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https://f1000research.com/articles/3-3/v2
|
13 Jan 14
|
{
"type": "Commentary",
"title": "Alternative ideas to increase the percentage of filled seats in nephrology fellowships",
"authors": [
"Tejas Desai"
],
"abstract": "Interest in nephrology has been decreasing for the last decade. In this opinion piece, the author provides four unconventional, outside-the-box strategies to increase the percentage of filled nephrology training positions.",
"keywords": [
"Interest in nephrology fellowship positions has been decreasing since 2002. Data from the National Residency Match Program (NRMP) show an increasing number of nephrology fellowship positions but a decreasing number of applicants",
"primarily from those who have graduated from US medical schools1–3. On December 4",
"2013",
"24% of all nephrology positions in the United States were unfilled [NRMP data",
"unpublished]. This percentage represents the largest failure of nephrology recruitment and a steady worsening since 20021",
"3. In the early years",
"this decline in interest by US medical graduates (USMGs) was mitigated by an increase in the number of applications by foreign medical graduates (FMGs). In recent years",
"fewer FMGs have applied to nephrology fellowships",
"creating an additional strain amongst nephrology fellowship programs4. Many reasons are offered to explain the declining interest in nephrology",
"each with their own merits and shortcomings. Common explanations include: 1) receiving lower salaries than other specialties2",
"5",
"2) experiencing poor job prospects upon completion of nephrology training2",
"5",
"3) working long hours2",
"3",
"5",
"4) caring for complicated",
"high acuity",
"and/or sick patients2",
"3",
"and/or 5) not witnessing positive breakthroughs in clinical research3. Leaders in the field of nephrology have addressed these challenges by offering complimentary membership to leading nephrology organizations",
"deeply discounted journal subscriptions",
"and fully funded hands-on research opportunities. Though praiseworthy",
"these initiatives have been a universal failure. Because conventional methods have not resulted in an increase in applications",
"nephrology educators are now forced to think “outside-the-box” to increase interest in this field. In this paper",
"I offer four unconventional solutions to consider that may increase the percentage of filled nephrology fellowship positions."
],
"content": "Introduction\n\nInterest in nephrology fellowship positions has been decreasing since 2002. Data from the National Residency Match Program (NRMP) show an increasing number of nephrology fellowship positions but a decreasing number of applicants, primarily from those who have graduated from US medical schools1–3. On December 4, 2013, 24% of all nephrology positions in the United States were unfilled [NRMP data, unpublished]. This percentage represents the largest failure of nephrology recruitment and a steady worsening since 20021,3. In the early years, this decline in interest by US medical graduates (USMGs) was mitigated by an increase in the number of applications by foreign medical graduates (FMGs). In recent years, fewer FMGs have applied to nephrology fellowships, creating an additional strain amongst nephrology fellowship programs4. Many reasons are offered to explain the declining interest in nephrology, each with their own merits and shortcomings. Common explanations include: 1) receiving lower salaries than other specialties2,5, 2) experiencing poor job prospects upon completion of nephrology training2,5, 3) working long hours2,3,5, 4) caring for complicated, high acuity, and/or sick patients2,3, and/or 5) not witnessing positive breakthroughs in clinical research3. Leaders in the field of nephrology have addressed these challenges by offering complimentary membership to leading nephrology organizations, deeply discounted journal subscriptions, and fully funded hands-on research opportunities. Though praiseworthy, these initiatives have been a universal failure. Because conventional methods have not resulted in an increase in applications, nephrology educators are now forced to think “outside-the-box” to increase interest in this field. In this paper, I offer four unconventional solutions to consider that may increase the percentage of filled nephrology fellowship positions.