muscles however formed links extend over a number of metameres. The multisegmentary muscular fibres have the muscle spindles, which are placed in parallel to them, as their point of reference. The muscle spindle is the connection between the unidirectional muscular fibres and the relative portion of the primordial fascia (Stecco, 2004). Only the presence of the muscle spindles, plus the activation of the muscular fibres in succession, allows for the arrest of movement at any angle of the entire joint range. Due to the muscle spindles and the Golgi tendon organs, the unidirectional fibres intervene in succession during the movement of a body part in one plane (Stecco, 2004). The evolution of peripheral receptors clearly demonstrates how the brain depends upon the fascia in order to be able to formulate the concept of space and time. To enable it to organise movement, in general the brain requires feedback concerning whatever is taking place in the periphery. Normally the free nerve endings are involved in deep somaesthetic activity or the perception of the body´s position and movement in space. This feedback transmits information to the CNS regarding the exact position of any given body segment. The fascia and, in particular, its directional sequences, have a predefined length and therefore can act as a type of measuring device in the periphery. Furthermore, because of its elasticity, it is also capable of applying stretch to neuroreceptors (Kent, 1997); (Stecco, 2004). According to the angle of the articulation and therefore, the fascial stretch, either a sequence, a diagonal or a spiral is activated with each step or movement. In other words the mind initiates a motor scheme and the fascia assists in the process of its realisation (Stecco, 2004). The functional meaning of this relationship between the activity of the muscle, the mobility of intra and extramuscular connective tissue and, function of intermuscular septa to achieve the formation of sequences, diagonals, and the spirals of movement, should be studied by also taking into account the presence of numerous embedded receptors that may play a role in peripheral coordination of movement. (Stecco, 2004; Stecco, 2007a; Stiwell, 1957; Turrina et al., 2012; Yahia et al., 1992).\n\n\nDiscussion\n\nIn the 1940s, when Doctor Herman Kabat began to develop the concept of Proprioceptive Neuromuscular Facilitation (PNF) (Voss et al., 2001), it was sustained that normal neuromuscular mechanisms were capable of developing a wide range of motor activities within the limits of the anatomical structure, the level of development, and innate and previously learnt neuromuscular responses. In other words, the countless combinations of movement that an individual employs to satisfy everyday needs are acquired by means of a well-established process of development and an abundance of learning situations. Neuromuscular mechanisms are assimilated and become efficient without paying attention to individual muscular activity, reflex activity, or the enormous amount of neurophysiologic reactions involved. In the introduction to the PNF method (Voss et al., 2001) it is stated that ‘movement patterns are mass movement patterns. Mass movement is a characteristic of normal motor activity and it is in consonance with Beevor’s Axiom: the brain knows nothing of individual muscle action, but knows only of movement. In normal motor activity, the varied combinations of movement, or mass movement, demand shortening and lengthening reactions from many muscles and in different degrees. Mass movement, which has the purpose of setting out a specific demand, has to consist of a special combination of movements, ideal to the specific sequence of muscles responsible for that movement and has to allow those muscles to contribute with their action components in a harmonic way’.\n\nMore recently, (Myers, 2009) has suggested that perhaps there are not in fact 600 muscles but a single connective tissues system with \"pockets, which gives muscles and muscles fibers to settle their spaces in different directions to perform its function.\n\nIn neurophysiology, we find that movement is programmed by the mind and carried out by the contraction of the muscle fibers. Nerve impulses generated from the brain determine the displacement of a certain segment and it is subject to a vast number of variables in the periphery (Stecco & Stecco, 2010).\n\nGiven that each muscle has fibers with different functions and innervations (Burke et al., 1974), according to the Fascial Manipulation® concept (Stecco, 2004), the musculoskeletal system could be divided into myofascial units (MFUs). Each MFU is said to be composed of motor units responsible for moving a segment in a specific direction and, along with the fascia surrounding these fibers, these forces generate movement vectors. A MFU necessarily includes the nerve components innervating these elements. Furthermore, the main vectors of each MFU generate other smaller, more distant vectors (Stecco, 2013).\n\nThe fascial context in which muscle fibers are inserted could potentially coordinate movement in the periphery. The MFU is the element that could synchronize the action of motor units innervated by the same axon. While the brain sends impulses to muscle fibers scattered within the muscle via the axon of a single motor neuron, diminishing the activity of the more distal fibers and increasing it in the most proximal ones in relation to changing positions of the limb or the required force presents neurological challenges. An elastic structure, sensitive to stretch, such as the fascia, could assist modulation of recruitment or inhibition via the activation of mechanoreceptors embedded in this tissue (Stecco & Stecco, 2004) The potential role of these mechanoreceptors in the coordination and synchronization of movement via their proprioceptive function is emphasized. As connective tissue continues in the tendons and the muscle insertions (Hujing, 2003; Huijing & Baan, 2001a; Huijing & Baan, 2001b; Stecco, 2009a; Stecco, 2009b), it also distributes and directs the muscular force to the bone in an appropriate way (Stecco & Stecco, 2010).\n\n\nNew questions for research\n\n1) If the brain is not aware of individual muscle activity, but knows and plans movement in terms of patterns (Beever’s axiom), should we continue thinking in terms of individual muscles bringing about movement?\n\n2) Is there a central command that sends an order through nerve impulses, all or nothing, and are there peripheral systems that regulate coordination and synchronization according to the requirements of movement patterns? Is the brain really in charge of everything?\n\n3) If the brain coordinates, regulates and synchronizes everything, what is the purpose of the peripheral nervous system, the fascia and its embedded mechanoreceptors?\n\nFrom this review, a new comprehension of the connective tissue in general, and more specifically, of the musculoskeletal system emerges:\n\n- The connective tissue drives musculoskeletal development from conception.\n\n- In the 1940s, prompted by his rehabilitative work with patients affected by poliomyelitis, Kabat initiated discussion concerning how the brain interprets movement in terms of finalized gestures or patterns of movement.\n\n- In the 70s, Stecco developed a comprehensive method (Fascial Manipulation®) based on similar reasoning, introducing the potential role of muscular fascia in musculoskeletal activity.\n\n- More recent studies concerning the anatomy and physiology of intramuscular and extramuscular connective tissue and its innervation suggest that the myo–fascial-skeletal system presents peripheral response mechanisms that are capable of responding to movement patterns.\n\n- These mechanisms include functions such as musculoskeletal coordination, synchronization, regulation of movement, and the transmission of contractile force from the muscle fibers and fascicles within muscles and as well as along body segments via myotendinous expansions and intramuscular septa. Lastly, the neurovascular tracts, wrapped in fascia, also transmit contractile force (Karassolik et al., 2009; Lindsay, 2008). As suggested by Langevin (2006), the fascial system represents a ‘body-wide proprioceptive/communicating organ’.\n\n\nConclusion\n\nConnective tissue guides our developmental phases during intrauterine life and continues to evolve through movement patterns. In addition, the brain apparently knows very little about individual muscle action, but recognizes movement patterns catalogued by learning. Muscle fibers and fascicles are oriented in a direction dictated by their connective tissue in a pre-established pattern, reinforced through movement patterns and modified to adjust to the individual, making it effectively redundant to name every muscle involved in the process. With ongoing research concerning the anatomy and physiology of the myo-fascial-skeletal system is it not time for a paradigm shift with regards to this system?",
"appendix": "Author contributions\n\n\n\nAuthor, Carolina Marchuk PT DO: in late 2014 began to read about the article and began to cross data of different authors, some old and many contemporaries, grew to investigate this idea of thinking different from musculoskeletal origin (Embryology) to its development functional (Anatomy and Physiology), as well as it was developed during our evolution, it was important to investigate the roles that the brain and mechanoreceptors, so it was that wrote and edited this review with the great contribution and ongoing monitoring of the co-author Carla Stecco, MD.\n\nWhen I read various articles or books I always ask: “Why?” and so I questioned to know and understand a little more about our locomotor function and disfunction.\n\nCo - Author, Prof. Carla Stecco MD: is Assistant Professor of Department of Molecular Medicine and one of the most Recognize Anatomist in the field of Fascia. She is Assistant professor of Department of Molecular Medicine to Padova University and is known worldwide for her studies in the connective tissue and she is the author and co-author of several books on fascias as well as the method created by her father (Luigi Stecco) Fascial Manipulation Method.\n\nCarla Stecco contributed to monitoring, ongoing review of the wording and great knowledge of the subject of this article as well as a significant contribution of bibliographic material.\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\n- Julie Ann Day PT, Fascial Manipulation© Teacher, for their suggestions and correction in the wording.\n\n- Cheryl Megalos PT and Fascial Manipulation© Teacher, for collaboration in the translation of this article.\n\n- Patricia Candia PT: Anatomy Teacher of the Degree in Physical Therapy from the University of Salvador, she belongs to the staff of the Department of Anatomy, School of Medicine, Universidad del Salvador, Buenos Aires Argentina.\n\n\nReferences\n\nBenjamin M: The fascia of the limbs and back--a review. J Anat. 2009; 214(1): 1–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlasi M, Blasi J, Domingo T, et al.: Anatomical and histological study of human deep fasciae development. Surg Radiol Anat. 2015; 37(6): 571–8. PubMed Abstract | Publisher Full Text\n\nBrand-Seberi B, Christ B: Genetic and epigenetic control of muscle development in vertebrates. Cell Tiss Res. 1999; 296(1): 199–212. PubMed Abstract | Publisher Full Text\n\nBurke RE, Levine DN, Salcman M: Motor units in cat soleus muscle: physiological, histochemical and morphological characteristics. J Physiol. 1974; 238(3): 503–514. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCinnamon Y, Kahane N, Kalcheim C: Characterization of the early development of specific hypaxial muscles from the ventrolateral myotome. Development. 1999; 126(19): 4305–4315. PubMed Abstract\n\nChevallier A, Kieny M, Mauger A: Limb-somite relationship: origin of the limb musculature. J Embryol Exp Morphol. 1977; 41: 245–58. 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PubMed Abstract | Publisher Full Text\n\nHuijing PA, Baan GC: Myofascial force transmission causes interaction between adjacent muscles and connective tissue: effects of blunt dissection and compartmental fasciotomy on length force characteristics of rat extensor digitorum longus muscle. Arch Physiol Biochem. 2001b; 109(2): 97–109. PubMed Abstract | Publisher Full Text\n\nHuijing PA, Maas H, Baan GC: Compartmental fasciotomy and isolating a muscle from neighboring muscles interfere with myofascial force transmission within the rat anterior crural compartment. J Morphol. 2003; 256(3): 306–321. PubMed Abstract | Publisher Full Text\n\nHuijing PA, Jaspers RT: Adaptation of muscle size and myofascial force transmission: a review and some new experimental results. Scand J Med Sci Sports. 2005; 15(6): 349–380. PubMed Abstract | Publisher Full Text\n\nHuijing PA: Epimuscular myofascial force transmission: a historical review and implications for new research. 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PubMed Abstract | Publisher Full Text\n\nLangevin HM, Bouffard NA, Badger GJ, et al.: Subcutaneous tissue fibroblast cytoskeletal remodeling induced by acupuncture: evidence for a mechanotransduction-based mechanism. J Cell Physiol. 2006b; 207(3): 767–774. PubMed Abstract | Publisher Full Text\n\nLangman´s Medical Embryology. 8th edition in Spanish. Ed. Lippincott Williams & Wilkins. 2001. ISBN 950-06-1367-0 / 84-7903-655-9.\n\nLindsay M: Fascia: clinical applications for health and human performance. Delmar Cengage Learning. New York. 2008. Reference Source\n\nMcCombe D, Brown T, Slavin J, et al.: The histochemical structure of the deep fascia and its structural response to surgery. J Hand Surg Br. 2001; 26(2): 89–97. PubMed Abstract | Publisher Full Text\n\nMyers TW: Vías Anatómicas. 2nd ed. Barcelona, España: Elsevier Masson. 2009: 36–44. Reference Source\n\nMonti RJ, Roy RR, Hodgson JA, et al.: Transmission of forces within mammalian skeletal muscles. J Biomech. 1999; 32(4): 371–380. PubMed Abstract | Publisher Full Text\n\nMoore K, Persaud T: The developing human. 6th edn. London: WB Saunders. 1999: 23–221.\n\nMoore MJ: The dual connective tissue system of rat soleus muscle. Muscle Nerve. 1983; 6(6): 416–422. PubMed Abstract | Publisher Full Text\n\nPappas GP, Asakawa DS, Delp SL, et al.: Nonuniform shortening in the biceps brachii during elbow flexion. J Appl Physiol (1985). 2002; 92(6): 2381–2389. PubMed Abstract | Publisher Full Text\n\nPetibois C, Gouspillou G, Wehbe K, et al.: Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue. Anal Bioanal Chem. 2006; 386(7–8): 1961–1966. PubMed Abstract | Publisher Full Text\n\nPlatzer W: Locomotor system. In: Kahle W, Leonhardt H, Platzer W, (Eds.), Color Atlas and Textbook of Human Anatomy. First ed, Georg Thieme Publishers, Stuttgart. 1978.\n\nPurslow PP: Muscle fascia and force transmission. J Bodyw Mov Ther. 2010; 14(4): 411–417. PubMed Abstract | Publisher Full Text\n\nRowe RW: Morphology of perimysial and endomysial connective tissue in skeletal muscle. Tissue Cell. 1981; 13(4): 681–690. PubMed Abstract | Publisher Full Text\n\nSadler TW: Langman Embriología Médica. 8va ed. Buenos Aires, Argentina: Editorial Médica Panamericana. Ed. Lippincot Williams & Wilkins. 2001; 181–185. Reference Source\n\nSanchis-Alfonso V, Roselló-Sastre E: Immunohistochemical analysis for neural markers of the lateral retinaculum in patients with isolated symptomatic patellofemoral malalignment. A neuroanatomic basis for anterior knee pain in the active young patient. Am J Sports Med. 2000; 28(5): 725–731. PubMed Abstract\n\nSakamoto Y: Histological features of endomysium, perimysium and epimysium in rat lateral pterygoid muscle. J Morphol. 1996; 227(1): 113–119. PubMed Abstract | Publisher Full Text\n\nSchultz L, Freitis R: The endless web. Berkeley, North Atlantic Books. 1996; 8–10.\n\nSnyder G: Fasciae: applied anatomy and physiology. Kirksville College of Ostheopaty, 1975.\n\nStandring S, Ellis H, Healy J, et al.: Gray’s Anatomy. 39th ed. Churchill Livingstone, London. 2005.\n\nStecco L: Fascial Manipulation for Musculoskeletal Pain. Ed Piccin Nuova Libraria S.p.A., Padova, Italy, ISBN 88-299-1697-8. 2004. Reference Source\n\nStecco C, Gagey O, Macchi V, et al.: Tendinous muscular insertions onto the deep fascia of the upper limb. First part: anatomical study . Morphologie. 2007a; 91(292): 29–37. PubMed Abstract | Publisher Full Text\n\nStecco C, Porzionato A, Macchi V, et al.: The expansions of the pectoral girdle muscles onto the brachial fascia: morphological aspects and spatial disposition. Cells Tissues Organs. 2008. 188(3): 320–9. PubMed Abstract | Publisher Full Text\n\nStecco A, Macchi V, Stecco C, et al.: Anatomical study of myofascial continuity in the anterior region of the upper limb. J Bodyw Mov Ther. 2009a; 13(1): 53–62. PubMed Abstract | Publisher Full Text\n\nStecco A, Macchi V, Masiero S, et al.: Pectoral and femoral fasciae: common aspects and regional specializations. Surg Radiol Anat. 2009b; 31(1): 35–42. PubMed Abstract | Publisher Full Text\n\nStecco L, Stecco A: Manipulación Fascial (parte teórica). En español. 2da. ed. Caracas, Venezuela: Amolca. 2013; 9–35. Reference Source\n\nStecco C, Stern R, Porzionato A, et al.: Hyaluronan within fascia in the etiology of myofascial pain. Surg Radiol Anat. 2011; 33(10): 891–896. PubMed Abstract | Publisher Full Text\n\nStilwell D: Regional variations in the innervation of deep fasciae and aponeuroses. Anat Rec. 1954; 23: 94–104.\n\nTidball JG, Law DJ: Dystrophin is required for normal thin filament-membrane associations at myotendinous junctions. Am J Pathol. 1991; 138(1): 17–21. PubMed Abstract | Free Full Text\n\nTrotter JA: Interfiber tension transmission in series-fibered muscles of the cat hindlimb. J Morphol. 1990; 206(3): 351–361. PubMed Abstract | Publisher Full Text\n\nTurrina A, Martínez-González MA, Stecco C: The muscular force transmission system: Role of the intramuscular connective tissue. J Bodyw Mov Ther. 2013; 17(1): 95–102. PubMed Abstract | Publisher Full Text\n\nVoss DE, Ionta MK, Myers BJ: Facilitación Neuromuscular Propioceptiva, patrones y técnicas. 3ra ed. Buenos Aires, Argentina: Editorial Médica Panamericana. 2001; 19–29. Reference Source\n\nYahia H, Rhalmi S, Newman N, et al.: Sensory innervation of human thoracolumbar fascia. An immunohistochemical study. Acta Orthop Scand. 1992; 63(2): 195–197. PubMed Abstract | Publisher Full Text\n\nYucesoy CA, Baan G, Huijing PA: Epimuscular myofascial force transmission occurs in the rat between the deep flexor muscles and their antagonistic muscles. J Electromyogr Kinesiol. 2010; 20(1): 118–126. PubMed Abstract | Publisher Full Text\n\nZhi Q, Huang R, Christ B, et al.: Participation of individual brachial somites in skeletal muscles of the avian distal wing. Anat Embryol (Berl). 1996; 194(4): 327–39. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "15179",
"date": "22 Jul 2016",
"name": "Benjamin Feldman",
"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 review sets out to challenge current thinking on the role of connective tissue. This is at its clearest in the “New questions for research” section where several concepts are listed that might guide new research or the interpretation of older research. Unfortunately the preceding text does not delineate with sufficient clarity or precision the context of potentially antiquated concepts or emerging ideas that would make these questions for new research seem important or relevant to the reader. Indeed, the promise in the title that this article will address a “paradigm shift” is simply not matched by a clear explanation of what old paradigm(s) are being questioned or what new paradigm(s) are being proposed. An explicit and specific synopsis of what concepts are being challenged and proposed is urgently needed and should appear prominently in the Abstract and/or Introduction and should perhaps be reiterated in the Discussion and/or Conclusion.\n\nMore explicit and conventional organization would benefit the article in other ways. While there is a titled section on “Anatomy and physiology”, the preceding text devoted to embryology falls in the titled “Introduction” section. This lack of symmetry and the immediate launch into embryological detail under the “Introduction” heading is confusing. Then there is a “Discussion” section in which the literature review continues, rather than switching to an overview or synthesis of what was presented in earlier sections, as normally occurs in a Discussion section. This inconsistent and non-conventional use of titled sections detracts from the article’s sense of focus and direction.\n\nFinally, there are a couple of needed improvements in the discussion of early development. 1) There are several Spanish words retained in Figure 2 where the following changes are needed: Corda to Chorda; Cefalic to Cephalic; Esclerotomo to Sclerotome; Miotomo to Myotome; Dermotomo to Dermatome. 2) The text discusses mesenchyme as a “special section” on the “sides of the paraxial mesoderm.” But the term “mesenchyme” does not appear in Figure 2. I think it would be more correct and more in line with Figure 2, to say that the mesenchyme (with the exception of head mesenchyme) is derived from or formed from lateral plate mesoderm (or “a special section flanking the paraxial mesoderm”) rather than defining it is an initial subdivision of the mesoderm.",
"responses": []
},
{
"id": "15642",
"date": "12 Aug 2016",
"name": "Simon Hughes",
"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 find this article both old-fashioned/out-dated in parts and filled with platitudes or assertions not substantiated by clear references. While some relevant literature is mentioned, the text fails to provide a coherent and balanced account for the naive reader attempting to understand the field.\n\nFor example, embryonic ages mentioned appear to refer to human development (although this is not stated), yet assertions concerning the role of TCF4, for example, are included in these paragraphs. As far as I am aware most work on Tcf4 function has been done in chick, mouse or other model organisms (including that quoted by Kardon and colleagues). Essentially no data is available showing a functional role of TCF4 in human muscle connective tissue development. Instead, according to OMIM, TCF4 mutations contribute to neural and epithelial phenotypes in Pitt-Hopkins Syndrome and Fuchs Endothelial Corneal Dystrophy. Thus, while muscle involvement is a reasonable hypothesis based on conservation in evolution, and while genetic variations in people may in future reveal a role, to my knowledge no clear link is as yet apparent. A useful and informative review would make these issues explicit.\n\nThere are sentences in this work, such as 'In this way, the method of Fascial Manipulation organized into six myofascial units, formed by mono and biarticular fibres as well as muscle spindles, were created for each segment.’, that are incomprehensible. For a start, this sentence is ungrammatical. But more seriously, it is meaningless. If Fascial Manipulation is a ‘method’, how can such a method be 'organized into six myofascial units’? The latter sound like anatomical structures in this sentence. At best, this is a very poorly thought-out sentence. At worst, it is an example of attempting to justify a medical treatment using obscurantist scientific jargon with no clear underlying meaning: blinding the naive with ‘science’.\n\nAnother example is the use a a picture in Fig 5 of what may be human gluteus maximus muscle to demonstrate an unclear point about 'bony fishes’ and to conclude 'Hence, these fibres lengthened and they connected to a number of segments’. As far as I can tell there are no segments in the anatomical sense in the gluteus maximus. The idea/view being suggested here is totally unclear. Such non sequiturs are scattered throughout the manuscript.\n\nBeevor may have said 'the brain knows nothing of individual muscle action’ in 1940, but in 2015/6 this is no longer a tenable view. It is quite clear that individual muscle spindles provide feedback to the CNS concerning individual muscle movement and tension and there is no reason to suppose that such information could not be transmitted to the brain. The mentioning of Fascial Manipulation and PNF without clear explanation is a huge omission. One is tempted to ask whether the publication of this manuscript is more about acquiring a recently dated ‘paper’ to quote in other literature than to really illuminate a biomedically important issue? Certainly, I find the statements on new paradigms and the broad philosophical brain/body statements (including the ‘New Questions’ the least valuable parts of this manuscript as they are either not well explained or have already been answered. For example, the answers to the two questions in New Question 2 are an unequivocal ‘yes’ and ‘yes’. For Question 3 there is an abundance of answers already in the literature: the motor axons of the PNS, for example, carry the information from CNS to muscle in terms of patterened series of action potentials.\n\nI concur with Ben Feldman, with the exception that I would be more severe on the author’s use of the term ‘mesenchyme’. In my understanding of modern cell biology, mesenchyme is used to describe a tissue that is not epithelial and has no specific relationship to particular regions of the body or developmental origin of the tissue. Thus mesenchymes in different tissue can be derived from any germ layer and simply reflect a apparent lack of apical/basal polarity in the cells so described.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-635
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https://f1000research.com/articles/3-221/v1
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12 Sep 14
|
{
"type": "Research Article",
"title": "Pandemic 2009 Influenza A (H1N1) virus infection in cancer and hematopoietic stem cell transplant recipients; a multicenter observational study.",
"authors": [
"Maria Cecilia Dignani",
"Patricia Costantini",
"Claudia Salgueira",
"Rosana Jordán",
"Graciela Guerrini",
"Alejandra Valledor",
"Fabián Herrera",
"Andrea Nenna",
"Claudia Mora",
"Inés Roccia-Rossi",
"Daniel Stecher",
"Edith Carbone",
"Ana Laborde",
"Ernesto Efron",
"Javier Altclas",
"Aníbal Calmaggi",
"José Cozzi",
"Patricia Costantini",
"Claudia Salgueira",
"Rosana Jordán",
"Graciela Guerrini",
"Alejandra Valledor",
"Fabián Herrera",
"Andrea Nenna",
"Claudia Mora",
"Inés Roccia-Rossi",
"Daniel Stecher",
"Edith Carbone",
"Ana Laborde",
"Ernesto Efron",
"Javier Altclas",
"Aníbal Calmaggi",
"José Cozzi"
],
"abstract": "Background: During March 2009 a novel Influenza A virus emerged in Mexico. We describe the clinical picture of the pandemic Influenza A (H1N1) Influenza in cancer patients during the 2009 influenza season.Methods: Twelve centers participated in a multicenter retrospective observational study of cancer patients with confirmed infection with the 2009 H1N1 Influenza A virus (influenza-like illness or pneumonia plus positive PCR for the 2009 H1N1 Influenza A virus in respiratory secretions). Clinical data were obtained by retrospective chart review and analyzed. Results: From May to August 2009, data of 65 patients were collected. Median age was 51 years, 57 % of the patients were female. Most patients (47) had onco-hematological cancers and 18 had solid tumors. Cancer treatment mainly consisted of chemotherapy (46), or stem cell transplantation (SCT) (16). Only 19 of 64 patients had received the 2009 seasonal Influenza vaccine. Clinical presentation included pneumonia (43) and upper respiratory tract infection (22). Forty five of 58 ambulatory patients were admitted. Mechanical ventilation was required in 12 patients (18%). Treatment included oseltamivir monotherapy or in combination with amantadine for a median of 7 days. The global 30-day mortality rate was 18%. All 12 deaths were among the non-vaccinated patients. No deaths were observed among the 19 vaccinated patients. Oxygen saturation <96% at presentation was a predictor of mortality (OR 19.5; 95%CI: 2.28 to 165.9).Conclusions: In our cancer patient population, the pandemic 2009 Influenza A (H1N1) virus was associated with high incidence of pneumonia (66%), and 30-day mortality (18.5%). Saturation <96% was significantly associated with death. No deaths were observed among vaccinated patients.",
"keywords": [
"Seasonal influenza is a known cause of morbidity and mortality among cancer and transplant patients. During influenza season",
"20 to 30% of stem cell transplant SCT recipients with respiratory symptoms can test positive for Influenza with a mortality rate of up to 28%1. Non-transplant cancer patients can also have a high mortality rate of up to 38%2",
"being higher in patients with lung",
"hematological and colorectal cancer",
"in patients that develop lower respiratory tract infections",
"and in patients with other co-morbid conditions. In Argentina",
"seasonal Influenza in onco-hematological patients is associated to a 12% incidence of pneumonia and to a 5% of 30-day mortality3."
],
"content": "Introduction\n\nSeasonal influenza is a known cause of morbidity and mortality among cancer and transplant patients. During influenza season, 20 to 30% of stem cell transplant SCT recipients with respiratory symptoms can test positive for Influenza with a mortality rate of up to 28%1. Non-transplant cancer patients can also have a high mortality rate of up to 38%2, being higher in patients with lung, hematological and colorectal cancer, in patients that develop lower respiratory tract infections, and in patients with other co-morbid conditions. In Argentina, seasonal Influenza in onco-hematological patients is associated to a 12% incidence of pneumonia and to a 5% of 30-day mortality3.\n\nIn March 2009 a novel Influenza A virus, later known as 2009 pandemic influenza A (H1N1), emerged in Mexico. The new strain initially spread among travelers to the USA and Canada, and subsequently infected people worldwide4. Clinical presentations ranged from mild symptoms to severe cases that lead to pneumonia and respiratory failure–related deaths.\n\nThe first cases of pandemic Influenza A (H1N1) in Argentina were reported in May 2009, in travelers returning from Mexico and the USA. From May to December 2009 there were 11931 cases of confirmed Influenza A H1N1 in Argentina, 617 deaths, and over 90% of the circulating respiratory viruses in adults were the novel Influenza A H1N15.\n\nData from different studies on the impact of this new virus in the adult cancer and SCT population are somewhat contradictory. Many studies from different countries were reported1,6–16. In these studies, the incidence of pneumonia ranges from 2011 to 52%8, while the reported mortality rate ranges from 0–10%6,11,12,14,16 to as high as 21–31%7–10,15.\n\nDuring the 2013 winter season, pandemic Influenza A H1N1 continued to circulate (FluNetDB, WHO, http://apps.who.int/globalatlas/dataQuery/default.asp).\n\nIn this study, we examined the effects and severity of pandemic H1N1 Influenza during the 2009 influenza season, in patients with cancer and SCT in two cities of Argentina.\n\n\nMethods\n\nThis is a multicenter retrospective observational study that included 12 medical centers. From May to August 2009, cancer and SCT patients older than 16 years, who presented a confirmed influenza infection by real-time PCR were included.\n\nThe following data were obtained anonymously: underlying illness, type and date of SCT, whether patients were or were not receiving immunosuppressive treatment, at the time of the influenza diseases, immunization for seasonal influenza, clinical presentation (influenza like or pneumonia), laboratory and radiology results, anti-viral treatment, and outcome. In addition, data on the time between the onset of symptoms and the initiation of antiviral therapy, need for ventilation support, and presence of co-infections were also collected. Hypoxemia was defined as an oxygen saturation value lower than 96%. We diagnosed lymphopenia when the absolute lymphocyte count was less than 1000/µL. The RT-PCR tests for pandemic Influenza A H1N1 virus were performed on nasopharyngeal swabs or bronchoalveolar lavage samples when available. Either of two PCR protocols were used for detection of the pandemic Influenza A H1N1 virus depending on test availability: the Real-time ready InfluenzaA/H1N1DetectionSet® Version June 2009 (Roche Diagnostics GmbH, Roche Applied Science68298 Mannheim, Germany) and the PCR protocol used by the WHO (CDC protocol of real-time RTPCR for influenza A H1N1 28 April 2009, revision 1, 30 April 2009).\n\nCategorical variables are shown as percentages and they are compared with the χ²-distribution test or Fisher test. Numeric variables such as median and range are compared with the Wilcoxon test. The association between baseline variables and events is presented as OR with the 95% CI. In all cases, statistical significance was assumed at a value of p=<0.05.\n\n\nResults\n\nFrom May to August 2009, 12 centers sent data of 65 cancer patients with 2009 H1N1 virus disease confirmed by positive PCR in BAL (3) or nasopharyngeal wash (62). The median age of the patients was 51 years (range 17 to 81), and 57% were female. The majority of patients (47) had onco-hematological cancer (72%) and 18 (28%) had solid tumors. Cancer treatment included chemotherapy (46), SCT (16), no treatment (2) and surgery (1). History of 2009 seasonal influenza vaccination was present in 19/64 patients (30%). No patient had received influenza chemoprophylaxis. The median time of patients follow up from the onset of symptoms was 61 days, range 5 to 259.\n\nData on overall clinical presentation and outcome are shown in Table 1. Pneumonia and pneumonia with oxygen saturation <96% were the most common clinical presentations (43/65, 66% and 30/65, 46%, respectively). Co-infections were present in a minority of cases (9/65, 14%) and only among patients with community acquired Influenza.\n\n*4 pneumonias (3 S. pneumoniae, 1 Moraxella catarrhalis), 3 bacteremia (K. pneumoniae, MRCNS, Streptococcus Group C); ** Influenza B and Parainfluenza 3 infection.\n\nPatients started treatment at a median of two days from onset of symptoms (range 0 to 45 days). Sixty eight percent (43/63) of patients started treatment within the 48h after the onset of symptoms. Some patients received combined antiviral treatment because of the potential circulation of seasonal Influenza A H1N1 known to be resistant to oseltamivir17.\n\nMost patients acquired the infection in the community (58, 89%) while 7 (11%) of infections were acquired in the hospital setting despite the implementation of adequate standard precautions and isolation measures during this outbreak. Detailed descriptions of the outcome of patients with community acquired (CAPIA) and nosocomially-acquired (NAPIA) pandemic Influenza A H1N1 infection patients are described in Figure 1 and Figure 2. The 30-day mortality was higher among patients with NAPIA (3/7, 43%) than among those with CAPIA (9/58, 15,5%).\n\nURTI: upper respiratory tract infection; ICU: Intensive care unit; MV: mechanical ventilation; ARDS: Acute respiratory distress syndrome.\n\nURTI: upper respiratory tract infection; ICU: Intensive care unit; MV: mechanical ventilation; ARDS: Acute respiratory distress syndrome.\n\nMost (45/58; 78%) of outpatients required hospital admission. Reasons for patient admission included mainly oxygen desaturation, but, in many cases, patients were admitted because of their severe state of immune suppression and the lack of information about this emergent virus, especially when the patient’s social environment prevented him/her from easy access to medical care.\n\nOutpatients who presented with upper respiratory tract symptoms (URTI), had the most benign course since the majority (11/19, 52%) resolved their infections with antiviral therapy in the outpatient setting, and, among the 8 (42%) who were admitted, none of them required ICU admission or developed signs of pneumonia. The 30-day mortality among CAPIA URTI was 0.\n\nOutpatients who presented with pneumonia had a more severe course since almost all of them (37/39, 96%) were admitted, 15/39 (38%) required ICU, 11/39 (28%) required mechanical ventilation (MV), and the 30-day mortality in this group was of 23% (9/39). The worst prognosis in this group was seen among those who presented with pneumonia and desaturation (25), leading to an admission rate of 100% (52% in ICU, 44% needed MV), and a 30-day mortality of 36%.\n\nPatients, who developed NAPIA, belonged to 3 different centers and started having symptoms at median of 20 days after admission (range 2 to 33). This group had the poorest prognosis since the 30-day mortality rate was 43% (3/7).\n\nOne of three (33%) NAPIA URTI progressed to pneumonia, while none of the 19 patients with CAPIA URTI did. Therefore, the overall progression from URTI to pneumonia was of 4.5% (1/22).\n\nThe 30-day mortality according to the clinical presentation and setting is best described in Table 2 for comparison. It is shown that having pneumonia at presentation and developing of the infection in the hospital setting tended to be associated with a higher 30-day mortality without achieving statistical significance.\n\nURTI: Upper respiratory tract infection; CAPIA: Community-acquired pandemic Influenza A infection; NAPIA: Nosocomially acquired pandemic Influenza A infection.\n\nBacterial complications were documented in 6 (9%) patients and included 3 bacteremias (CVC related MRCNS, Acinetobacter baumanii, and GNR that was only seen in direct examination), 2 pneumonias (MRSA, S. pneumoniae) and 1 meningitis (Ps. aeruginosa). The median time from the onset of symptoms to the development of a bacterial complication was 11 days, range 0–34. Bacterial complications developed only among patients who presented with pneumonia (6/43, 14%) by the pandemic Influenza A H1N1. No bacterial complication developed among the 22 patients who presented with URTI.\n\nNon-infectious complications developed in 14 (22%) patients. They included: renal failure (5), respiratory failure (5), shock (3), hypokalemia (3), nonbacterial infections (3) (CMV reactivation, candidiasis by C. glabrata, and PCP) and bleeding (2) (lung and brain). Most patients presented more than one complication.\n\nNo deaths were observed among patients who had been vaccinated against seasonal influenza in the same year. We could not collect data on the date of vaccination. However, we do know that the first case was detected on May 12th, while the seasonal influenza vaccine was available since March. Therefore, there is a high probability that at least 14 days might have passed between vaccination and the onset of symptoms.\n\nThe presence of any co-infection (bacterial or viral) at onset of symptoms and the delay in treatment were not associated to death or mechanical ventilation. By univariate analysis lack of history of vaccination, and the following baseline characteristics: pneumonia, oxygen saturation <96%, and lymphocyte count <800 cells/μL, were associated to 30-day mortality and mechanical ventilation. By multivariate analysis only lack of history of vaccination (OR did not apply because none died in the vaccinated group) and baseline oxygen saturation <96% (OR 19.5; 95% CI 2.28-165.9; P=0,007) were associated to mechanical ventilation and death. There might be a bias regarding the apparent benefit of vaccination because in cancer patients, immunization is usually advised when the period of major immunosuppression has finished.\n\n\nDiscussion\n\nOur study shows the clinical course of the infection by the 2009 pandemic Influenza A H1N1 virus in 65 cancer patients from 12 institutions located in two cities of Argentina. Eleven percent of these infections were nosocomially acquired. Overall we found a high rate of pneumonia (66%) and mortality (18%). The clinical course was less severe in those who presented with an URTI in the outpatient setting in contrast to those who presented with pneumonia and desaturation especially in the hospital setting. We also found that the best predictors of death were oxygen desaturation at presentation and lack of vaccination against seasonal Influenza.\n\nThe incidence of pneumonia we found is higher than the one reported with seasonal influenza in cancer patients (5–44%)3,18–20 and it is also higher than the incidence of lower respiratory tract infections (LRTI) caused by the 2009 pandemic Influenza A H1N1 in the hospitalized general population (40–44%)21, in solid organ transplant recipients (23%)16, in HCT recipients (21–56%)1,6,8,12–14,22 and in patients with hematological malignancies (48%)23. Only one small study that includes 15 confirmed cases of 2009 pandemic Influenza A H1N1 infection in onco-hematological patients reports a higher incidence of pneumonia (87%)15.\n\nThe 30-day mortality rate we show in this study is more than three times the one observed in Argentina in the same patient population when looking at infections by other respiratory viruses such as Adenovirus, Influenza, Parainfluenza, and RSV (5%)3. However, it is similar to the mortality rate reported in hematological patients with pneumonia by Influenza, Parainfluenza, Picornavirus and RSV at a US institution (15%)24.\n\nIt is well known that seasonal influenza-induced pneumonia is independently associated with mortality after HCT (adjusted HR 2.6; 95% CI 1.40-4.86)20. The pandemic Influenza A H1N1 virus is an independent risk factor for progression to LRTI (OR 5.64; 95% CI 1.3-25) and hypoxemia (OR 5.91; 95% 1.4–24) compared with seasonal influenza virus in HCT recipients1. In addition, immunosuppression was a main risk factor for early mortality among 337 Argentinean patients admitted to ICU with influenza like illness and respiratory failure that required mechanical ventilation. Mortality was highly associated with refractory hypoxemia25. These data explain our high mortality rate observed among the 17 patients who were admitted to ICU (11/17; 65%) or among the 12 patients who developed respiratory failure (11/12; 92%). These values are comparable to those reported in the same type of population1,8,12,22, but are higher than those reported in other populations, which ranged from 0–24%21,26–29. Indeed, the overall mortality rate observed among Argentinean patients admitted to ICU and requiring mechanical ventilation was 46%25. To further support the high mortality of patients with pandemic 2009 Influenza pneumonia, we identified hypoxemia at onset of symptoms as an independent predictor of mortality.\n\nLymphocytopenia has been described as a risk factor for progression from upper to lower viral respiratory tract infection in cancer patients24,30, and profound lymphopenia (<100 cell/µL) was reported as a significant risk factor for requirement of mechanical ventilation and death in HCT recipients infected with seasonal influenza virus30. In our study, having fewer than 800 lymphocytes/µL at presentation was a predictor for the need for mechanical ventilation and death in a univariate but not in a multivariate analysis. We did not analyze a lower value such as <100 of lymphocytes due to the small number of patients included with this value.\n\nIt is noteworthy that co-infections or bacterial complications developed in less than 15% of patients.\n\nNeuraminidase inhibitor therapy appears to be effective in preventing progression to LRTI2,30 and hypoxemia30 when instituted early after onset of symptoms. It was reported that delaying therapy in cancer patients with the pandemic Influenza A H1N1 virus infection was significantly associated with death16. Early initiation of antiviral therapy in these patients may attenuate the severity of disease21,27. In our series, antiviral therapy was started early after a median of two days after the onset of symptoms, with a range from 0–45 days. We did not find any correlation between days from onset of symptoms to therapy or diagnosis to therapy by univariate or multivariate analysis.\n\nIt is known that patients with URTI can be treated as outpatients and can recover completely from their infection31. In our series half of the outpatients with URTI remained as such, while the other half was admitted but did not require ICU. There is a possibility that most of the admitted patients could have been managed as outpatients as well.\n\nThe global progression from URTI to pneumonia in our study was of 4.5% (1/22). This single patient had a nosocomial infection and died 21 days later with sepsis, respiratory failure and neutropenia. This is according to reports of progression to LRTI that may occur even after one week of symptoms20.\n\nIn contrast, patients with LRTI required hospitalization with a high number of them requiring admission to ICU for ventilation support. The dismal outcome seen in these patients despite treatment with oseltamivir probably indicates that this high-risk group needs to be treated differently from patients with isolated URTI. Some authors have suggested an initial treatment with high dose of oseltamivir and/or combination therapy approaches in the case of respiratory failure22. Higher doses could also be considered in a setting of profound lymphocytopenia30. All our patients received standard dose of oseltamivir (75 mg po twice a day) for a minimum of 10 days based on data on slower viral clearance30.\n\nNosocomial outbreaks of seasonal32 and pandemic 2009 Influenza A H1N1 infection33 can develop even in the setting of appropriate infection control measures. Seven (11%) of our patients had hospital-acquired influenza. Three of them (43%) died. This mortality rate is higher than the previously reported 13–27%, however, the number of patients in our report is too small to make any conclusion.\n\nSeasonal influenza vaccination is recommended yearly for all patients with cancer and HSCT recipients34. In our study all deaths occurred among the non-vaccinated patients, while there were no deaths among the vaccinated patients. Individuals vaccinated against seasonal Influenza A or with previous seasonal influenza infection may benefit from preexisting cross-reactive memory CD4+ T cells and CD8+ T cells reducing their susceptibility to Influenza A H1N1 infection or explaining, at least in part, the unexpected mild illness in the community35–39. Whether the trivalent seasonal Influenza vaccine is protective against the pandemic Influenza A H1N1 virus in cancer patients is still a matter of debate38.\n\nIn conclusion, we report a series of cancer patients with the pandemic Influenza A H1N1 infection with a high incidence of hospitalization, severe pneumonia, ICU admission, mechanical ventilation, and 30-day mortality. In our series hypoxemia and lack of vaccination with seasonal trivalent influenza vaccine were predictors of mechanical ventilation requirement and death. A larger study is needed to evaluate the possibility of cross protection with the seasonal influenza vaccination.\n\n\nData availability\n\nF1000Research: Dataset 1. Baseline characteristics, clinical presentation, treatment and outcome of 65 cancer or SCT patients doi: 10.5256/f1000research.5251.d10027640\n\n\nConsent\n\nEthical committee approval was not required in Argentina at the time of the study.",