\n\n\nIdea 1: Offer combined nephrology-critical care fellowships positions\n\nAnecdotal evidence suggests that applicants are increasingly attracted to critical care medicine6. In an informal and unscientific survey of internal medicine residents and medical students, critical care medicine offers the following attractive features: 1) salaries that are well regarded by students and residents, 2) clearly defined work hours, and 3) excellent job prospects upon fellowship completion. At our institution, we have begun offering a one-year critical care medicine fellowship position to qualified candidates. Whether by chance or design, many applicants are requesting an integrated nephrology-critical care fellowship position. It is conceivable that combining nephrology with critical care training into a single 3-year consolidated fellowship may attract individuals who are “on the fence” regarding nephrology fellowship alone. The anticipated candidate for such a hybrid fellowship would be one who has a true scientific interest in nephrology and whose concerns about employment prospects, financial compensation, and/or the work hours can be alleviated by the critical care component of the training.\n\n\nIdea 2: Training programs should exit The Match\n\nIn 2009 nephrology fellowship programs entered the US National Residency Match Program (NRMP; The Match)7,8. The Match, which began in 1952, was intended to centralize the application process for graduate medical positions9. The matching algorithm provides applicants with enough time to make a duress-free decision about the fellowship position they want8,9. One can debate whether applicants still need this protection, but it is increasingly evident that for unfilled nephrology training programs, The Match is harmful. Known as “the scramble”, unfilled nephrology programs enter into an unregulated, pressure-filled and disorganized ‘free-for-all’ to find individuals for their positions. Increasingly, nephrology programs are entering the scramble because the number of positions being offered (supply) is greater than the number of applicants (demand)4. This ‘supply-demand’ imbalance places a select few programs in an undesirable, stress-filled environment in which they must fill training positions in the shortest amount of time. Put colloquially, the scramble allows any applicant of any quality, at the eleventh hour and from out of left field, to secure a nephrology fellowship position. The Match, whose intentions are to protect the applicants, actually selectively hurts nephrology fellowship programs. For those programs that cannot fill their training positions through The Match, the resulting scramble period opens their gates for un- or under-qualified applicants. Exiting The Match would return all nephrology fellowships to a pre-2010 state, in which every nephrology program would be on a level playing field. Each program would have greater control over recruitment. Indeed programs that exit The Match can develop innovative recruitment strategies; ones that are currently suppressed by The Match rules and cannot be executed in the scramble because of time constraints. Innovative recruitment would allow all nephrology programs to search for the best candidates instead of a few programs competing for the available candidates.\n\n\nIdea 3: Extend clinical nephrology fellowship training to three years\n\nExtending clinical nephrology fellowship training to three years (from its current duration of two) is counterintuitive. Some programs offer 3-year research-track positions (less than 20% of all positions), but these are few and geared primarily to individuals who want a career in research7. One would expect applicants to view a 2-year clinical training program more favorably than a 3-year program. The current 2-year duration has not been attractive enough to increase the number of applications to nephrology fellowship programs. Fellows in 2-year clinical training programs use their first year to develop their clinical skills and pass their American Board of Internal Medicine (ABIM) exam. In their second and final year, fellows focus on ABIM-specialty board preparations and searching for a job. As a result, 2-year clinical training programs offer limited time to experience the rewards of research and scholarly work. Many nephrology educators have considered the lack of research exposure and scholarly activities for fellows as having negative impacts on recruitment3. Two-year clinical training programs may turn residents away who want to participate in a modest amount of research, but do not want a career heavily focused on it. The value of a 3-year training program is in its middle (second) year, where fellows can enhance their training by learning about and partaking in scholarly activities (Figure 1). No longer having the pressures of taking and preparing for board exams, learning clinical nephrology and finding a job, fellows can enjoy research in a stress-reduced time period. Residents considering nephrology may now be attracted to training programs that will expose them to a greater degree of scholarly work in a less stressful environment.