"appendix": "Author contributions\n\n\n\nM. Cecilia Dignani: Idea, coordination, database building, patient inclusion, analysis of results and manuscript writing.\n\nPatricia E. Costantini, Claudia C. Salgueira, Rosana Jordán and José A. Cozzi: Patient inclusion, database building, analysis and revision of results, and manuscript writing.\n\nGraciela M. Guerrini, Alejandra Valledor, Fabián A. Herrera, Andrea V. Nenna, Claudia A. Mora, Inés Roccia-Rossi, and Aníbal R. Calmaggi: Patient inclusion, analysis and revision of results and writing the manuscript.\n\nDaniel R. Stecher, Edith A. Carbone, Ana R. Laborde, Ernesto D. Efron, and Javier D. Altclas: Patient inclusion, revision of results and manuscript writing.\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\nThe abstract of this manuscript has been presented partially at the 16th Symposium on Infections in the Immunocompromised Host 27–30 June 2010, Budapest, Hungary. Abstract P82. Published in:\n\nClinical Microbiology and Infection, 2010; Vol. 16 S3: S36.\n\nThe authors acknowledge the kind assistance provided by Dr. Jorge Thierer in reviewing the statistical analysis.\n\n\nReferences\n\nChoi SM, Boudreault AA, Xie H, et al.: Differences in clinical outcomes after 2009 influenza A/H1N1 and seasonal influenza among hematopoietic cell transplant recipients. Blood. 2011; 117(19): 5050–5056. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChemaly RF, Torres HA, Aguilera EA, et al.: Neuraminidase inhibitors improve outcome of patients with leukemia and influenza: an observational study. Clin Infect Dis. 2007; 44(7): 964–967. PubMed Abstract | Publisher Full Text\n\nSosa Avila L, Dignani MC, Fernandez I, et al.: Infecciones por virus respiratorios en pacientes con enfermedades oncohematológicas y receptores de trasplante de células hematopoyéticas. IX Congreso Argentino de la Sociedad Argentina de Infectología. Mar del Plata. 2009.\n\nDawood FS, Jain S, Finelli L, et al.: Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med. 2009; 360(25): 2605–2615. PubMed Abstract | Publisher Full Text\n\nMinisterio de Salud, Presidencia de la Nación de la República Argentina. Influenza Pandémica (H1N1) 2009: Informe Semana Epidemiológica N°. 2010; 52. Reference Source\n\nRedelman-Sidi G, Sepkowitz KA, Huang CK, et al.: 2009 H1N1 influenza infection in cancer patients and hematopoietic stem cell transplant recipients. J Infect. 2010; 60(4): 257–263. PubMed Abstract | Publisher Full Text\n\nTramontana AR, George B, Hurt AC, et al.: Oseltamivir resistance in adult oncology and hematology patients infected with pandemic (H1N1) 2009 virus, Australia. Emerg Infect Dis. 2010; 16(7): 1068–1075. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEspinosa-Aguilar L, Green JS, Forrest GN, et al.: Novel H1N1 influenza in hematopoietic stem cell transplantation recipients: two centers’ experiences. Biol Blood Marrow Transplant. 2011; 17(4): 566–573. PubMed Abstract | Publisher Full Text\n\nSouza TM, Salluh JI, Bozza FA, et al.: H1N1pdm influenza infection in hospitalized cancer patients: clinical evolution and viral analysis. PLoS One. 2010; 5(11): e14158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGeorge B, Ferguson P, Kerridge I, et al.: The Clinical Impact of Infection with Swine Flu (H1N109) Strain of Influenza Virus in Hematopoietic Stem Cell Transplant Recipients. Biol Blood Marrow Transplant. 2011; 17(1): 147–153. PubMed Abstract | Publisher Full Text\n\nSuyani E, Aki Z, Guzel O, et al.: H1N1 infection in a cohort of hematopoietic stem cell transplant recipients: prompt antiviral therapy might be life saving. Transpl Infect Dis. 2011; 13(2): 208–212. PubMed Abstract | Publisher Full Text\n\nLjungman P, de la Camara R, Perez-Bercoff L, et al.: Outcome of pandemic H1N1 infections in hematopoietic stem cell transplant recipients. Haematologica. 2011; 96(8): 1231–1235. PubMed Abstract | Publisher Full Text | Free Full Text\n\nProtheroe RE, Kirkland KE, Pearce RM, et al.: The clinical features and outcome of 2009 H1N1 influenza infection in allo-SCT patients: a British Society of Blood and Marrow Transplantation study. Bone Marrow Transplant. 2012; 47(1): 88–94. PubMed Abstract | Publisher Full Text\n\nRihani R, Hayajneh W, Sultan I, et al.: Infections with the 2009 H1N1 influenza virus among hematopoietic SCT recipients: a single center experience. Bone Marrow Transplant. 2011; 46(11): 1430–1436. PubMed Abstract | Publisher Full Text\n\nMinnema BJ, Husain S, Mazzulli T, et al.: Clinical characteristics and outcome associated with pandemic (2009) H1N1 influenza infection in patients with hematologic malignancies: a retrospective cohort study. Leuk Lymphoma. 2013; 54(6): 1250–1255. PubMed Abstract | Publisher Full Text\n\nChemaly RF, Vigil KJ, Saad M, et al.: A multicenter study of pandemic influenza A (H1N1) infection in patients with solid tumors in 3 countries: early therapy improves outcomes. Cancer. 2012; 118(18): 4627–4633. PubMed Abstract | Publisher Full Text\n\nInfluenza Project Team. Oseltamivir resistance in human seasonal influenza viruses (A/H1N1) in EU and EFTA countries: an update. Euro Surveill. 2008; 13(6): pii: 8032. PubMed Abstract\n\nMartino R, Ramila E, Rabella N, et al.: Respiratory virus infections in adults with hematologic malignancies: a prospective study. Clin Infect Dis. 2003; 36(1): 1–8. PubMed Abstract | Publisher Full Text\n\nMartino R, Porras RP, Rabella N, et al.: Prospective study of the incidence, clinical features, and outcome of symptomatic upper and lower respiratory tract infections by respiratory viruses in adult recipients of hematopoietic stem cell transplants for hematologic malignancies. Biol Blood Marrow Transplant. 2005; 11(10): 781–796. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNichols WG, Guthrie KA, Corey L, et al.: Influenza infections after hematopoietic stem cell transplantation: risk factors, mortality, and the effect of antiviral therapy. Clin Infect Dis. 2004; 39(9): 1300–1306. PubMed Abstract | Publisher Full Text\n\nJain S, Kamimoto L, Bramley AM, et al.: Hospitalized patients with 2009 H1N1 influenza in the United States, April–June 2009. N Engl J Med. 2009; 361(20): 1935–1944. PubMed Abstract | Publisher Full Text\n\nCasper C, Englund J, Boeckh M: How I treat influenza in patients with hematologic malignancies. Blood. 2010; 115(7): 1331–1342. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGirmenia C, Mercanti C, Federico V, et al.: Management of the 2009 A/H1N1 influenza pandemic in patients with hematologic diseases: a prospective experience at an Italian center. Acta Haematol. 2011; 126(1): 1–7. PubMed Abstract | Publisher Full Text\n\nChemaly RF, Ghosh S, Bodey GP, et al.: Respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center. Medicine (Baltimore). 2006; 85(5): 278–287. PubMed Abstract | Publisher Full Text\n\nEstenssoro E, Rios FG, Apezteguia C, et al.: Pandemic 2009 influenza A in Argentina: a study of 337 patients on mechanical ventilation. Am J Respir Crit Care Med. 2010; 182(1): 41–48. PubMed Abstract | Publisher Full Text\n\nCao B, Li XW, Mao Y, et al.: Clinical features of the initial cases of 2009 pandemic influenza A (H1N1) virus infection in China. N Engl J Med. 2009; 361(26): 2507–2517. PubMed Abstract | Publisher Full Text\n\nDominguez-Cherit G, Lapinsky SE, Macias AE, et al.: Critically Ill patients with 2009 influenza A(H1N1) in Mexico. JAMA. 2009; 302(17): 1880–1887. PubMed Abstract | Publisher Full Text\n\nLouie JK, Acosta M, Winter K, et al.: Factors associated with death or hospitalization due to pandemic 2009 influenza A(H1N1) infection in California. JAMA. 2009; 302(17): 1896–1902. PubMed Abstract | Publisher Full Text\n\nRello J, Rodriguez A, Ibanez P, et al.: Intensive care adult patients with severe respiratory failure caused by Influenza A (H1N1)v in Spain. Crit Care. 2009; 13(5): R148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoudreault AA, Xie H, Leisenring W, et al.: Impact of corticosteroid treatment and antiviral therapy on clinical outcomes in hematopoietic cell transplant patients infected with influenza virus. Biol Blood Marrow Transplant. 2011; 17(7): 979–986. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhanna N, Steffen I, Studt JD, et al.: Outcome of influenza infections in outpatients after allogeneic hematopoietic stem cell transplantation. Transpl Infect Dis. 2009; 11(2): 100–105. PubMed Abstract | Publisher Full Text\n\nHansen S, Stamm-Balderjahn S, Zuschneid I, et al.: Closure of medical departments during nosocomial outbreaks: data from a systematic analysis of the literature. J Hosp Infect. 2007; 65(4): 348–353. PubMed Abstract | Publisher Full Text\n\nEnstone JE, Myles PR, Openshaw PJ, et al.: Nosocomial pandemic (H1N1) 2009, United Kingdom, 2009–2010. Emerg Infect Dis. 2011; 17(4): 592–598. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEngelhard D, Mohty B, de la Camara R, et al.: European guidelines for prevention and management of influenza in hematopoietic stem cell transplantation and leukemia patients: summary of ECIL-4 (2011), on behalf of ECIL, a joint venture of EBMT, EORTC, ICHS, and ELN. Transplant Infect Dis. 2013; 15(3): 219–232. PubMed Abstract | Publisher Full Text\n\nDuvvuri VR, Moghadas SM, Guo H, et al.: Highly conserved cross-reactive CD4+ T-cell HA-epitopes of seasonal and the 2009 pandemic influenza viruses. Influenza Other Respir Viruses. 2010; 4(5): 249–258. PubMed Abstract | Publisher Full Text\n\nOrellano PW, Reynoso JI, Carlino O, et al.: Protection of trivalent inactivated influenza vaccine against hospitalizations among pandemic influenza A (H1N1) cases in Argentina. Vaccine. 2010; 28(32): 5288–5291. PubMed Abstract | Publisher Full Text\n\nCowling BJ, Ng S, Ma ES, et al.: Protective efficacy of seasonal influenza vaccination against seasonal and pandemic influenza virus infection during 2009 in Hong Kong. Clin Infect Dis. 2010; 51(12): 1370–1379. PubMed Abstract | Publisher Full Text\n\nGarcia-Garcia L, Valdespino-Gomez JL, Lazcano-Ponce E, et al.: Partial protection of seasonal trivalent inactivated vaccine against novel pandemic influenza A/H1N1 2009: case-control study in Mexico City. BMJ. 2009; 339: b3928. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTu W, Mao H, Zheng J, et al.: Cytotoxic T lymphocytes established by seasonal human influenza cross-react against 2009 pandemic H1N1 influenza virus. J Virol. 2010; 84(13): 6527–6535. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDignani MC, Costantini P, Salgueira C, et al.: Baseline characteristics, clinical presentation, treatment and outcome of 65 cancer or SCT patients. F1000Research. 2014. Data Source"
}
|
[
{
"id": "6116",
"date": "01 Oct 2014",
"name": "Per Ljungman",
"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\nOne of several reports on outcome of the H1N1 pandemic in 2009. A couple of issues:Does the report include all patients fulfilling the inclusion criteria at the 12 hospitals? Why was the cut-off for lymphocytopenia chosen as <800? Most previous studies have chosen a lower cut-off.",
"responses": [
{
"c_id": "1021",
"date": "02 Oct 2014",
"name": "María Cecilia Dignani",
"role": "Reader Comment",
"response": "The report does include all patients fulfilling the inclusion criteria at the 12 hospitals. We do know though, that several patients may have had the infection but were not included just because the PCR was not done or the results were not available at the time of the study period. We did not find a statistical significance at a lower levels of lymphocytopenia. We only found a significance when the 800 cut-off was analyzed."
}
]
},
{
"id": "6327",
"date": "17 Nov 2014",
"name": "Pablo Bonvehí",
"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 that shows the impact of pandemic 2009 influenza A (H1N1) in a highly vulnerable population for influenza complications such as immunocompromised oncohaematological patients.The paper is well written and I would like to make some comments or questions to the authors:Did you observe any difference in time elapsed since the initiation of symptoms between those with community acquired pandemic 2009 influenza A (H1N1) who presented with upper respiratory tract infections and those who presented with pneumonia? Previous vaccination against seasonal influenza showed that some degree of preexisting immunity to pandemic 2009 influenza A (H1N1) strains exists, especially among adults aged >60 years. Have you found any difference in progression from UTRI to pneumonia or to death between those older or younger than 69 years of age?",
"responses": [
{
"c_id": "1113",
"date": "08 Dec 2014",
"name": "María Cecilia Dignani",
"role": "Author Response",
"response": "Question 1:Patients with community acquired 2009 Influenza A (H1N1) who presented with URTI (N=19) had symptoms for a median of 2 days (range 1-11). Patients who presented with pneumonia (N=39) had symptoms for a median 3 days (range 1-20).There was a trend towards a longer period with symptoms in patients who presented with pneumonia.Question 2:Patients older than 60 years old who had been vaccinated against seasonal Influenza (N=10) had 60% rate of pneumonia and 0 mortality at day 30 of treatment. Patients with the same age that had not been vaccinated (N=4) had 100% rate of pneumonia and 25% mortality. Patients younger than 60 years old that had been vaccinated (N=9) had 55% rate of pneumonia and 0 mortality at day 30 of treatment. Patients with the same age that had not been vaccinated (N=31) had 68% rate of pneumonia and 26% mortality. Vaccination against seasonal Influenza seemed to have some degree of protection against death in all age groups."
}
]
}
] | 1
|
https://f1000research.com/articles/3-221
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https://f1000research.com/articles/4-626/v1
|
25 Aug 15
|
{
"type": "Review",
"title": "Sevoflurane",
"authors": [
"Stefan De Hert",
"Anneliese Moerman",
"Anneliese Moerman"
],
"abstract": "Sevoflurane has been available for clinical practice for about 20 years. Nowadays, its pharmacodynamic and pharmacokinetic properties together with its absence of major adverse side effects on the different organ systems have made this drug accepted worldwide as a safe and reliable anesthetic agent for clinical practice in various settings.",
"keywords": [
"Sevoflurane",
"anesthetic",
"anesthesiology",
"pharmacodynamic",
"pharmacokinetic"
],
"content": "Introduction\n\nAlthough sevoflurane was synthesized in the early 1970s1, it was not released for clinical use until the early 1990s. This was related partly to the expensive synthesis and the initial concern of apparent toxic effects2, which later appeared to be a consequence of a flawed experimental design3. Nowadays, its pharmacodynamic and pharmacokinetic properties together with its absence of major adverse side effects on the different organ systems have made this drug accepted worldwide as a safe and reliable anesthetic agent for clinical practice in various settings.\n\n\nPhysicochemical properties\n\nSevoflurane (1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy)propane) is a colorless, volatile, and non-flammable liquid with a characteristic smell. It is stable at room temperature and has a boiling point of 58.6°C and a vapor pressure of 157 mm Hg. Hence, in contrast to desflurane, it can be used in standard vaporizers3. Sevoflurane has an oil/gas partition coefficient of 47.2 and its minimal alveolar concentration (MAC), which is the percentage that is necessary to prevent movement in 50% of patients during skin incision, is 2.05%4,5. As a consequence, its potency is considerably lower than that of the older inhalational agents such as halothane and isoflurane, but it is about three times more potent than desflurane.\n\nUpon contact with alkaline carbon dioxide (CO2) absorbers, sevoflurane undergoes degradation6–8. The most important degradation product is fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether, better known as compound A. In experimental studies, compound A has been reported to be nephrotoxic9,10. Although the clinical implications of these findings remained unclear11, the safety issue related to compound A formation led to intense debates for many years before the issue was resolved12.\n\nIn 1996, Abbott Laboratories voluntarily recalled one lot of sevoflurane because evaluation of a bottle of sevoflurane revealed an uncharacteristically pungent odor13,14. This was caused by the formation of hydrogen fluoride as a consequence of a Lewis acid-fluorocarbon reaction. Even in minute amounts, this substance is highly reactive and toxic and can cause respiratory irritation and pulmonary hemorrhage15. Subsequently, the offending Lewis acid (ferric oxide)-containing part was removed from the sevoflurane handling equipment, and a Lewis acid inhibitor (water) was added to the final product16,17. Although Abbott adapted the production process to create a “high water” sevoflurane (>300 parts per million), the manufacturers that subsequently launched sevoflurane (Minrad and Baxter) did not18–20.\n\n\nPharmacological properties\n\nIt is beyond the scope of this review to discuss in detail the pharmacological properties of sevoflurane. Several excellent review articles have addressed this topic21–25.\n\nMAC values of sevoflurane decrease with age, from 3.3% in neonates and 2.5% in infants and young adults to 1.58% to 2.05% in middle-aged adults and 1.45% in adults who are more than 70 years old26–31. In the presence of 65% nitrous oxide in the inspired gas mixture, MAC values for sevoflurane decrease by about 50% in adults32.\n\nGender does not influence the MAC of sevoflurane, but there is some evidence suggesting that ethnic factors may play a role: MAC values reported in US studies were consistently higher (2.05% to 2.6%)5,32 than those reported for Japanese adults (1.58% to 1.71%)30,31.\n\nAs for the volatile anesthetic uptake, distribution and elimination are best described by a five-compartment mammillary model33. This model consists of the lungs, the vessel-rich group of organs, muscle, fat adjacent to the vessel-rich organs, and peripheral fat. In general, an inverse relationship exists between the blood/gas partition coefficient of a volatile anesthetic and the time required for the inspired and alveolar concentrations to reach equilibrium. Sevoflurane has a low blood/gas partition coefficient (0.69), resulting in a swifter equilibration of the alveolar-to-inspiratory fraction (FA/FI ratio) than with enflurane and isoflurane but slightly slower than with nitrous oxide and desflurane33,34.\n\nBecause of its pleasant odor and the absence of irritation to the airways, sevoflurane can be used for inhalational induction both in children and in adults35. Studies have shown that induction is as rapid as36,37 or even swifter than38–40 with halothane.\n\nElimination of a volatile anesthetic is also related to its blood solubility. Between 95% and 98% of sevoflurane is eliminated through the lung. The driving force for this elimination is the difference in partial pressures between the inspired gas mix and the pulmonary capillary blood. In humans, 2% to 5% of the absorbed dose of sevoflurane is metabolized by the liver, resulting in the formation of inorganic fluoride and the organic fluoride metabolite hexafluoroisopropanol41. The latter is conjugated with glucuronic acid and excreted rapidly via the kidneys. The biotransformation of sevoflurane occurs predominantly through cytochrome P450(CYP)2E142,43. Serum inorganic fluoride concentrations after sevoflurane anesthesia are dose-dependent, reaching 10 to 20 µmol/L after 1 to 2 MAC hours and up to 20 to 90 µmol/L with prolonged exposure41. Although most studies could not show nephrotoxic effects after sevoflurane anesthesia44, some controversial reports45 of mild renal dysfunction after the use of sevoflurane resulted in a recommendation by the US Food and Drug Administration for caution in the use of sevoflurane in patients with coexisting renal disease. Interestingly, the majority of data report no differences in pharmacokinetics between patients with and those without kidney diseases46,47. Percutaneous losses account for less than 1% of the total uptake of sevoflurane48.\n\n\nEffects on vital systems\n\nLike the effects of other anesthetic agents, those of sevoflurane on the vital systems are mostly depressant.\n\nA decrease in ventilation leading to apnea at concentrations of between 1.5 and 2.0 MAC can be observed. The ventilatory depression with sevoflurane is the result of a combination of central depression of medullary respiratory neurons49 and depression of diaphragmatic function50 and contractility51.\n\nSevoflurane provides bronchodilation and attenuates bronchial smooth muscle constriction by histamine or acetylcholine and can be safely used in patients with asthma21. Hypoxic pulmonary vasoconstriction is inhibited by sevoflurane in a dose-dependent manner and is not mediated by cyclo-oxygenase21–23.\n\nSevoflurane decreases blood pressure in a dose-dependent manner by decreasing total peripheral resistance. At clinically relevant concentrations, cardiac output is usually preserved21–23. Heart rate remains unchanged or even decreases. Coronary blood flow remains preserved and regional blood flow to other vascular beds appears to be maintained at least when systemic hemodynamics are preserved. For sevoflurane (unlike for desflurane), no sympathetic nervous system activation is observed21–23. Although sevoflurane has been reported to prolong the QT and the QTc interval52, it has no effect on the normal cardiac conduction pathways and therefore is considered a safe agent that can also be used in cardiac electrophysiological procedures25.\n\nSevoflurane is a cerebral vasodilator. In neurosurgical patients, sevoflurane decreased middle cerebral artery flow velocity and caused no epileptiform electroencephalogram activity and no increase of intracranial pressure53. Cerebral autoregulation is maintained at low concentrations of sevoflurane54, but higher doses seem to decrease autoregulatory capacity55.\n\n\nSafety\n\nOverall, sevoflurane is considered to be a safe and reliable agent that has also been used in uncommon medical conditions such as pheochromocytoma, acute intermittent porphyria, carnitine deficiency, muscular dystrophy, multiple sclerosis, primary aldosteronism, and myotonic dystrophy23.\n\nIn the early years of its use, a number of reports on malignant hyperthermia with sevoflurane were published. In many of them, it was difficult to isolate the potential effects of sevoflurane from the influence of the concurrent use of other triggers such as succinylcholine. Animal studies have suggested that the malignant hyperthermia trigger of sevoflurane was substantially lower than that of other volatile anesthetic agents56. However, a recent Japanese database study did not find evidence that sevoflurane would be a weaker triggering agent for malignant hyperthermia57. Since its introduction in clinical practice, sevoflurane has been safely used in millions of people, and reports of sevoflurane-related malignant hyperthermia are scarce. Nevertheless, it seems wise to avoid exposure to sevoflurane in patients with a known susceptibility.\n\nIn the early years of clinical sevoflurane use, it was reported that sevoflurane in the presence of the CO2 absorbers soda lime (calcium, sodium, and potassium hydroxide mixture), or baralyme (barium, sodium, calcium, and potassium hydroxide mixture) degrades to compound A. This degradation occurs in the anesthesia machine as a result of the extraction of an acidic proton (from the inhalational anesthetic) by a strong base (soda lime or baralyme)58. The rate of degradation at a given temperature and moisture level is two to four times greater with baralyme compared with soda lime58,59. The order of solubility of inhalation anesthetics in dry soda lime is sevoflurane > enflurane > desflurane ≥ halothane > isoflurane4. As a consequence, more sevoflurane is absorbed into the CO2 absorber than is observed with other inhalational anesthetics. The production and subsequent inhalation of compound A correlate inversely with the inflow rate60 and directly with the absorbent temperature61. In addition, low fresh gas flows of sevoflurane are associated with increased temperatures in the CO2 absorber62. Therefore, compound A production can be limited by decreasing the temperature of the absorbent63. For these reasons, US and Canadian package labels and licensing authorities have recommended minimal fresh gas inflow rates of 1 or 2 L/min, although other licensing authorities have not made such a recommendation. Compound A production can also be reduced by the amount of absorbent (smaller canisters)64 and adapting the composition of the absorbent by eliminating potassium and sodium hydroxide65–67. The clinical implications of compound A production have been a point of debate for many years68, but the introduction of the new-generation absorbers11 has made this issue largely obsolete.\n\n\nSpecial populations\n\nThere seems to be no significant difference in sevoflurane pharmacokinetics between children and adults24. Because of its pleasant odor, lack of airway irritation, and maintenance of stable hemodynamics, sevoflurane is the agent of choice for mask induction. In general, complications upon emergence are infrequent, although some studies mention a significantly higher incidence of excitement/agitation with sevoflurane69,70. However, this observation has been linked to the fact that the prompt recovery from anesthesia with sevoflurane also facilitates earlier awareness of postoperative pain69,71. This causal relationship was confirmed in a number of studies demonstrating that adequate pain treatment was associated with significantly fewer episodes of emergence agitation72,73.\n\nAmbulatory surgery has increased rapidly in recent years and this has put an emphasis on the use of short-acting drugs in anesthetic practice, allowing fast recovery and early mobilization. Differences in early recovery between sevoflurane, desflurane, and propofol have been reported to be small but in favor of the inhaled anesthetics74, although the clinical implications of these small differences are debatable. Postoperative nausea and vomiting are higher with volatile anesthetics than with propofol, but adequate anti-emetic prophylaxis can prevent or blunt this side effect.\n\nThe prevalence of obesity is increasing dramatically, not only in industrialized countries but also in developing ones. As a consequence, we encounter a growing number of morbidly obese patients who need different types of surgery. Obese patients are traditionally reported to have slower emergence from anesthesia because of a delayed release of volatile anesthetics from the excess fat tissue. However, comparable recovery times have been reported in obese and non-obese subjects after anesthetic procedures lasting 2 to 4 hours75.\n\nThe new inhalation drugs have a much lower lipid solubility compared with the older volatile anesthetic agents, resulting in a more rapid and consistent recovery profile76. For sevoflurane, no significant differences in FA/FI ratio have been observed, but the wash-out curve—that is, the alveolar-to-expiratory fraction (FA/FAO ratio)—was reported to be slower in obese patients compared with non-obese patients. However, 5 minutes after sevoflurane discontinuation, no differences in wash-out were observed between obese and non-obese patients77. Several studies have compared kinetic profiles of sevoflurane and desflurane. Some studies observed more rapid emergence from anesthesia with desflurane but this was not confirmed in other studies (reviewed in 78). Finally, an advantage of sevoflurane in this setting is that it allows progressive induction of anesthesia via face mask79.\n\n\nOrgan protection\n\nThe finding in the late 1990s that sevoflurane was capable of limiting the extent of myocardial infarction after myocardial ischemia triggered a new research direction investigating potential cardioprotective effects of volatile anesthetic agents. Experimental studies have clearly indicated that volatile anesthetic agents are capable of protecting the myocardium against the consequences of ischemia-reperfusion injury by decreasing the extent of myocardial damage, decreasing the extent of reperfusion injury, and better preserving myocardial function. Subsequent research was directed toward unraveling the underlying mechanisms and intracellular pathways of these cardioprotective effects80–85.\n\nAlthough the experimental evidence of cardioprotection with volatile anesthetic agents was quite straightforward, the implications for clinical practice remain a point of debate. The potential cardioprotective effects related to the use of volatile anesthetics were first explored in the setting of cardiac surgery. In coronary artery surgery patients, the results of preconditioning protocols were conflicting: some authors demonstrated a protective effect whereas others failed to observe such an effect. Later, it became clear that this might be attributed to the preconditioning protocol used. It also seems that the administration of the volatile anesthetic agent throughout the entire procedure results in a more pronounced protective effect than when administered intermittently. It is beyond the scope of this review to discuss these studies in detail. The interested reader is referred to a number of reviews on the topic86–95.\n\nFor non-cardiac surgery, the potential clinical implication of the cardioprotective properties of volatile anesthetics is even more debatable. One small study in vascular surgery patients observed a lower incidence of cardiac complications in patients treated with sevoflurane compared with those anesthetized with propofol96. Others, however, observed no difference in the extent of myocardial damage when comparing a volatile anesthetic regimen with a total intravenous regimen97,98. This clearly indicates that only in the presence of myocardial ischemia/reperfusion injury can a potential beneficial effect of volatile anesthetics be expected99. Interestingly, in the study by Lurati Buse and colleagues98, in which 385 patients were randomly assigned to receive anesthesia with either sevoflurane or propofol, the incidences of perioperative myocardial ischemia were comparable (40.8% in the sevoflurane group and 40.3% in the propofol group). Within 12 months, 14 patients had a major cardiac event in the sevoflurane-treated group (7.6%) and 17 in the propofol-treated group (8.5%). However, given that a potential cardioprotective effect of volatile anesthetic agents relates to a modulation of the extent of myocardial ischemia/reperfusion injury, the analysis of the major cardiac events needs to be focused on the occurrence of these events in the subgroup of patients who had perioperative myocardial ischemia. To further clarify this issue, the authors performed an additional analysis of their data. They found that the incidence of major cardiac complications in patients with evidence of perioperative myocardial ischemia was similar in both groups: 8 out of 67 patients (11.9%) in the sevoflurane group and 9 out of 72 patients (12.9%) in the propofol group. The remaining 6 and 8 patients with postoperative major cardiac complications had not shown any evidence of perioperative myocardial ischemia (Seeberger M, unpublished observations).\n\nClinical studies on the protective effects of sevoflurane on other organ systems are scarce and limited to a small number of patients. Three studies from the same group suggest protective effects after liver100,101 and lung102 ischemia with sevoflurane, but the potential implications on long-term outcome remain to be established.\n\n\nNeurotoxicity in the young and aged brain\n\nPreclinical evidence in rodents and non-human primates has caused concern regarding the safety of anesthesia in infants and children. Indeed, animal studies suggest that neurodegeneration with possible cognitive sequelae may constitute a potential long-term risk of anesthesia in neonatal and young pediatric patients (reviewed in 103,104). No hard clinical data suggest that the use of anesthetics in the neonate or young child is associated with signs of developmental neurotoxicity103. It has been argued that the increased risk of poor outcome in some human cohort studies is because of the inflammation and stress associated with the surgery rather than the anesthetic105. It is expected that the results of two ongoing large-scale studies—the Multi-site Randomized Controlled Trial comparing Regional and General Anesthesia for Effects on Neurodevelopment Outcome and Apnea in Infants (GAS) study and the Pediatric Anesthesia and NeuroDevelopment Assessment (PANDA) study—will give more insight into the problem104.\n\nSimilarly, experimental studies, observing effects of anesthetic agents on memory formation and the induction of neurodegenerative changes on a cellular level, have raised concerns about the effects of anesthesia and surgery on the elderly brain. The incidence of postoperative cognitive dysfunction (POCD) varies according to the definitions used in the various studies but is reported to be higher in major surgery (reviewed in 106–108). Whether general anesthesia contributes to POCD remains uncertain. A recent meta-analysis of 26 randomized trials comparing general to regional anesthesia was unable to identify general anesthesia as an independent risk factor for POCD109. It is conceivable that surgical trauma and underlying pathology are of greater importance. Given the complexity and still-unknown elements of the pathogenesis of POCD, further research on the topic is needed.\n\n\nConclusions\n\nSince its introduction in clinical practice, sevoflurane has gained wide acceptance as an anesthetic for various types of surgery. Its ease of administration, versatility, and stable hemodynamic profile make it a safe and easily applicable anesthetic agent.",
"appendix": "Competing interests\n\n\n\nStefan De Hert has received speakers fees from Abbott and AbbVie. Anneliese Moerman declares that she has 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\nWallin RF, Regan BM, Napoli MD, et al.: Sevoflurane: a new inhalational anesthetic agent. Anesth Analg. 1975; 54(6): 758–66. PubMed Abstract | Publisher Full Text\n\nHitt B, Mazze R, Cook T, et al.: Thermoregulatory defect in rats during anesthesia. Anesth Analg. 1977; 56(1): 9–15. PubMed Abstract | Publisher Full Text\n\nPatel SS, Goa KL: Sevoflurane. A review of its pharmacodynamic and pharmacokinetic properties and its clinical use in general anaesthesia. Drugs. 1996; 51(4): 658–700. PubMed Abstract | Publisher Full Text\n\nStrum DP, Eger EI 2nd: Partition coefficients for sevoflurane in human blood, saline, and olive oil. Anesth Analg. 1987; 66(7): 654–6. 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Anesthesiology. 1999; 91(5): 1342–8. PubMed Abstract | Publisher Full Text\n\nStabernack CR, Brown R, Laster MJ, et al.: Absorbents differ enormously in their capacity to produce compound A and carbon monoxide. Anesth Analg. 2000; 90(6): 1428–35. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEger EI 2nd: Compound A: does it matter? Can J Anesth. 2001; 48(5): 427–30. PubMed Abstract | Publisher Full Text\n\nLerman J, Davis PJ, Welborn LG, et al.: Induction, recovery, and safety characteristics of sevoflurane in children undergoing ambulatory surgery. A comparison with halothane. Anesthesiology. 1996; 84(6): 1332–40. PubMed Abstract | Publisher Full Text\n\nPicard V, Dumont L, Pellegrini M: Quality of recovery in children: sevoflurane versus propofol. Acta Anaesthesiol Scand. 2000; 44(3): 307–10. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWelborn LG, Hannallah RS, Norden JM, et al.: Comparison of emergence and recovery characteristics of sevoflurane, desflurane, and halothane in pediatric ambulatory patients. Anesth Analg. 1996; 83(5): 917–20. PubMed Abstract | Publisher Full Text\n\nDavis PJ, Greenberg JA, Gendelman M, et al.: Recovery characteristics of sevoflurane and halothane in preschool-aged children undergoing bilateral myringotomy and pressure equalization tube insertion. Anesth Analg. 1999; 88(1): 34–8. PubMed Abstract | Publisher Full Text\n\nJohannesson GP, Florén M, Lindahl SG: Sevoflurane for ENT-surgery in children. A comparison with halothane. Acta Anaesthesiol Scand. 1995; 39(4): 546–550. PubMed Abstract | Publisher Full Text\n\nGupta A, Stierer T, Zuckerman R, et al.: Comparison of recovery profile after ambulatory anesthesia with propofol, isoflurane, sevoflurane and desflurane: a systematic review. Anesth Analg. 2004; 98(3): 632–41. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCork RC, Vaughan RW, Bentley JB, et al.: General anesthesia for morbidly obese patients--an examination of postoperative outcomes. Anesthesiology. 1981; 54(4): 310–3. PubMed Abstract | Publisher Full Text\n\nSollazzi L, Perilli V, Modesti C, et al.: Volatile anesthesia in bariatric surgery. Obes Surg. 2001; 11(5): 623–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCasati A, Marchetti C, Spreafico E, et al.: Effects of obesity on wash-in and wash-out kinetics of sevoflurane. Eur J Anaesthesiol. 2004; 21(3): 243–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLemmens HJ: Perioperative pharmacology in morbid obesity. Curr Opin Anaesthesiol. 2010; 23(4): 485–91. PubMed Abstract | Publisher Full Text\n\nSalihoglu Z, Karaca S, Kose Y, et al.: Total intravenous anesthesia versus single breath technique and anesthesia maintenance with sevoflurane for bariatric operations. Obes Surg. 2001; 11(4): 496–501. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZaugg M, Lucchinetti E, Uecker M, et al.: Anaesthetics and cardiac preconditioning. Part I. Signalling and cytoprotective mechanisms. Br J Anaesth. 2003; 91(4): 551–65. PubMed Abstract | Publisher Full Text\n\nTanaka K, Ludwig LM, Kersten JR, et al.: Mechanisms of cardioprotection by volatile anesthetics. Anesthesiology. 2004; 100(3): 707–21. PubMed Abstract | Publisher Full Text\n\nStowe DF, Kevin LG: Cardiac preconditioning by volatile anesthetic agents: a defining role for altered mitochondrial bioenergetics. Antioxid Redox Signal. 2004; 6(2): 439–48. PubMed Abstract | Publisher Full Text\n\nBienengraeber MW, Weihrauch D, Kersten JR, et al.: Cardioprotection by volatile anesthetics. Vascul Pharmacol. 2005; 42(5–6): 243–52. PubMed Abstract | Publisher Full Text\n\nPratt PF Jr, Wang C, Weihrauch D, et al.: Cardioprotection by volatile anesthetics: new applications for old drugs? Curr Opin Anaesthesiol. 2006; 19(4): 397–403. PubMed Abstract | Publisher Full Text\n\nPagel PS, Hudetz JA: Delayed cardioprotection by inhaled anesthetics. J Cardiothorac Vasc Anesth. 2011; 25(6): 1125–40. PubMed Abstract | Publisher Full Text\n\nZaugg M, Lucchinetti E, Garcia C, et al.: Anaesthetics and cardiac preconditioning. Part II. Clinical implications. Br J Anaesth. 2003; 91(4): 556–76. PubMed Abstract | Publisher Full Text\n\nDe Hert SG, Turani F, Mathur S, et al.: Cardioprotection with volatile anesthetics: mechanisms and clinical implications. Anesth Analg. 2005; 100(6): 1584–93. PubMed Abstract | Publisher Full Text\n\nDe Hert SG: The concept of anaesthetic-induced cardioprotection: clinical relevance. Best Pract Res Clin Anaesthesiol. 2005; 19(3): 445–59. PubMed Abstract | Publisher Full Text\n\nDe Hert SG: Anesthetic preconditioning: how important is it in today’s cardiac anesthesia? J Cardiothorac Vasc Anesth. 2006; 20(4): 473–6. PubMed Abstract | Publisher Full Text\n\nDe Hert S: Cardioprotection in anaesthesia. Minerva Anaesthesiol. 2008; 74: 259–70.\n\nDe Hert S, Preckel B, Schlack WS: Update on inhalational anaesthetics. Curr Opin Anaesthesiol. 2009; 22(4): 491–5. PubMed Abstract | Publisher Full Text\n\nFrässdorf J, De Hert S, Schlack W: Anaesthesia and myocardial ischaemia/reperfusion injury. Br J Anaesth. 2009; 103(1): 89–98. PubMed Abstract | Publisher Full Text\n\nDe Hert SG: Is anaesthetic cardioprotection clinically relevant? Another futile search for a magic bullet? Eur J Anaesthesiol. 2011; 28(9): 616–7. PubMed Abstract | Publisher Full Text\n\nBein B: Clinical application of the cardioprotective effects of volatile anaesthetics: PRO--get an extra benefit from a proven anaesthetic free of charge. Eur J Anaesthesiol. 2011; 28(9): 620–2. PubMed Abstract | Publisher Full Text\n\nVan Rompaey N, Barvais L: Clinical application of the cardioprotective effects of volatile anaesthetics: CON--total intravenous anaesthesia or not total intravenous anaesthesia to anaesthetise a cardiac patient? Eur J Anaesthesiol. 2011; 28(9): 623–7. PubMed Abstract | Publisher Full Text\n\nVan der Linden PJ, Dierick A, Wilmin S, et al.: A randomized controlled trial comparing an intraoperative goal-directed strategy with routine clinical practice in patients undergoing peripheral arterial surgery. Eur J Anaesthesiol. 2010; 27(9): 788–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZangrillo A, Testa V, Aldrovandi V, et al.: Volatile agents for cardiac protection in noncardiac surgery: a randomized controlled study. J Cardiothorac Vasc Anesth. 2011; 25(6): 902–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLurati Buse GA, Schumacher P, Seeberger E, et al.: Randomized comparison of sevoflurane versus propofol to reduce perioperative myocardial ischemia in patients undergoing noncardiac surgery. Circulation. 2012; 126(23): 2696–704. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDe Hert SG: Cardioprotection by volatile anesthetics: what about noncardiac surgery? J Cardiothorac Vasc Anesth. 2011; 25(6): 899–901. PubMed Abstract | Publisher Full Text\n\nBeck-Schimmer B, Breitenstein S, Urech S, et al.: A randomized controlled trial on pharmacological preconditioning in liver surgery using a volatile anesthetic. Ann Surg. 2008; 248(6): 909–18. PubMed Abstract | F1000 Recommendation\n\nBeck-Schimmer B, Breitenstein S, Bonvini JM, et al.: Protection of pharmacological postconditioning in liver surgery: results of a prospective randomized controlled trial. Ann Surg. 2012; 256(5): 837–44; discussion 844–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDe Conno E, Steurer MP, Wittlinger M, et al.: Anesthetic-induced improvement of the inflammatory response to one-lung ventilation. Anesthesiology. 2009; 110(6): 1316–26. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMellon RD, Simone AF, Rappaport BA: Use of anesthetic agents in neonates and young children. Anesth Analg. 2007; 104(3): 509–20. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSun L: Early childhood general anaesthesia exposure and neurocognitive development. Br J Anaesth. 2010; 105(Suppl 1): i61–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavidson AJ: Neurotoxicity and the need for anesthesia in the newborn: does the emperor have no clothes? Anesthesiology. 2012; 116(3): 507–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHussain M, Berger M, Eckenhoff RG, et al.: General anesthetic and the risk of dementia in elderly patients: current insights. Clin Interv Aging. 2014; 9: 1619–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStrøm C, Rasmussen LS, Sieber FE: Should general anaesthesia be avoided in the elderly? Anaesthesia. 2014; 69(Suppl 1): 35–44. PubMed Abstract | Publisher Full Text\n\nMashour GA, Woodrum DT, Avidan MS: Neurological complications of surgery and anaesthesia. Br J Anaesth. 2015; 114(2): 194–203. PubMed Abstract | Publisher Full Text\n\nGuay J: General anaesthesia does not contribute to long-term post-operative cognitive dysfunction in adults: A meta-analysis. Indian J Anaesth. 2011; 55(4): 358–63. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "10110",
"date": "25 Aug 2015",
"name": "Roderic Eckenhoff",
"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",
"responses": []
},
{
"id": "10111",
"date": "25 Aug 2015",
"name": "Manfred Seeberger",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-626
|
https://f1000research.com/articles/4-623/v1
|
25 Aug 15
|
{
"type": "Research Note",
"title": "Rapid assessment of iron in blood plasma and serum by spectrophotometry with cloud-point extraction",
"authors": [
"Tatyana Samarina",
"Mikhail Proskurnin",
"Tatyana Samarina"
],
"abstract": "Rapid photometric assessment of iron in blood plasma and serum by a simple procedure after the extraction of iron(II) complex with 1-nitroso-2-naphthol in the micellar phase of a nonionic surfactant at the cloud point upon heating (pH range is 4.5–6.3) is proposed. The procedure trueness was verified using a standard reference protocol using bathophenanthroline. The advantages of the procedure are higher sensitivity than the reference protocol: the limit of detection is 0.03 μg/mL, the limit of quantitation is 0.1 μg/mL, the determination range is 0.1 – 2.8 μg/mL (RSD 0.02–0.10). Copper does not interfere with the iron assessment.",
"keywords": [
"iron assessment",
"plasma",
"serum",
"cloud-point extraction",
"spectrophotometry"
],
"content": "Introduction\n\nIron level in blood plasma is affected by many physiological and pathological conditions1. Plasma iron is determined in diagnosing hemochromatosis2,3, acute iron poisoning4, active cirrhosis5, or hepatitis6, which lead to increased levels of transferrin, an iron(III)-binding glycoprotein that transports iron in the human body7. Only 0.1% of the total iron is present in the blood plasma2, thus its assessment should be rather sensitive, precise, and rapid.\n\nIron in plasma/serum is determined by spectrophotometry or atomic-absorption spectroscopy8,9 after the recovery of transferrin-bound iron(III) from acidic solutions using chelatants or detergents10. Highly sensitive and specific though labour-extensive radioisotope11 and immunological12 assays for iron in blood plasma are seldom used due to the need for special equipment and expensive reagents. Spectrophotometric methods are most frequent in clinical practice and based on the formation of iron chelates with bathophenanthroline recommended as a reference method13–15 or its sulfonated analogue14,16, ferrozine17, Ferene S18–20, or Chromazurol S21. However, they are not always sensitive and (e.g. ferrozine) result in overestimation compared to bathophenanthroline22.\n\nWe report rapid photometric determination of iron in blood plasma and serum by a simple procedure after the extraction of iron(II) complex with 1-nitroso-2-naphthol into the micellar phase of a nonionic surfactant at the cloud point upon heating.\n\n\nMethods\n\nAn Agilent Cary 60 spectrophotometer (USA; optical path length, 1 cm) and an inoLab pH Level 1 pH-meter (Germany) with a glass pH-selective electrode (precision ±5%) were used. Solutions were mixed with a Biosan MMS 3000 automixer with a micro-stirrer. Mass-spectrometry measurements were performed on a quadrupole Agilent 7500c ICP-MS (Germany) in a time-resolved analysis mode. The sample introduction system consisted of a robust Babbington nebulizer with a Scott spray chamber (Agilent Technologies) cooled by a Peltier element (2°C). The data were acquired and processed with ICP-MS ChemStation (version G1834B) software (Agilent Technologies).\n\nA GSO 7765-2000 Russian certified reference sample of Fe(III) (1.00 mg/mL in 0.1 M HCl) was used for calibration. 1-nitroso-2-naphthol (Reakhim, Russia) purified as in 23, ascorbic acid (Fluka, China), neonol (AF-neonol 9–12, Elarum, Russia), sodium and ammonium acetates, HCl, trichloroacetic acid (all from KhimMed, Russia), bathophenanthroline (ReaKhim, Russia), and ethanol (Ferien, Russia) were used.\n\nBuffer solutions (pH 4) were prepared by adding the necessary amount of a 1M sodium acetate solution to 0.1 M hydrochloric acid. Chemically pure chloroform (KomponentReaktive, Russia) pre-washed with water from hydrochloric acid was used as a micellar phase diluent.\n\nBlood samples were provided by 2 healthy volunteers. All tests were made in 3 replicates. To obtain native serum, a sample was put in a clean glass test tube and left for 1 h at room temperature to form a clot. The clot was separated from the walls with a glass tip and the sample was centrifuged for 15 min at 1500 rpm. The resulting serum was transferred into a clean test tube. For the decomposition of the iron(III) complex with transferrin, 0.5 ml of serum/plasma in a glass test tube was mixed with 1 ml of 2M HCl, next, 1 ml of fresh 2.5% ascorbic acid solution was added. The sample was diluted to 5 ml and mixed thoroughly.\n\nA 1 ml portion of the test or a calibration solution is mixed with 1 ml of a 0.001M reagent solution in 5% neonol, 0.5 ml of 1M sodium acetate, and 8.5 ml of 5% neonol in a glass test tube. In the blank, 1 ml of distilled water was added instead of plasma/serum. Solutions were stirred in a boiling water bath for 15 min. Blood proteins denaturise and form a viscous white precipitate in the upper phase. Next, test tubes are cooled for 1 min in a cold-water stream, and the upper phase is removed by decanting. The lower, micellar, phase (0.6 mL) is diluted to 1.5 ml of chloroform and absorbance is measured at 715 nm against the blank.\n\n0.7 ml of the test sample was mixed with 0.1 ml of 1% ascorbic acid, 0.35 ml of 1M HCl, and after stirring, with 0.2 ml of 20% trichloroacetic acid and centrifuged at 1500 rpm. A 0.7-ml supernatant of the reaction mixture is transferred into a test tube, 0.6 ml of saturated ammonium acetate and 0.7 ml of a bathophenanthroline solution in ethanol are added. After 1 min, absorbance is measured at 536 nm against the blank.\n\n\nResults and discussion\n\nThe conditions for iron preconcentration with 1-nitroso-2-naphthol into a neonol micellar phase in the cloud point were selected as reported elsewhere24. The iron recovery is 98 ± 2%. The optimum pH range is 4.5–6.3; the limit of detection is 0.03 µg/mL, the determination range is 0.1 – 2.8 µg/mL (RSD 0.02–0.10). The verification of the procedure using the reference protocol (bathophenanthroline) and an independent method (ICP-MS, isotope 54Fe) shows insignificantly different results (Table 1).\n\na ICP-MS\n\nb Reference protocol with bathophenanthroline\n\nThe own colour of the reagent does not affect the blank. It is noteworthy that copper(II), existing in significant quantities in plasma and serum25, does not interfere with the determination as the absorbance maximum of copper complex with nitroso-naphthols lies at 430–490 nm. This avoids using toxic and corrosive thioglycolic acid as a masking reagent. In addition, sample procedure provides the denaturisation of proteins and their removal at the stage of phase separation. Finally, the advantage of the proposed procedure over the bathophenanthroline protocol is much higher sensitivity: while the reference protocol assumes the determination at the boundary of the spectrophotometer working range, the results for our procedure correspond to its middle. Moreover, the separation does not exceed 15 min, which is promising for the development of rapid assessment protocols. It is also noteworthy that the extraction occurs under rather soft conditions, and the pH interval of complex formation in the nonionic surfactant is rather wide (ca. 1 pH both in acidic and alkaline ranges).\n\n\nData availability\n\nF1000Research: Dataset 1. Raw dataset for Samarina et al., 2015 'Rapid assessment of iron in blood plasma and serum by spectrophotometry with cloud-point extraction', 10.5256/f1000research.6716.d10075726",
"appendix": "Author contributions\n\n\n\nMP and TS conceived the study and carried out the research. TS designed the experiments. TS and MP prepared the first draft of the manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe work is supported by The Russian Science Foundation, grant no. 14-23-00012 (MP).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful to Agilent Technologies — Russia and its CEO, Dr. Konstantin Evdokimov, for Agilent equipment used in this study. Samples of blood plasma courtesy of Dr. I. F. Seregina (M.V. Lomonosov Moscow State University).\n\n\nReferences\n\nAndrews NC: Iron metabolism: iron deficiency and iron overload. Annu Rev Genomics Hum Genet. 2000; 1: 75–98. PubMed Abstract | Publisher Full Text\n\nHider RC, Kong X: Iron: effect of overload and deficiency. In: A. Sigel, H. Sigel, RKO. Sigel (Eds.) Interrelations between Essential. Interrelations between Essential Metal Ions and Human Diseases. Springer, Netherlands. 2013; 13: 229–294. PubMed Abstract | Publisher Full Text\n\nKowdley KV: Iron, hemochromatosis, and hepatocellular carcinoma. Gastroenterology. 2004; 127(5 Suppl 1): S79–S86. PubMed Abstract | Publisher Full Text\n\nBanner W Jr, Tong TG: Iron poisoning. Pediatr Clin North Am. 1986; 33(2): 393–409. PubMed Abstract\n\nStål P: Iron as a hepatotoxin. Dig Dis. 1995; 13(4): 205–222. PubMed Abstract\n\nSilva IS, Perez RM, Oliveira PV, et al.: Iron overload in patients with chronic hepatitis C virus infection: clinical and histological study. J Gastroenterol Hepatol. 2005; 20(2): 243–248. PubMed Abstract | Publisher Full Text\n\nWheby MS, Umpierre G: Effect of Transferrin Saturation on Iron Absorption in Man. N Engl J Med. 1964; 271: 1391–1395. PubMed Abstract | Publisher Full Text\n\nJittangprasert P, Wilairat P, Pootrakul P: Comparison of colorimetry and electrothermal atomic absorption spectroscopy for the quantification of non-transferrin bound iron in human sera. Southeast Asian J Trop Med Public Health. 2004; 35(4): 1039–1044. PubMed Abstract\n\nFavier A, Maljournal B, Decoux G, et al.: Microanalysis of serum iron by atomatic absorption spectrophotometry in a graphite oven: improvement and evaluation of this method. Ann Biol Clin (Paris). 1983; 41(1): 45–50. PubMed Abstract\n\nKoshiishi I, Mamura Y, Liu J, et al.: Evaluation of an acidic deproteinization for the measurement of ascorbate and dehydroascorbate in plasma samples. Clin Chem. 1998; 44(4): 863–868. PubMed Abstract\n\nFraser CG, Petersen PH, Ricos C, et al.: Proposed quality specifications for the imprecision and inaccuracy of analytical systems for clinical chemistry. Eur J Clin Chem Clin Biochem. 1992; 30(5): 311–317. PubMed Abstract | Publisher Full Text\n\nLevy AL, Vitacca P: Direct Determination and Binding Capacity of Serum Iron. Clin Chem. 1961; 7(3): 241–248. Reference Source\n\nLewis SM: International Committee for Standardization in Hematology: proposed recommendations for measurement of serum iron in human blood. Am J Clin Pathol. 1971; 56(4): 543–545. PubMed Abstract\n\nRecommendations for measurement of serum iron in human blood. Br J Haematol. 1978; 38(2): 291–294. PubMed Abstract | Publisher Full Text\n\nDerman DP, Green A, Bothwell TH, et al.: A systematic evaluation of bathophenanthroline, ferrozine and ferene in an ICSH-based method for the measurement of serum iron. Ann Clin Biochem. 1989; 26(Pt 2): 144–147. PubMed Abstract | Publisher Full Text\n\nThomas B, Gautam A, Prasad BR, et al.: Evaluation of micronutrient (zinc, copper and iron) levels in periodontitis patients with and without diabetes mellitus type 2: a biochemical study. Indian J Dent Res. 2013; 24(4): 468–73. PubMed Abstract | Publisher Full Text\n\nCarter P: Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Anal Biochem. 1971; 40(2): 450–458. PubMed Abstract | Publisher Full Text\n\nPieroni L, Khalil L, Charlotte F, et al.: Comparison of bathophenanthroline sulfonate and ferene as chromogens in colorimetric measurement of low hepatic iron concentration. Clin Chem. 2001; 47(11): 2059–2061. PubMed Abstract\n\nRevised recommendations for the measurements of the serum iron in human blood. Iron Panel of the International Committee for Standardization in Haematology. Br J Haematol. 1990; 75(4): 615–616. PubMed Abstract | Publisher Full Text\n\nCharlier C, Plomteux G, Vernet M, et al.: Modification of the selected method for the determination of serum iron. Substitution of bathophenanthroline by ferene S. Ann Biol Clin (Paris). 1992; 50(3): 191–202. PubMed Abstract\n\nBrivio G, Brega A, Torelli G: Determination of iron and iron-binding capacity in serum without blank sample. Ric Clin Lab. 1986; 16(4): 523–532. PubMed Abstract\n\nLauber K: Determination of serum iron; a comparison of two methods: Teepol/dithionite/bathophenanthroline versus guanidine/ascorbic acid/Ferrozine (author's transl). J Clin Chem Clin Biochem. 1980; 18(2): 147–148. PubMed Abstract\n\nUmland F, Janssen A, Thierig D, et al.: Theorie und pratische anwendung von komplexbildnern. Akademische Verlagsgesellschaft Frankfurt am Main. 1971. Reference Source\n\nSamarina TO, Ivanov VM, Figurovskaya VN: Optical and chromaticity parameters of transition metal complexes with 1-nitroso-2-naphthol-3,6-disulfonic acid in the presence of surfactants. J Anal Chem (Russ.). 2012; 67(4): 321–329. Publisher Full Text\n\nYamashita S, Abe A, Noma A: Sensitive, direct procedures for simultaneous determinations of iron and copper in serum, with use of 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol (nitro-PAPS) as ligand. Clin Chem. 1992; 38(7): 1373–1375. PubMed Abstract\n\nSamarina T, Proskurnin M: Dataset 1 in: Rapid assessment of iron in blood plasma and serum by spectrophotometry with cloud-point extraction. F1000Research. 2015. Data Source"
}
|
[
{
"id": "12443",
"date": "22 Feb 2016",
"name": "Jean-Paul Canselier",
"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 should better specify the nature of the surfactant 'Neonol AF 9-12', that is a polyethoxylated nonylphenol with ca. 12 ethylene oxide units, and the conditions of the cloud point extraction. In fact, the surfactant cloud point is rather high (ca. 86°C?) and cooling down must be very sudden (quenching). Also, be sure that the conditions for iron preconcentration into the Neonol micellar phase are reported in ref.24.In addition, the paper deals with complexation with 1-nitroso-2 naphtol whereas ref.24 describes complexation with 1-nitroso-2 naphtol-3,6-disulfonic acid.The past tense ('was, were' instead of' is, are') should be used in the § 'Procedure with cloud point extraction' and 'Reference procedure with bathophenanthroline'.",
"responses": []
},
{
"id": "13727",
"date": "09 May 2016",
"name": "Massoud Kaykhaii",
"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 method for the determination of iron in blood serum and plasma by a simple photometric procedure based on the cloud point extraction of a complex formed between ferrous ions and 1-nitroso-2-naphthol. Since spectrophotometric instrumentations own merits of simplicity, cheapness, portability and so on, this makes the paper interesting.The authors claim that this is a “rapid” assessment of iron; therefore, it is better that the total analysis time be specified. Also it’s better to have the complex formation reaction between Iron (II) ions and the ligand. Moreover, I prefer to see a table in which a comparison between the proposed method with other spectrophotometry methods for the determination of Fe(II) in blood samples is included.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-623
|
https://f1000research.com/articles/4-622/v1
|
25 Aug 15
|
{
"type": "Review",
"title": "Viscoelastic Properties of Hyaluronan in Physiological Conditions",
"authors": [
"Mary K. Cowman",
"Tannin A. Schmidt",
"Preeti Raghavan",
"Antonio Stecco",
"Tannin A. Schmidt",
"Preeti Raghavan",
"Antonio Stecco"
],
"abstract": "Hyaluronan (HA) is a high molecular weight glycosaminoglycan of the extracellular matrix (ECM), which is particularly abundant in soft connective tissues. Solutions of HA can be highly viscous with non-Newtonian flow properties. These properties affect the movement of HA-containing fluid layers within and underlying the deep fascia. Changes in the concentration, molecular weight, or even covalent modification of HA in inflammatory conditions, as well as changes in binding interactions with other macromolecules, can have dramatic effects on the sliding movement of fascia. The high molecular weight and the semi-flexible chain of HA are key factors leading to the high viscosity of dilute solutions, and real HA solutions show additional nonideality and greatly increased viscosity due to mutual macromolecular crowding. The shear rate dependence of the viscosity, and the viscoelasticity of HA solutions, depend on the relaxation time of the molecule, which in turn depends on the HA concentration and molecular weight. Temperature can also have an effect on these properties. High viscosity can additionally affect the lubricating function of HA solutions. Immobility can increase the concentration of HA, increase the viscosity, and reduce lubrication and gliding of the layers of connective tissue and muscle. Over time, these changes can alter both muscle structure and function. Inflammation can further increase the viscosity of HA-containing fluids if the HA is modified via covalent attachment of heavy chains derived from Inter-α-Inhibitor. Hyaluronidase hydrolyzes HA, thus reducing its molecular weight, lowering the viscosity of the extracellular matrix fluid and making outflow easier. It can also disrupt any aggregates or gel-like structures that result from HA being modified. Hyaluronidase is used medically primarily as a dispersion agent, but may also be useful in conditions where altered viscosity of the fascia is desired, such as in the treatment of muscle stiffness.",
"keywords": [
"hyaluronan",
"viscosity",
"viscoelasticity",
"lubrication",
"fascia"
],
"content": "Introduction\n\nHyaluronan (HA) is a high molecular weight glycosaminoglycan polymer of the extracellular matrix (ECM) in vertebrate tissues1. It is composed of disaccharides of alternating D-glucuronic acid and N-acetyl D-glucosamine connected by β-1,3 and β-1,4 glycosidic bonds, respectively. In most healthy tissues, HA has an average molecular weight of approximately 6–8 million2. HA has a high turnover rate, but homeostasis is normally maintained by similar rates of synthesis and degradation3,4. It can be enzymatically cleaved by hyaluronidases, or chemically degraded by hydroxyl radicals and peroxynitrite during inflammation5–7. It has a wide variety of physiological functions in the mammalian body, including maintenance of a viscoelastic cushion to protect tissues, control of tissue hydration and water transport, lubrication of biointerfaces, creation of large assemblies with proteins and proteoglycans in the ECM, and receptor-mediated signaling roles in cell detachment, mitosis, migration, tumor development, and inflammation3,8,9. HA is ubiquitous, but is particularly abundant in soft connective tissues, including between deep fascia and muscle, within muscle10–12, and also between the collagen layers that compose the deep fascia. This tissue is a multilayered structure formed by two to three layers of densely packed collagen fibers13,14, spaced by a layer of loose connective tissue (containing adipose cells, sulfated glycosaminoglycans and HA)15–17. The proposed function of HA is to facilitate smooth gliding between these structures during movement, and in the transmission of force generated from muscle contraction.\n\nThe aim of the present study was to examine more closely the viscoelastic properties of HA in association with these structures, and to evaluate if the above mechanisms can be affected by HA viscoelastic variations.\n\n\nReview of the field\n\nSolutions of high molecular weight hyaluronan can be highly viscous with non-Newtonian flow properties (see for example the review by Cowman and Matsuoka18 and references therein). These properties may affect the movement of HA-containing fluid layers within and underlying the deep fascia. Additionally, the concentration and molecular weight of HA affects its contribution to the lubrication of biological interfaces. Changes in the concentration, molecular weight, or even covalent modification of HA in inflammatory conditions, as well as changes in binding interactions with other macromolecules, can have dramatic effects on the sliding movement of fascia.\n\nFor a semi-flexible polymer such as HA, the volume occupied by each chain is very large. Most of the volume is water, not bound by the polymer, and the polymer shape is constantly changing, but the water still contributes to the effective size of each molecule because the solvent movement is affected by frictional interaction with closely spaced polymer segments. Due to its rapid chain motions, the time-average shape of the molecule can be described as a sphere, with greatest density of chain segments near the center. Furthermore, the effective sphere-like volume of a wormlike HA chain in a good solvent grows approximately as the molecular weight raised to the power of 1.8 (=M1.8). This means that, the larger the polymer, the lower the average density because the volume grows faster than the mass. For HA, with molecular weight normally in the millions, this leads to extremely large chain volumes. In contrast, the volume of a compactly folded globular protein chain increases only in direct proportion to the number of amino acids and is therefore proportional to the molecular weight to the first power. The expanded shape of a flexible polymer in solution is a key reason for the high viscosity of \"unfolded\" polymer solutions.\n\nThe hydrodynamic volume of HA chains is usually studied at an ionic strength that is close to physiological. At that ionic strength, the charges due to the carboxylate groups on the HA chain are almost completely screened from each other, and the repulsion between them does not significantly expand the coil volume. In solutions with lower salt concentrations than about 0.15 M NaCl, the electrostatic repulsion would increase the hydrodynamic volume of individual HA molecules, and also increase repulsion between molecules.\n\nThe specific viscosity, ηsp, of an ideal polymer solution is proportional to the fraction of the solution volume that is filled with polymer chains. The Stokes-Einstein equation expresses the specific viscosity of a dilute solution of spherical particles (determined from the solution viscosity, η, and that of the pure solvent, η0) as proportional to the product of the number of spherical particles per unit of solution volume, n, and the volumes of the particles themselves, V. This product corresponds to the volume occupied by all the particles, divided by the solution volume, or the volume fraction, φ, of the solution that is occupied by particles. The occupied volume fraction can also be expressed in terms of the mass concentration of the polymer (c, in g polymer/cm3 of solution) multiplied by the specific volume of the polymer (in cm3 occupied/g). The specific volume (inverse of the density) is proportional to the intrinsic viscosity [η]. As discussed above, the density of the polymer domain decreases with increasing molecular weight, so the intrinsic viscosity is a sensitive measure of the molecular weight. For HA in neutral aqueous salt solution at physiological ionic strength, the intrinsic viscosity is proportional to M0.819.\n\n\n\nFrom Equation 1, we can see that the occupied volume fraction, φ, is equal to 0.4c[η]. When the product c[η] is 2.5, the volume fraction is 1, and the solution can be considered to reach the \"coil overlap\" point. This is the nominal point at which the chains fill the solution and are forced to touch each other, although they already interact at lower concentrations/hydrodynamic volumes, and can interpenetrate at higher concentrations/hydrodynamic volumes because the coil volumes contain mostly solvent. An ideal solution should be much more dilute than the critical concentration for coil overlap. Experimentally, the coil overlap point is usually identified as the value of c[η] above which the specific viscosity begins to dramatically increase.\n\nIn order to estimate the concentration at which a HA solution might exceed coil overlap, we can consider the hydrodynamic size of the polymer at different molecular weights (Figure 1)20. Some example chain parameters are given below (Table 1)20. For HA with a molecular weight of 6 million, overlap requires a HA concentration of only about 320 µg/cm3 (=2.5/7700). For HA with a molecular weight of 1 million, the coil overlap concentration would be about 1400 µg/cm3. For comparison, the concentration of HA in human synovial fluid is usually 2000–3000 µg/ml, and the average molecular weight is close to 6 million, so the HA chains are well above the coil overlap point.\n\nHyaluronan chains with molecular weight of (from left to right) 0.1, 0.5, 1, 3 and 6 million have hydrodynamic diameters of approximately 50, 140, 210, 400, and 600 nm, respectively in physiological saline solution. The diameter of a small globular protein would be on the order of a few nm. Adapted from Cowman and Matsuoka20.\n\nThe chain contour length, L, is calculated as M/ML, where ML is the mass per unit length, approximately 401 nm-1, for the sodium salt form. The intrinsic viscosity of hyaluronan is related to M by the equation [η] = 0.029 M0.80. The specific volume of the polymer, Vs, was obtained from the intrinsic viscosity, as [η]/2.5. Chain hydrodynamic diameter was approximated by the root mean square end-to-end distance, <r2>1/2, of the polymer chain, which is equal to ([η]M/Φ)1/3, where Φ is the Flory constant with the empirical value of 2.1 x 1023. Coil overlap concentration was calculated as 2.5/[η], or equivalently, 1 / Vs. Adapted from Cowman and Matsuoka20.\n\nBased on the ideal model for HA in solution, specific viscosity should be equal to c[η]. A comparison of the observed behavior of HA solutions to the ideal case (Figure 2) shows that the ideal model is far from sufficient. Well below the coil overlap point of c[η]=2.5, HA solutions are already significantly non-ideal. Crowding between molecules increases the viscosity above that expected for the ideal solution.\n\nExperimental data for hyaluronan in physiological saline, plotted using the fitted equation ηsp = c[η] + 0.42(c[η])2 + 7.77 x 10-3 (c[η])4.18 reported by Berriaud and coworkers21, shows a marked increase in viscosity with increasing concentration and intrinsic viscosity. (Note that this data represents low shear conditions, where hyaluronan chains are not distorted or aligned with flow.) The experimental data can be compared with predictions based on theory. For an ideal case in which the hyaluronan molecules act independently, the specific viscosity would simply be equal to the product c[η]. When the molecules become crowded, the effective concentration increases, leading to a significant nonideality contribution, predicted by the last three terms of the mutual macromolecular crowding equation, (Equation 2 in text).\n\nWhen polymer molecules in solution begin to restrict the space available for movement of other chains, the solution is no longer dilute, and our simple model needs modification. In Figure 2, a comparison of the curve from experimental data21 can be seen to deviate from the ideal case at values of c[η] well below the nominal coil overlap point of 2.5.\n\nWe have developed a theory for crowding between flexibly coiled macromolecules like HA18,22–24. It is based on the theory for gel filtration, developed by Ogston and Laurent25–27. The Ogston-Laurent theory for excluded volume provides a rational basis for understanding how proteins can be affected by a random suspension of fibers. A globular (spherical) protein is excluded from the space (a cylindrical shell) surrounding a fiber, by its own radius, and the thickness of the fiber. The center of the spherical protein defines its position, and the center cannot approach the fiber more closely than the sum of the radius of the sphere and the finite radius of the fiber. The probability of a protein finding space in a random suspension of such fibers (corresponding to the interior of a gel bead) is exponentially decreased as a function of the excluded volume. More fibers, or bigger proteins, mean less available space, and lower probability of being inside the beads. A similar picture can be imagined for the crowding of globular proteins by HA chains (Figure 3).\n\nA illustrates the ability of small globular proteins to penetrate most of the hydrodynamic domain of the hyaluronan polymer. B shows the size of the excluded volume for a globular protein in the presence of a segment of a linear polymer as a crowding agent. The cross section of the cylindrical excluded volume has a radius equal to the sum of the radius of the crowding polymer and the thickness of a cylindrical shell determined by the radius of the globular protein. This figure has been reproduced with permission from23 Cowman et al. (2012) in Structure and Function of Biomatrix. Control of Cell Behavior and Gene Expression. Ed. E.A. Balazs, pp.45–66. Copyright 2012 Matrix Biology Institute.\n\nWe adapted the Ogston-Laurent excluded volume concept to the problem of mutual exclusion (mutual macromolecular crowding) that occurs between coiled HA chains in solution. Each chain crowds the others, by an amount related to the hydrodynamic volume it occupies, rather than just its physical chain length and thickness (Figure 4).\n\nThe effective hydrodynamic domain of each chain is modeled as a sphere, the volume of which is dependent on the molecular weight to the 1.8 power. This figure has been reproduced with permission from23 Cowman et al. (2012) in Structure and Function of Biomatrix. Control of Cell behavior and Gene Expression. Ed. E.A. Balazs, pp.45–66. Copyright 2012 Matrix Biology Institute.\n\nThe reduced probability of finding space for movement as a function of increased total concentration makes the effective concentration of the HA greater. The effective concentration is exponentially increased with HA real concentration and with the intrinsic viscosity. Since intrinsic viscosity is a measure of hydrodynamic volume, it is connected with molecular weight. The viscosity of HA solutions should then increase exponentially with concentration and molecular weight (as measured by intrinsic viscosity). We expanded the exponential term into a series. The first four terms of the series provide an excellent approximation of the observed specific viscosity. The first term is the ideal case, and the next three terms provide the nonideality contribution as shown in Figure 2. The sum of the two matches the experimental observation well18.\n\n\n\nNow the extremely high viscosity of HA solutions can be successfully rationalized on the basis of mutual macromolecular crowding, which increases the effective concentration of the HA, and substantially increases the viscosity. There is no need to invoke intermolecular association or ordered structures of the HA molecules. It is also of great interest to note that, depending on the starting point on the curve, increasing or decreasing the HA concentration or intrinsic viscosity (and thus molecular weight) can have enormous impact (e.g., varying as the third or fourth power of the change). The absence of ordered structures in pure semi-dilute HA solutions is also supported by HA diffusion coefficients measured by the confocal Fluorescence Recovery After Photobleaching (FRAP) studies of Hardingham and coworkers28,29.\n\nThe large hydrodynamic volume of HA chains depends on the stiffness of the chain, which is due to steric hindrance to rotation about the linkages between sugar residues, and to the dynamically forming and breaking hydrogen bonds across those linkages. With increasing temperature, rotations about the linkages are easier, and the chains gain flexibility. This shrinks the molecular volume, and consequently reduces the viscosity. It is possible to predict the extent of viscosity reduction, based on Equation 2, and the known dependence of the intrinsic viscosity on temperature. Data from Cleland30 and Fouissac, Milas, and Rinaudo31 show that the intrinsic viscosity of high molecular weight HA is decreased by about 25% as the temperature is increased from 25° to 65°C. Hoefling et al.32 showed that incorporating the 25% decrease in intrinsic viscosity into Equation 2 above gave an excellent prediction of the 2–3 fold experimental change in specific viscosity of semidilute HA solutions over that temperature range. There is no need to propose a change from an ordered conformation to a disordered one, because a modest increase in chain flexibility explains the marked solution viscosity change with temperature.\n\nThe viscosity of HA solutions is not very sensitive to pH in the physiological range. At very high pH, above about pH 11, the rotational freedom at the glycosidic linkages is greatly increased due to breakage of residual hydrogen bonds, and the chain volume shrinks, reducing the solution viscosity33–35. At low pH of about 2.5, at physiological ionic strength, an interesting viscoelastic putty (nearly like a gel) is formed as a result of interchain association33,35–37. But between pH values of 6.5–8.0, the expansion of the hyaluronan chains is nearly constant, and the intrinsic viscosity is not changed38.\n\nWhen a solution containing flexible polymers is flowing, the molecules can become distorted and stretched in the direction of the flow. The more slowly the molecules recover to their undisturbed shapes, relative to the rate of shear, the more they become aligned with the flow. The aligned molecules have a reduced contribution to the solution viscosity. In steady shear conditions, the solution viscosity is highest when the rate of shear is low, and molecules can reorient and relax to the undisturbed shape as rapidly as they move. But with increasing shear rate, the molecules cannot relax fast enough, and the viscosity drops. Figure 5 shows a typical example of the viscosity of a semi-dilute solution of HA in physiological solution39.\n\nData from three consecutive runs are shown. This figure has been reproduced with permission from39 Cowman et al. (2011) Anal. Biochem., 417, 50–56. Copyright 2011 Elsevier Inc.\n\nThis shear rate effect is seen for both dilute and semi-dilute solutions. In dilute solutions, the relaxation time depends on the molecular volume (proportional to the molar volume, [η]M) and the solvent viscosity. The more viscous the solvent, the longer the time needed for relaxation. In semi-dilute, or crowded solutions, the relaxation time is much more strongly increased because molecules must find space to move past each other. Again, the probability of finding space is exponentially related to the excluded volume. The higher the molecular weight, or the higher the concentration, the longer the relaxation time, and the more dramatic the loss of viscosity (shear thinning) with increasing shear rate18.