\n\nDr. Argyropoulos is a nephrologist and pharmaceutical consultant in applied clinical research. He practices in Athens, Greece and can be reached on Twitter @ChristosArgyrop.\n\n\nIdea 4: Decrease the number of positions offered\n\nThe most obvious and immediate solution is to limit the number of fellowship positions available. Smaller fellowship programs tremble at this idea. They believe that their small programs would be the most likely to shut down first, consistent with the commonly accepted but unproven rationale that the value of a fellowship program is directly proportional to its size. An alternative strategy exists that does not place the entire burden on small programs. Decrease the number of fellowship positions based on the geographic prevalence of chronic kidney disease (CKD) and/or end-stage renal disease (ESRD). Since the number of fellowship positions offered is loosely based on the anticipated need of kidney doctors, which in turn is based on the incidence and prevalence of CKD and ESRD in a geographic location, than any reduction in fellowship numbers should be based on these factors. Such a systematic method would ensure that programs would be cut based on the populations they serve and not based on their size, prestige, or other fellowship-specific factors. This method would be an equitable distribution of the pain associated with fellowship reduction and would not be prejudicial.\n\n\nConclusion\n\nIn this exploration of potential policies, I have described four possible methods by which nephrology programs can decrease the percentage of unfilled fellowship positions. Although the effectiveness of these strategies has not been scientifically studied, each idea can be tested through a Plan-Do-Study-Act cycle (PDSA)10. In a PDSA cycle, ideas can be tested on a small scale and temporary basis to determine outcomes. Those that result in unintended negative outcomes would not systemically affect all nephrology training programs when tested through a PDSA cycle. Because recruitment occurs yearly and the implementation of these ideas would require a major investment of resources, the PDSA cycle offers the best method by which these strategies can be tested.\n\nFinally, these ideas do not address the disinterest that medical students and residents have for nephrology. Increasing interest would be the best and most long-lasting way in which to increase the number of applications to nephrology training programs. For years, national organizations and individual nephrology divisions have tried to cultivate a passion for nephrology in residents and medical students5. As of 2013, however, those efforts have failed to increase the number of applications to our training programs. Perhaps more time is needed before one can determine if these endeavors have worked. In the interim, common sense ideas may need to be implemented to stave off the “tide” of disinterest in nephrology.",
"appendix": "Competing interests\n\n\n\nThe author is the creator of Nephrology On-Demand (http://www.myNOD.org). He does not receive any funding from this website.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe opinions expressed in this manuscript are those of the author and not necessarily the opinions of his institution.\n\n\nReferences\n\nDesai T, Ferris M, Christiano C, et al.: Predicting the number of US medical graduates entering adult nephrology fellowships using search term analysis. Am J Kidney Dis. 2012; 59(3): 467–469. PubMed Abstract | Publisher Full Text\n\nFewer U.S. Medical Students Choosing Nephrology. Renal Business Today. 2011. Reference Source\n\nParker MG, Ibrahim T, Shaffer R, et al.: The future nephrology workforce: will there be one? Clin J Am Soc Nephrol. 2011; 6(6): 1501–1506. PubMed Abstract | Publisher Full Text\n\nAmerican Society of Nephrology 2013 Meetings. Nephrology On-Demand. 2013. Reference Source\n\nParker MG, Pivert KA, Ibrahim T, et al.: Recruiting the next generation of nephrologists. Adv Chronic Kidney Dis. 2013; 20(4): 326–335. PubMed Abstract | Publisher Full Text\n\nDual Certification Resources: Nephrology and Critical Care Medicine. Reference Source\n\nKohan DE, Rosenberg ME: Nephrology training programs and applicants: a very good match. Clin J Am Soc Nephrol. 2009; 4(1): 242–247. PubMed Abstract | Publisher Full Text\n\nPeranson E, Randlett RR: The NRMP matching algorithm revisited: Theory versus practice. National Resident Matching Program. Acad Med. 1995; 70(6): 477–484. PubMed Abstract\n\n“National Resident Matching Program”. Wikipedia, The Free Encyclopedia. Wikimedia Foundation. Reference Source\n\nTaylor MJ, McNicholas C, Nicolay C, et al.: Systematic review of the application of the plan-do-study-act method to improve quality in healthcare. BMJ Qual Saf. 2013. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3086",
"date": "13 Jan 2014",
"name": "Manish Ponda",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well-written and I agree with many of Dr. Desai's points. The topic is highly relevant to the field of nephrology.A few comments/suggestions: For idea #1, it should be recognized that the critical care field will need to accept renal/critical care trainees into practice. If these combined-program trainees fail to find jobs, the combined program will eventual fail to attract trainees.#2 - Exiting the Match could be voluntary; also programs should be able to have some spots off-Match .#3 - Many programs already have NIH-sponsored T32 grants that allow for the support of a 3rd year of research. It is also common for programs to provide institutional support (non-NIH) for a 3rd year for interested candidates. Unfortunately, there is very little interest from prospective renal fellows even with these options available. Indeed, historically 3-year programs are now offering 2-year spots because of a lack of interest. Is there any data from NRMP to compare the success of 3-year vs. 2-year programs in terms of filling spots? The supply/demand imbalance may be inherent to nephrology itself, which I think Dr. Desai correctly identifies as a core issue in ideas #2/#4 and the Introduction.",
"responses": []
},
{
"id": "3084",
"date": "27 Jan 2014",
"name": "Paul Phelan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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. Desai has written an article regarding novel ways to increase the number of unfilled Nephrology fellowship positions. This is a well written piece by an author who has a track record of interest and research in this area. His paper is timely, coming at a point where interest in the specialty is at an all-time low, and new ideas are sorely needed.Specifically regarding his ideas:Combined critical care fellowships: this seems a good idea on the face of it. However, do we have any evidence that trainees want to pursue a combined fellowship like this? Programs exiting the match: While this will certainly help some programs, it is not realistic that competitive programs would ever consider this. I personally think this is a good idea, as having been trained abroad where fellowship is longer I can see advantages to increased experience before exiting training. However, trainees on the most part would likely not be keen on this, and it may further decrease interest. Decrease the number of positions. We certainly want quality applicants and it is not in anybody's interest to have substandard candidates getting positions just because there is a lack of demand. However this does not address the fundamental problem of why trainees are not choosing Nephrology.Overall, this is a well written and thought-out article coming at a time of critical need for innovation to publicise Nephrology to trainees. While not everybody (including this reviewer) will agree with all the ideas in the paper, we need outside-the box thinking and innovative ideas so the editorial is welcome.",
"responses": []
},
{
"id": "3085",
"date": "03 Feb 2014",