\n\nAnother consequence of the long relaxation time of large HA molecules in semi-dilute solutions is a transition from viscous behavior to elastic behavior as a function of increasing rate of deformation (Figure 6)40. If a solution is cyclically deformed, then slow rates allow the molecules to keep up with changes and flow. But rapid cyclic deformation does not allow the molecules to relax in shape, and instead they behave elastically, stretching and recoiling without flow. This behavior is called viscoelastic. For HA, it plays an important role in its protection of the articular joints under rapid motion. For HA in other tissues such as fascia, it can inhibit flow if the concentration and molecular weight are large enough that elastic behavior dominates under normal rates of motion.\n\nThis figure has been reproduced with permission from40 Gibbs et al. Biopolymers 1968, 6, 777–791. Copyright 1968 John Wiley & Sons, Inc.\n\nThe three main modes of lubrication are boundary, fluid film or hydrodynamic, and mixed. In boundary mode lubrication, surface-to-surface contact occurs between articulating surfaces, and molecules bound to the surface mediate friction. In fluid film lubrication, a thick (relative to the surface roughness of the articulating surfaces) viscous fluid film supports the load and separates the surfaces allowing motion with little resistance to shear. Mixed mode lubrication is where both boundary and fluid film mode lubrication are operative. The conditions under which each mode operate are classically defined by a Stribeck curve (Figure 7), which demonstrates how a friction coefficient (µ = friction force divided by normal force) varies with (velocity × viscosity/load)41. Boundary mode lubrication occurs at the left end of the curve (low velocity and high loads with a small film thickness), whereas fluid film lubrication occurs at right end of the curve (high velocity and low loads with a large film thickness). While this curve was generated using classic hard, non-porous, engineering materials (e.g. steel), and may not be completely applicable to soft, porous, hydrated tissues or materials42, it is still useful in understanding general conditions under which different modes of lubrication are operative.\n\nThe schematic shows boundary, mixed, and hydrodynamic lubrication regimes. This figure has been reproduced with permission from41 Coles et al. (2010) Curr. Opin. Colloid Interface Sci. 15, 406–416. Copyright 2010 Elsevier Ltd.\n\nThe viscosity of HA solutions can affect the mode of lubrication in which HA reduces friction as its relative effectiveness in reducing friction at tissue biointerfaces. For example, in a boundary mode of lubrication at a cartilage-cartilage biointerface, onto which HA is able to bind, relative effectiveness of friction reduction (especially static friction, the resistant to start up motion) has been shown to be dependent on the molecular weight of HA, with higher molecular weight resulting in lower friction (Figure 8)43. This has been speculated to be due to a ‘viscous boundary layer’ of HA at the surface of cartilage44.\n\nRegression lines are shown for (A) mean static µstatic, Neq friction values at pre-sliding duration, Tps = 1,200 s, and (B) mean kinetic <µkinetic, Neq> friction values at Tps = 1.2 s obtained vs log molecular weight of hyaluronan. Mean values in phosphate-buffered saline (PBS) and synovial fluid (SF) are shown for reference. This figure has been reproduced with permission from43 Kwiecinski et al. (2011) Osteoarthritis Cartilage 19, 1356–1362. Copyright 2011 Osteoarthritis Research Society International.\n\nConversely, in fluid film lubrication where a thick film of HA separates articulating surfaces, friction would be predicted to increase with viscosity, potentially reaching very high levels. In deep fascia, the thickness of the HA-containing fluid layer is apparently on the order of tens of microns, which is large compared with the diameter of the molecules and even the roughness of the surfaces. In such a case, if HA concentration and/or molecular weight are high, the resistance to flow due to high viscosity can negatively affect lubrication.\n\nLubricin is a lubricating mucin-like glycoprotein present in various body fluids, such as synovial fluid (essentially a 2–3 mg/ml solution of high molecular weight HA), that can alter the viscosity of HA solutions. We have shown that lubricin is able to reduce the viscosity of a high molecular weight HA solution when both components are present at physiological concentrations (Figure 9)45, potentially by binding and shrinking the hydrodynamic domains of HA molecules, enabling them to flow more easily. In cases where HA concentration becomes high and viscous flow is reduced, lubricin could facilitate increased motion and thus decreased friction.\n\nShear rate dependent specific viscosity at 25°C of hyaluronan at 3.3 mg/ml alone and with 450 μg/ml rhPRG4, shown in black. Predicted specific viscosity (experimental hyaluronan + experimental rhPRG4) shown in red. This figure has been reproduced with permission from45 Ludwig et al. (2014) Biorheology 51, 409–422. Copyright 2014 IOS Press and the authors.\n\nA common observation in inflamed tissues is an increase in the concentration of HA2. The HA content of injured skeletal muscle is known to be elevated46. Stecco et al.17 documented, with a highly specific HA-binding peptide, the deposition of HA inside the loose connective tissue in three different fasciae of the body: fascia lata, rectus abdominis sheet and sternocleidomastoid (SCM) fascia. Stecco et al.20 also documented an increase of the thickness of the loose connective tissue in the SCM fascia in patients complaining of chronic neck pain syndrome. If the HA content of fascia is increased, the viscosity and elasticity of the HA-containing fluid would be increased, and its fluid film lubricating properties reduced.\n\nPossibly more important might be the covalent modification of HA by heavy chain domains derived from plasma inter-α-inhibitor (IαI). An increase in the expression of TSG-6 protein is commonly observed during inflammation. TSG-6 acts catalytically to transfer heavy chain (HC) domains from the chondroitin sulfate chain of IαI to HA47–49. This transfer is normally a protective function that can stabilize the pericellular coat of cells. The HC domains can dimerize, and effectively act to hold HA chains noncovalently together50,51. It could be imagined that the HA, modified by HC, becomes gel-like and immobile in the deep fascia. HC-modified HA can also be found aggregated into fibers or cables52,53. An increase in both HA and TSG-6 has been reported in cultured vascular smooth muscle cells subjected to mechanical strain54, and proliferating smooth muscle cells in rat neointima after injury express high levels of TSG-655. Recently, an increase in HA, TSG-6, and HC-modified HA was observed in damaged mouse skeletal muscle tissue46.\n\n\nDiscussion\n\nThe fascia assumes a fundamental role with its two components: dense connective tissue (collagen fibers type I and III) and loose connective tissue (adipose cells, GAGs (glycosaminoglycans), and HA). HA is an important component of the loose connective tissue in fascia. In this review, we have considered the physico-chemical properties of HA solutions, and how they depend on factors such as concentration, molecular weight, and modification by covalent linkage to HC derived from IαI, or noncovalent interactions with proteins such as lubricin.\n\nImmobilization of a limb or body segment can lead to an increase in the concentration of HA within and between the fascial and muscular compartments, which can increase the fluid viscosity. The increased fluid viscosity within the loose connective tissue can in turn decrease the gliding between the layers of collagen fibers, which may be perceived by the subject as stiffness56. Changes documented in rat soleus muscle due to one week of immobilization include increase in HA concentration and shortening of sarcomere length57. These changes were postulated to increase the number of cross bridges attached during contraction58,59. In the early stages of this process, the arrangement of collagen fibrils in the endomysium may remain longitudinal, however, by about 4 weeks, the collagen fibrils became arranged circumferentially, which signals pre-contracture. Thus subtle changes in the turnover of HA and in the properties of the extracellular matrix with immobility can lead to structural and eventually functional changes in the muscles with significant consequences on movement60.\n\nThe interdependence of mechanoreceptor activation and viscoelasticity of the surrounding tissue has been previously noted61–65. Due to the fundamental role of HA in determining the viscoelasticity of fluids in soft connective tissues, its alteration could therefore modify the activation of the receptors, producing non-specific musculoskeletal pain.\n\nThe increased HA content of fascia and the underlying muscle may result from increased HA synthesis, due to a stimulation of the fibroblast-like cells that were previously suggested to be the biosynthetic source of hyaluronan17. It may also reflect impaired turnover via flow toward the lymph. A high HA concentration would increase the viscosity of the HA-containing fluids. When the viscosity of the fluid in the loose connective tissue increases due to increased HA concentration or its covalent modification, the dense connective tissue can spread the stiffness throughout the surrounding areas, driving even further the sensation of muscle stiffness. Deep friction manipulation may aid outflow of HA if the effective shear rate within the fluid layers generates a drop in the viscosity. This may explain the reduced perception of stiffness that is reported by both therapist and patient during this manual treatment. This review suggests a basis for the typical finding in manual therapy: the more chronic is the stiffness, the higher the concentration of HA may be, and the greater the effort and time required for manual treatments65.\n\nThere are a number of HA-cleaving enzymes6. For medical applications, a preparation containing a recombinant fragment of human PH20 hyaluronidase is currently available. It hydrolyzes HA (and susceptible linkages in chondroitin sulfate glycosaminoglycans) by splitting the glycosidic bond between C1 of an N-acetylhexosamine moiety and C4 of a glucuronic acid moiety. It reduces the molecular weight, and would be expected to lower the viscosity of the extracellular matrix fluid and thus make outflow easier. It can also disrupt aggregates or gel made by HA crosslinked via HC chains. The products of enzymatic cleavage may include small oligosaccharides of HA, which have been reported to trigger specific inflammatory responses and have a pro-inflammatory effect66,67. That possibility has been disputed68. In any case, the presence of fragments generated by the action of hyaluronidase is expected to be short lived as restored flow can wash the small polymers away. Hyaluronidase is used primarily as a dispersion agent, but may now be considered for use in conditions where altered viscosity of the fascia is desired, such as in muscle stiffness.\n\n\nConclusion\n\nThe physico–chemical properties of HA are modulated by its concentration, molecular weight, solvent ionic composition, temperature, and covalent or noncovalent binding of proteins and other species. If HA is forced to exist in a highly crowded environment, or more generally if its density within the loose connective tissue inside the fascia is increased as a result of injury or other pathological process, the behavior of the whole deep fascia and of the underlying connective tissue epimysium and perimysium could be compromised. Treatments that address the role of HA may hold promise.",
"appendix": "Author contributions\n\n\n\nMC, TS, PR and AS contributed to the writing of the manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMC was supported in part by a grant from the Endre A. Balazs Foundation. TS, PR, and AS declared that no grants were involved in supporting their contributions.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nLaurent TC, Fraser JR: Hyaluronan. FASEB J. 1992; 6(7): 2397–404. PubMed Abstract\n\nCowman MK, Lee HG, Schwertfeger KL, et al.: The Content and Size of Hyaluronan in Biological Fluids and Tissues. Front Immunol. 2015; 6: 261. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFraser JR, Laurent TC, Laurent UB: Hyaluronan: its nature, distribution, functions and turnover. 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EA Balazs, Editor. Matrix Biology Institute: Edgewater, NJ, 2012; 45–66.\n\nCowman MK, Mendichi R: Methods for Determination of Hyaluronan Molecular Weight. In Chemistry and Biology of Hyaluronan, HG Garg and CA Hales, Editors. Elsevier: Amsterdam, 2004; 41–69. Publisher Full Text\n\nOgston AG: The Spaces in a Uniform Random Suspension of Fibres. Trans Faraday Soc. 1958; 54(11): 1754–1757. Publisher Full Text\n\nLaurent TC, Killander J: A theory of gel filtration and its experimental verification. J Chromatogr. 1964; 14(3): 317–330. Publisher Full Text\n\nLaurent TC: The interaction between polysaccharides and other macromolecules. 9. The exclusion of molecules from hyaluronic acid gels and solutions. Biochem J. 1964; 93(1): 106–12. PubMed Abstract | Free Full Text\n\nGribbon P, Heng BC, Hardingham TE: The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching. Biophys J. 1999; 77(4): 2210–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGribbon P, Heng BC, Hardingham TE: The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain-chain association. Biochem J. 2000; 350(Pt 1): 329–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCleland RL: Effect of Temperature on the Limiting Viscosity Number of Hyaluronic Acid and Chondroitin 4-Sulfate. Biopolymers. 1979; 18(7): 1821–1828. Publisher Full Text\n\nFouissac E, Milas M, Rinaudo M: Shear-rate, Concentration, Molecular Weight, and Temperature Viscosity Dependencies of Hyaluronate, a Wormlike Polyelectrolyte. Macromolecules. 1993; 26(25): 6945–6951. Publisher Full Text\n\nHoefling JM, Cowman MK, Matsuoka S, et al.: Temperature Effect on the Dynamic Rheological Characteristics of Hyaluronan, Hylan A and Synvisc®. In Hyaluronan, JF Kennedy, Phillips GO, Williams PA, Hascall VC, Editor. Woodhead Publishing Ltd, Cambridge, 2002; 103–108. Publisher Full Text\n\nMorris ER, Rees DA, Welsh EJ: Conformation and dynamic interactions in hyaluronate solutions. J Mol Biol. 1980; 138(2): 383–400. PubMed Abstract | Publisher Full Text\n\nMathews MB, Decker L: Conformation of hyaluronate in neutral and alkaline solutions. Biochim Biophys Acta. 1977; 498(1): 259–63. PubMed Abstract | Publisher Full Text\n\nGatej I, Popa M, Rinaudo M: Role of the pH on hyaluronan behavior in aqueous solution. Biomacromolecules. 2005; 6(1): 61–7. PubMed Abstract | Publisher Full Text\n\nPigman W, Hawkins W, Gramling E, et al.: Factors affecting the viscosity of hyaluronic acid and synovial fluid. Arch Biochem Biophys. 1960; 89: 184–93. PubMed Abstract | Publisher Full Text\n\nBalazs EA: Sediment volume and viscoelastic behavior of hyaluronic acid solutions. Fed Proc. 1966; 25(6): 1817–22. PubMed Abstract\n\nBalazs EA, Cowman MK, Briller SO, et al.: On the Limiting Viscosity Number of Hyaluronate in Potassium Phosphate Buffers Between pH 6.5 and 8. Biopolymers. 1983; 22(2): 589–591. Publisher Full Text\n\nCowman MK, Chen CC, Pandya M, et al.: Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan. Anal Biochem. 2011; 417(1): 50–6. PubMed Abstract | Publisher Full Text\n\nGibbs DA, Merrill EW, Smith KA, et al.: Rheology of hyaluronic acid. Biopolymers. 1968; 6(6): 777–91. PubMed Abstract | Publisher Full Text\n\nColes JM, Chang DP, Zauscher S: Molecular mechanisms of aqueous boundary lubrication by mucinous glycoproteins. Curr Opin Colloid In Sci. 2010; 15(6): 406–416. Publisher Full Text\n\nDunn AC, Sawyer WG, Angelini TE: Gemini Interfaces in Aqueous Lubrication with Hydrogels. Tribol Lett. 2014; 54(1): 59–66. Publisher Full Text\n\nKwiecinski JJ, Dorosz SG, Ludwig TE, et al.: The effect of molecular weight on hyaluronan's cartilage boundary lubricating ability--alone and in combination with proteoglycan 4. Osteoarthritis Cartilage. 2011; 19(11): 1356–62. PubMed Abstract | Publisher Full Text\n\nYakubov GE, McColl J, Bongaerts JH, et al.: Viscous boundary lubrication of hydrophobic surfaces by mucin. Langmuir. 2009; 25(4): 2313–21. PubMed Abstract | Publisher Full Text\n\nLudwig TE, Cowman MK, Jay GD, et al.: Effects of concentration and structure on proteoglycan 4 rheology and interaction with hyaluronan. Biorheology. 2014; 51(6): 409–22. PubMed Abstract | Publisher Full Text\n\nTorihashi S, Ho M, Kawakubo Y, et al.: Acute and Temporal Expression of TNF-α-stimulated Gene 6 Product,TSG-6,in Mesenchymal Stem Cells Creates Microenvironments Required for Their Successul Transplantation into the Muscle Tissue. J Biol Chem. 2015. PubMed Abstract | Publisher Full Text\n\nMilner CM, Higman VA, Day AJ: TSG-6: a pluripotent inflammatory mediator? Biochem Soc Trans. 2006; 34(Pt 3): 446–50. PubMed Abstract | Publisher Full Text\n\nSanggaard KW, Sonne-Schmidt CS, Krogager TP, et al.: TSG-6 transfers proteins between glycosaminoglycans via a Ser28-mediated covalent catalytic mechanism. J Biol Chem. 2008; 283(49): 33919–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang L, Yoneda M, Kimata K: A serum-derived hyaluronan-associated protein (SHAP) is the heavy chain of the inter alpha-trypsin inhibitor. J Biol Chem. 1993; 268(35): 26725–30. PubMed Abstract\n\nYingsung W, Zhuo L, Morgelin M, et al.: Molecular heterogeneity of the SHAP-hyaluronan complex. Isolation and characterization of the complex in synovial fluid from patients with rheumatoid arthritis. J Biol Chem. 2003; 278(35): 32710–8. PubMed Abstract | Publisher Full Text\n\nHe H, Li W, Tseng DY, et al.: Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-alpha-inhibitor (HC*HA) purified from extracts of human amniotic membrane. J Biol Chem. 2009; 284(30): 20136–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde la Motte CA, Hascall VC, Drazba J, et al.: Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function. Am J Pathol. 2003; 163(1): 121–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMajors AK, Austin RC, de la Motte CA, et al.: Endoplasmic reticulum stress induces hyaluronan deposition and leukocyte adhesion. J Biol Chem. 2003; 278(47): 47223–31. PubMed Abstract | Publisher Full Text\n\nLee RT, Yamamoto C, Feng Y, et al.: Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells. J Biol Chem. 2001; 276(17): 13847–51. PubMed Abstract | Publisher Full Text\n\nYe L, Mora R, Akhayani N, et al.: Growth factor and cytokine-regulated hyaluronan-binding protein TSG-6 is localized to the injury-induced rat neointima and confers enhanced growth in vascular smooth muscle cells. Circ Res. 1997; 81(3): 289–96. PubMed Abstract | Publisher Full Text\n\nStecco A, Gesi M, Stecco C, et al.: Fascial components of the myofascial pain syndrome. Curr Pain Headache Rep. 2013; 17(8): 352. PubMed Abstract | Publisher Full Text\n\nOkita M, Yoshimura T, Nakano J, et al.: Effects of reduced joint mobility on sarcomere length, collagen fibril arrangement in the endomysium, and hyaluronan in rat soleus muscle. J Muscle Res Cell Motil. 2004; 25(2): 159–66. PubMed Abstract | Publisher Full Text\n\nLieber RL, Steinman S, Barash IA, et al.: Structural and functional changes in spastic skeletal muscle. Muscle Nerve. 2004; 29(5): 615–27. PubMed Abstract | Publisher Full Text\n\nJulian FJ, Morgan DL: Tension, stiffness, unloaded shortening speed and potentiation of frog muscle fibres at sarcomere lengths below optimum. J Physiol. 1981; 319(1): 205–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStecco A, Stecco C, Raghavan P: Peripheral mechanisms contributing to spasticity and implications for treatment. Curr Phys Med Rehabil Rep. 2014; 2(2): 121–227. Publisher Full Text\n\nSong Z, Banks RW, Bewick GS: Modelling the mechanoreceptor's dynamic behaviour. J Anat. 2015; 227(2): 243–54. PubMed Abstract | Publisher Full Text\n\nBell J, Holmes M: Model of the dynamics of receptor potential in a mechanoreceptor. Math Biosci. 1992; 110(2): 139–74. PubMed Abstract | Publisher Full Text\n\nSuslak TJ, Armstrong JD, Jarman AP: A general mathematical model of transduction events in mechano-sensory stretch receptors. Network. 2011; 22(1–4): 133–42. PubMed Abstract\n\nLoewenstein WR, Skalak R: Mechanical transmission in a Pacinian corpuscle. An analysis and a theory. J Physiol. 1966; 182(2): 346–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwerup C, Rydqvist B: A mathematical model of the crustacean stretch receptor neuron. Biomechanics of the receptor muscle, mechanosensitive ion channels, and macrotransducer properties. J Neurophysiol. 1996; 76(4): 2211–20. PubMed Abstract\n\nJiang D, Liang J, Noble PW: Hyaluronan as an immune regulator in human diseases. Physiol Rev. 2011; 91(1): 221–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStern R, Asari AA, Sugahara KN: Hyaluronan fragments: an information-rich system. Eur J Cell Biol. 2006; 85(8): 699–715. PubMed Abstract | Publisher Full Text\n\nHuang Z, Zhao C, Chen Y, et al.: Recombinant human hyaluronidase PH20 does not stimulate an acute inflammatory response and inhibits lipopolysaccharide-induced neutrophil recruitment in the air pouch model of inflammation. J Immunol. 2014; 192(11): 5285–95. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10135",
"date": "02 Sep 2015",
"name": "Timothy E. Hardingham",
"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 review of the current biophysics and biology of hyaluronan. It presents an analysis of the complex non-ideal behaviour of hyaluronan characterised by different biophysical techniques and interprets this technical information in a way that enables the non-specialist to understand the consequences these properties have in biology. It's up to date and presents a very readable insight into the complex behaviour of this essentially simple biopolymer.",
"responses": []
},
{
"id": "10133",
"date": "14 Sep 2015",
"name": "John Sandy",
"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 expertly constructed review article on hyaluronan (HA) provides an in-depth analysis of the relationship between its structural and physicochemical properties. The article also contains a well-informed update on the effects of modifications, such as protein binding, cross-linking and molecular weight on these properties. In particular, this is discussed in relation to its role in regulating the viscoelasticity of loose connective tissues (fascia) required for effective organ separation. For example, the authors point out that abnormally high HA-dependent viscosity in muscle fascia can lead to local and more generalized stiffness, and potentially mechanosensor-mediated pain. Indeed, this section could be expanded to underline the importance of future research on the effects of structural modifications on catabolic processing of HA, both within the extracellular matrix and in fluid compartments. On the general topic of hyaluronan-mediated effects at interfaces, readers will also find interesting the papers from Richter and colleagues who are examining the effects of end-grafted HA on the properties of glycoconjugate cell coats (Richter, et al., 2007), lipid membranes (Attili, et al., 2012), nanoparticles (Bano, et al., 2015) and pulmonary surfactants (Lopez-Rodriguez, et al., 2013).We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Competing Interests: No competing interests were disclosed.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-622
|
https://f1000research.com/articles/4-620/v1
|
24 Aug 15
|
{
"type": "Opinion Article",
"title": "Hypothesis: Is frequent, commercial jet travel by the general public a risk factor for developing cutaneous melanoma?",
"authors": [
"Harvey Arbesman"
],
"abstract": "Melanoma incidence has been increasing worldwide over the past 50 years and various risk factors have been identified. Interestingly, multiple studies have shown a multifold increased risk of developing melanoma in jet pilots and airline crew. There has also been a dramatic increase in the availability and frequency of jet travel by the general population during this time period.. Therefore, it is hypothesized that frequent commercial jet travel may represent an additional risk factor for the development of cutaneous melanoma in susceptible individuals of the general public.",
"keywords": [
"melanoma",
"risk factor",
"epidemiology",
"jet travel",
"cosmic radiation"
],
"content": "Background\n\nThe incidence of cutaneous melanoma in light pigmented individuals worldwide has been steadily increasing over the past 4–5 decades1–4. Sun exposure, including intermittent exposure, is an important environmental risk factor for melanoma, along with history of sunburn, residence in equatorial latitudes and tanning bed usage5–11. Genetic risk factors include red hair, family history of melanoma, dysplastic nevi, lightly pigmented skin, tendency to burn, inability to tan, and DNA repair defects12. Phenotypic expressions of gene/environmental interactions are risk factors and include melanocytic nevi (increased total number, multiple atypical [dysplastic], and congenital [particularly large axial lesions with multiple satellites]), ephelides, and personal history of melanoma12,13. Non-solar occupational risk factors have also been shown to be associated with the development of melanoma14. Since no specific currently recognized risk factor adequately explains the rapidly increasing incidence among the general population, the identification of new risk factors that could play a role in melanoma prevention is needed.\n\nStudies from many countries have shown a significant increase in the risk of melanoma in commercial and military pilots; these range from approximately 2 to10 fold15–20. An increased risk has also been shown in some studies of cabin crew21–23. Recently a meta-analysis was published that also showed an increased risk in pilots and cabin crew24.\n\nThough it is unclear why aircrew have an increased risk of melanoma, one proposed explanation regarding their increased risk has focused on the exposure to cosmic ionizing radiation present at cruising altitudes of 30,000 feet or higher25–34. Currently, commercial jets frequently cruise at an altitude that results in cosmic radiation exposure26,28,30–33. In addition, the cosmic radiation exposure is increased with higher altitude flights and long-haul routes25. Cosmic ionizing radiation contains multiple particles that can damage DNA28,32. Epidemiologic studies have reported a possible relationship between melanoma and exposure to ionizing radiation in other occupational settings35–37. A recent systematic review regarding cosmic radiation and cancer assessed the role cosmic radiation plays in the development of cancer as compared with other lifestyle factors25.\n\nAnother proposed explanation for the documented increased risk among pilots is their increased exposure to UV light during flight24,38. Recently an analysis was done and found greater amounts of UVA exposure through the windows of jets39. At this time, it is unclear whether increased UVA exposure, cosmic ionizing radiation, circadian rhythm disturbance and/or other undetermined factors are contributing to this increased risk of melanoma in pilots and cabin crew26.\n\n\nPresentation of the hypothesis\n\nIt is hypothesized that individuals of the general population who frequently travel by jet plane may also have an increased risk of developing cutaneous melanoma in a similar fashion as pilots and cabin crew. This hypothesized risk factor may play a role in the development of melanoma due to a variety of potentially harmful exposures associated with frequent jet travel that could interact synergistically with other known genetic and environmental risk factors in susceptible individuals. This hypothesis is based on the findings that both pilots and cabin crew, generally healthier than the general public18, and with a prevalence of skin cancer risk factors similar to that of the general public21,40, have a dramatically higher risk of melanoma.\n\nSince the late 1950s, commercial jets have begun to cruise at an altitude of 30,000 feet or higher. This major change in the flying altitude of air travel is consistent with the temporal nature of the rapid increase in the incidence of melanoma. During the past 50 years, the availability and frequency of jet travel among the general public have increased dramatically. In addition, with the deregulation of the airline industry in the 1970s, and the resulting decrease in fares, a higher percentage of the overall population began to experience jet travel. An ecologic study found an association between accessibility to air travel and the incidence rate of melanoma41.\n\nA widely accepted epidemiologic finding consistent with the proposed hypothesis is that intermittent sun exposure is an independent risk factor for melanoma5,11. The correlation between melanoma incidence and ‘sun holidays’42–46 has been primarily interpreted as secondary to intermittent sun exposure. The proposed hypothesis suggests that the increased risk associated with ‘sun holidays,’ may also be related to jet travel and the resultant exposures associated with jet travel to those vacation destinations. Higher socioeconomic status (SES) is also a risk factor for melanoma development42,47; and higher SES is associated with increased jet travel and ‘sun holidays,’44,45,48 all findings consistent with the hypothesis.\n\n\nTesting the hypothesis\n\nA case-control methodology could be utilized to test this hypothesis. One would assess jet travel histories in melanoma patients and comparable controls, controlling for known risk factors such as age, skin type, genetic host factors and sun exposure history. One would obtain assessments of subjects’ jet travel history in terms of frequency, duration of flights and altitude49,50. Assessment of travel routes, season of travel and increased sunspot activity during flights should also be undertaken. It would be necessary to disentangle intermittent sun exposure history from jet travel history.\n\n\nImplications of the hypothesis\n\nIt is hypothesized that frequent commercial jet travel by the general public may increase the risk of developing melanoma due to various harmful exposures associated with frequent jet travel. This melanoma and jet travel hypothesis has potential for reducing melanoma-associated morbidity and mortality and warrants properly designed analytic epidemiologic evaluation to assess the validity of this hypothesis. In addition, if demonstrated to be a risk factor, evaluation of the underlying mechanisms behind this increased risk may lead to the expansion of basic science research of etiologic factors of melanoma and of cancer in general.",
"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\nAcknowledgements\n\nI thank Joshua Arbesman, MD, James Marshall, PhD and Thomas Helm, MD for fruitful discussions about the hypothesis and reading drafts of the manuscript.\n\n\nReferences\n\nErdmann F, Lortet-Tieulent J, Schüz J, et al.: International trends in the incidence of malignant melanoma 1953-2008--are recent generations at higher or lower risk? Int J Cancer. 2013; 132(2): 385–400. PubMed Abstract | Publisher Full Text\n\nHolterhues C, Hollestein LM, Nijsten T, et al.: Burden of disease due to cutaneous melanoma has increased in the Netherlands since 1991. Br J Dermatol. 2013; 169(2): 389–97. PubMed Abstract | Publisher Full Text\n\nErdei E, Torres SM: A new understanding in the epidemiology of melanoma. Expert Rev Anticancer Ther. 2010; 10(11): 1811–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Vries E, Bray FI, Coebergh JW, et al.: Changing epidemiology of malignant cutaneous melanoma in Europe 1953-1997: rising trends in incidence and mortality but recent stabilizations in western Europe and decreases in Scandinavia. Int J Cancer. 2003; 107(1): 119–26. PubMed Abstract | Publisher Full Text\n\nChang YM, Barrett JH, Bishop DT, et al.: Sun exposure and melanoma risk at different latitudes: a pooled analysis of 5700 cases and 7216 controls. Int J Epidemiol. 2009; 38(3): 814–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGandini S, Sera F, Cattaruzza MS, et al.: Meta-analysis of risk factors for cutaneous melanoma: II. Sun exposure. Eur J Cancer. 2005; 41(1): 45–60. PubMed Abstract | Publisher Full Text\n\nSolomon CC, White E, Kristal AR, et al.: Melanoma and lifetime UV radiation. Cancer Causes Control. 2004; 15(9): 893–902. 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PubMed Abstract | Publisher Full Text\n\nGandini S, Sera F, Cattaruzza MS, et al.: Meta-analysis of risk factors for cutaneous melanoma: I. Common and atypical naevi. Eur J Cancer. 2005; 41(1): 28–44. PubMed Abstract | Publisher Full Text\n\nFortes C, de Vries E: Nonsolar occupational risk factors for cutaneous melanoma. Int J Dermatol. 2008; 47(4): 319–28. PubMed Abstract | Publisher Full Text\n\nYong LC, Pinkerton LE, Yiin JH, et al.: Mortality among a cohort of U.S. commercial airline cockpit crew. Am J Ind Med. 2014; 57(8): 906–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHammer GP, Auvinen A, De Stavola BL, et al.: Mortality from cancer and other causes in commercial airline crews: a joint analysis of cohorts from 10 countries. Occup Environ Med. 2014; 71(5): 313–22. 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PubMed Abstract | Publisher Full Text\n\nGrajewski B, Pinkerton LE: Exposure assessment at 30 000 feet: challenges and future directions. Ann Occup Hyg. 2013; 57(6): 692–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchüz J: Airline crew cohorts: is there more to learn regarding their cancer risk? Occup Environ Med. 2014; 71(5): 307. PubMed Abstract | Publisher Full Text\n\nSigurdson AJ, Ron E: Cosmic radiation exposure and cancer risk among flight crew. Cancer Invest. 2004; 22(5): 743–61. PubMed Abstract | Publisher Full Text\n\nLim MK: Cosmic rays: are air crew at risk? Occup Environ Med. 2002; 59(7): 428–32; discussion 432–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFriedberg W, Copeland K, Duke FE, et al.: Radiation exposure during air travel: guidance provided by the Federal Aviation Administration for air carrier crews. Health Phys. 2000; 79(5): 591–5. PubMed Abstract | Publisher Full Text\n\nKendall GM: Factors affecting cosmic-ray doses at aircraft altitudes. Health Phys. 2000; 79(5): 560–2. PubMed Abstract | Publisher Full Text\n\nGoldhagen P: Overview of aircraft radiation exposure and recent ER-2 measurements. Health Phys. 2000; 79(5): 526–44. PubMed Abstract | Publisher Full Text\n\nTownsend LW: Invited editorial: Radiation exposures of aircrew in high altitude flight. J Radiol Prot. 2001; 21(1): 5–8. PubMed Abstract | Publisher Full Text\n\nMenzel HG, O'Sullivan D, Beck P, et al.: European measurements of aircraft crew exposure to cosmic radiation. Health Phys. 2000; 79(5): 563–7. PubMed Abstract | Publisher Full Text\n\nFink CA, Bates MN: Melanoma and ionizing radiation: is there a causal relationship? Radiat Res. 2005; 164(5): 701–10. PubMed Abstract | Publisher Full Text\n\nFreedman DM, Sigurdson A, Rao RS, et al.: Risk of melanoma among radiologic technologists in the United States. Int J Cancer. 2003; 103(4): 556–62. PubMed Abstract | Publisher Full Text\n\nCaldwell GG, Kelley D, Zack M, et al.: Mortality and cancer frequency among military nuclear test (Smoky) participants, 1957 through 1979. JAMA. 1983; 250(5): 620–4. PubMed Abstract | Publisher Full Text\n\nChorley AC, Evans BJ, Benwell MJ: Civilian pilot exposure to ultraviolet and blue light and pilot use of sunglasses. Aviat Space Environ Med. 2011; 82(9): 895–900. PubMed Abstract | Publisher Full Text\n\nSanlorenzo M, Vujic I, Posch C, et al.: The risk of melanoma in pilots and cabin crew: UV measurements in flying airplanes. JAMA Dermatol. 2015; 151(4): 450–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRafnsson V, Hrafnkelsson J, Tulinius H, et al.: Risk factors for cutaneous malignant melanoma among aircrews and a random sample of the population. Occup Environ Med. 2003; 60(11): 815–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgredano YZ, Chan JL, Kimball RC, et al.: Accessibility to air travel correlates strongly with increasing melanoma incidence. Melanoma Res. 2006; 16(1): 77–81. PubMed Abstract | Publisher Full Text\n\nIdorn LW, Wulf HC: Socioeconomic status and cutaneous malignant melanoma in Northern Europe. Br J Dermatol. 2014; 170(4): 787–93. PubMed Abstract | Publisher Full Text\n\nVeierød MB, Adami HO, Lund E, et al.: Sun and solarium exposure and melanoma risk: effects of age, pigmentary characteristics, and nevi. Cancer Epidemiol Biomarkers Prev. 2010; 19(1): 111–20. PubMed Abstract | Publisher Full Text\n\nBentham G, Aase A: Incidence of malignant melanoma of the skin in Norway, 1955-1989: associations with solar ultraviolet radiation, income and holidays abroad. Int J Epidemiol. 1996; 25(6): 1132–8. PubMed Abstract | Publisher Full Text\n\nWesterdahl J, Olsson H, Ingvar C, et al.: Southern travelling habits with special reference to tumour site in Swedish melanoma patients. Anticancer Res. 1992; 12(5): 1539–42. PubMed Abstract\n\nOsterlind A, Tucker MA, Stone BJ, et al.: The Danish case-control study of cutaneous malignant melanoma. II. Importance of UV-light exposure. Int J Cancer. 1988; 42(3): 319–24. PubMed Abstract | Publisher Full Text\n\nClegg LX, Reichman ME, Miller BA, et al.: Impact of socioeconomic status on cancer incidence and stage at diagnosis: selected findings from the surveillance, epidemiology, and end results: National Longitudinal Mortality Study. Cancer Causes Control. 2009; 20(4): 417–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeierød MB, Weiderpass E, Thörn M, et al.: A prospective study of pigmentation, sun exposure, and risk of cutaneous malignant melanoma in women. J Natl Cancer Inst. 2003; 95(20): 1530–8. PubMed Abstract | Publisher Full Text\n\nHammer GP, Zeeb H, Tveten U, et al.: Comparing different methods of estimating cosmic radiation exposure of airline personnel. Radiat Environ Biophys. 2000; 39(4): 227–31. PubMed Abstract | Publisher Full Text\n\nKojo K, Aspholm R, Auvinen A: Occupational radiation dose estimation for Finnish aircraft cabin attendants. Scand J Work Environ Health. 2004; 30(2): 157–63. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10189",
"date": "02 Sep 2015",
"name": "Desiree Ratner",
"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 hypothesis, “Is frequent, commercial jet travel by the general public a risk factor for developing cutaneous melanoma?”, provides an interesting perspective on possible reasons for the increasing incidence of melanoma worldwide. The author cites multiple studies documenting a significant increase in melanoma risk among commercial and military pilots as well as cabin crew. Possible reasons for this increase include exposure to cosmic ionizing radiation at cruising altitudes of 30,000 feet or higher, particularly on long haul routes, as well as increased exposure to UV light during flight. Given that a significant portion of the world’s population now takes advantage of the increased availability and frequency of jet travel, it is possible that the increased incidence of melanoma may be, in part, due to increased exposure to cosmic rays and/or UV exposure during high altitude jet travel. The author suggests that a case-control methodology be used to test his hypothesis, by assessing jet travel histories in melanoma patients and comparable controls, controlling for known risk factors such as age, skin type, and exposure history. It would be necessary to look at frequency of air travel, duration of flights, and altitude, and to disentangle patients’ intermittent sun exposure history from their jet travel history. If the hypothesis that frequent commercial jet travel represents an additional risk factor for developing cutaneous melanoma proves to be true, not only will it be necessary to look at the mechanisms behind this increased risk, but it will also be necessary to develop appropriate screening and preventive measures to decrease the morbidity and mortality to the greatest degree possible from air travel associated melanoma.",
"responses": []
},
{
"id": "10379",
"date": "29 Sep 2015",
"name": "Robyn Lucas",
"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 raise the hypothesis that commercial jet travel by the general public may increase the risk of developing cutaneous melanoma through increased exposure to cosmic radiation or UVA radiation. Previous studies have shown an increased risk of melanoma in flight crews and pilots. The hypothesis in relation to UVA seems unlikely, given that most air travellers are well-clothed. The hypothesis in relation to cosmic radiation needs stronger evidence of the measured levels within aircraft. The author proposes a case-control study, but this seems unlikely to provide valid results - the most likely explanation of an increased risk of melanoma in flight crews and pilots is higher sun exposure at the locations that they fly to, especially of the intermittent high dose pattern that confers particular risk of melanoma. However sun exposure is poorly reported (as would be the measure of exposure in a case-control study) while air travel likely to be well-reported (and able to be validated). It will be impossible to clearly separate these \"exposures\". There is likely to be a very high correlation between a history of air travel and of sunny holidays in people with melanoma, such that a history of air travel is a proxy for high dose intermittent sun exposure. Findings resulting from such a study are highly likely to be spurious associations. It is an interesting hypothesis - a start would be to measure levels of cosmic radiation inside passenger aircraft, more clearly make the link between cosmic radiation and melanoma, and assess the body sites affected in cabin crew and pilots who develop melanoma.",
"responses": []
},
{
"id": "11326",