"name": "Edgar Lerma",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this paper is well written and is very timely, particularly in recognition of the recent decline in interest in choosing Nephrology as a subspecialty among medical students and trainees. It is with great interest that I read this and offer a few comments -Idea 1: Offer combined nephrology-critical care fellowship positionsNowadays, additional sub-specialization in fields such as Transplant Nephrology, Interventional Nephrology, etc. requires an additional year of training. I would suspect that a combined Nephrology-Critical Care Fellowship position would follow the same format.Similarly, I do not think that there are currently enough practice opportunities that will cater to both specializations, whether academic or private practice for those coming out of training.Idea 2: Training programs should exit the MatchI do not think that this would solve the problem. I don't think that applicants gear towards any specialty (Nephrology or others) based on a program's being in the Match or not. My impression is that the Match evens out the playing field not only for the applicants but for the programs as well.In the pre-Match era, I would think that those programs coming from well-established institutions would tend to fill their positions much earlier (than would less well-recognized programs) with the 'best candidates' that they could identify during the allotted time period.Issue 3: Extend clinical nephrology fellowship training to three yearsI am afraid that extending the training to three years may come in as a disincentive to potential applicants, especially since majority of trainees tend to go into private practice (as opposed to academics). Perhaps, those who are more academic and research oriented may be attracted to this opportunity as it gives them an extra year of research and scholarly activity which is required for subsequent promotion in the faculty ranks.Similarly, recognizing that majority of US medical school graduates have debts to pay, they would probably prefer to start reaching their earning potentials much earlier; an extra year of training may be counter to this.Issue 4: Decrease the number of positions offeredIn my opinion, this is perhaps a viable solution to the problem at hand. The author has recognized the potential 'conflict of interest' that may arise when deciding which Fellowship programs should decrease their offered positions.",
"responses": []
}
] | 2
|
https://f1000research.com/articles/3-3
|
https://f1000research.com/articles/3-5/v1
|
09 Jan 14
|
{
"type": "Case Report",
"title": "BiSpectral Index (BIS) monitoring may detect critical hypotension before automated non-invasive blood pressure (NIBP) measurement during general anaesthesia; a case report.",
"authors": [
"Matthew M. J. Smith"
],
"abstract": "A patient undergoing general anaesthesia for neurosurgery exhibited an unexpected sudden decrease in the BiSpectral Index (BIS) value to near-zero. This prompted the detection of profound hypotension using non-invasive blood pressure (NIBP) measurement and expedited urgent assessment and treatment, with the patient making a full recovery. Widely regarded as a ‘depth of anaesthesia’ monitor, this case demonstrates the potential extra clinical benefit BIS may have in the detection of critical incidents such as anaphylaxis during general anaesthesia.",
"keywords": [
"BIS",
"bispectral index",
"hypotension",
"anaesthesia",
"anaphylaxis",
"anaphylactoid",
"TIVA",
"neurosurgery"
],
"content": "Presentation and clinical findings\n\nA retired, 68 year old man of white British origin presented to the neurosciences unit with a subdural haematoma of unknown aetiology. With no past medical or family history of note, he was scheduled for neurosurgical intervention via burr holes under general anaesthesia. Prior to the operation, his Glasgow Coma Score was 15/15 and he was alert and oriented. The patient was in good general health, normotensive, and had no regular medications or known drug allergies. A total intravenous anaesthesia (TIVA) technique was chosen, as is usual for such cases in our institution.\n\nBefore induction of anaesthesia, we instituted standard monitoring according to guidelines published by the Association of Anaesthetists of Great Britain and Ireland. A BIS Quattro sensor (Covidien LLC, Mansfield, USA) was also applied to the forehead on the non-pathological side and connected to a BIS ‘VISTA’ monitor (Covidien LLC, Mansfield, USA).\n\nTarget-controlled infusions of propofol (3µg/ml) and remifentanil (3ng/ml) were used to induce unconsciousness. To maintain normotension, a 4mg/h infusion of metaraminol was simultaneously started. Once the BIS value had fallen to 60, a 40mg dose of atracurium was given to facilitate tracheal intubation. During this period cardiovascular stability was maintained as measured by pulse rate and regular (every 2.5m) NIBP readings.\n\nThe patient was prepared for the operating theatre and transferred into the operating room, where we noticed that the BIS value had dramatically fallen to 04 with an almost isoelectric real-time EEG reading. This triggered the anaesthetist to immediately re-measure the NIBP, which revealed a blood pressure of 44/26.\n\nPossible anaphylaxis to atracurium was suspected. Immediate treatment was initiated with 250ml 0.9% saline, 6mg ephedrine and 0.5mg of metaraminol IV, whilst adrenaline was prepared. These interim measures were enough to restore the blood pressure and BIS back to their expected values, and in the event no adrenaline was administered. Subsequent acute care included institution of invasive blood pressure monitoring, and treatment with hydrocortisone 100mg, chlorphenamine 10mg, and ranitidine 50mg IV. Moderate flushing and urticaria became evident some 15 minutes after the onset of the hypotension, however the patient remained otherwise stable and the rest of surgery and recovery were uneventful. Blood samples for mast-cell tryptase were taken as per local guidelines.\n\n\nTimeline\n\nFigure 1 below illustrates the timeline of the case. Anaesthetic induction occurs at 0940. The period of low BIS and hypotension is encircled in dashed green. The subsequent rise in BIS was coincident with restoration of normotension.\n\n\nDiagnosis and follow up\n\nThe patient had an uncomplicated post-operative recovery and was discharged and sent home. He was referred to the immunology clinic for follow-up allergy testing. Mast cell tryptase results were all within the norm, making suspicion of anaphylaxis unlikely; however a raised IgE (147IU/L) plus the clinical signs seen during anaesthesia raised the possibility of an anaphylactoid drug reaction. A non-immune-mediated cause could not be ruled out for the critical incident.\n\n\nDiscussion\n\nBIS uses the frontal electroencephalogram and proprietary algorithms to quantify level of consciousness on a scale of arbitrary units from 0 (isoelectric raw signal) to 100 (awake). Intended to guide the hypnotic aspect of general anaesthesia, previous reports have however suggested that BIS may be used as a crude marker of cerebral hypoperfusion1–5. Positron Emission Tomography scanning of anaesthetised patients has correlated the cerebral metabolic rate for oxygen consumption with BIS6, strengthening the case for this.\n\nDuring the events described, the drop in BIS was detected before that of any other monitored variable or clinical change. It prompted rapid reassessment, discovery of cardiovascular collapse, and timely intervention. Had the patient not been BIS monitored, the hypotension would not have been revealed until the next ‘scheduled’ cycling of the NIBP, which may have led to further harm. We feel this demonstrates the potential extra clinical utility that BIS may have in the detection of critical incidents over and above its perception as solely a ‘depth of anaesthesia’ monitor.\n\n\nConsent\n\nInformed written consent for publication of clinical details and clinical images was obtained from the patient at the time of allergy clinic referral.",
"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\nMS would like to acknowledge Dr. Ajay Raithatha and Dr. Matt Wiles for their encouragement to write this report.\n\n\nReferences\n\nMérat S, Lévecque JP, Le Gulluche Y, et al.: [BIS monitoring may allow the detection of severe cerebral ischemia]. Can J Anaesth. 2001; 48(11): 1066–1069. PubMed Abstract\n\nKertai MD, Whitlock EL, Avidan MS: Brain monitoring with electroencephalography and the electroencephalogram-derived bispectral index during cardiac surgery. Anesth Analg. 2012; 114(3): 533–546. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMyles PS: Bispectral index monitoring in ischemic-hypoxic brain injury. J Extra Corpor Technol. 2009; 41(1): P15–19. PubMed Abstract\n\nEstruch-Pérez MJ, Ausina-Aguilar A, Barberá-Alacreu M, et al.: Bispectral index changes in carotid surgery. Ann Vasc Surg. 2010; 24(3): 393–399. PubMed Abstract | Publisher Full Text\n\nWin NN, Kohase H, Miyamoto T, et al.: Decreased bispectral index as an indicator of syncope before hypotension and bradycardia in two patients with needle phobia. Br J Anaesth. 2003; 91(5): 749–752. PubMed Abstract\n\nAlkire MT: Quantitative EEG correlations with brain glucose metabolic rate during anesthesia in volunteers. Anesthesiology. 1998; 89(2): 323–333. PubMed Abstract"