"date": "30 Nov 2015",
"name": "Gabriella Fabbrocini",
"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 hypothesis put forward by the author is interesting, yet hard to prove. it is extremely difficult to separate the weight of individual exposures: the cosmic radiation at high altitudes and that related to sun exposure of the trip. The bibliography is extensive and still valid. Considering the bases from which originates the article, or the increased incidence of melanoma in the last decades and the multifactoriality, it might be interesting to start the case control study that the author sets out to accomplish. It would be necessary to consider some essential factors, including the frequency of air travel, the duration of the flight, altitude, intermittent sun exposure of patients during their trip.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-620
|
https://f1000research.com/articles/4-617/v1
|
24 Aug 15
|
{
"type": "Systematic Review",
"title": "Dermatoglyphic meta-analysis indicates early epigenetic outcomes & possible implications on genomic zygosity in type-2 diabetes",
"authors": [
"Seile Yohannes"
],
"abstract": "Background: Dermatoglyphic studies, particularly those arising from the Dutch Hunger Winter Families Cohort, indicate an involvement of prenatal epigenetic insults in type-2 diabetes. However, the exact orchestration of this association is not fully understood. Herein is described a meta-analysis performed based on a belief that such an approach could shed some light as to the role of genetic & epigenetic influences in the etiology of type-2 diabetes.\nMethodology/principal findings: The study incorporated reports identified from PubMed, Medline, & Google Scholar databases for eligible case-control studies that assessed dermatoglyphics in type-2 diabetes cases relative to controls. Over 44,000 fingerprints & 2300 palm prints from around 4400 individuals were included in the analysis. Decreased loops patterns [OR= 0.76; 95% CI= (0.59, 0.98)], increased non-loop patterns [OR= 1.31; 95% CI= (1.02, 1.68)], and reduced absolute finger ridge counts [OR= -0.19; 95% CI= (-0.33, -0.04)] were significant findings among the diabetic group. These results are indicative of mild developmental deviances, with epigenetic insults significantly linked to early gestation wherein critical events &signaling pathways of the endocrine pancreas development are witnessed. Further, the increased loop patterns with decreased non-loop patterns were deemed as possible indicators of decreased genomic heterozygosity with concurrently increased homozygosity in the diabetic group, linked to reduced buffering capacities during prenatal development.\nConclusions: Epigenetic insults primarily during the 1st trimester, to a lesser extent between the early-to-mid 2ndtrimester, but least likely linked to those beyond the mid-second trimester are evident in type-2 diabetes. It is recommended that future research aimed at expounding the prenatal origins of T2DM, as well as developing novel therapeutic methods, should focus on the early stages of endocrine pancreatic development.",
"keywords": [
"dermatoglyphics",
"diabetes",
"epigenetics",
"prenatal development",
"heterozygosity",
"developmental homeostasis",
"genetic predisposition"
],
"content": "Introduction\n\nThe dawn of the 21st century has witnessed a trend of epidemiologic shift from acute infections to chronic degenerative disorders of intricate etiologies markedly obscure to comprehend. One such multifaceted forerunner deemed as a major global public health issue is diabetes mellitus, recording an estimated global prevalence of 8.3% in 2013 that is projected to rise to an alarming figure of over 590 million afflicted individuals by 20351. Type-2 diabetes mellitus (T2DM) accounts for greater than 90% of all diabetics2. It is a polygenic multi-factorial disorder entailing multiple endogenous & exogenous risk factors3,4.\n\nThe estimated heritability of T2DM underscores the contribution of non-genetic components in its etiologic complexity5. Several prenatal environmental agents have been hypothesized as putative risk factors for T2DM, including: maternal dietary patterns, objective hardship or stress related to natural disasters, season of birth (related to vitamin D or melatonin metabolisms), urbanization, medications such as corticosteroids during pregnancy, use of pesticides, and so on6–10.\n\nIn line with this, the Dutch Hunger Winter Families Cohort studies investigate the concept of the thrifty phenotype hypothesis, linking gestational hunger to the offspring’s adulthood insulin resistance, glucose intolerance, and subsequent development of diabetes11,12. Such outcomes have in turn been shown to be associated with epigenetic alterations, including changes in the normal pattern of DNA methylation among genes essential for growth & development4,13–15. Yet, further studies from varied populations originating from varied environmental settings are necessitated in order to comprehend such etiologic complexity.\n\nA currently emerging area of study with multifarious findings of peculiar phenotypes among a broad array of chronic disorders including T2DM is dermatoglyphics (DG). It refers to the systematic study of the friction ridge features on the palmar & plantar surfaces, which are formed as complex polygenic multi-factorial traits16,17.\n\nDevelopment of DG begins during the 6th week of gestation, and their basic structure remains unaffected by subsequent environmental insults following full establishment during the 24th week18. Owing to this & several other advantageous features of DG, prospects of serving as markers to explore the nature & timing of intra-uterine irregularities have been assessed19. Developmental noise associated with chromosomal/gene abnormalities, environmental pressures, or a combination of these, have been shown to be detectable via DG20.\n\nDespite their pervasiveness, studies exploring DG in T2DM that entail strong deductions fortified by models that elucidate the underlying developmental mechanism in play thereby vindicating such associations, are distinctively vague. The manifold DG variable types, methodological discrepancies, as well as poorly designed or implemented studies have been deemed citable attributes to the prevalent contradicting findings21.\n\nIn contrast, a momentous cluster of researchers stand-out from among this crowd, which view peculiar DG as ripple-effects of early prenatal insults behind the structural & functional defects intrinsic of T2DM per se, including pancreatic endocrine system (β-cell functioning) alterations22–26. Though the core of such studies contends that DG are indicative of possible genetic predispositions or epigenetic prenatal outcomes, the exact orchestration of this association is not fully understood. This impedes the strides in the etiological understanding of T2DM, rendering the concept of employability of DG in genetic epidemiology to still be subject to further debate & research.\n\nHerein is described an exhaustive meta-analysis based on a hypothesis that an advanced statistical approach would lead to better conclusive results that will shed some light as to the role of genetic & epigenetic influences in the etiology of T2DM.\n\n\nMethods\n\nThe protocols for this work adhered to the principles of the Cochrane’s guidelines for systematic reviews27, as well as the preferred reporting items for systematic reviews and meta-analyses ‘PRISMA’28. The PRISMA checklist of the study is depicted in Supplementary file S1.\n\nA literature search of studies published up to July 2015 which report the association between diabetes and dermatoglyphics was performed, wherein PubMed, PubMed Central, Medline, & Google Scholar databases were exhaustively searched using synonyms and combinations of the terms, as summarized in Table 1. As a secondary option, reference lists of initially identified studies were hand-searched for putative studies, which were, once identified, looked up in the archives of journals they were published in. The data search was performed three times to ensure exhaustiveness, twice before the analysis, and a final cross-checking search after the analysis was completed based on the studies identified from the two initial searches.\n\nThe eligibility of each of the studies strictly adhered to the following criteria: (1) the article reports on an original peer-reviewed analysis; (2) it is available as a full text article published in English; (3) the article includes relevant raw data for pair-wise comparisons, mainly odds ratios (OR), confidence intervals (CI), mean values, and standard deviations (SD), or at least reported sufficient data that can be employed to estimate these parameters, including count or frequency data, accurate percentage frequencies, or standard errors of mean values.\n\nFor studies with some missing raw data or manifesting some inconsistencies hindering accurate extraction of data for meta-analysis, an attempt was made to contact the authors, which was done twice for each corresponding author within a 30 day interval via email addresses available on their articles. Data from responsive authors within the 30 days from the second contact (60 days in total) were included in the analysis.\n\nThe continuous dermatoglyphic variables included by a recent dermatoglyphic meta-analysis on schizophrenia were adopted29, and included herein in addition to a few more relevant continuous variables, as well as the inclusion of dichotomous type variables (pattern type distribution).\n\nThereby, three major classes of variables, each with respective subclasses, were included: Fingerprint patterns: based on the three pattern classification of fingerprint patterns as: arch, loop, and whorl patterns. Ridge counts: a count of ridges between a triradius and a core (in fingerprint patterns), or between two triradii (for palmar ridge counts). Thus the total finger ridge count (TFRC), absolute finger ridge count (AFRC), and total palmar a-b ridge count (TABRC) were assessed. Palmar Angles: angles formed by joining distal palmar inter-digital triradii a/d with the proximal palmar axial triradii t, yielding the ATD, DAT, and ADT angles30,31.\n\nQualitative and quantitative DG variables reported by each study were extracted carefully and summarized in coded spreadsheets. Pattern frequencies or means and SDs were pooled from left- & right-hand data, as well as from male and female data for each study as per previous reports29. Whenever necessary, percentage frequencies were converted to counts, and SDs calculated from reported standard error of means by multiplying standard errors of means from within a treatment group by the square root of the sample size27.\n\nCochrane RevMan 5.332 & MetaXL 2.2 software were used for analyses. Hedge’s g effect sizes for each continuous variable & ORs for each dichotomous variable, together with respective 95% confidence intervals, z-scores, & p-values were estimated27.\n\nHeterogeneity across studies was assessed following the I2 statistics33. The pooled effect size estimations were done using the fixed-effect model if heterogeneity was non-significant or the random-effects model if significant heterogeneity was shown to exist34–36.\n\nPublication bias was investigated by visual inspections of funnel plots. Finally, sensitivity analyses for each class were performed to evaluate the influences of selected study and participant characteristics on study results.\n\n\nResults\n\nThe literature search (Figure 1), which was to the best of my knowledge, exhaustive and comprehensive, yielded a total of 27 eligible studies after excluding unavailable articles, non-open-access reports, articles not in English, reviews and/or short reports, as well as those with unavailable or insufficient data. Of the 27 studies, respectively 20, 9, 4, 5, 10, 4 and 4 studies reported valid & usable data on the TFRC, AFRC, A-B RC, ATD, DAT, & ADT variables, which were incorporated into the meta-analysis. The characteristics of the 27 eligible studies37–63 are included in Table 2, while the excluded studies are elaborated in Table S1 within the Supplementary file S2.\n\nAuthorship, year of publication, country of origin of the sampled population, and sample sizes of the diabetic & healthy groups within the sampled population.\n\naType-2 diabetes mellitus.\n\nThe pooled effect sizes for all three fingerprint pattern datasets showed that significant heterogeneity was prominent (Figure 2–Figure 4). The OR datasets ranged from 0.24 to 2.27, 0.34 to 3.35, and 0.55 to 5.99 for the loops, whorls, and arches respectively. The pooled OR of the loops resulted in a significant effect size (OR = 0.76, 95% C.I. = 0.59–0.98, z = 2.15, p = 0.03), with decreased occurrence of loops among diabetic patients. In contrast, the pooled estimates for the whorls & arches resulted in non-significant effect sizes, with an OR of 1.30 (95% C.I. = 0.98–1.72, z = 1.83, p = 0.07) for whorls, and an OR of 1.19 (95% C.I. = 0.98–1.72, z = 1.12, p = 0.26) for the arches, indicating higher prevalence of both patterns among diabetic patients.\n\nAs is evident from the forest plot of TFRC effect sizes (Figure 5), the pooled effect sizes for the TFRC dataset were significantly heterogeneous (I2 = 85%, p<.001). The pooled estimate resulted in an insignificant effect size (g = -0.03, 95% C.I. = −0.29–0.22, p = 0.79), with slightly lower TFRC among diabetic patients.\n\nThe forest plot of AFRC effect sizes (Figure 6) reveals no heterogeneity within the dataset, with the pooled estimate yielding a significant effect size (g = 0.19, 95% C.I. = -0.33–-0.04, z = 2.58, p = 0.010), with lower means prevalent among diabetic patients.\n\nThe pooled effect sizes for the palmar TABRC dataset (Figure 7) was significantly heterogeneous (I2 = 68%, p = 0.01), with the pooled estimate yielding slightly decreased but insignificant means among the diabetic subjects (g = -0.02, 95% C.I. = -0.25–-0.21, z = 0.18, p = 0.85).\n\nAs is evident from the forest plot of TFRC effect sizes (Figure 8), pooled effect sizes for the palmar ATD angle dataset was significantly heterogeneous (I2 = 91%, p<.001). The pooled estimate resulted in a significant effect size (g = 0.16, 95% C.I. = -0.16–0.48, z = 0.99, p = 0.32), with higher ATD angles among diabetics.\n\nAs depicted in Figure 9, significant heterogeneity was observed for the DAT angle dataset (I2 = 72%, p = 0.01), with the pooled estimate showing an insignificant effect size (g = -0.05, 95% C.I. = -0.35–0.25, z = 0.33, p = 0.74), with lower values expected among the group of diabetics.\n\nSimilar to the DAT angle outcome, the ADT (TDA) angle dataset (Figure 10) was also significantly heterogeneous (I2 = 85%, p<0.001), yet the pooled OR being insignificant. Herein, the pooled effect size indicated lower values of the ADT angle among diabetic patients (g = -0.27, 95% C.I. = -0.68–0.13, z = 1.32, p = 0.19).\n\nAnalysis of publication bias via funnel plots and related tests are depicted in Supplementary file S3, which indicate that it is unlikely that any significant publication bias occurred, but the necessity of considering several underlying factors from various angles is imperative, as discussed in the later sections. Sensitivity analysis for each dermatoglyphic variable & T2DM revealed that none of the studies influenced the results substantially.\n\n\nDiscussion\n\nThe current study is supportive of T2DM being a polygenic multi-factorial disorder in which both genetic & environmental epigenetic insults cumulatively contribute to the disorder. The overall trend of reduction in digital & palmar ridge counts, considered to be directly proportional to the growth rates during the 1st two trimesters of gestation, indicate abnormally delayed growth in diabetic cases than expected under normal conditions64.\n\nSimilarly, the ATD angle is related to the axial triradius t position that migrates from the centre to the lower proximal portion of the palm during the early stages of gestation65. Thus, distally deviating t positions imply prematurely halted t migrations or delayed developmental outcomes, resulting in increased ATD angles. The current analysis has observed increased (albeit insignificant) ATD increases among T2DM cases. Such findings evidence mild distortions of development during the early phases of gestational development.\n\nOne advantage of DG is that each variable is formed over a given gestational timeframe, thus enabling an estimation of the relative timing of developmental insults. This entails an assumption that the stressors specifically affecting an organ or system linked to the etiology of the disorder in question also had parallel bearings on the development of DG.\n\nRegarding the fingerprint discrepancies, the current analysis has revealed that individuals with T2DM manifested significantly reduced loop patterns, counterbalanced by elevated whorl & arch frequencies. Evidences attest that pattern determination occurs much earlier than the ridge proliferation, possibly influenced by the volar pad’s shape, the embryonic epidermal axon development, or a combination of both18,66. This asserts that patterns develop as early as weeks 6–11, thus indicating that the results of fingerprint discrepancies in this study evidence mid-to-late 1st trimester insults on the developing fetus.\n\nFurther, for comparison purposes, the arch & whorl patterns were pooled together regarding them as a common group of the “non-loops” & analyzed, which yielded heterogeneous but significant effect sizes [OR = 1.31; 95% C.I. = 1.02–1.68, z = 2.15, p = 0.03; I2 = 98%, P < 0.01], as depicted in the Supplementary file S3.\n\nSimilarly, slightly reduced digital & palmar ridge counts were found to be characteristic of T2DM. Finger ridge counts are indicative of the rate of fetal growth during ridge development which occurs from the weeks 10.5 to 17, while palmar ridge counts (TABRC) are indicative of slightly later outcomes occurring over a wider timeframe and prone to reflect effects of non-shared environmental factors18,64,65. Thus, the insignificant findings of TFRC & TABRC reductions, coupled with the significantly reduced AFRC (though the number of studies assessing this variable is low), are indicative of disruptive events occurring during gestation roughly between weeks 11 & 20, but of lesser effects as compared to the weeks prior to this.\n\nUnlike disorders like schizophrenia, wherein epigenetic stressors have been attributed to prevail throughout the entirety of the gestational timeframe29, the results of significant fingerprint pattern deviations and the insignificant TFRC, TABRC, and palmar angle discrepancies suggest that the insults must primarily be abundant during the early stages of gestation in T2DM. More specifically, events occurring during the mid-to-late 1st trimester have been underscored.\n\nThis stage of development witnesses key events of the endocrine pancreas development, whereby epigenetic insults could have maximum effects to result in structural & functional pancreatic defects. This period includes critical events in the prenatal development of the pancreas, including: development & fusion of ventral & dorsal primordial pancreatic buds (weeks 4–8), differentiation of the first insulin producing β-cells (weeks 7–9), as well as the clustering of endocrine cells & initiation of their subsequent association with the developing ducts of the pancreas (weeks 10–12)67. Important tissues developing parallel with the pancreas (the Notochord & Mesenchyme), as well as key signaling pathways (Hedgehog & Notch) have also been shown to influence pancreatic organogenesis & underlying β-cell functioning67,68.\n\nThree lines of research suggest that fingerprint loops are associated with heterozygosity while arches & whorls are linked to homozygosity: (1) Population admixtures, which are known to increase genomic heterozygosity, have revealed significant loop frequency increases that are counterbalanced by non-loop (especially arch) pattern suppressions69,70; (2) The occurrence of inbreeding or endogamy, an opposite scenario to that of admixture & attributed to increased homozygosity, have been shown to decrease the loops while elevating the frequency of both non-loops71,72; (3) The classical inheritance model of fingerprint patterns attributes two of the seven genes with cumulative bearings on multiple (all) fingers, both of which are dominant, favor the non-loop outcomes in the homozygous states. Further, loops have been shown to be an attribute of heterozygous conditions for most of the remaining five genes in this model16.\n\nAs advocated by the “heterozygote advantage” hypothesis73, increased genomic homozygosity is associated with less viability, while an improved overall genetic fitness is attributed to increased heterozygosity. The decreased loop frequency together with the increased whorl & arch frequencies presumably represent increased homozygosity paralleled by a decreased heterozygosity in the T2DM, thus implying an overall decreased fitness in the former.\n\nThis can be interpreted via the concept of developmental homeostasis. An organism is regarded as developmentally stable based on how adequately it buffered against epigenetic disturbances during prenatal development, a capacity dependent on the interaction of genes with environmental conditions74. With respect to the genotypic influences, homozygosity has been positively associated with decreased buffering capacity, while more genetically heterozygous organisms have been shown to be more resistant to developmental noise75.\n\nAll in all, this zygosity-fitness-fingerprint correlation is in accordance with a string of reports on DG in T2DM & associated factors such as obesity that incorporate variables that are more potent indicators of developmental deviations associated with reduced buffering capacities, including fluctuating asymmetry, dR45 (ridge-count difference between the 4th & 5th fingertips), and Md15 (mean RC both thumbs minus the mean RC of both little fingers)22–26.\n\nSupplementary evidence also originates from the previously discussed admixture changes. Heterozygosis underlying admixture was shown to be directly proportional to mean TFRC70, from which it can be hypothesized that relative reductions in TFRC are indicative of increased homozygosis. The current analysis has shown that the T2DM group manifested reduced TFRC means which, though yielding non-significant pooled effect sizes, are in agreement with this hypothesis.\n\n\nLimitations\n\nEven with the exhaustive search protocols and stringent analysis methods characteristic of the current analysis, certain limitations of the study per se, as well as individual studies included in the analysis need be outlined:\n\n(1) Gender & ethnic discrepancies in DG are inherent features of normal populations76,77. Since the individual reports included consisted of samples of mixed gender and/or ethnic affiliations, there lies a possibility that such polymorphisms masked some discrepancies possessing discriminating powers between the cases & controls that would otherwise have been evident.\n\n(2) Certain studies had underscored the presence of gender wise and/or right-left discrepancies among cases & controls for some of the dermatoglyphic variables. However, only the pooled data (for the right & left sides, as well as the males & females) was used in this analysis since the majority of the studies didn’t provide such stratified data. This poses the possibility of neglecting markers of true discriminating powers for a specific gender or side.\n\n(3) It is known that T2DM is very heterogeneous, with distinct subtypes with parallel variations in underlying genetic determinants, such as maturity-onset diabetes of youth (MODY), latent adult-onset autoimmune diabetes, & diabetes secondary to rare genetic disorders shown to be part of the syndrome3,8,13. Specific details, such as the ages-of-onset and other relevant specifications important in the determination of the relative homogeneity of the participants are virtually absent from all of the studies. This could be one possible explanation for the heterogeneity of the findings within a given variable type among the studies.\n\n(4) Similarly, the co-morbidity of disorders closely associated with DM, such as hypertension and cardiac disorders, were taken into consideration by a very limited number of studies only. This occurrence has been given little coverage, even though such co-morbidity is expected to be a prevalent event78, and the fact that each of these disorders are determined by distinct genetic & developmental outcomes parallel with the underlying deviations in the DG manifestations being distinct79.\n\n(5) A number of reports were not included as they were not available as free (open-access) articles. A few were also noted to bear inconsistent or incomplete data, with corresponding authors unavailable or unwilling to aid in alleviating this obstacle.\n\n\nConclusions & recommendations\n\nT2DM is orchestrated by the cumulative effects of a polygenic system with significant contributions from both pre- & postnatal environmental factors. Results of the current meta-analysis are indicative of epigenetic insults significantly linked to the 1st trimester, to a lesser extent on factors of the mid-second trimester, but least likely linked to those beyond the mid-second trimester in T2DM. Further, reduced heterozygosis possibly associated with a decreased buffering capacity to epigenetic stressors (developmental noise) during prenatal development has also been noted among the diabetic group. Based on these findings, the author recommends that future research aimed at expounding the prenatal origins of T2DM, as well as developing novel therapeutic methods, should focus on the early stages of endocrine pancreatic development.\n\nFurther, the Dutch Hunger Winter Families Cohort studies80 have indicated the possibility of such a link between T2DM & prenatal environment, evident in the Md15 dermatoglyphic variable, to be associated with seasonal factors. An involvement of seasonal circumstances such as vitamin D or melatonin levels, as well as fetal toxicity caused by-products of surface water chlorine treatment has been hypothesized26. It is recommended to undertake parallel studies from other global areas less prone to seasonality before ascertaining such hypotheses.\n\nFinally, the author highlights on the necessity of advancing the currently existing knowledge on the molecular genetics of DG per se, as this would profoundly enhance their applicability as research tools to assess the genetic epidemiology of chronic disorders.",
"appendix": "Competing interests\n\n\n\nThe author declares no competing interests.\n\n\nGrant information\n\nNo grants/funding were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are highly grateful to all the academic & administrative staff of the University of Jigjiga for all their support & constructive comments forwarded during this analysis.\n\n\nSupplementary materials\n\nPRISMA Checklist\n\nClick here to access the data.\n\nStudies excluded from the meta-analysis, with the respective justifications\n\nClick here to access the data.\n\nFunnel-plots for testing publication bias\n\nClick here to access the data.\n\n\nReferences\n\nInternational Diabetes Federation. IDF Diabetes Atlas (6th ed.). ISBN: 2-930229. 2013; 32–49. Reference Source\n\nRubino F: Is type 2 diabetes an operable intestinal disease? A provocative yet reasonable hypothesis. Diabetes Care. 2008; 31(Suppl 2): S290–6. 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IFAU Institute for Evaluation of Labour Market & Education Policy, 2012; 6: 1–44. Publisher Full Text\n\nYajnik CS: Commentary: Thrifty phenotype: 20 years later. Int J Epidemiol. 2013; 42(5): 1227–1229. PubMed Abstract | Publisher Full Text\n\nToperoff G, Aran D, Kark JD, et al.: Genome-wide survey reveals predisposing diabetes type 2-related DNA methylation variations in human peripheral blood. Hum Mol Genet. 2012; 21(2): 371–383. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTobi EW, Slieker RC, Stein AD, et al.: Early gestation as the critical time-window for changes in the prenatal environment to affect the adult human blood methylome. Int J Epidemiol. 2015: 1–13. PubMed Abstract | Publisher Full Text\n\nGreenhill C: Diabetes: DNA methylation affects T2DM risk. Nat Rev Endocrinol. 2015. PubMed Abstract | Publisher Full Text\n\nSlatis HM, Katznelson MB, Bonné-Tamir B: The inheritance of fingerprint patterns. Am J Hum Genet. 1976; 28(3): 280–289. PubMed Abstract | Free Full Text\n\nMedland S, Loesch DZ, Mdzewski B, et al.: Linkage analysis of a model quantitative trait in humans: finger ridge count shows significant multivariate linkage to 5q14.1. PLoS Genet. 2007; 3(9): 1736–1743. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBabler WJ: Embryologic development of epidermal ridges and their configurations. Birth Defects Orig Artic Ser. 1991; 27(2): 95–112. PubMed Abstract\n\nLivshits G, Kobyliansky E: Fluctuating asymmetry as a possible measure of developmental homeostasis in humans. Hum Biol. 1991; 63(4): 441–466. PubMed Abstract\n\nMartín B, Fañanás L, Gutiérrez B, et al.: Dermatoglyphic Profile in 22q Deletion Syndrome. Am J Med Genet B Neuropsychiatr Genet. 2004; 128B(1): 46–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYohannes S, Alebie G, Assefa L: Dermatoglyphics in diabetes: a prospective diagnostic aid & early preventive tool. Practical Diabetes. 2015; 32(2): 1–3. Publisher Full Text\n\nKahn HS, Ravindranath R, Valdez R, et al.: Fingerprint ridge-count difference between adjacent fingertips (dR45) predicts upper-body tissue distribution: evidence for early gestational programming. Int J Epidemiol. 2001; 153(4): 338–344. PubMed Abstract | Publisher Full Text\n\nRavindranath R, Joseph AM, Bosco SI, et al.: Fluctuating asymmetry in Dermatoglyphics of non-insulin dependent diabetes mellitus in Bangalore-based population. Ind J of Hum Genet. 2005; 11(3): 149–153. Publisher Full Text\n\nKahn HS, Graff M, Stein AD, et al.: A fingerprint characteristic associated with the early prenatal environment. Am J Hum Biol. 2008; 20(1): 59–65. PubMed Abstract | Publisher Full Text\n\nKahn HS, Graff M, Stein AD, et al.: A fingerprint marker from early gestation associated with diabetes in middle age: The Dutch Hunger Winter Families Study. Int J Epidemiol. 2009; 38(1): 101–109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKahn HS, Stein AD, Lumey LH: Prenatal environmental exposures that may influence β-cell function or insulin sensitivity in middle age. J Dev Orig Health Dis. 2010; 1(5): 300–309. PubMed Abstract | Publisher Full Text\n\nAlderson P, Green S, Higgins JPT: Cochrane reviewers’ handbook 4.2.2. In: Cochrane Library, Issue 1. Chichester: Wiley. 2004. Reference Source\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGolembo-Smith S, Walder DJ, Daly MP, et al.: The presentation of dermatoglyphic abnormalities in schizophrenia: a meta-analytic review. Schizophr Res. 2012; 142(1–3): 1–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCummins H, Midlo C: Finger prints, palms and soles : an introduction to dermatoglyphics. Dover Publications, New York. 1961. Reference Source\n\nMiller JR: Dermatoglyphics. J Invest Dermatol. 1973; 60(6): 435–442. PubMed Abstract | Publisher Full Text\n\nThe Cochrane Collaboration. Review Manager (RevMan) [Computer program]. Version 5.3. Copenhagen: The Nordic Cochrane Centre. 2014. Reference Source\n\nHiggins JP, Thompson SG, Deeks JJ, et al.: Measuring inconsistency in meta-analyses. BMJ. 2003; 327(7414): 557–560. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMantel N, Haenszel W: Statistical aspects of the analysis of data from the retrospective analysis of disease. J Natl Cancer Inst. 1959; 22(4): 719–748. PubMed Abstract\n\nDerSimonian R, Laird N: Meta-analysis in clinical trials. Control Clin Trials. 1986; 7(3): 177–188. PubMed Abstract | Publisher Full Text\n\nHunter JE, Schmidt FL: Methods of meta-analysis: Correcting error and bias in research findings (2nd ed.). Thousand Oaks, CA: Sage. 2004. Reference Source\n\nBala A, Deswal A, Sarmah PC, et al.: Palmar Dermatoglyphics patterns in diabetes mellitus and diabetic with hypertension patients in Gangtok region. Int J Adv Res. 2015; 3(4): 1117–1125. Reference Source\n\nBurute P, Kazi SN, Swamy V, et al.: Role of Dermatoglyphic Fingertip Patterns in the prediction of Maturity Onset Diabetes Mellitus (Type II). IOSR-JDMS. 2013; 8(1): 1–5. Reference Source\n\nDesai SD, Hadimani GA: Dermatoglyphics and Health. Anatomica Karnataka. 2013; 7(1): 1–9. Reference Source\n\nFuller IC: Dermatoglyphics: A Diagnostic Aid? J Med Genet. 1973; 10(2): 165–169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGabriel SO, Babajide MO: Dermatoglyphic patterns in diabetes mellitus in a south eastern Nigerian population. Afr J of Appl Zool & Environ Biol. 2004; 6: 6–10. Publisher Full Text\n\nKarim KJ, Saleem MA: Dermatoglyphics Study of Finger Prints Pattern’s Variations of a Group of Type II Diabetic Mellitus Patients in Erbil City. Zanco Journal of Pure and Applied Sciences. 2014; 26(4): 11–16. Reference Source\n\nMarera DO, Oyieko W, Agumba G: Variation in Dermatoglyphic patterns among diabetics in Western Uganda population. African Journal of Science & Research 2015; 7(3): 20–5. Reference Source\n\nMehta AA, Mehta AA: Study of fingerprint patterns in type II diabetes mellitus. Int J Anat Res. 2015; 3(2): 1046–1048. Publisher Full Text\n\nMittal M, Lala BS: Dermatoglyphics: An Economical Tool for Prediction of Diabetes Mellitus. Int J Med Health Sci. 2013; 2(3): 292–297. Reference Source\n\nNayak V, Shrivastava U, Kumar S, et al.: Dermatoglyphic study of diabetes mellitus Type 2 in Maharashtrian population. Inter J Medical Sci Res Prac. 2015; 2(2): 66–69. Reference Source\n\nOjha P, Gupta G: Dermatoglyphic Study: A Comparison in Hands of Type 2 Diabetes Mellitus Patients and Normal Persons of Udaipur Region. JEMDS. 2014; 3(47): 1358–11368. Publisher Full Text\n\nPathan FKJ, Gosavi AG: Dermatoglyphics in Type II Diabetes Mellitus. MIMSR Medical College, Latur’s Journal of Medical Education & Research. 2011; 1(1): 6–8. Reference Source\n\nPathan FKJ, Hashmi RN: Variations of Dermatoglyphic Features in Non Insulin Dependent Diabetes Mellitus. IJRTST. 2013; 8(1): 16–19. Reference Source\n\nRajnigandha V, Mangala P, Latha P, et al.: Digito-Palmar Complex in Non-Insulin Dependent Diabetes Mellitus. Turk J Med Sci. 2006; 36(6): 353–355. Reference Source\n\nRakate NS, Zambare BR: Fingertip Patterns: A diagnostic tool to predict diabetes mellitus. Natl J Med Dent Res. 2014; 2(3): 49–53. Reference Source\n\nRakate NS, Zambare BR: Comparative study of the Dermatoglyphic patterns in type II diabetes mellitus patients with non diabetics. Int J Med Res Health Sci. 2013; 2(4): 955–959. Publisher Full Text\n\nRavindranath R, Thomas IM: Finger ridge count and finger print pattern in maturity onset diabetes mellitus. Indian J Med Sci. 1995; 49(7): 153–156. PubMed Abstract\n\nSachdev B: Biometric Screening Method for Predicting Type 2 Diabetes Mellitus among Selected Tribal Population of Rajasthan. Int J Cur Bio Med Sci. 2012; 2(1): 191–194.\n\nSengupta S, Boruah J: Palmar dermatoglyphics in diabetes mellitus. The Bulletin of the Department of Anthropology, Dibrugarh University. 1996; 24: 73.\n\nSharma MK, Sharma H: Dermatoglyphics: A Diagnostic Tool to Predict Diabetes. JCDR. 2012; 6(3): 327–332. Reference Source\n\nSrivastava S, Rajasekar SS: Comparison of Digital and Palmar Dermatoglyphic Patterns in Diabetic and Non-Diabetic individuals. IOSR-JDMS. 2014; 13(7): 93–95. Reference Source\n\nSudagar M, Radha K, Duraipandian K, et al.: Study of palmar patterns in diabetic patients. Int J Adv Med. 2014; 1(2): 117–122. Publisher Full Text\n\nSumathi S, Desai SD: Study of Dermatoglyphics in Patients with Type II Diabetes Mellitus Essential Hypertension in the Age Group between 35-55 Years. Analytica Medica. 2007; 10(2): 22–28. Reference Source\n\nTaiwo IA, Adebanjo OO: Evaluation of association between digital dermatoglyphic traits and type-2 diabetes in Lagos, Nigeria. Nig Q J Hosp Med. 2012; 22(3): 191–199. PubMed Abstract\n\nTrivedi PN, Singel TC, Kukadiya UC, et al.: Correlation of atd angle with Non-Insulin Dependent Diabetes Mellitus in Gujarati population. JRMDS. 2014; 2(2): 47–51. Publisher Full Text\n\nUdoaka AL, Lawyer-Egbe K: Dermatoglyphic Patterns of Diabetic Mellitus Patients of Ijaw Origin in Port Harcourt, Nigeria. Niger J Health Biomed Sci. 2009; 8(2): 72–82. Publisher Full Text\n\nUmana UE, Bello R, Timbuak J, et al.: Dermatoglyphic and Cheiloscopic Patterns among Diabetic Patients: A Study in Ahmadu Bello University Teaching Hospital Zaria, Nigeria. J Biol Life Sci. 2013; 4(2): 206–214. Publisher Full Text\n\nCohen-Bendahan CC, van de Beek C, Berenbaum SA: Prenatal sex hormone effects on child and adult sex-typed behavior: methods and findings. Neurosci Biobehav Rev. 2005; 29(2): 353–384. PubMed Abstract | Publisher Full Text\n\nDavid TJ: Embryonic migration during the prenatal development of palm print patterns. Med Hypotheses. 1981; 7(5): 639–644. PubMed Abstract | Publisher Full Text\n\nMoore SJ, Munger BL: The early ontogeny of the afferent nerves and papillary ridges in human digital glabrous skin. Brain Res Dev Brain Res. 1989; 48(1): 119–141. PubMed Abstract | Publisher Full Text\n\nGittes GK: Developmental biology of the pancreas: a comprehensive review. Dev Biol. 2009; 326(1): 4–35. PubMed Abstract | Publisher Full Text\n\nGittes GK, Rutter WJ: Onset of cell-specific gene expression in the developing mouse pancreas. Proc Natl Acad Sci U S A. 1992; 89(3): 1128–1132. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGutierez SB, Lucenario JLS, Yebes MJT: Dermatoglyphic studies among the Dumagat-Remontado tribal population of the Philippines. J Anthropol. 2012; 1–6. Publisher Full Text\n\nCheng X, Li H, Gupta S, et al.: Dermatoglyphic changes during the population admixture between Kam and Han Chinese. Homo. 2009; 60(2): 143–157. PubMed Abstract | Publisher Full Text\n\nBadaruddoza: Effects of inbreeding on the finger print patterns. Anthropologist. 2000; 2(3): 193–195. Reference Source\n\nŢarcă A: Dermatoglyphic indicators of illness risk for endogamous populations. J Prev Med. 2002; 10(1): 47–53. Reference Source\n\nMerrell DJ: Ecological Genetics. University of Minnesota Press, Minneapolis, 1981. Reference Source\n\nKobyliansky E, Bejerano M, Katznelson MB, et al.: Relationship between genetic anomalies of different levels and deviations in dermatoglyphic traits- Dermatoglyphic sexual dimorphism in control healthy group of Israeli Jews. Studies in Historical Anthropology. 2006; 4: 61–121. Reference Source\n\nZaidi ZF: Body asymmetries: incidence, etiology and clinical implications. Aust J Basic Appl Sci. 2011; 5(9): 2157–2191. Reference Source\n\nZhang HG, Chen YF, Ding M, et al.: Dermatoglyphics from all Chinese ethnic groups reveal geographic patterning. PLoS One. 2010; 5(1): e8783. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYohannes S, Bekele E: Ethiopian population dermatoglyphic study reveals linguistic stratification of diversity. PLoS One. 2015; 10(6): e0126897. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMannino DM, Thorn D, Swensen A, et al.: Prevalence and outcomes of diabetes, hypertension and cardiovascular disease in COPD. Eur Respir J. 2008; 32(4): 962–969. PubMed Abstract | Publisher Full Text\n\nEberechi DU, Gabriel OS, Peter OD: A comparative study of the digital pattern, position of triradii, b-c and a-d palmar distances of diabetic subjects and essential hypertensive individuals in River State. Int J Adv Biotechnol Res. 2012; 3(2): 615–620. Reference Source\n\nLumey LH, Stein AD, Kahn HS, et al.: Cohort profile: the Dutch Hunger Winter families study. Int J Epidemiol. 2007; 36(6): 1196–204. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10489",
"date": "11 Nov 2015",
"name": "Buddhika Wijerathne",
"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 main objective of this paper is to make a systematic review and meta-analysis of the association between dermatoglyphics and Type 2 Diabetes Mellitus. The systematic review was conducted in appropriate manner. It is true that poor study designs, incorrect sample selections, overlooked confounding variables, made the reliability of many appraised studies questionable. The author has mentioned this in the limitation section. However, as this article is a systematic review and meta-analysis it is better to use a checklist like STROBE to assess the methodological quality of the observational studies. (http://www.strobe-statement.org/). In results section it mentioned “Of the 27 studies, respectively 20, 9, 4, 5, 10, 4 and 4 studies reported valid & usable data on the”What are these numbers? Are they references?ATD angle is not a good variable in Dermatoglyphics studies for various reasons and therefore needs a little extension of discussion. In a recent review on hypertension and dermatoglyphics we have discussed this issue in detail. DOI: 10.1186/s40101-015-0065-3.",
"responses": []
},
{
"id": "17932",
"date": "12 Dec 2016",
"name": "Tarimobo Otobo",
"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 properly framed and reflective of the study, with a precise summary. The study design and methodology meet the standards of meta-analysis. The use of dermatoglyphics as a surrogate marker of developmental instabilities is well documented in literature. However, the correlation is equivocal. But the author has been able to present a balanced and objective report within the limitations of the analyzed reports and this study.\nThe article has been reported with a reasonable depth of knowledge, providing the necessary information for replication.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-617
|
https://f1000research.com/articles/4-601/v1
|
20 Aug 15
|
{
"type": "Review",
"title": "Congenital Adrenal Hyperplasia",
"authors": [
"Phyllis W. Speiser"