}
|
[
{
"id": "3052",
"date": "17 Jan 2014",
"name": "Ehab Farag",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a nice case, I approve this with no reservations.",
"responses": []
},
{
"id": "4222",
"date": "25 Mar 2014",
"name": "Sumio Hoka",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 showing an immediate response of BIS monitor to circulatory failure. BIS monitors could express not only the depth of anesthesia but also the state of brain ischemia.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-5
|
https://f1000research.com/articles/2-259/v1
|
26 Nov 13
|
{
"type": "Case Report",
"title": "Severe bilateral amyotrophic neuralgia associated with major dysphagia secondary to acute hepatitis E",
"authors": [
"Xavier Moisset",
"Nicolas Vitello",
"Elodie Bicilli",
"Romain Courtin",
"Anna Ferrier",
"Frederic Taithe",
"Clément Lahaye",
"Ali Ait Hssain",
"Cyril Garrouste",
"Clavelou Pierre",
"Nicolas Vitello",
"Elodie Bicilli",
"Romain Courtin",
"Anna Ferrier",
"Frederic Taithe",
"Clément Lahaye",
"Ali Ait Hssain",
"Cyril Garrouste",
"Clavelou Pierre"
],
"abstract": "Introduction: Several acute neurological syndromes can be triggered by immune events. Hepatitis E virus (HEV), an emerging infectious disease, can be one of these triggers.Case report: We report the case of a 36-year-old man that presented nausea and a dull abdominal pain for a week and then felt an acute neuralgic pain involving both shoulders that lasted for 8 to 10 hours. Immediately after, the patient presented a severe bilateral muscular weakness of the proximal part of both upper limbs, corresponding to an amyotrophic neuralgia. Two days after the shoulder pain, the patient presented a dysphagia necessitating tube feeding. A blood sample confirmed hepatitis caused by hepatitis E virus (HEV; genotype 3F). Oral feeding resumed progressively after five months. The patient was fully independent for the activities of daily living but was still unable to work after six months.Conclusion: Amyotrophic neuralgia and hepatitis E are both under-diagnosed. It is noteworthy that HEV can trigger amyotrophic neuralgia. Antiviral drugs, oral steroids and intravenous immunoglobulins can be proposed, but the optimal treatment has not yet been determined.",
"keywords": [
"Amyotrophic neuralgia",
"Hepatitis E",
"dysphagia"
],
"content": "Introduction\n\nNeurological syndromes such as Guillain-Barré Syndrome, transverse myelitis, encephalitis or amyotrophic neuralgia can be triggered by immune events. Hepatitis E virus (HEV), discovered in the 1980s, can be one of these triggers. Epidemics of hepatitis E occur periodically throughout the developing world, but autochthonous HEV infections have also been reported in most developed countries during the last decade. Several HEV-associated neurological syndromes have been described but are probably under-diagnosed1.\n\n\nCase report\n\nWe report the case of a 36-year-old French, Caucasian truck driver, without any significant medical history. The clinical symptoms started in May 2012 with nausea and a dull abdominal pain. No sign of chronic liver disease or of portal hypertension was noted. High liver enzymes were diagnosed after assay for: alanine aminotransferase (ALT) 1707 µmol/L (normal range: N<78), aspartate aminotransferase (AST) 554 µmol/L (N<37), gamma-glutamyltranspeptidase (GGT) 737 U/L (N<95) and alkaline phosphatase at 311 U/L (N<136). Total bilirubin level was at 54 µmol/L (N<17). There was no hepatitis A, B or C, no HIV and no sign of autoimmune disease. Liver ultrasound was normal.\n\nAround one week after the first digestive symptoms, the patient felt an acute neuralgic pain involving both shoulders that lasted for 8 to 10 hours. Immediately after, the patient presented a severe bilateral muscular weakness of the proximal part of both upper limbs. Two days after the shoulder pain, the patient presented with hypophonia and dysphagia. The MRI did not show any brain abnormality. The spinal cord and the brachial plexus were unharmed. The cerebrospinal fluid (CSF) was normal (2 white blood cells/mm3; CSF Protein= 0.37 g/L) and there was no intrathecal antibody synthesis. Antiganglioside antibodies were negative (GM1, GM2, GD1a, GD1b, GQ1b). Electromyography (EMG) and nerve conduction studies (NCS) showed normal amplitudes and conduction velocity but bilateral denervation in the supraspinatus, infraspinatus, subscapularis and deltoid muscles. An acute hepatitis E infection was suspected due to the presence of IgM and confirmed by PCR (genotype 3f). The initial serum HEV RNA count was 5.2 log-copies/ml. The PCR was negative in the CSF.