],
"abstract": "Congenital adrenal hyperplasia associated with deficiency of steroid 21-hydroxylase is the most common inborn error in adrenal function and the most common cause of adrenal insufficiency in the pediatric age group. As patients now survive into adulthood, adult health-care providers must also be familiar with this condition. Over the past several years, F1000 has published numerous commentaries updating research and practical guidelines for this condition. The purposes of this review are to summarize basic information defining congenital adrenal hyperplasia and to highlight current knowledge and controversies in management.",
"keywords": [
"Congenital",
"adrenal hyperplasia",
"glucocorticoids"
],
"content": "Introduction\n\nIn most patients with classic congenital adrenal hyperplasia (CAH), both cortisol and aldosterone production are impaired while adrenal androgen production is excessive. As a result of the lack of the vital hormones cortisol and aldosterone, patients are susceptible to potentially lethal adrenal insufficiency if untreated. Thus, emergency and critical care personnel must consider the diagnosis in patients presenting in shock. Excess androgen production, a side effect of 21-hydroxylase deficiency, causes genital ambiguity in females along with various endocrinologic, gynecologic, and reproductive complications. Men with CAH may also have reproductive and endocrine problems, most notably testicular adrenal rest tumors and oligospermia.\n\nIn this context, I will present data and arguments supporting the need for informed treatment of patients across the life span and across primary and specialty care practices. The main themes to be discussed will be prenatal and neonatal diagnosis, optimization of growth during childhood, and quality of life (QOL), including reproductive health in adult life.\n\n\nPrenatal and neonatal diagnosis\n\nCAH is a monogenic autosomal recessive disease caused by mutations or deletions in CYP21A2, the gene encoding steroid 21-hydroxylase1,2. All newborns in the United States and in many developed countries are screened for 21-hydroxylase deficiency among other disorders diagnosed by obtaining heel-stick blood on filter paper3. At present, the diagnosis is most often made by immunoassays for 17-hydroxyprogesterone. The assay methods have changed over time in an effort to improve the rather poor positive predictive value of these tests. Innovations in this regard have included stratifying cut-points by birth weight, introducing ratios of various analytes, and using the more specific methods of tandem mass spectrometry. In some screening programs, a second blood sample is obtained after several weeks to capture potentially missed cases. It is believed that newborn screening has significantly improved infant morbidity and mortality because of earlier diagnosis and treatment of these babies at risk for sudden death due to adrenal insufficiency4. It has further been estimated that the cost-benefit ratio for screening is comparable to that of other inborn errors of metabolism for which screening has been mandated4,5.\n\nGenotyping for screening purposes so far has not been deemed cost-effective. Rather, genotyping is most often performed when the hormonal diagnosis is in question or when genetic counseling is indicated6. A common scenario is that the family with a proband affected with CAH is seeking prenatal diagnosis for a fetus. If one knows the proband’s genotype, fetal diagnosis can be accomplished during the first trimester by several approaches. Whereas in the past chorionic sampling was performed at about 10 weeks’ gestation, the novel approach of extracting fetal DNA from the maternal circulation at as early as 5 to 6 weeks holds promise for earlier anticipatory guidance7. Another reproductive option for couples at risk for this and other well-characterized monogenic disorders is pre-implantation genetic screening to avoid producing a second affected child, although this procedure is considered by some to be eugenic and is quite expensive and still not widely available8.\n\nPrenatal treatment of the fetus via dexamethasone administration to the pregnant mother is potentially fraught with unknown long-term risks based on both human and animal studies9. The Endocrine Society and other medical groups have deemed this practice experimental6. Swedish investigators have placed a moratorium on the practice10.\n\n\nOptimization of growth during childhood\n\nThe primary goal of treating classic CAH is to reduce the excess adrenal androgen production and replace the deficient hormones, namely cortisol and aldosterone. Proper treatment will prevent both adrenal crisis and ongoing virilization. Daily oral medications, including glucocorticoids, mineralocorticoids, and salt supplements, are prescribed at the time of diagnosis in infancy and titrated according to blood levels of adrenal steroids, plasma renin activity, and electrolytes measured periodically, along with annual bone age x-rays. Diets of older children and adults contain more than enough sodium, obviating the need for supplemental salt. Statural growth and weight gain are also measured regularly since overtreatment or undertreatment may be associated with inappropriate growth. The preferred treatment in children and adults is hydrocortisone, the least potent glucocorticoid6. Owing to its short half-life, this drug is given in two or three daily doses. Currently being studied are newer long-acting or slowly released (or both) glucocorticoid preparations that could enhance adherence and more closely mimic physiologic cortisol secretion by the healthy adrenal cortex11,12. Retrospective systematic reviews and meta-analysis have revealed that exceeding about 17 mg/m2 per day of hydrocortisone equivalents, especially during early childhood or adolescence, is associated with poorer height outcomes13. During major life-threatening stress, surgery, or serious illness, patients with CAH require larger or more frequent doses (or both) of glucocorticoids and additional fluids. It is therefore crucial to educate parents of young children, and re-educate patients at the transition to adult care, about stress dosing. Unfortunately, mortality rates for CAH remain unacceptably high and are thought to be due largely to failure to appropriately deliver glucocorticoids during serious illness14. Additionally, many adults with CAH are lost to follow-up, even in countries where socialized medical care is a basic benefit for all15.\n\n\nQuality of life and reproductive health in adults\n\nQOL for patients with CAH has been reported as being suboptimal to poor16,17. With CAH, as with many chronic illnesses, various factors contribute to these measures. In fact, there has been a dearth of validated CAH-specific QOL instruments. A major negative factor has been sexual function among women who underwent complex genital reconstruction by older surgical techniques18. Newer approaches seem to provide better outcomes, but detailed long-term follow-up studies are lacking. Despite exposure to prenatal androgens, women with CAH most often have female core gender identity and behavior19,20. Transgender individuals have been reported but are relatively rare21.\n\nFertility and fecundity are reduced in both men and women with CAH compared with controls22,23. Women who are more severely affected are less likely to attempt pregnancy24. Additionally, adequate control of adrenal hormones, particularly follicular phase progesterone, is key in both conception and fetal retention24. Pregnant women with classic CAH must be managed by high-risk practitioners and receive adjustments to steroid doses and careful fetal monitoring. Interestingly, female fetuses cannot be easily virilized by transplacental passage of maternal androgens and this is due to a very efficient placental aromatase enzyme system.\n\n\nSummary\n\nThe issues to be resolved in coming years will be reduction of fetal and neonatal morbidities and mortality associated with CAH by improved diagnostic methods as discussed above. Patient education is key to guarantee continued utilization of medical services and decreased mortality in later life. Stunted growth may be avoided by improving available steroid treatment options, thereby improving adherence. Finally, long-term outcome studies of newer genital surgery techniques will help guide management across the life span.\n\n\nAbbreviations\n\nCAH, congenital adrenal hyperplasia; QOL, quality of life.",
"appendix": "Competing interests\n\n\n\nThe author declares that she has no competing interests.\n\n\nReferences\n\nSpeiser PW, White PC: Congenital adrenal hyperplasia. N Engl J Med. 2003; 349(8): 776–88. PubMed Abstract | Publisher Full Text\n\nAuchus RJ, Arlt W: Approach to the patient: the adult with congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2013; 98(7): 2645–55. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWhite PC: Optimizing newborn screening for congenital adrenal hyperplasia. J Pediatr. 2013; 163(1): 10–2. PubMed Abstract | Publisher Full Text\n\nGidlöf S, Falhammar H, Thilén A, et al.: One hundred years of congenital adrenal hyperplasia in Sweden: a retrospective, population-based cohort study. Lancet Diabetes Endocrinol. 2013; 1(1): 35–42. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTajima T, Fujikura K, Fukushi M, et al.: Neonatal screening for congenital adrenal hyperplasia in Japan. Pediatr Endocrinol Rev. 2012; 10(Suppl 1): 72–8. PubMed Abstract | F1000 Recommendation\n\nSpeiser PW, Azziz R, Baskin LS, et al.: Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2010; 95(9): 4133–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNew MI, Tong YK, Yuen T, et al.: Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-free fetal DNA in maternal plasma. J Clin Endocrinol Metab. 2014; 99(6): E1022–30. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSparrow R: Gender eugenics? The ethics of PGD for intersex conditions. Am J Bioeth. 2013; 13(10): 29–38. PubMed Abstract | Publisher Full Text\n\nMiller WL, Witchel SF: Prenatal treatment of congenital adrenal hyperplasia: risks outweigh benefits. Am J Obstet Gynecol. 2013; 208(5): 354–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHirvikoski T, Nordenström A, Wedell A, et al.: Prenatal dexamethasone treatment of children at risk for congenital adrenal hyperplasia: the Swedish experience and standpoint. J Clin Endocrinol Metab. 2012; 97(6): 1881–3. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVerma S, Vanryzin C, Sinaii N, et al.: A pharmacokinetic and pharmacodynamic study of delayed- and extended-release hydrocortisone (Chronocort) vs. conventional hydrocortisone (Cortef) in the treatment of congenital adrenal hyperplasia. Clin Endocrinol (Oxf). 2010; 72(4): 441–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMallappa A, Sinaii N, Kumar P, et al.: A phase 2 study of Chronocort, a modified-release formulation of hydrocortisone, in the treatment of adults with classic congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2015; 100(3): 1137–45. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMuthusamy K, Elamin MB, Smushkin G, et al.: Clinical review: Adult height in patients with congenital adrenal hyperplasia: a systematic review and metaanalysis. J Clin Endocrinol Metab. 2010; 95(9): 4161–72. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFalhammar H, Frisén L, Norrby C, et al.: Increased mortality in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Endocrinol Metab. 2014; 99(12): E2715–21. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nArlt W, Willis DS, Wild SH, et al.: Health status of adults with congenital adrenal hyperplasia: a cohort study of 203 patients. J Clin Endocrinol Metab. 2010; 95(11): 5110–21. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGilban DL, Alves Junior PA, Beserra IC: Health related quality of life of children and adolescents with congenital adrenal hyperplasia in Brazil. Health Qual Life Outcomes. 2014; 12: 107. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nStrandqvist A, Falhammar H, Lichtenstein P, et al.: Suboptimal psychosocial outcomes in patients with congenital adrenal hyperplasia: epidemiological studies in a nonbiased national cohort in Sweden. J Clin Endocrinol Metab. 2014; 99(4): 1425–32. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNordenström A, Frisén L, Falhammar H, et al.: Sexual function and surgical outcome in women with congenital adrenal hyperplasia due to CYP21A2 deficiency: clinical perspective and the patients' perception. J Clin Endocrinol Metab. 2010; 95(8): 3633–40. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBerenbaum SA, Bailey JM: Effects on gender identity of prenatal androgens and genital appearance: evidence from girls with congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2003; 88(3): 1102–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMeyer-Bahlburg HF, Dolezal C, Baker SW, et al.: Prenatal androgenization affects gender-related behavior but not gender identity in 5-12-year-old girls with congenital adrenal hyperplasia. Arch Sex Behav. 2004; 33(2): 97–104. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMeyer-Bahlburg HF, Gruen RS, New MI, et al.: Gender change from female to male in classical congenital adrenal hyperplasia. Horm Behav. 1996; 30(4): 319–32. PubMed Abstract | Publisher Full Text\n\nFalhammar H, Nyström HF, Ekström U, et al.: Fertility, sexuality and testicular adrenal rest tumors in adult males with congenital adrenal hyperplasia. Eur J Endocrinol. 2012; 166(3): 441–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHagenfeldt K, Janson PO, Holmdahl G, et al.: Fertility and pregnancy outcome in women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Hum Reprod. 2008; 23(7): 1607–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCasteràs A, De Silva P, Rumsby G, et al.: Reassessing fecundity in women with classical congenital adrenal hyperplasia (CAH): normal pregnancy rate but reduced fertility rate. Clin Endocrinol (Oxf). 2009; 70(6): 833–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "10038",
"date": "20 Aug 2015",
"name": "Perrin 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",
"responses": []
},
{
"id": "10039",
"date": "20 Aug 2015",
"name": "Richard Auchus",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-601
|
https://f1000research.com/articles/4-592/v1
|
20 Aug 15
|
{
"type": "Review",
"title": "Recent advances in understanding/managing eosinophilic esophagitis in adults",
"authors": [
"David A. Katzka"
],
"abstract": "It is an exciting time for research in eosinophilic esophagitis (EoE). As a new and increasingly prevalent disease, it is receiving considerable attention in the medical world, resulting in a flood of new insights. Clearly, a genetic predisposition seems likely with the identification of abnormalities in thymic stromal lymphopoietin (TSLP), calpain14, and eotaxin-3 genes. There are also well-defined abnormalities described in esophageal epithelial barrier function in these patients. The relationship between gastroesophageal reflux disease (GERD) and EoE remains unclear, but emerging data suggest that the concept of proton pump inhibitor responsive esophageal eosinophilia (PPIREE) may retain less importance, as this subset of patients becomes a likely subset of EoE in general. Finally, we approach the looming issue of long-term maintenance therapy. Although we lack adequate specific data on how to provide long-term pharmacologic treatment, studies clearly show that for most patients, this is a progressive disease that warrants such consideration.",
"keywords": [
"eosinophilic",
"esophagitis"
],
"content": "Introduction\n\nIt is not often in medicine that we have the excitement of researching and understanding a new disease, but this has occurred with eosinophilic esophagitis (EoE). Neither described nor understood until the 1990s, a wealth of information on EoE has emerged over the past two decades. It is remarkable that within such a relatively short period of time, major advances in understanding its molecular and clinical pathogenesis, disease characteristics, and treatment options have been defined. Nevertheless, a need for greater understanding of this disease, including basic questions with regard to diagnosis and treatment, remains. The goal of this review is to present many of the known highlights of EoE with identification of the key questions that need to be answered in the future.\n\n\nPathophysiology\n\nEoE appears to fall within the paradigm of an allergic disease with a genetic predisposition. In children, several genetic abnormalities have been identified, including in the eotaxin-3 gene and thymic stromal lymphopoietin (TSLP)1–5. More than just association, these abnormalities may have clear functional consequences. For example, the eotaxin-3 gene codes for eotaxin, which is a strong chemoattractant for eosinophils6. TSLP, on the other hand, is a key promotor of T(H)2 responses found in EoE3. There are also reported abnormalities in the epidermal differentiation complex (EDC) genes that code for filaggrin, an important protein regulator of the epithelial barrier7 and the calpain 14 gene8. These data are not surprising, as allergic disorders, such as asthma, atopic dermatitis, and seasonal allergies, commonly affect multiple family members and are thought to also have a genetic predisposition.\n\nThe proof that EoE is an allergy-based disease comes from several types of data. The first is in animal models in which EoE has been recapitulated in a mouse model9. Interestingly, there appears to be a “two-hit” pathway in which animal priming with first airway disease (by administration of intratracheal Aspergillus) or skin disease (by subcutaneous injection with ovalbumin) is needed to induce esophageal eosinophilia10. With this model, a careful analysis of the Th2 allergy pathway found in EoE has revealed multiple levels of involvement including an initial exposure of food antigens to dendritic cell recognition in the esophageal epithelium, epithelial secretion of key cytokines and eosinophil attractants, and recruitment of eosinophils and mast cells leading to esophageal inflammation and fibrosis11,12. Data in adults that corroborate these hypotheses include confirmation of the Th2 reaction histologically, identification of elevation of similar cytokines including eotaxin, and the frequent presence of extraesophageal allergies in patients13–15. This last point includes the finding that airway allergies commonly precede the onset of EoE. Some of the most compelling data suggesting that EoE is an allergy-based disease come from response to therapy. Specifically, medications such as steroids, which are useful in allergic diseases, are effective in inducing histologic remission in EoE16–18. Moreover, removal of food antigens by using an elemental diet (i.e. a diet devoid of food antigens) leads to histologic remission in close to 100% of EoE patients19–22.\n\nOther important mechanisms have also been postulated as being contributory to EoE. One area of investigation includes how food antigens putatively gain access to the esophageal epithelium. Towards this end, investigators have demonstrated that patients with EoE have altered esophageal epithelial permeability, as shown through Ussing chambers containing biopsies and by an endoscopically placed mucosal impedance probe23. Histologically, the epithelium in EoE demonstrates the presence of dilated intercellular spaces consistent with these physiologic findings. Furthermore, abnormalities in tight junction proteins, an important regulator of intercellular space diameter, have been shown in EoE tissue, some of which may become normal with the use of steroids6. Thus, a clear pathway of how antigen penetration into esophageal epithelium, with subsequent recognition by the dendritic cell, is evolving.\n\nIn adults, an extremely important aspect of pathophysiology that is being explored is the mechanism of fibrosis in the esophageal wall with subsequent ring and stricture formation that commonly accompanies EoE. Investigators have clearly shown that in EoE, there is a secretion of fibrogenic peptides with esophageal remodeling24 that may improve with steroids25. The potential for this fibrosis is presumably related to ongoing uncontrolled esophageal inflammation. Furthermore, a recent fascinating study shows that IgG4, a known immunoglobulin associated with fibrosis in other diseases26, may also be important in EoE.\n\nFinally, there are compelling data that EoE is a genetic disease. Demographic data show that a personal and family history of allergies is common in patients with EoE14. It has also been described in first-degree relatives both vertically and horizontally27,28. As previously noted, two genetic abnormalities have been identified in up to 50% of children.\n\n\nDiagnosis\n\nAs in an early description of any new disease, a classic set of characteristics emerges. In EoE, it became readily apparent that these characteristics include its occurrence in white male children and young adults, a presentation with dysphagia, and an endoscopic appearance of rings, furrows, white exudates and strictures associated with esophageal eosinophilia29–32. As recognition of EoE has evolved, it has become clear the these characteristics are not entirely specific, particularly in patients with gastroesophageal reflux disease (GERD)33. Indeed, the greatest diagnostic challenge in EoE is distinguishing it from GERD. This task is formidable for several reasons. Firstly, as discussed, clinical features overlap. Secondly, GERD is a common disease and may co-exist with EoE. Thirdly, recent data suggest that GERD may potentiate EoE but cause the initial dilation of intercellular spaces and the subsequent increase in esophageal permeability23. Fourthly, the results of ambulatory pH monitoring do not lead to accurate differentiation of these two diseases in all patients34. Fifthly, histologically there are no obvious routine histologic markers nor special stains (e.g. of mast or eosinophil-derived proteins) that distinguish these two diseases35,36. Finally, proton pump inhibitors (PPIs) are therapeutic not only in GERD but also in EoE, where they may have cytokine-blocking effects independent of the acid-suppressing mechanism37,38. As a result, this has led to a newly defined category of patients, labeled PPI-responsive esophageal eosinophilia (PPIREE). Whether this truly represents a different phenotype of disease or a combination of EoE and GERD is unclear, although preliminary data suggest that PPIREE patients may eventually progress to EoE. It is the author’s opinion that categorization should be based on the terms EoE, GERD, and an overlap of the two39.\n\nJust as the typical characteristics of EoE are not diagnostic, we are starting to observe patients with atypical features of EoE. For example, whereas it was assumed that all adults with EoE have dysphagia, some patients are now being seen with heartburn or nausea as their presenting symptom40. This is common in children where the isolated inflammatory response might be expected to produce such symptoms but was not expected in adults40. Similarly, approximately 5% of adult patients will have a normal endoscopic appearance. These data must be considered carefully, however, as endoscopy is relatively insensitive to the detection of esophageal strictures when compared to barium esophagography18. Thus, a normal endoscopic appearance may not reflect a truly normal esophagus.\n\nWith the lack of a gold standard for the diagnosis of EoE, establishing the diagnosis at this point still necessitates a combination of compatible clinical, endoscopic (and perhaps, radiologic), and histologic criteria, as recently defined30. Needless to say, this definition will continue to evolve.\n\n\nTreatment\n\nBefore discussing specific treatment options, it is important to understand some of the fundamental questions on treatment that have not been answered. The most essential question is what is the desired endpoint of treatment? The present options are symptoms, histology or both. Although symptom resolution is a desirable endpoint of any therapy, it is problematic in EoE where symptom resolution correlates poorly to other important endpoints, such as histology18. This is not surprising in adults, as short-term eradication of the esophageal epithelial inflammatory response does not lead to improvement of the fibrotic strictures that typically generate the symptoms of dysphagia. Another confounding issue in assessing symptoms is the lack of a standardized and comprehensive means of accurately measuring response. A study in press from the Swiss Eosinophilic Esophagitis Group will contribute greatly to resolving this problem, but, at this point, many physicians do not appreciate the subtle and adaptive behaviors that EoE patients adopt to cope with their disease and which can lead to inaccurate assessment of symptoms41. The third issue is in the way we define treatment groups. For the past 5 years, patients with esophageal eosinophilia who respond to PPIs are termed as either GERD patients or PPI-responsive eosinophilia. As data continue to accumulate in this area, particularly histologic and cytokine data, it appears that those patients with PPIREE, that is patients with the phenotype of EoE who do respond to PPIs, are likely a subset of EoE and will be referred to in that manner.\n\nThe use of histology as a therapeutic endpoint also leads to several potential pitfalls. The first is in defining if the measurement of eosinophils is the most important (if not the only) correct histologic parameter to follow. Other pathologic features accompany esophageal eosinophilia, such as the elongation of rete pegs, basal zone hyperplasia, an increased number of mast cells, and dilation of intercellular spaces42. It is unclear if all of these findings need to become normal in order to define a complete response. It also needs to be determined to what level eosinophils need to be reduced (or eliminated) to constitute a biologically meaningful response. Thus, in reading studies on EoE evaluating therapy, endpoints vary with different levels of eosinophil reduction including absolute numbers (e.g. <5) or percentage reduction. The main reason for this inability to establish a firm therapeutic histologic endpoint is the lack of knowledge of what is the natural history of EoE in the presence of these varied but sustained eosinophil levels. More specifically, we lack the knowledge of whether reduction to 15, 5 or 0 eosinophils per high power field prevents fibrosis and the progression of disease to strictures. Similarly, pathologists debate whether this measurement of eosinophils should be maximum or average per high power field and if all fields examined should be held to the same standard. With this in mind, the gold standard at this time remains elimination of all esophageal eosinophils, though, realistically, this is hard to achieve in all patients.\n\nWith these caveats in mind, medical treatment consists of pharmacologic and diet therapy. The first medication that should be considered in a patient with EoE is a PPI. Although one might define a response as indicating a patient with PPIREE, in a patient with classic features of EoE, I still consider this a first-line response. In patients with a typical phenotype of EoE, studies suggest a 30–60% initial response to PPI, but likely closer to 30%30,43.\n\nFrom a theoretical point of view, diet therapy for EoE appears most logical. This logic stems from the knowledge that food antigens trigger the T(H)2 allergic response and therefore preventing exposure to these foods putatively eliminates the disease. Unfortunately, determining specific foods that initiate this response in individual patients is difficult, owing to the lack of reliable non-invasive tests to identify culpable food antigens. In a recent meta-analysis, antigen determination in EoE through routine allergy testing in adults was only 45% accurate44. As a result, endoscopy followed by biopsy remains the only test at this time that reliably evaluates the effects of foods on esophageal inflammation. This can lead to up to ten endoscopies required for individual patients to find these foods using a series of additions and withdrawals of potential dietary constituents followed by esophageal biopsies45. Although newer methods of esophageal mucosa sampling are being developed, they are not ready for general use at this time. On the other hand, a recent meta-analysis of diet therapy demonstrated that six-food elimination and elemental diets led to remission in 75 and 95% of patients44. Although the six-food elimination diet has appeared unattractive to patients given the commonality of the foods eliminated, variations of this diet are gaining more popularity. For example, preliminary work is being performed for four- and two-food elimination diets46. Even so, as the population embraces current trends of avoiding gluten and dairy (the two most common food antigens that trigger EoE), elimination diets are becoming more acceptable to patients.\n\nAlthough not a pharmacologic therapy, endoscopic dilation is an important part of therapy in EoE, particularly in adults where strictures are common. Early literature that evaluated dilation in patients with EoE suggested a high rate of perforation, warning against its use or at least suggesting marked caution be taken47. With the publication of far larger studies, a recent review suggests that the frequency of perforation is 1%, with most patients who sustain perforation responding to non-operative therapy48. On the other hand, these studies demonstrate that dilation should be done gradually and carefully and perhaps over multiple sessions. With this in mind, dilation alone has been shown to be as effective in reducing symptoms of EoE as steroids in 2-year follow-up, although mechanical therapy directed at strictures does not improve histology49. Rather than being viewed as an either/or therapy, in most adult patients therapeutic response is commonly achieved with a combination of medical and dilation therapy. This is particularly true in patients with marked esophageal narrowing, severe symptoms, or a history of food impaction.\n\nOne of the ongoing debates occurring in the field of EoE at present is the role of maintenance therapy. On the one hand, although EoE is not known to be associated with malignancy, there is concern that it leads to morbidity and chronic use of steroids. Furthermore, relapse is almost universal, there is a significant effect on quality of life, and severe complications such as food impaction or Boerhaave’s syndrome49,50 may occur. Recent data also suggest that untreated inflammation eventually leads to stricture formation in most patients50. As a result, physicians are starting to define subsets of patients who might benefit from maintenance therapy. These include patients with EoE who respond to PPIs, patients effectively managed with diet therapy, and those who have a rapid relapse of symptoms and/or eosinophilia, severe stricturing disease such as in small caliber esophagus, or a history of food impactions or perforation. Whether all patients will ultimately require maintenance therapy is unclear.\n\nIf it is decided that chronic pharmacologic maintenance therapy is needed, unfortunately it is not clear what dose of medication to use. For PPIs, there are no randomized studies examining their role in maintenance, let alone whether once- or twice-daily dosing is effective. For topical steroids, there is only one randomized trial demonstrating that 0.5 mg of budesonide daily is suboptimally effective51. At this point, anecdotal advice that varies from 1 mg every other day to 1 mg twice daily has been used to establish symptomatic and/or histologic remission.\n\n\nFuture directions\n\nThere are numerous areas that need to be elucidated further in the field of EoE, but several are more pressing than others. The first is to find a “gold standard” for the diagnosis of EoE. As discussed, consensus guidelines dictate that the diagnosis is made with use of compatible clinical, endoscopic, and histologic characteristics. Genetic testing, as discussed previously, may fulfill this role, although positive testing is not found in all patients. The second area is in developing a test that is less expensive and invasive than endoscopy to assess histologic activity. This is important for monitoring therapy. This is particularly true in patients undergoing diet therapy where it can take up to ten endoscopies to determine the specific foods to which the patient is allergic. Candidate devices include the esophageal string test52 and the cytosponge53. Both are office-based procedures that do not require anesthesia. The hope for an easier test (such as through blood samples) is present, but results have not approached a level of accuracy amenable for practice. Finally, identification of subtypes of EoE, likely based on phenotype and/or genomic differences, will be helpful to define prognosis and predict the need for more intensive immediate and long-term therapy. Already, recent studies from the laboratories of Dr Marc Rothenberg (who has been pivotal in finding these genetic variations) have demonstrated an abnormal molecular profile in EoE patients which may also predict response to steroids54,55.\n\n\nConclusion\n\nEoE is a newly identified disease in which tremendous progress has been made in a matter of a couple of decades. From a classic description of an ostensibly rare disease, EoE is now a disease whose mechanism is well understood and has been well characterized, clinically, endoscopically, histologically, and genetically. Excellent and relatively safe treatment options are already available. Future clinical research on EoE is likely to concentrate mostly on the identification of a gold standard for diagnosis, the determination of factors that predict severe disease and response to treatment, and the role of maintenance therapy as we gain further understanding of the long-term complications of this disease.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nReferences\n\nSherrill JD, Rothenberg ME: Genetic dissection of eosinophilic esophagitis provides insight into disease pathogenesis and treatment strategies. J Allergy Clin Immunol. 2011; 128(1): 23–32; quiz 33–4. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLu TX, Sherrill JD, Wen T, et al.: MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers. J Allergy Clin Immunol. 2012; 129(4): 1064–75.e9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRothenberg ME, Spergel JM, Sherrill JD, et al.: Common variants at 5q22 associate with pediatric eosinophilic esophagitis. Nat Genet. 2010; 42(4): 289–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlanchard C, Wang N, Stringer KF, et al.: Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis. J Clin Invest. 2006; 116(2): 536–47. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSherrill JD, Gao PS, Stucke EM, et al.: Variants of thymic stromal lymphopoietin and its receptor associate with eosinophilic esophagitis. J Allergy Clin Immunol. 2010; 126(1): 160–5.e3. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKatzka DA, Tadi R, Smyrk TC, et al.: Effects of topical steroids on tight junction proteins and spongiosis in esophageal epithelia of patients with eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2014; 12(11): 1824–9.e1. PubMed Abstract | Publisher Full Text\n\nBlanchard C, Stucke EM, Burwinkel K, et al.: Coordinate interaction between IL-13 and epithelial differentiation cluster genes in eosinophilic esophagitis. J Immunol. 2010; 184(7): 4033–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKottyan LC, Davis BP, Sherrill JD, et al.: Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease. Nat Genet. 2014; 46(8): 895–900. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRothenberg ME: Biology and treatment of eosinophilic esophagitis. Gastroenterology. 2009; 137(4): 1238–49. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMishra A, Hogan SP, Brandt EB, et al.: An etiological role for aeroallergens and eosinophils in experimental esophagitis. J Clin Invest. 2001; 107(1): 83–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlanchard C, Wang N, Rothenberg ME: Eosinophilic esophagitis: pathogenesis, genetics, and therapy. J Allergy Clin Immunol. 2006; 118(5): 1054–9. PubMed Abstract | Publisher Full Text\n\nBlanchard C, Rothenberg ME: Basic pathogenesis of eosinophilic esophagitis. Gastrointest Endosc Clin N Am. 2008; 18(1): 133–43; x. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbonia JP, Rothenberg ME: Eosinophilic esophagitis: rapidly advancing insights. Annu Rev Med. 2012; 63: 421–34. PubMed Abstract | Publisher Full Text\n\nRoy-Ghanta S, Larosa DF, Katzka DA: Atopic characteristics of adult patients with eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2008; 6(5): 531–5. PubMed Abstract | Publisher Full Text\n\nAtkins D, Furuta GT: Mucosal immunology, eosinophilic esophagitis, and other intestinal inflammatory diseases. J Allergy Clin Immunol. 2010; 125(2 Suppl 2): S255–61. PubMed Abstract | Publisher Full Text\n\nSchaefer ET, Fitzgerald JF, Molleston JP, et al.: Comparison of oral prednisone and topical fluticasone in the treatment of eosinophilic esophagitis: a randomized trial in children. Clin Gastroenterol Hepatol. 2008; 6(2): 165–73. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLucendo AJ, De Rezende L, Comas C, et al.: Treatment with topical steroids downregulates IL-5, eotaxin-1/CCL11, and eotaxin-3/CCL26 gene expression in eosinophilic esophagitis. Am J Gastroenterol. 2008; 103(9): 2184–93. PubMed Abstract | Publisher Full Text\n\nAlexander JA, Jung KW, Arora AS, et al.: Swallowed fluticasone improves histologic but not symptomatic response of adults with eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2012; 10(7): 742–749.e1. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKelly KJ, Lazenby AJ, Rowe PC, et al.: Eosinophilic esophagitis attributed to gastroesophageal reflux: improvement with an amino acid-based formula. Gastroenterology. 1995; 109(5): 1503–12. PubMed Abstract | Publisher Full Text\n\nPeterson KA, Byrne KR, Vinson LA, et al.: Elemental diet induces histologic response in adult eosinophilic esophagitis. Am J Gastroenterol. 2013; 108(5): 759–66. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWechsler JB, Schwartz S, Amsden K, et al.: Elimination diets in the management of eosinophilic esophagitis. J Asthma Allergy. 2014; 7: 85–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarkowitz JE, Spergel JM, Ruchelli E, et al.: Elemental diet is an effective treatment for eosinophilic esophagitis in children and adolescents. Am J Gastroenterol. 2003; 98(4): 777–82. PubMed Abstract | Publisher Full Text\n\nvan Rhijn BD, Weijenborg PW, Verheij J, et al.: Proton pump inhibitors partially restore mucosal integrity in patients with proton pump inhibitor-responsive esophageal eosinophilia but not eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2014; 12(11): 1815–23.e2. PubMed Abstract | Publisher Full Text\n\nAceves SS, Newbury RO, Dohil R, et al.: Esophageal remodeling in pediatric eosinophilic esophagitis. J Allergy Clin Immunol. 2007; 119(1): 206–12. PubMed Abstract | Publisher Full Text\n\nAceves SS, Newbury RO, Chen D, et al.: Resolution of remodeling in eosinophilic esophagitis correlates with epithelial response to topical corticosteroids. Allergy. 2010; 65(1): 109–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClayton F, Fang JC, Gleich GJ, et al.: Eosinophilic esophagitis in adults is associated with IgG4 and not mediated by IgE. Gastroenterology. 2014; 147(3): 602–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAlexander ES, Martin LJ, Collins MH, et al.: Twin and family studies reveal strong environmental and weaker genetic cues explaining heritability of eosinophilic esophagitis. J Allergy Clin Immunol. 2014; 134(5): 1084–1092.e1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatel SM, Falchuk KR: Three brothers with dysphagia caused by eosinophilic esophagitis. Gastrointest Endosc. 2005; 61(1): 165–7. PubMed Abstract | Publisher Full Text\n\nLiacouras CA, Furuta GT, Hirano I, et al.: Eosinophilic esophagitis: updated consensus recommendations for children and adults. J Allergy Clin Immunol. 2011; 128(1): 3–20.e6; quiz 21–2. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDellon ES, Gonsalves N, Hirano I, et al.: ACG clinical guideline: Evidenced based approach to the diagnosis and management of esophageal eosinophilia and eosinophilic esophagitis (EoE). Am J Gastroenterol. 2013; 108(5): 679–92; quiz 693. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKapel RC, Miller JK, Torres C, et al.: Eosinophilic esophagitis: a prevalent disease in the United States that affects all age groups. Gastroenterology. 2008; 134(5): 1316–21. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMarkowitz JE, Liacouras CA: Ten years of eosinophilic oesophagitis: small steps or giant leaps? Dig Liver Dis. 2006; 38(4): 251–3. PubMed Abstract | Publisher Full Text\n\nSpechler SJ, Genta RM, Souza RF: Thoughts on the complex relationship between gastroesophageal reflux disease and eosinophilic esophagitis. Am J Gastroenterol. 2007; 102(6): 1301–6. PubMed Abstract | Publisher Full Text\n\nFrancis DL, Foxx-Orenstein A, Arora AS, et al.: Results of ambulatory pH monitoring do not reliably predict response to therapy in patients with eosinophilic oesophagitis. Aliment Pharmacol Ther. 2012; 35(2): 300–7. PubMed Abstract | Publisher Full Text\n\nSridhara S, Ravi K, Smyrk TC, et al.: Increased numbers of eosinophils, rather than only etiology, predict histologic changes in patients with esophageal eosinophilia. Clin Gastroenterol Hepatol. 2012; 10(7): 735–41. PubMed Abstract | Publisher Full Text\n\nDellon ES, Speck O, Woodward K, et al.: Markers of eosinophilic inflammation for diagnosis of eosinophilic esophagitis and proton pump inhibitor-responsive esophageal eosinophilia: a prospective study. Clin Gastroenterol Hepatol. 2014; 12(12): 2015–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang X, Cheng E, Huo X, et al.: Omeprazole blocks STAT6 binding to the eotaxin-3 promoter in eosinophilic esophagitis cells. PLoS One. 2012; 7(11): e50037. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCheng E, Zhang X, Huo X, et al.: Omeprazole blocks eotaxin-3 expression by oesophageal squamous cells from patients with eosinophilic oesophagitis and GORD. Gut. 2013; 62(6): 824–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlexander J, Katzka D: Editorial: expanding a narrow perspective on narrow calibre oesophagus in eosinophilic oesophagitis--authors' reply. Aliment Pharmacol Ther. 2015; 41(1): 148–9. PubMed Abstract | Publisher Full Text\n\nDellon ES, Gibbs WB, Fritchie KJ, et al.: Clinical, endoscopic, and histologic findings distinguish eosinophilic esophagitis from gastroesophageal reflux disease. Clin Gastroenterol Hepatol. 2009; 7(12): 1305–13; quiz 1261. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchoepfer A, Safroneeva E: Activity assessment of eosinophilic esophagitis. Dig Dis. 2014; 32(1–2): 98–101. PubMed Abstract | Publisher Full Text\n\nOdze RD: Histologic features of gastroesophageal reflux disease and eosinophilic esophagitis. Gastroenterol Hepatol (N Y). 2012; 8(7): 472–3. PubMed Abstract | Free Full Text\n\nMolina-Infante J, Ferrando-Lamana L, Ripoll C, et al.: Esophageal eosinophilic infiltration responds to proton pump inhibition in most adults. Clin Gastroenterol Hepatol. 2011; 9(2): 110–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nArias A, González-Cervera J, Tenias JM, et al.: Efficacy of dietary interventions for inducing histologic remission in patients with eosinophilic esophagitis: a systematic review and meta-analysis. Gastroenterology. 2014; 146(7): 1639–48. PubMed Abstract | Publisher Full Text\n\nLucendo AJ, Arias Á, González-Cervera J, et al.: Empiric 6-food elimination diet induced and maintained prolonged remission in patients with adult eosinophilic esophagitis: a prospective study on the food cause of the disease. J Allergy Clin Immunol. 2013; 131(3): 797–804. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMolina-Infante J, Arias A, Barrio J, et al.: Four-food group elimination diet for adult eosinophilic esophagitis: A prospective multicenter study. J Allergy Clin Immunol. 2014; 134(5): 1093–9.e1. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCohen MS, Kaufman AB, Palazzo JP, et al.: An audit of endoscopic complications in adult eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2007; 5(10): 1149–53. PubMed Abstract | Publisher Full Text\n\nMoawad FJ, Cheatham JG, DeZee KJ: Meta-analysis: the safety and efficacy of dilation in eosinophilic oesophagitis. Aliment Pharmacol Ther. 2013; 38(7): 713–20. PubMed Abstract | Publisher Full Text\n\nLipka S, Keshishian J, Boyce HW, et al.: The natural history of steroid-naïve eosinophilic esophagitis in adults treated with endoscopic dilation and proton pump inhibitor therapy over a mean duration of nearly 14 years. Gastrointest Endosc. 2014; 80(4): 592–8. PubMed Abstract | Publisher Full Text\n\nSchoepfer AM, Safroneeva E, Bussmann C, et al.: Delay in diagnosis of eosinophilic esophagitis increases risk for stricture formation in a time-dependent manner. Gastroenterology. 2013; 145(6): 1230–6.e1–2. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nStraumann A, Conus S, Degen L, et al.: Long-term budesonide maintenance treatment is partially effective for patients with eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2011; 9(5): 400–9.e1. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFuruta GT, Kagalwalla AF, Lee JJ, et al.: The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis. Gut. 2013; 62(10): 1395–405. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKatzka DA, Geno DM, Ravi A, et al.: Accuracy, safety, and tolerability of tissue collection by Cytosponge vs endoscopy for evaluation of eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2015; 13(1): 77–83.e2. PubMed Abstract | Publisher Full Text\n\nWen T, Stucke EM, Grotjan TM, et al.: Molecular diagnosis of eosinophilic esophagitis by gene expression profiling. Gastroenterology. 2013; 145(6): 1289–99. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWen T, Dellon ES, Moawad FJ, et al.: Transcriptome analysis of proton pump inhibitor-responsive esophageal eosinophilia reveals proton pump inhibitor-reversible allergic inflammation. J Allergy Clin Immunol. 2015; 135(1): 187–197.e4. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "10032",
"date": "20 Aug 2015",
"name": "Chris Liacouras",
"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",
"responses": []
},
{
"id": "10033",
"date": "20 Aug 2015",
"name": "Marc Rothenberg",
"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\nNo further comments provided.",
"responses": []
},
{
"id": "10034",
"date": "20 Aug 2015",
"name": "Jonathan Markowitz",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-592
|
https://f1000research.com/articles/4-590/v1
|
20 Aug 15
|
{
"type": "Correspondence",
"title": "Murine Cep290 phenotypes are modified by genetic backgrounds and provide an impetus for investigating disease modifier alleles",
"authors": [
"Simon A. Ramsbottom",
"Colin Miles",
"John A. Sayer",
"Colin Miles",
"John A. Sayer"
],
"abstract": "The study of primary cilia is of broad interest both in terms of disease pathogenesis and the fundamental biological role of these structures. Murine models of ciliopathies provide valuable tools for the study of these diseases. However, it is important to consider the precise phenotype of murine models and how dependant it is upon genetic background. Here we compare and contrast murine models of Cep290, a frequent genetic cause of Joubert syndrome in order to refine our concept of genotype-phenotype correlations.",
"keywords": [
"Joubert syndrome",
"Cep290",
"cilia",
"modifier",
"cystic kidney",
"nephronophthisis"
],
"content": "Background\n\nThe role of cilia and the diseases associated with aberrant cilial formation and function, termed ciliopathies, have in the last ten years become a major area of study1. Within the field of human genetics, the understanding of the importance of these organelles has given rise to a profound shift in the way that the study and treatment of a range of diseases is undertaken. Many syndromes, although known to be pleiotropic in their manifestation, were previously considered to be discrete entities requiring specific individualised treatment. The discovery that many of these syndromes appear to have a large degree of commonality in their disease mechanism via the malformation or mis-localisation of cilia, has lead to the thinking that they may indeed exist within a spectrum, and subsequently that they may respond to similar treatments.\n\nJoubert syndrome (JBTS) is an autosomal recessive ciliopathy which gives rise to cerebellar vermis aplasia, hypotonia, ataxia and developmental delay. It is often associated with retinal degeneration, leading to blindness. It may also be associated with a cystic renal phenotype known as nephronophthisis (NPHP). The most common genetic lesion among patients with JBTS who present with the cerebello-oculo-renal phenotype is CEP290 (OMIM 610142)2. Mutations in the CEP290 gene are associated with numerous disorders including Leber congenital amaurosis3, Senior-Loken syndrome, JBTS4,5 and Meckel syndrome6. There have been over 100 different mutation sites reported in human patients7. Work attempting to understand genotype-phenotype correlations of CEP290 mutations based on total amount of protein has recently been reported8. A lack of correlation between genotype and phenotype may also be dependent on oligogenic inheritance and a mutational load in ciliary genes9. While this diversity has been suggested to contribute to these differential outcomes, recent work utilising mouse models of JBTS has indicated that there may be underlying mechanisms that modify the disease phenotype, even when the exact Cep290 mutation site is conserved.\n\nIn early 2015, Rachel et al. described a Cep290KO mouse model of JBTS, which exhibits retinal degeneration and hydrocephalus in juvenile mice, with a slowly progressive renal pathology resulting in cysts in adult mice10. While the retinal degeneration and cerebral phenotypes broadly reflect those observed in JBTS patients, the murine kidney phenotype is unusual, as in humans it typically presents in teenage years and young adults as opposed to being of late onset. This lack of a significant renal aspect in the pathology may simply reflect the diversity in the JBTS phenotype. It should be noted however that while a renal phenotype in JBTS is not universal, patients with mutations in CEP290, as opposed to other JBTS genes, do more commonly have renal disease (nephronophthisis)11.\n\nRachel et al. report that a significant number (80%) of Cep290KO mice do not survive past weaning, suggesting that the reported phenotype is that of less affected individuals. Additionally, to obtain viable mice, the authors propagated the mutation within a mixed background of C57BL/6 and 129/SvJ; mice could not be bred purely on either line past the N3 generation. While this genetic diversity in the population may echo that of a human scenario, it can be difficult to interpret data with the lack of experimental control that this diversity introduces. Furthermore it is likely that to fully ascertain an accurate picture of the disease model, it is necessary to use a significantly greater number of animals, which is not desirable. To investigate a wider phenotypic spectrum of Cep290-related disorders, Rachel et al. also use a gene trap (gt) model of Cep290, in which the pGT0xfT2 gene trap vector is inserted in intron 25, resulting in the introduction of a premature stop codon10. This lesion is similar to that of a number of common mutation sites within human CEP290, and so potentially provides a good model of the human disease. Cep290gt were backcrossed onto 129SvJ and Rachel et al. reported a significant level of lethality, as was reported for the Cep290KO model, with the vast majority of Cep290gt mice dying in utero between E12 and E14. Surviving mice displayed massively dilated kidneys, with loss of tubules and the formation of large cysts, a phenotype more similar to that of infantile nephronophthisis. As with the Cep290KO model however, the large mortality rate within the population needs to be considered, as those mice that do survive may not be fully representative.\n\nThis gene trap model (Cep290gt) is similar to a murine model reported in 2014, described as Cep290LacZ12. In this study, a similarly high level of in utero mortality was reported when Cep290LacZ mice were backcrossed on to a C57BL/6 background. On the 129/Ola background however mice were fertile and viable beyond 12 months12. Although the two studies generated mice from the same embryonic gene trap cell line (CC0582 [SIGTR, http://www.sanger.ac.uk/resources/mouse/sigtr/]), differences in the phenotype can be observed. While the retinal degeneration and ventriculomegaly were broadly similar between the two studies, kidneys of Cep290LacZ mice were not massively dilated as described by Rachel et al., but had progressive formation of cysts reminiscent of human nephronophthisis12. Cysts were observed in newborn mice, and became progressively larger over the first 4 weeks, instead of developing after 12 months. This disparity in the viability and phenotype of mice containing the same genetic alteration indicates that there are significant genetic modifiers specific to each strain of mice which can adversely affect the way in which this disease presents. The genetic diversity present between human populations therefore may provide some answers as to the broad range of phenotypes seen in JBTS patients. In the case of mutations such as those in Cep290 which do have pleiotropic effects, the difference between the outcomes in different mouse strains provides an opportunity to study how genetic variability affects the disease phenotype. By utilising strains with known discrete polymorphisms it should be possible to identify variants that correlate with specific outcomes, allowing for a much greater understanding of the role that specific genetic modifiers play in human disease and allowing therapies to be directed at these genetic modifiers.",
"appendix": "Author contributions\n\n\n\nSAR prepared the first draft. SAR, CMG and JAS 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\nWork was funded by the Medical Research Council (MR/M012212/1), support to all authors.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBenmerah A, Durand B, Giles RH, et al.: The more we know, the more we have to discover: an exciting future for understanding cilia and ciliopathies. Cilia. 2015; 4: 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nValente EM, Brancati F, Boltshauser E, et al.: Clinical utility gene card for: Joubert syndrome--update 2013. Eur J Hum Genet. 2013; 21(10). PubMed Abstract | Publisher Full Text | Free Full Text\n\nden Hollander AI, Koenekoop RK, Yzer S, et al.: Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am J Hum Genet. 2006; 79(3): 556–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSayer JA, Otto EA, O'Toole JF, et al.: The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. Nat Genet. 2006; 38(6): 674–81. PubMed Abstract | Publisher Full Text\n\nBrancati F, Barrano G, Silhavy JL, et al.: CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders. Am J Hum Genet. 2007; 81(1): 104–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrank V, den Hollander AI, Brüchle NO, et al.: Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome. Hum Mutat. 2008; 29(1): 45–52. PubMed Abstract | Publisher Full Text\n\nCoppieters F, Lefever S, Leroy BP, et al.: CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Hum Mutat. 2010; 31(10): 1097–108. PubMed Abstract | Publisher Full Text\n\nDrivas TG, Wojno AP, Tucker BA, et al.: Basal exon skipping and genetic pleiotropy: A predictive model of disease pathogenesis. Sci Transl Med. 2015; 7(291): 291ra97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaghloul NA, Katsanis N: Functional modules, mutational load and human genetic disease. Trends Genet. 2010; 26(4): 168–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRachel RA, Yamamoto EA, Dewanjee MK, et al.: CEP290 alleles in mice disrupt tissue-specific cilia biogenesis and recapitulate features of syndromic ciliopathies. Hum Mol Genet. 2015; 24(13): 3775–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBachmann-Gagescu R, Dempsey JC, Phelps IG, et al.: Joubert syndrome: a model for untangling recessive disorders with extreme genetic heterogeneity. J Med Genet. 2015; 52(8): 514–22. PubMed Abstract | Publisher Full Text\n\nHynes AM, Giles RH, Srivastava S, et al.: Murine Joubert syndrome reveals Hedgehog signaling defects as a potential therapeutic target for nephronophthisis. Proc Natl Acad Sci U S A. 2014; 111(27): 9893–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "10029",
"date": "25 Aug 2015",
"name": "Eugen Boltshauser",
"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 correspondence Ramsbottom and colleagues refer to a publication by Rachel et al. (2015) in which phenotypes in mice strains with mutations in Cep290 (knock-out and a gen trap model) were studied, and a comparison with patients with Joubert syndrome (JBTS) with CEP290 mutations is made. In brief: mice develop a retinal degeneration, hydrocephalus, and a rather late onset progressive renal pathology, while JBTS patients develop kidney disease in childhood or early adulthood. In addition the majority of knock-out mice do not survive past weaning, thus the survivors are probably not really representative of the phenotype, Ramsbottom et al argue that differences between the outcomes in different mouse strains provide an opportunity to study how genetic variability affects the disease phenotype. This point is well taken. Looking at affected children (in my professional activity) even within the same sibship, with identical mutations, there is often marked intrafamilial variability of the phenotype, pointing to the relevance of genetic modifiers and probable interaction with other cilia-genes. Studies of mouse strains (and zebra fish) have greatly contributed to understanding Joubert related genes, however the animal phenotype may differ from humans - as illustrated by the lack of hydrocephalus in CEP290 mutated JBTS patients.",
"responses": []
},
{
"id": "10185",
"date": "22 Sep 2015",
"name": "Russell J. Ferland",
"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 recent years, it has become increasingly more evident that many ciliopathies exist not as separate diseases, but instead fall along a spectrum. Meckel syndrome 1 (MKS1) mutations may cause the severe Meckel Gruber syndrome, but recently, has been reported by Romani et al. (2014) to also be responsible for cases of the relatively more mild Joubert syndrome (JBTS). Our research on the centrosome and spindle pole associated protein 1 (CSPP1) gene supports the notion that mutations within the same gene can carry a wide range of phenotypes (Tuz et al., 2015). We identified 18 individuals with JBTS from 14 families with biallelic truncating mutations in CSPP1, and saw no evidence of a correlation between phenotype and the site of truncation. Surprisingly, individuals with the most N-terminal truncations displayed the least severe phenotype. Our findings also revealed that siblings with the same mutations had varying phenotypes, suggesting that other factors outside the mutation of the ciliopathy gene itself plays a role in the ultimate phenotype of the affected individual.The phenotypic heterogeneity of individuals with JBTS with the same mutations has not gone unnoticed. Recently, Ben-Omran et al. (2015) described two Qatari families with histories of consanguineous marriages with the same CSPP1 mutation, but yet, the probands displayed clinical heterogeneity. Ben-Omran and colleagues (2015) argue that highly inbred families likely have less diversity in genetic modifiers, and attribute their findings of clinical heterogeneity to unidentified stochastic events that occur in development. They correctly acknowledge that a disadvantage of their study is the lack of identical twins.Murine ciliopathy models provide one way of controlling for the contribution of genetic background to the eventual phenotype of the affected individual. In this correspondence from Ramsbottom, Miles, and Sayer, the authors provide important insight into the role of genetic background in modifying transgenic ciliopathy phenotypes. Here, the authors reflect on a recent publication in the journal Human Molecular Genetics by Rachel et al. (2015) which describes different ciliopathy phenotypes in mice with different Cep290 mutations (a constitutive deletion of Cep290 (proposed null) and a genetrap allele (proposed truncated Cep290)). Moreover, the phenotypes were clearly modified by the genetic background of the mice. Further support for this came from comparisons of these phenotypes to other published Cep290 transgenic mice with different genetic alterations. Genetic background and the modifiers that can be in these backgrounds is a significant consideration that needs to be appreciated in the cilia field as it has in many other fields, such as behavioral neuroscience. In fact, we have published work in which we have shown that C57BL/6J mice with a targeted deletion of Ahi1, another gene causing Joubert syndrome, die within 12 hours after birth, but if these mice are backcrossed to FVB, Balbc/J, or Swiss Webster mice (>10 generations), then post-natal survivability is significantly enhanced and most mice can reach adulthood. This correspondence serves as a very important reminder that the genetic background on which you are studying your ciliopathy phenotype can result in different phenotypes dependent on the mouse strain and therefore needs to be considered in interpreting one's results. Consequently, identification of such differing ciliopathy phenotypes on different genetic backgrounds also serve as an important tool for not only potentially identifying modifiers of ciliopathy phenotypes, but may also result in the identification of new ciliopathy genes.Ramsbottom, Miles, and Sayer argue that a difference in genetic background could account for disparities in some phenotypes amongst individuals with the same mutation, a finding that is supported by our work in humans and mice. However, as the authors point out, murine models of ciliopathies do not always recapitulate the findings in affected individuals with JBTS. Thus, a possible difference between murine and human regulation of ciliopathy phenotype must also be considered.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-590
|
https://f1000research.com/articles/4-589/v1
|
20 Aug 15
|
{
"type": "Research Article",
"title": "Molecular Dynamics Simulations of the Temperature Induced Unfolding of Crambin Follow the Arrhenius Equation.",
"authors": [
"Andrew R. Dalby",
"Mohd Shahir Shamsir",
"Mohd Shahir Shamsir"
],
"abstract": "Molecular dynamics simulations have been used extensively to model the folding and unfolding of proteins. The rates of folding and unfolding should follow the Arrhenius equation over a limited range of temperatures. This study shows that molecular dynamic simulations of the unfolding of crambin between 500K and 560K do follow the Arrhenius equation. They also show that while there is a large amount of variation between the simulations the average values for the rate show a very high degree of correlation.",
"keywords": [
"Molecular dynamics",
"Arrhenius",
"unfolding rate"
],
"content": "Introduction\n\nMolecular dynamics (MD) simulations have become an important tool in understanding chemical and biochemical processes at the molecular level. Through the use of Newtonian mechanics and an empirically derived force-field, simulations have been used to investigate the interactions of drug molecules and their targets as well as to predict the behaviour of proteins and peptides (Vasquez et al., 1994). Originally simulations were carried out in vacuo, but now with the increasing power of computers, simulations are usually carried out using periodic boundary conditions in water.\n\nIncreasingly realistic simulations lead to a better understanding of processes such as protein folding and unfolding at the molecular level (Lindorff-Larsen et al., 2012; Scheraga et al., 2007). The literature on molecular dynamics simulations for protein folding and unfolding is extensive. This can include very large simulations that can be simplified using coarse-graining, down to simulations of small proteins or peptides. One area where MD simulations have been particularly widely used is in the simulation of prion proteins and the protein mis-folding diseases associated with them (Shamsir & Dalby, 2005; Shamsir & Dalby, 2007; van der Kamp & Daggett, 2010; van der Kamp & Daggett, 2011).\n\nMolecular dynamics simulations give us a detailed view of protein folding and unfolding pathways and their rates of unfolding (Daggett, 2002). Temperature is often used to accelerate protein unfolding and it has been shown that this does not affect the protein unfolding pathway (Day et al., 2002). It is often difficult to relate the simulated results to experimental data. A review of simulated folding times has shown that the times predicted by MD and the experimental rates for thermal unfolding are in good agreement (Snow et al., 2005). Atomic force microscopy data is another possibility and this has been used to investigate protein folding of T4 lysozyme (Peng & Li, 2008).\n\nThe rate of protein folding at increasing temperatures should be described by the Arrhenius equation over a limited range of temperatures (Alberty):\n\n\n\nWhere k is the rate of the reaction, A is a constant (pre-exponential factor) Ea is the energy of activation, T is the Temperature in Kelvin and R is the Universal Gas Constant.\n\nA rearrangement of the Arrhenius equation taking natural logarithms gives the linear function:\n\n\n\nA plot of the natural logarithm of the rate ln(k) against 1/Temperature will be a straight line if the simulations obey the Arrhenius equation.\n\nThe Arrhenius equation has been used in solid-state chemistry calculations but currently no studies have tested whether it is valid in MD simulations of protein folding (Huwe et al., 1999). This paper presents a MD simulation study using a small protein (crambin) to test whether the models do agree with the predicted linear behaviour.\n\n\nMaterials and methods\n\nCrambin was chosen as the model protein for simulation of its size as it only has 46 amino acids (Caves et al., 1998). A high resolution crystal structure of crambin (3NIR.pdb) was downloaded from the RCSB Protein Databank (Rose et al., 2011; Schmidt et al., 2011). Molecular dynamics simulations were carried out using Gromacs 4.6.4 on an Ubuntu 12.04 machine with GPU acceleration (Hess et al., 2008).\n\nThe protein was solvated using periodic boundary conditions and a surrounding distance of 4nm around the protein. Simulations were run at 500K to 560K at 10K intervals using the OPLS forcefield. At temperatures of 570K and above the simulations fail to complete. The models were initially equilibrated using the canonical (nvt) and isothermal-isobaric (npt) ensembles. At 500K the simulations were run for 10ns with a time-step of 2fs. At 560K the simulations were run for 1ns with a time-step of 2fs. Secondary structure was calculated using DSSP (Dodge et al., 1998) (the data is available in dssp_files.zip). RMSD deviations from the original crystal structure were calculated in Gromacs and displayed in Grace (the data is available in rmsd_grace_datafiles.zip). All of the scripts for equilibration dynamics and analysis can be found in the accompanying data files (the Gromacs files are in gromacs_files.zip and the shell script to run all the simulations and analysis is gromacs_runs_complete_analysis.sh).\n\nThe statistical analysis of the rate data was carried out using SPSS version 22 (IBM_Corp., 2013). The line of best fit to the mean data was fitted using linear regression (the data files are available as md_arrhenius_crambin_averaged.sav, md_arrhenius_crambin_averaged.spv). The line of best fit for the complete dataset was calculated using the general linear model (the data files are available as md_arrhenius_crambin.sav, md_arrhenius_crambin.spv). All of the scripts and data for the statistical analysis are available in the supplementary materials.\n\n\nResults\n\nOver these time periods crambin did not unfold as much as had been expected. As it is such a small protein it seems to be particularly stable to rises in temperature. There were three possible end points that could be used in determining the rate:\n\n1) The unfolding of the C-terminal final bend (Figure 1, the green region from residues 36-38).\n\n2) The loss of the beta sheet (Figure 1, the two red regions residues 2-4 and 33-34).\n\n3) The increase of the root mean squared deviation (RMSD) to 0.4nm from the crystal structure.\n\nIt was not clear which of the measures would be the most reliable and so time was taken for all three. There were however missing values because the end points were not reached during the simulations. This was particularly apparent in the 540K simulations, which seem to be anomalous as can be seen in the boxplots for the reaction times from the simulations (Figure 2A–C). The boxplots show the expected downward trend, although there is considerable variation between the times taken for the repeated simulations. This variability declines at higher temperatures as rates become faster. A summary of the times to the three different end points are given in Table 1–Table 3.\n\nA: Boxplot of the times for the loss of the final bend from the secondary structure in picoseconds. Outliers are labelled. B: Boxplot of the times for the loss of the beta sheet in picoseconds. Outliers are labelled as circles or numbers if they are extreme. C: Boxplot of the times for the RMSD to go above 0.4nm from the crystal structure in picoseconds.\n\nThe Arrhenius plot can be constructed using either the mean values for the different temperatures or all of the values for the repeats. In both cases a linear model is produced but the correlation is much stronger for the averaged values, which also give a narrower confidence interval for the line of best fit (Figure 3A–C). The wider confidence intervals for all of the data and the extent of the scatter can be seen in Figure 4A–C.\n\nA: The Arrhenius plot for the unfolding of the final bend using the averaged data. B: The Arrhenius plot for the unfolding of the beta sheet using the averaged data. C: The Arrhenius plot for the increase of the RMSD by 0.4nm from the crystal structure using the averaged data.\n\nA: The Arrhenius plot for the unfolding of the final bend using the complete data. B: The Arrhenius plot for the unfolding of the beta sheet using the complete data. C: The Arrhenius plot for the increase of the RMSD by 0.4nm from the crystal structure using the complete data.\n\nThe parameters for the lines of best fit using all of the data and only the mean values are given in Table 4 and Table 5.\n\n\nDiscussion\n\nAll three of the end points used produce a linear model in the Arrhenius plot with a high degree of correlation. This suggests that in these cases the MD simulations are following Arrhenius behaviour and that these models can be used to predict rates of unfolding.\n\nOf the three end points the RMSD variation is the easiest to calculate and least ambiguous, but it is also difficult to understand what this signifies at the protein level, when compared to using the disruption of secondary structure as a metric. The other advantage of using the unfolding of the secondary structure is that experimental values are available for unfolding of proteins and so if an appropriate end point can be found in the simulations then the gradients of the Arrhenius plot can be used to calculate the activation energy for unfolding.\n\nThis study also highlights the high degree of variability between the trajectories of different simulations. This variability is very high at lower temperatures. Simulations were repeated ten times and this resulted in values for the standard errors for the time of unfolding that could be up to 25% of the time. These standard errors are large and so the number of simulations that are run needs to be increased in order to reduce them. As the standard errors falls with the square root of the sample size this would mean a 4-fold increase in the number of simulations is needed to reduce the standard error by half. This would suggest that more reliable results could be obtained by carrying out 40 simulations at each of the temperatures, which is a considerable additional computational burden.\n\nThe large amount of variation also affected the quality of the lines of best fit to the Arrhenius equation. The coefficient of determination was much lower when considering all of the data but nonetheless there is clear evidence of the simulations following Arrhenius behaviour. The confidence intervals for the linear models using all the data and the average data are similar. The variation in the gradient is too large for making comparisons with experimentally derived energies of unfolding and this is another reason why a larger number of simulations will be needed in future studies.\n\nThere was a surprising degree of agreement in the slopes and intercepts of the bend loss and beta sheet loss end-points. This suggests that the energies involved in stabilising the beta sheet and final bend are similar. This consistency is encouraging and suggests that detailed energy predictions will be possible from MD simulations.\n\nCrambin was not an ideal case for using in this study. Although it is very small and allows the simulations to be run in a shorter time, the protein does not unfold very much and so longer time-scales are needed within the simulations. Prion protein is another possible model that could be used to test unfolding as this has been shown to unfold over short simulation times (< 10ns) (Shamsir & Dalby, 2005). The other alternative is longer simulations times in order to produce clearer and less ambiguous end-points for the simulations.\n\n\nData and software availability\n\nAll of the code and data required for carrying out the simulations is available from http://dx.doi.org/10.5281/zenodo.20550. The shell script used to perform the simulations is available from http://dx.doi.org/10.5281/zenodo.20544. The dssp plots and the RMSD graphs for the simulations are available from http://dx.doi.org/10.5281/zenodo.20548. The SPSS data files and output files that include the details of how the analysis was carried out are available from http://dx.doi.org/10.5281/zenodo.20549. The software is released under a MIT license.",
"appendix": "Author contributions\n\n\n\nARD conceived the study and designed the experiments. ARD and MSS carried out the simulations and the analysis. ARD and MSS were involved in all of the versions 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\nThe author(s) declared that no grants were involved in supporting the work.\n\n\nReferences\n\nAlberty R: Physical Chemistry. John Wiley and Sons: New York. 1987. Reference Source\n\nCaves LS, Evanseck JD, Karplus M: Locally accessible conformations of proteins: multiple molecular dynamics simulations of crambin. Protein Sci. 1998; 7(3): 649–666. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaggett V: Molecular dynamics simulations of the protein unfolding/folding reaction. Acc Chem Res. 2002; 35(6): 422–429. PubMed Abstract | Publisher Full Text\n\nDay R, Bennion BJ, Ham S, et al.: Increasing temperature accelerates protein unfolding without changing the pathway of unfolding. J Mol Biol. 2002; 322(1): 189–203. PubMed Abstract | Publisher Full Text\n\nDodge C, Schneider R, Sander C: The HSSP database of protein structure-sequence alignments and family profiles. Nucleic Acids Res. 1998; 26(1): 313–315. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHess B, Kutzner C, Van Der Spoel D, et al.: GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput. 2008; 4(3): 435–447. Publisher Full Text\n\nHuwe A, Kremer F, Behrens P, et al.: Molecular Dynamics in Confining Space: From the Single Molecule to the Liquid State. Phys Rev Lett. 1999; 82(11): 2338. Publisher Full Text\n\nIBM_Corp: IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp. 2013. Reference Source\n\nLindorff-Larsen K, Trbovic N, Maragakis P, et al.: Structure and dynamics of an unfolded protein examined by molecular dynamics simulation. J Am Chem Soc. 2012; 134(8): 3787–3791. PubMed Abstract | Publisher Full Text\n\nPeng Q, Li H: Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: evidence for a kinetic partitioning mechanism. Proc Natl Acad Sci U S A. 2008; 105(6): 1885–1890. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRose PW, Beran B, Bi C, et al.: The RCSB Protein Data Bank: redesigned web site and web services. Nucleic Acids Res. 2011; 39(Database issue): D392–D401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScheraga HA, Khalili M, Liwo A: Protein-folding dynamics: overview of molecular simulation techniques. Annu Rev Phys Chem. 2007; 58: 57–83. PubMed Abstract | Publisher Full Text\n\nSchmidt A, Teeter M, Weckert E, et al.: Crystal structure of small protein crambin at 0.48 Å resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011; 67(Pt 4): 424–428. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShamsir MS, Dalby AR: One gene, two diseases and three conformations: molecular dynamics simulations of mutants of human prion protein at room temperature and elevated temperatures. Proteins. 2005; 59(2): 275–290. PubMed Abstract | Publisher Full Text\n\nShamsir MS, Dalby AR: Beta-Sheet containment by flanking prolines: Molecular dynamic simulations of the inhibition of beta-sheet elongation by proline residues in human prion protein. Biophys J. 2007; 92(6): 2080–2089. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSnow CD, Sorin EJ, Rhee YM, et al.: How well can simulation predict protein folding kinetics and thermodynamics? Annu Rev Biophys Biomol Struct. 2005; 34: 43–69. PubMed Abstract | Publisher Full Text\n\nvan der Kamp MW, Daggett V: Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding. J Mol Biol. 2010; 404(4): 732–748. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan der Kamp MW, Daggett V: Molecular dynamics as an approach to study prion protein misfolding and the effect of pathogenic mutations. Top Curr Chem. 2011; 305: 169–197. PubMed Abstract | Publisher Full Text\n\nVasquez M, Nemethy G, Scheraga HA: Conformational energy calculations on polypeptides and proteins. Chem Rev. 1994; 94(8): 2183–2239. Publisher Full Text"
}
|
[
{
"id": "10439",
"date": "28 Sep 2015",
"name": "Melchor Sanchez-Martinez",
"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 research article entitled 'Molecular Dynamics Simulations of the Temperature Induced Unfolding of Crambin Follow the Arrhenius Equation' by Dalby and Shamshir shows how the Molecular Dynamics simulations of crambin unfolding do follow the Arrhenius equation. It is addressing an interesting point as that Molecular Dynamics simulations of protein folding is used to exhibiting an non-Arrhenius behaviour and protein unfolding follow an Arrhenius equation, it is widely accepted. Testing if it is true is a really nice and interesting work.In the article, there is a comprehensive explanation of study design, methods and analysis. The conclusions are well explained and justified on the basis of the results, Furthermore, the authors have made the data fully available, including the scripts to reproduce their work, being able to be revised by the author. That point makes the work more trustable and robust.As a consequence of that, the manuscript is recommended to be approved. However there are some minor changes and comments that the authors may consider to improving the study.The authors stated that \"The Arrhenius equation has been used in solid-state chemistry calculations but currently no studies have tested whether it is valid in MD simulations of protein folding\". This phrase should be rewritten and clarified, because as it is written, it seems that this is the first time that the Arrhenius equation is used in protein folding simulations and that's not true because its usage is widely established in protein folding.To gain statistical significance, the authors performed 10 replicas, which is a good number. However more replicas changing the force-field, for instance using Amberff99SB-ILDN, would be a good option to gain more statistics and at the same time ensure to avoid the force-field dependence of the results, as in folding/unfolding processes it is very relevant (see for instance Lindorff-Larsen et al., 2012). Simulations with one or two more force-fields, performing 10 replicas for each one, would be appreciated.To explore if the protein is unfolded or not, three different metrics were employed. One of them is the increase of the root mean squared deviation (RMSD) to 0.4nm from the crystal structure. Why 0.4 and not another value? There are some references to that in the literature or it is just an observed trend in the simulations? If it is an observed trend, would change varying the force-field. In Shimada et al., 2001, they identified important structural regions of Crambin. Some of them are used by the authors as unfolding indicators, although the aminoacid numbers are not exactly the same. Thus, I wondering if helix 1 (residues 6-18,sequence: SIVARSNFNVCRL) could be also a good indicator of Crambin unfolding with the employed PDB structure, and if it is, if there is a reason to not be used.As the authors stated longer simulations of 40ns or even longer should show a more realistic and picture of Crambin unfolding. It is true that it constitutes a considerable additional computational time, but will probably result in stronger and more sound results. Thus I encourage the authors to do it, at least, in the future.",
"responses": [
{
"c_id": "1634",
"date": "01 Oct 2015",
"name": "Andrew Dalby",
"role": "Author Response",
"response": "I would like to thank the referee for his helpful comments.We definitely need to correct that omission in referring to other work using Arrhenius on proteins, although it has mostly been on short segments of protein or peptides rather than a complete protein. In writing the introduction I was surprised by the lack of literature on using Arrhenius in a biological simulation context, except for enzyme simulations. I had searched for Arrhenius behaviour in molecular dynamics and as stated I only found references to its use in simulations of materials. It seems that the problem was I should have searched just for Arrhenius and in using behaviour it needs to be the US spelling, behavior to return the protein modelling literature! The work of Baker, Karplus and Kuriyan, and Pande are particularly important in establishing the field. The more recent work by Best and Mittal is another excellent example which incorporates the effects of forcefields.I agree with the point about the forcefield but this adds a confounding variables and also increases the variance of the simulations, which increases the uncertainty in the gradient and has a negative impact on the confidence intervals for predicted energy barriers. An alternative is to use the technique to calculate relative differences in energy barriers between protein variants using the same forcefield. As you are calculating a difference the forcefield effects can be considered to cancel out. This was the idea behind free energy perturbation calculations."
}
]
},
{
"id": "10438",
"date": "14 Oct 2015",
"name": "Dmitry Nerukh",
"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 two main critical points:The conceptual one is that the loss of the beta sheet or the bend could not be, strictly speaking, classified as \"unfolding\". There is very little in the text that describes how specifically the authors quantified the moment of this loss of structure. Looking at Fig. 1 it can be seen that both motifs come back for some periods of time. Is this a loss of structure or just equilibrium fluctuations? If the latter, then there are no observable unfolding event in the data. As authors rightfully admit, the uncertainty of these loss of structure times is very high, especially at low temperatures. This is most likely because the length of simulations, 1-10ns, is too short (as again, the authors mention this in the text) and also because the algorithm of determining these times is not statistically robust. For proteins, even small ones like crambin, typical time scales for structural rearrangements is of the order of tens of nanoseconds. Even dialanin takes hundreds of nanoseconds to accumulate statistically sound number of its very simple conformational transitions.Taking into account these points, the main conclusion of the authors based on data shown on Fig. 2-4 appears unsubstantiated to me.Overall, the paper is interesting and worth indexing if the authors more rigorously calculate the \"loss of structure\" times. I would consider building a statistical network on the major conformational states, in the spirit of Markov States Model. Then, the average transition times would faithfully represent the \"unfolding\" events.",
"responses": []
},
{
"id": "10967",
"date": "28 Oct 2015",
"name": "Adrian J. Mulholland",
"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've looked at this paper and have some comments and concerns, some of which reflect the other reviewer's comments.\n\nI believe that there have been previous demonstrations of Arrhenius behaviour in unfolding simulations, including for crambin (see e.g. Ferrara et al. J. Phys. Chem. B 2000, 104, 5000-5010) and other proteins (e.g. Piana et al. PNAS 109 17845–17850, doi: 10.1073/pnas.1201811109). Consideration of protein stability is crucial here (see Scalley and Baker Proc Natl Acad Sci U S A. 1997 Sep 30; 94(20): 10636–10640) and as the current simulations are run at high temperatures where the protein is unstable, this is an issue. The dielectric behaviour of the water model at these high temperatures is also a concern for me.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-589
|
https://f1000research.com/articles/4-586/v1
|
19 Aug 15
|
{
"type": "Case Report",
"title": "Case Report: Traumatic vein of Labbé hemorrhagic infarction - a distinct neurosurgical entity",
"authors": [
"Sunil Munakomi",
"Bijoy Kumar",
"Bijoy Kumar"
],
"abstract": "Traumatic vein of Labbé haemorrhagic infarction is a distinct neurosurgical entity which requires special attention due to the important nature of the area it drains and its higher propensity for early uncal herniation. Herein we discuss the case of a 55 year-old male presenting with altered sensorium following a road traffic accident. His computerized tomogram (CT) head was suggestive of traumatic vein of Labbé haemorrhagic infarction which was subsequently confirmed by magnetic resonance (MR) venography. Due to impending herniation, he underwent urgent craniotomy and evacuation of hematoma.The patient made an uneventful recovery and was subsequently discharged home.This diagnosis should always be kept in mind for a patient with petrous bone fracture, transverse sinus thrombosis and hematoma in the mid and posterior temporal lobe.",
"keywords": [
"Trauma",
"vein of Labbé",
"outcome"
],
"content": "Introduction\n\nDural venous sinus thrombosis after blunt head trauma has been reported in few case series1–6. Only a few studies have been done on outcome following traumatic vein of Labbé haemorrhagic infarction7. It is an important neurosurgical entity because of the involvement of the area that it drains in language comprehension and processing, as well as possessing a higher propensity for causing early uncal herniation with a sometimes fatal outcome8–10. As such, stringent monitoring of the patients and early surgical evacuation if required is the key to the successful management of this condition. One of the important differential diagnoses for vein of Labbé infarction is traumatic temporal artery damage wherein damage to the medial temporal region including the insular territory is also seen. Another entity to be excluded is transverse sinus thrombosis. This case highlights the importance of close observation of patients with petrous bone fracture and transverse sinus thrombosis for evolving vein of Labbé haemorrhagic infarction and early uncal herniation. There have only been a few studies highlighting this clinical entity and the cumulative advice from these is the suggestion of performing cerebral venographic studies in suspected cases so as to make a timely and correct surgical decision.\n\n\nCase report\n\nA 55-year-old Nepalese male from a remote village in Nawalparasi, Nepal was brought to the emergency room after being hit by a moving car. Medical history turned up no significant past medical illnesses or surgical interventions. At the time of arrival, his Glasgow coma scale (GCS) was E3M5V4. Vital parameters like blood pressure (130/80), pulse rate (76/min), respiratory rate (23/min) and oxygen saturation (99% at room air) were within normal range. Both of his pupils were equally sized and equally reactive to light. A primary and secondary injury survey did not reveal other systemic injuries. An urgent head CT scan revealed the presence of a hyperdense lesion in the right temporal region with mild effacement of the ipsilateral ambient cisterns and widening of the cerebello-pontine cisterns suggestive of early uncal herniation (Figure 1). We made the provisional diagnosis of traumatic vein of Labbé hemorrhagic infarction with traumatic contusions as the main differential diagnosis. Screening MR venography proved the findings of traumatic vein of Labbé haemorrhagic infarction through identification of the absence of vein of Labbé on the right side (Figure 2).\n\nDue to the risk of imminent herniation, patient’s relatives were counseled regarding the benefits and risks involved in the surgical management. After verbal and written consent, the patient was taken up for surgery. Fronto-temporo-parietal flap craniotomy was performed. Durotomy was done and the posterior temporal corticostomy with evacuation of the hematoma was undertaken. Brain was lax and pulsatile at the end of the procedure. Patient was extubated the following morning after a repeat CT showed resolution in the herniation effect without any untoward post-operative events. Patient was started on the antiepileptic Sodium Valproate (300 mg intravenously, every 8 hours) for seizure prophylaxis and was advised to continue on this regiment for at least 6 months. Patient showed remarkable improvement, attaining a GCS score of E4M5V5 but with deficits in the prosody of his speech, attributable to the involvement of his right temporal lobe. The patient was early ambulated after the second post-operative day so as to prevent complications like chest infection and deep venous thrombosis due to prolonged immobilization. He was discharged home on the seventh post-operative day, after removal of his wound stitches, with the advice of taking the antiepileptic regularly. The patient was followed up in the outpatient clinic 3weeks later. Patient still had some deficits to prosody of his speech but language fluency and content were normal. Repeat CT showed complete resolution in hematoma and mass effects (Figure 3). Patient was advised to continue with the antiepileptic medication (Sodium Valproate 300mg via oral route three times daily) for 6 months.\n\n\nDiscussion\n\nNamed after the French surgeon Charles Labbé, the vein of Labbé (also known as the inferior anastomotic vein) crosses the temporal lobe between the Sylvian fissure and the transverse sinus and connects the superficial middle cerebral vein and the transverse sinus.\n\nSince there is higher propensity for early uncal herniation and concurrent rapid neurological deterioration, any traumatic temporal lobe lesion poses an enigma for neurosurgeons.\n\nImpact injury and counterblow are the main causes of injuries to the vein of Labbé, which can consequently lead to serious traumatic cerebral infarction with its associated poor prognosis7. Temporal bone fracture was associated in 15 of all the 16 cases in a study by Long et al.7\n\nIn a study by Giannetti et al.11, CT scan findings such as mediolateral diameter of the lesion, location of the hematoma, status of the ambient cisterns and position of the midline structures were used as a criteria to decide which patients would benefit from early surgery. In this case, we used the location of the hematoma, its volume and features of obliteration of ambient cisterns to assess the need to surgically evacuate the hematoma.\n\nIn previous studies of patients with blunt head trauma who have skull fractures extending to a dural venous sinus or jugular bulb, multi-detector CT venography identified dural venous sinus thrombosis (DVST) in 40.7% of cases, and of these 55% were occlusive12. There is a high risk of evolution of vein of Labbé haemorrhagic infarction in the subsets of patients with petrous bone fracture. So proper monitoring is justified for any signs and symptoms of increased intracranial pressure.\n\nAlso given the nature of the area of brain that vein of Labbé drains (language processing and comprehension), there is a need for long-term follow up of these patients to determine any neurological sequelae.\n\n\nConclusion\n\nA high index of suspicion needs to be kept in patients with petrous bone fractures for probable vein of Labbé hemorrhagic infarction following transverse sinus thrombosis. In those with traumatic venous infarction, stringent monitoring needs to be taken for evidence of early uncal herniation. In case of lesions more than 25ml, anisocoria, uncal herniation and asymmetric ambient cisterns, early surgical evacuation is justified.\n\n\nConsent\n\nWritten consent for publication of clinical data and images was sought and received from the son of the patient.",
"appendix": "Author contributions\n\n\n\nSM wrote and formatted the paper. BMK revised and edited the final format. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declared 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\nStiefel D, Eich G, Sacher P: Posttraumatic dural sinus thrombosis in children. Eur J Pediatr Surg. 2000; 10(1): 41–44. PubMed Abstract | Publisher Full Text\n\nMiller JD, Jennett WB: Complications of depressed skull fracture. Lancet. 1968; 2(7576): 991–995. PubMed Abstract | Publisher Full Text\n\nCarlucci GA: Injury to the longitudinal sinus accompanying a depressed fracture of the skull. Am J Surg. 1939; 45(1): 120–124. Publisher Full Text\n\nKaplan A: Compound depressed fractures of the skull involving the superior longitudinal sinus. Am J Surg. 1947; 74(1): 80–85. PubMed Abstract | Publisher Full Text\n\nReilly HP Jr, Erbengi A, Sachs E Jr, et al.: Penetration of the sagittal sinus by a depressed skull fracture. Roentgenographic diagnosis in an asymptomatic boy. JAMA. 1967; 202(8): 841–842. PubMed Abstract | Publisher Full Text\n\nKinal ME: Traumatic thrombosis of dural venous sinuses in closed head injuries. J Neurosurg. 1967; 27(2): 142–145. Publisher Full Text\n\nLong LS, Xin ZC, Wang WM, et al.: [Clinic analysis of 16 patients of craniocerebral trauma with Labbé vein injury]. Zhonghua Wai Ke Za Zhi. 2011; 49(11): 1022–5. PubMed Abstract\n\nHeiskanen O, Vapalahti M: Temporal lobe contusion and haematoma. Acta Neurochir (Wien). 1972; 27(1): 29–35. PubMed Abstract | Publisher Full Text\n\nMaurice-Williams RS: Temporal lobe swelling: a common treatable complication of head injury. Br J Surg. 1976; 63(3): 169–172. PubMed Abstract | Publisher Full Text\n\nMcLaurin RL, Helmer F: The syndrome of temporal-lobe contusion. J Neurosurg. 1965; 23(3): 296–304. PubMed Abstract | Publisher Full Text\n\nGiannetti AV: Post-traumatic temporal lobe lesions: natural history and treatment. Arq Neuro-Psiquiatr. 1998; 56(4): 859–859. Publisher Full Text\n\nDelgado Almandoz JE, Kelly HR, Schaefer PW, et al.: Prevalence of traumatic dural venous sinus thrombosis in high-risk acute blunt head trauma patients evaluated with multidetector CT venography. Radiology. 2010; 255(2): 570–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10009",
"date": "04 Sep 2015",
"name": "Carlos Bagley",
"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 very interesting article on a challenging, and potentially devastating, clinical entity. The patient in this manuscript had an excellent outcome, it would be valuable to present factors that are associated with good versus poor outcomes. In addition, although the alternative causes of a similar clinical picture were mentioned, more information could be presented regarding how one might differentiate one underlying cause from another, the management of each cause, and the associated outcomes.",
"responses": []
},
{
"id": "10171",
"date": "14 Sep 2015",
"name": "Hazem Al-Khawaja",
"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\nSunil Munakomi and Bijoy Kumar presented an interesting case report about a traumatic vein of Labbé haemorrhagic infarction. However, several aspects need to be considered before acceptance.Major: lack of specificity. The first CT brain presented is an urgent early CT scan, showing multi traumatic injuries, with contra coup contusions. It is not evident that the right-sided haemorrhagic defects are infarctions or contusions.Furthermore, the CT brain after decompressive craniectomy shows no residual infarction in the region of vein of labbé. This is strange since the treatment does not provide any recovery of the thrombosis in the vein of labbé. The case presented is a result of early decompression in a TBI patient.Major: The surgery that is performed is far from delicate and not in line with TBI guidelines.The bone flap that is performed is very small; removal of such small flap is a high risk for developing mushroom like herniation of the brain after decompression. Furthermore the authors performed haemorrhage removal during surgery. The haemorrhage is within the brain, meaning they removed contusions sq. infarction. This part of treatment is only performed in highly complicated cases where there is no choice but to remove the brain.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-586
|
https://f1000research.com/articles/4-585/v1
|
19 Aug 15
|
{
"type": "Review",
"title": "Androgenetic alopecia: new insights into the pathogenesis and mechanism of hair loss",
"authors": [
"Rodney Sinclair",
"Niloufar Torkamani",
"Leslie Jones",
"Niloufar Torkamani",
"Leslie Jones"
],
"abstract": "The hair follicle is a complete mini-organ that lends itself as a model for investigation of a variety of complex biological phenomena, including stem cell biology, organ regeneration and cloning. The arrector pili muscle inserts into the hair follicle at the level of the bulge- the epithelial stem cell niche. The arrector pili muscle has been previously thought to be merely a bystander and not to have an active role in hair disease.Computer generated 3D reconstructions of the arrector pili muscle have helped explain why women with androgenetic alopecia (AGA) experience diffuse hair loss rather than the patterned baldness seen in men. Loss of attachment between the bulge stem cell population and the arrector pili muscle also explains why miniaturization is irreversible in AGA but not alopecia areata.A new model for the progression of AGA is presented.",
"keywords": [
"Androgenetic",
"alopecia",
"follicle"
],
"content": "Introduction\n\nAndrogenetic alopecia (AGA) affects both genders and is characterised by hair loss in a distinctive and reproducible pattern from the scalp1. Bitemporal recession affects 98.6% of men and 64.4% of women, whereas mid-frontal hair loss (Figure 1) affects nearly two thirds of women over the age of 80 years, and three quarters of men over 80 years have mid-frontal and vertex hair loss2. Local and systemic androgens transform large terminal follicles into smaller vellus-like ones3. Follicular miniaturization is the histological hallmark of AGA4,5.\n\nDiffuse hair thinning and sometimes increased hair shedding (Figure 2) precede the clinical appearance of baldness by a number of years6. This is because the process of follicular miniaturization which occurs in AGA does not simultaneously affect all follicles within a follicular unit (FU). Instead, there is a hierarchy of follicular miniaturization within FUs, and secondary follicles are affected initially and primary follicles are miniaturized last7.\n\nStage 1 is normal. Stage 2 shows widening of the central part. Stage 3 shows widening of the central part and loss of volume lateral to the part line. Stage 4 shows the development of a bald spot anteriorly. Stage 5 shows advanced hair loss.\n\nWomen are asked which image best corresponds to the amount of hair shed on an average day. Grades 1 to 4 are considered normal for women with long hair. Grades 5 and 6 indicate excessive shedding. Seventy percent of women with female pattern hair loss have excessive shedding.\n\n\nHistology of follicles in androgenetic alopecia\n\nScalp hairs arise from FUs that are best seen on horizontal scalp biopsy. FUs comprise a primary follicle that gives rise to an arrector pili muscle (APM), a sebaceous gland, and multiple secondary follicles that arise distal to the APM (Figure 3). Hairs from secondary follicles commonly emerge from a single infundibulum (Figure 4). In contrast, hairs over the beard, trunk, and limbs do not give rise to secondary hairs and exist singly or in groups of three, known as Mejeres trios (Figure 5). Miniaturization occurs initially in the secondary follicles, leading to the reduction in hair density that precedes visible baldness (Figure 6). Baldness ensues when all of the hairs within an FU are miniaturized.\n\nFollicles exist within follicular units comprising arrector pili muscle, sebaceous gland, and derived secondary hairs, some of which have miniaturized to become secondary vellus hairs. The image in the upper right depicts the level of the follicle where the horizontal sections have been cut.\n\nMultiple hair fibres can be seen to emerge from a single infundibulum.\n\n\nRole of the arrector pili muscle: New findings and implications for androgenetic alopecia\n\nOne intriguing question is that identical hair follicle miniaturization is seen histologically in lesions of alopecia areata. In this condition, miniaturization of all follicles occurs simultaneously, and unlike AGA, miniaturization occurring in alopecia areata is potentially fully reversible.\n\nThis apparent paradox may be explained by examination of the APM and in particular its proximal attachment to the hair follicle bulge8. The APM is a small band of smooth muscle that runs from the hair follicle to the adjacent upper dermis and epidermis. This muscle contributes to thermoregulation and sebum secretion. The APM arises proximally at the hair follicle at the bulge, which is an epithelial stem cell niche. Three-dimensional reconstructions of scalp biopsy specimens demonstrate that preservation of the APM predicts reversible hair loss (Figure 7) and that, conversely, loss of attachment between the APM and hair follicle bulge is associated with irreversible or partially reversible hair loss (Figure 8).\n\n(a) 3-dimensional reconstruction of the follicular unit with the muscles coloured red and follicles blue rotated to the left and (b) to the right.\n\nThe APM plays a significant role in maintaining hair follicle integrity. Restoration of the APM in transplanted hair follicle units has been shown to induce the regeneration of the neurofollicular and neuromuscular junction in the follicle bulge in single FU transplants in patients with AGA9.\n\nThe discovery that progressive muscle volume loss and fat infiltration of the APM leading to total or near total loss of the muscle attachment to the primary follicle bulge in AGA samples10 led to the hypothesis that maintenance of the attachment between the APM and the bulge might differentiate between reversible and irreversible hair follicle miniaturization. These features were exclusive to AGA and not seen in alopecia areata, a disorder associated with reversible hair follicle miniaturization11. The finding that the APM is preserved in telogen effluvium and alopecia areata supports this view.\n\nIt appears likely that the interaction between the mesenchyme-derived APM and the follicle bulge epithelium is essential for the integrity of the pilosebaceous unit, much in the same way as the interaction between the mesenchymal-derived dermal papilla and the epithelial hair follicle matrix.\n\nFollicle cycling is associated with the movement of cells between the dermal papilla and dermal sheath12. It is thought that disruption of this process in AGA causes a loss of cells from the dermal sheath and then the dermal papilla that leads to hair follicle miniaturization (Figure 9). Cells from the dermal papilla and dermal sheath are capable of undergoing both smooth muscle and adipose differentiation in vitro. Cells from the follicle mesenchyme might also contribute to maintenance of the APM, and the muscle degeneration seen in AGA could be caused by the loss of a progenitor cell population that maintains both the APM and the dermal papilla.\n\nThe sheath cells (solid cells) that surround the follicle are an integral part of the follicle dermis (a). If they are functionally lost (dotted cells indicated by arrows) from the follicle (b), then dermal papilla cells (outline only) move from the papilla to replace them (c). As a result, the papilla and the follicle become smaller. Reproduced with permission from John Wiley & Sons, Inc.12.\n\n\nResearch summary\n\nIn conclusion, we propose a new model for AGA (Figure 10). In early stages of hair loss, the APM remains attached to the primary follicle but loses its attachment to some of the regressing secondary follicles in some FUs. Miniaturization of secondary follicles and detachment of the APM from these follicles extend to the rest of the FUs. At this stage, patients may complain of hair thinning and loss of volume in their pony tail without visible baldness.\n\nIn androgenetic alopecia, miniaturization occurs initially in the secondary follicles. This leads to a reduction in hair density that precedes visible baldness. Bald scalp becomes visible only when all of the hairs within a follicular unit are miniaturized. With miniaturization, the muscle initially loses attachment to the secondary follicles. When primary follicles eventually miniaturize and lose muscle attachment, the hair loss becomes irreversible.\n\nWith further progression, miniaturization continues and the muscle loses attachment to the secondary follicles in affected FUs completely. Primary follicles eventually miniaturize and this leads to visible baldness. When primary follicles lose muscle attachment, the hair loss becomes irreversible. Hopefully, this model facilitates a clearer understanding of normal physiological hair growth and also alterations to hair growth in hair loss conditions.",
"appendix": "Competing interests\n\n\n\nThe authors declared that they have 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\nSinclair R: Male pattern androgenetic alopecia. BMJ. 1998; 317(7162): 865–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGan DC, Sinclair RD: Prevalence of male and female pattern hair loss in Maryborough. J Investig Dermatol Symp Proc. 2005; 10(3): 184–9. PubMed Abstract | Publisher Full Text\n\nEllis JA, Sinclair R, Harrap SB: Androgenetic alopecia: pathogenesis and potential for therapy. Expert Rev Mol Med. 2002; 4(22): 1–11. PubMed Abstract | Publisher Full Text\n\nSinclair R, Jolley D, Mallari R, et al.: The reliability of horizontally sectioned scalp biopsies in the diagnosis of chronic diffuse telogen hair loss in women. J Am Acad Dermatol. 2004; 51(2): 189–99. PubMed Abstract | Publisher Full Text\n\nYazdabadi A, Magee J, Harrison S, et al.: The Ludwig pattern of androgenetic alopecia is due to a hierarchy of androgen sensitivity within follicular units that leads to selective miniaturization and a reduction in the number of terminal hairs per follicular unit. Br J Dermatol. 2008; 159(6): 1300–2. PubMed Abstract | Publisher Full Text\n\nMessenger AG, Sinclair R: Follicular miniaturization in female pattern hair loss: clinicopathological correlations. Br J Dermatol. 2006; 155(5): 926–30. PubMed Abstract | Publisher Full Text\n\nSinclair R: Hair Shedding In Women: How much is too much? Br J Dermatol. 2015. PubMed Abstract | Publisher Full Text\n\nTorkamani N, Rufaut NW, Jones L, et al.: Beyond goosebumps: does the arrector pili muscle have a role in hair loss? Int J Trichology. 2014; 6(3): 88–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSato A, Toyoshima K, Toki H, et al.: Single follicular unit transplantation reconstructs arrector pili muscle and nerve connections and restores functional hair follicle piloerection. J Dermatol. 2012; 39(8): 682–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTorkamani N, Rufaut NW, Jones L, et al.: Destruction of the arrector pili muscle and fat infiltration in androgenic alopecia. Br J Dermatol. 2014; 170(6): 1291–8. PubMed Abstract | Publisher Full Text\n\nYazdabadi A, Whiting D, Rufaut N, et al.: Miniaturized Hairs Maintain Contact with the Arrector Pili Muscle in Alopecia Areata but not in Androgenetic Alopecia: A Model for Reversible Miniaturization and Potential for Hair Regrowth. Int J Trichology. 2012; 4(3): 154–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJahoda CA: Cellular and developmental aspects of androgenetic alopecia. Exp Dermatol. 1998; 7(5): 235–48. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10008",
"date": "19 Aug 2015",
"name": "Satoshi Itami",
"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",
"responses": []
},
{
"id": "10013",
"date": "19 Aug 2015",
"name": "Matthew Harries",
"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",
"responses": []
},
{
"id": "10014",
"date": "19 Aug 2015",
"name": "Lynne Goldberg",
"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 presents a novel concept that incorporates a three dimensional view into the understanding of androgenetic alopecia, and introduces the arrector pili muscle as a new player in the game.",
"responses": []
},
{
"id": "10015",
"date": "19 Aug 2015",
"name": "Victoria M.L. Jolliffe",
"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",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-585
|
https://f1000research.com/articles/3-313/v1
|
22 Dec 14
|
{
"type": "Software Tool Article",
"title": "PRN: a preprint service for catalyzing R-fMRI and neuroscience related studies",
"authors": [
"Chao-gan Yan",
"Qingyang Li",
"Lei Gao",
"Qingyang Li",
"Lei Gao"
],
"abstract": "Sharing drafts of scientific manuscripts on preprint hosting services for early exposure and pre-publication feedback is a well-accepted practice in fields such as physics, astronomy, or mathematics. The field of neuroscience, however, has yet to adopt the preprint model. A reason for this reluctance might partly be the lack of central preprint services for the field of neuroscience. To address this issue, we announce the launch of Preprints of the R-fMRI Network (PRN), a community funded preprint hosting service. PRN provides free-submission and free hosting of manuscripts for resting state functional magnetic resonance imaging (R-fMRI) and neuroscience related studies. Submissions will be peer viewed and receive feedback from readers and a panel of invited consultants of the R-fMRI Network. All manuscripts and feedback will be freely available online with citable permanent URL for open-access. The goal of PRN is to supplement the “peer reviewed” journal publication system – by more rapidly communicating the latest research achievements throughout the world. We hope PRN will help the field to embrace the preprint model and thus further accelerate R-fMRI and neuroscience related studies, eventually enhancing human mental health.",
"keywords": [
"Free-submission",
"Neuroscience",
"Open-access",
"“Peer viewed”",
"Preprint-hosting",
"R-fMRI"
],
"content": "Introduction\n\nBefore submitting manuscripts to traditional journals for peer review and publication, researchers in some fields routinely distribute the manuscripts as preprints within their field. In this way, they receive early feedback, which may help in preparing articles for definitive submission as well as rapidly propagating novel ideas to their fields. The well-known central repository for preprints, arXiv (http://arXiv.org), was founded in 1991 by Dr. Paul Ginsparg for the field of physics. It gradually expanded to include astronomy, mathematics, computer science, nonlinear science, quantitative biology, and statistics as scientists in these fields began to embrace preprints (Ginsparg, 2011). arXiv now hosts close to one million fulltext preprints (983,739 as of November 1, 2014). Registered users of arXiv can submit manuscripts (multiple versions are allowed) and all users can freely browse, view and cite any articles. Although arXiv lacks rating systems or a feedback mechanism to let users recommend papers of interest to peers or to provide feedback to authors, it is still an invaluable resource for the fields it serves.\n\nHowever, researchers’ attitude toward preprints, varies depending on the field. The field of neuroscience has yet to adopt the practice of releasing preprints. Instead, neuroscientists commonly circulate their manuscripts to collaborators and colleagues for feedback before submission, but distribution is private and limited to small groups. The reason for such limited sharing might partly be the lack of central preprint services for the field. Only in 2013 did two preprint services dedicated to biology emerge for the field of life science (Callaway, 2013; Van Noorden, 2012). The two preprint services, PeerJ Preprints (https://peerj.com/preprints/) started by PeerJ, Inc. and bioRxiv (http://biorxiv.org) launched by Cold Spring Harbor Laboratory, are providing preprint hosting services with online feedback and comment systems. It is expected that early feedback will be helpful for authors in revising and improving their articles for later peer review process of traditional journals. Furthermore, commenters can be acknowledged for their contributions in later publication. However, it is only the dawn of neuroscience preprints -- bioRxiv and PeerJ Preprints have only received 56 and 38 neuroscience papers, respectively (as of 11/1/2014, see Table 1). More efforts to facilitate adoption of the preprint model appear to be needed.\n\n*: Number of articles returned by searching the key word “neuroscience” on arxiv.org.\n\n**: Number of articles in the neuroscience sub-category of the corresponding websites.\n\n***: Number of articles returned by searching the key word “fMRI” on corresponding websites.\n\nA subfield of neuroscience, neuroimaging, especially that which focuses on resting-state functional magnetic resonance imaging (R-fMRI), has emerged as field which is embracing innovations such as open data sharing (e.g., ADHD-200-Consortium, 2012; Biswal et al., 2010; Di Martino et al., 2014; Hall et al., 2012; Mennes et al., 2013; Milham, 2012; Mueller et al., 2005; Satterthwaite et al., 2014; Van Essen et al., 2013; Zuo et al., 2014), open software sharing (e.g., Bellec et al., 2012; Rubinov & Sporns, 2010; Sikka et al., 2014; Song et al., 2011; Taylor & Saad, 2013; Whitfield-Gabrieli & Nieto-Castanon, 2012; Xia et al., 2013; Chao-Gan & Yu-Feng, 2010; Zang et al., 2012; Zuo & Xing, 2014) and sharing of learning resources (e.g., Training Course in fMRI (http://sitemaker.umich.edu/fmri.training.course) and The R-fMRI Course (http://rfmri.org/Course)). As a method to investigate ongoing brain activity in basic, translational and clinical neuroscience studies, R-fMRI has become an increasingly prevalent research area especially in recent years (Fornito & Bullmore, 2012; Fox & Raichle, 2007; Kelly et al., 2012; Van Dijk et al., 2010) considering its sensitivity to characterize developmental, aging and pathological features (Andrews-Hanna et al., 2007; Fair et al., 2008; Greicius, 2008; Zuo et al., 2010), subject-friendly data collection procedures in clinical samples, and high comparability and consistency across studies and sites (ADHD-200-Consortium, 2012; Biswal et al., 2010; Mennes et al., 2013; Tomasi & Volkow, 2012). This field has expanded exponentially, now exceeding more than 1000 studies published per year (Figure 1). Given the emerging traditions of openness in this field, and an increasing number of researchers involved, we believe that the field can benefit from a preprint service that provides peer viewing and commenting.\n\nAccordingly, we are announcing a preprint publication model for catalyzing R-fMRI and related neuroscience studies. We have designed PRN as a community funded, open-access, free-submission, “peer viewed,” preprint service. The goal of PRN is to supplement the “peer reviewed” journal publication system by supporting more rapid communication of the latest research observations throughout the world.\n\n\nImplementation\n\nWe have implemented the PRN service based on the success of The R-fMRI Network (RFMRI.ORG). The R-fMRI Network (RFMRI.ORG) has been designed as a framework to support R-fMRI studies. The R-fMRI Network comprises R-fMRI researchers (the nodes) who are connected by sharing (the edges) with each other. Through the network, imagers can efficiently share ideas, comments, resources, tools, experiences, data, and increasing knowledge of the brain. Researchers (nodes) with basic neuroscience, methodological, or clinical backgrounds can connect with each other in the network. The R-fMRI Network currently has more than 5000 registered members, aiming to enhance collaborations among researchers, especially to translate our knowledge of basic neuroscience and methodology to clinical applications (bench to bedside).\n\nThe R-fMRI Network (RFMRI.ORG) is designed with a forum system and an integrated mailing list based on drupal (http://drupal.org) and mailman (http://www.gnu.org/software/mailman/). As an online forum system, The R-fMRI Network allows researchers to propose research ideas, discuss controversial issues, request help in using software, share experiences, report preliminary results, initiate collaborations and even seek jobs. The R-fMRI Network hosts several instances of R-fMRI software (e.g., DPABI, DPARSF and GraphVar), online learning resources, open data links, and gathers the latest R-fMRI related studies from PubMed. All new posts are sent to all R-fMRI Network registered users via an integrated mailing list, and users can comment on any post by directly replying to the mailing list.\n\nThe PRN has been built based on the existing infrastructure of RFMRI.ORG. Submission of a manuscript is as easy as posting a forum post with the paper title as the post title, manuscript title page and abstract as the post content and a PDF version of the fulltext manuscript as an attachment of the post. The preprint manuscript will have a permanent online URL with a convenient commenting system as in the forum system, and with mailing list immediate notification to all registered users. Furthermore, PRN has been empowered with the following features.\n\n\nFeatures\n\nAll submissions to PRN are preprint submissions, thus authors can freely revise and submit unrevised or revised manuscripts to formal “peer reviewed” traditional journals which allow preprints. PRN only checks the format of manuscripts, and contacts the corresponding author to confirm his/her approval of submission. As a preprint service, PRN has no peer review process and no editing service.\n\nAll PRN articles are freely available online after submission. Readers can freely read, download and comment on articles. Like other posts at the R-fMRI Network, all submissions are dated, citable with a permanent URL and indexed by Google. Furthermore, each PRN submission has a unique URL with a time stamp such as http://rfmri.org/PRN_140828001.\n\nThe PRN does not ask the copyright of the work to be transferred, however, the PRN requires sufficient rights to distribute submitted articles in perpetuity as documented at http://rfmri.org/PRN_140831001. In general, the authors should grant the PRN a non-exclusive and irrevocable license to distribute the article, or certify the work is either under Creative Commons Attribution license, or the Creative Commons Attribution-Noncommercial-ShareAlike license.\n\nUnlike other open-access journals, submission to PRN is free of charge.\n\nArticles at PRN will be peer viewed by interested readers and also by consultants. The PRN has enrolled a panel of consultants – each obligated to comment on three PRN papers per six-month period. On a monthly basis, PRN will rate “consultants’ choice” and “readers’ choice” articles. Furthermore, PRN will rate the most active articles, i.e., those which elicited the most comments and revisions – as a way to spur feedback and revision of articles.\n\nThe PRN is a community funded effort. We encourage all researchers to make a small contribution at http://rfmri.org/HelpUs to help the PRN effort, but this is completely voluntary.\n\n\nCompatibility with traditional formal journals\n\nA major concern is that traditional formal journals may refuse to publish manuscripts which were previously made available online on a preprint server. To address this concern, a cross-field discussion on preprints has been initiated with editors-in-chief of journals in neuroscience, physics and mathematics. An editor-in-chief in physics responded that arXiv is invaluable for doing research in physics, and is scanned by most physicists every day. Several editors-in-chief of Neuroscience journals have confirmed that their journals do accept preprint manuscripts. Based on the information of Sherpa-Romeo (http://www.sherpa.ac.uk/romeo), we have organized a table of PRN compatible journals (http://rfmri.org/PRN_20140921001). The authors should pay a close attention to the table (http://rfmri.org/PRN_20140921001) before submitting preprint manuscripts to PRN, to avoid jeopardizing their subsequent submission to PRN-incompatible journals.\n\n\nConclusions\n\nWe have launched PRN as a preprint service for catalyzing R-fMRI and related neuroscience studies. By empowering this preprint system with an online commenting system and mailing list notification system to promote the newest studies to the R-fMRI community, as well as inviting R-fMRI experts as consultants to comment on preprint manuscripts, we hope PRN will help the field embrace the preprint model and thus accelerate R-fMRI and neuroscience related studies, eventually enhancing human mental health.",
"appendix": "Author contributions\n\n\n\nConceived and designed the experiments: CY. Performed the experiments: CY QL LG. Analyzed the data: CY QL LG. Contributed reagents/materials/analysis tools: CY QL. Wrote the paper: CY QL LG.\n\n\nCompeting interests\n\n\n\nThe authors declare that PRN receives technical support and hosting service from My Research Network (RNET.PW).\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Drs. Charles E. Schroeder, F. Xavier Castellanos and Yu-Feng Zang for their assistance and support for the PRN effort. This work is supported by the community contributors (http://rfmri.org/Contributors).\n\n\nReferences\n\nADHD-200 Consortium: The ADHD-200 Consortium: A Model to Advance the Translational Potential of Neuroimaging in Clinical Neuroscience. Front Syst Neurosci. 2012; 6: 62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews-Hanna JR, Snyder AZ, Vincent JL, et al.: Disruption of large-scale brain systems in advanced aging. Neuron. 2007; 56(5): 924–935. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBellec P, Lavoie-Courchesne S, Dickinson P, et al.: The pipeline system for Octave and Matlab (PSOM): a lightweight scripting framework and execution engine for scientific workflows. Front Neuroinform. 2012; 6: 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiswal BB, Mennes M, Zuo XN, et al.: Toward discovery science of human brain function. Proc Natl Acad Sci U S A. 2010; 107(10): 4734–4739. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCallaway E: Preprints come to life. Nature. 2013; 503(7475): 180. PubMed Abstract | Publisher Full Text\n\nChao-Gan Y, Yu-Feng Z: DPARSF: A MATLAB Toolbox for “Pipeline” Data Analysis of Resting-State fMRI. Front Syst Neurosci. 2010; 4: 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDi Martino A, Yan CG, Li Q, et al.: The autism brain imaging data exchange: towards a large-scale evaluation of the intrinsic brain architecture in autism. Mol Psychiatry. 2014; 19(6): 659–667. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFair DA, Cohen AL, Dosenbach NU, et al.: The maturing architecture of the brain’s default network. Proc Natl Acad Sci U S A. 2008; 105(10): 4028–4032. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFornito A, Bullmore ET: Connectomic intermediate phenotypes for psychiatric disorders. Front Psychiatry 2012; 3: 32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFox MD, Raichle ME: Spontaneous fluctuations in brain activity observed with functional magnetic resonance imaging. Nat Rev Neurosci. 2007; 8(9): 700–711. PubMed Abstract | Publisher Full Text\n\nGinsparg P: ArXiv at 20. Nature. 2011; 476(7359): 145–147. PubMed Abstract | Publisher Full Text\n\nGreicius M: Resting-state functional connectivity in neuropsychiatric disorders. Curr Opin Neurol. 2008; 21(4): 424–430. PubMed Abstract | Publisher Full Text\n\nHall D, Huerta MF, McAuliffe MJ, et al.: Sharing heterogeneous data: the national database for autism research. Neuroinformatics. 2012; 10(4): 331–339. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKelly C, Biswal BB, Craddock RC, et al.: Characterizing variation in the functional connectome: promise and pitfalls. Trends Cogn Sci. 2012; 16(3): 181–188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMennes M, Biswal BB, Castellanos FX, et al.: Making data sharing work: the FCP/INDI experience. Neuroimage. 2013; 82: 683–691. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilham MP: Open neuroscience solutions for the connectome-wide association era. Neuron. 2012; 73(2): 214–218. PubMed Abstract | Publisher Full Text\n\nMueller SG, Weiner MW, Thal LJ, et al.: Ways toward an early diagnosis in Alzheimer’s disease: the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Alzheimers Dement. 2005; 1(1): 55–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRubinov M, Sporns O: Complex network measures of brain connectivity: uses and interpretations. Neuroimage. 2010; 52(3): 1059–1069. PubMed Abstract | Publisher Full Text\n\nSatterthwaite TD, Elliott MA, Ruparel K, et al.: Neuroimaging of the Philadelphia neurodevelopmental cohort. Neuroimage. 2014; 86: 544–553. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSikka S, Cheung B, Khanuja R, et al.: Towards Automated Analysis of Connectomes: The Configurable Pipeline for the Analysis of Connectomes (C-PAC). 5th INCF Congress of Neuroinformatics, Munich, Germany. 2014. 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(9): e25031. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTaylor PA, Saad ZS: FATCAT: (an efficient) Functional and Tractographic Connectivity Analysis Toolbox. Brain Connect. 2013; 3(5): 523–535. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTomasi D, Volkow ND: Abnormal functional connectivity in children with attention-deficit/hyperactivity disorder. Biol Psychiatry. 2012; 71(5): 443–450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Dijk KR, Hedden T, Venkataraman A, et al.: Intrinsic functional connectivity as a tool for human connectomics: theory, properties, and optimization. J Neurophysiol. 2010; 103(1): 297–321. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Essen DC, Smith SM, Barch DM, et al.: The WU-Minn Human Connectome Project: an overview. Neuroimage. 2013; 80: 62–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Noorden R: Journal offers flat fee for ‘all you can publish’. Nature. 2012; 486(7402): 166. PubMed Abstract | Publisher Full Text\n\nWhitfield-Gabrieli S, Nieto-Castanon A: Conn: a functional connectivity toolbox for correlated and anticorrelated brain networks. Brain Connect. 2012; 2(3): 125–141. PubMed Abstract | Publisher Full Text\n\nXia M, Wang J, He Y: BrainNet Viewer: a network visualization tool for human brain connectomics. PLoS One. 2013; 8(7): e68910. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZang ZX, Yan CG, Dong ZY, et al.: Granger causality analysis implementation on MATLAB: a graphic user interface toolkit for fMRI data processing. J Neurosci Methods. 2012; 203(2): 418–426. PubMed Abstract | Publisher Full Text\n\nZuo XN, Anderson JS, Milham MP, et al.: An open science resource for establishing reliability and reproducibility in functional connectomics. Scientific Data. 2014; 1. Publisher Full Text\n\nZuo XN, Kelly C, Di Martino A, et al.: Growing together and growing apart: regional and sex differences in the lifespan developmental trajectories of functional homotopy. J Neurosci. 2010; 30(45): 15034–15043. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZuo XN, Xing XX: Test-retest reliabilities of resting-state FMRI measurements in human brain functional connectomics: a systems neuroscience perspective. Neurosci Biobehav Rev. 2014; 45: 100–118. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "7125",
"date": "07 Jan 2015",
"name": "Ze Wang",
"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 found it is a great idea to have this kind of service for neuroimaging. One comment that can improve the acceptability of this preprint service is: should the service require authors to notice the normal journals that this manuscript has been archived in PRN? And should PRN share the review comments for the accepted preprints with the standard journals?",
"responses": [
{
"c_id": "1180",
"date": "15 Jan 2015",
"name": "Chaogan Yan",
"role": "Author Response",
"response": "Thank you very much for your comments!We request the authors to reveal that the papers have been archived in PRN while submitting to preprint compatible journals.We will share the comments with traditional journals, and encourage the authors to to enclose with their submission. The PRN is trying to work together with publons (publons.com) to get credits for the commenters/reviewers.Thanks,Chao-Gan and The PRN Team"
}
]
},
{
"id": "7780",
"date": "09 Mar 2015",
"name": "Wei Gao",
"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 done for this initiative! Glad to see this is happening for the community of resting state fMRI.",
"responses": []
},
{
"id": "7782",
"date": "18 Mar 2015",
"name": "Krzysztof Gorgolewski",
"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 wholeheartedly support novel approaches to publishing, especially those run by scientists for scientists and free of commercial interests. Even though I believe a news article would be a better place for PRN to advertise itself, I am happy to review this paper as a \"Software Tool Article\". I believe addressing the following issues will make the manuscript better:Discussion of \"peer review\" is very confusing. Putting the term in inverted quotes does not improve readers understanding. In one paragraph you write \"As a preprint service, PRN has no peer review process and no editing service.\" just to follow by \"Articles at PRN will be peer viewed by interested readers and also by consultants.\" There is a lot of future tense used in the paper. This combined with no signs or examples of the platform being used gives the impression that you are describing features you plan to implement rather than existing and mature software solution. It would be good to show how does this service compare to using other existing preprint servers combined with peer review platforms such as Publons or PubPeer. Could anyone review a preprint using your platform? Will the review be public and signed or anonymous? Are the preprints indexed by Google Scholar (in contrast to just Google Web)? I could not find a link to the code of your platform (this is a formal F1000Research requirement).",
"responses": [
{
"c_id": "1510",
"date": "19 Aug 2015",
"name": "Chaogan Yan",
"role": "Author Response",
"response": "1. Thank you very much for pointing out this confusion. In the revised manuscript, we have revised all “peer viewing” into “open discussion”.2. We have revised the future tense to past tense and present tense, as the features have already been implemented.3. Comparing with arXiv, we have an online commenting system. Comparing with bioRxiv and PeerJ Preprints, we focus on a specific research field (R-fMRI), and invite a panel of consultants to comment (notifications will be sent to all ~5000 R-fMRI members). We are in the process of negotiating with Publons to give credits to the members who give review comments.4. Yes, we had launched an online review and comment system.5. Review comments are public and signed. They are intended to help the authors to improve their manuscript(s) for further submission to formal journals. These early feedback would be helpful for authors in revising and improving their articles for later peer review process of traditional scientific journals.6. We are making efforts to let the preprints to be indexed by Google Scholar. It’s still in progress.7. The codes have been released through Github (https://github.com/Chaogan-Yan/rfmri.org), we have included this link in the revised manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/3-313
|
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