\n\nA treatment with intravenous immunoglobulins (Tegeline®, LFB laboratory, France; 0.4 g/kg/day) was given for 5 days. Ribavirin (600 mg/day) was also introduced. Nine days after ribavirin initiation, the PCR showed 2.02 log-copies/ml and was negative after 18 days. After three weeks, the patient still required nasogastric tube-feeding and a gastric feeding tube was placed endoscopically. There was no contraction of the shoulder girdle muscles. Oral feeding resumed progressively after five months. After six months follow-up and intensive rehabilitation, there was a 3/5 muscular strength in the affected muscles, corresponding to a movement possible against gravity, but not against resistance by the examiner. The patient was fully independent for the activities of daily living but still unable to work.\n\n\nDiscussion\n\nThis is the first report of both severe bilateral amyotrophic neuralgia and dysphagia caused by an acute hepatitis E infection. Amyotrophic neuralgia (AN) is a peripheral neuropathy consisting of multiple symptoms including abrupt onset of shoulder pain, usually unilaterally, followed by motor weakness, with an annual incidence above 2 per 100,000 inhabitants2. Concomitant involvement of other peripheral nervous system structures (such as the lumbosacral plexus or phrenic nerve) is described. AN can be triggered by immune events but also by trauma or surgery. Many patients are left with residual disabilities that affect their ability to work and their everyday life. It is noteworthy that a particularly severely affected subgroup presents sign of liver dysfunction, as seen in HEV infections2. The only validated treatment is corticosteroids but this may have been dangerous in this case of acute hepatitis E3. Some authors have also reported a positive effect of intravenous immunoglobulins and this was the option we selected4–6.\n\nHEV-associated neurological syndromes include both central and peripheral nervous system involvement7. Such cases have been described in the Asian sub-continent (probably due to HEV1) but also in Western Europe with acute and chronic HEV3 infection. For patients with chronic HEV infections, neurological symptoms completely resolved or significantly improved when viral clearance was achieved1. This is the reason why we tried antiviral treatment. Unfortunately, although viral clearance was achieved quickly, this did not lead to fast clinical improvements.\n\nAn alternative diagnosis of pharyngeal-cervical-brachial variant of Guillain-Barré syndrome could have been made. This pathology is characterized by oropharyngeal, neck, and upper limb muscle involvement. However, in the present case, this diagnosis was excluded (no neck involvement, atypical EMG, no albuminocytologic dissociation of the cerebrospinal fluid and negative GQ1b antibody).\n\n\nConclusion\n\nPost-infectious neurological diseases following HEV infection must be recognized to avoid unnecessary and potentially invasive procedures. Further studies are needed in order to determine the optimal treatment. In the meantime, antiviral drugs, steroids and IV-immunoglobulins are all possibilities.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nXM: wrote the manuscript. NV: revised the first draft. EB, RC and CL: managed the patient in the rheumatology and neurology departments. AF: suggested HEV diagnosis. FT: did the EMG. AAH: managed the patient in the intensive care unit. FT, CG and PC: decided on the treatment plan. All authors were involved in the revision of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nKamar N, Bendall R, Legrand-Abravanel F, et al.: Hepatitis E. Lancet. 2012; 379(9835): 2477–2488. PubMed Abstract | Publisher Full Text\n\nVan Alfen N: Clinical and pathophysiological concepts of neuralgic amyotrophy. Nat Rev Neurol. 2011; 7(6): 315–322. PubMed Abstract | Publisher Full Text\n\nVan Alfen N, Van Engelen BG, Hughes RA: Treatment for idiopathic and hereditary neuralgic amyotrophy (brachial neuritis). Cochrane Database Syst Rev. 2009; 8(3): CD006976. PubMed Abstract | Publisher Full Text\n\nJohnson NE, Petraglia AL, Huang JH, et al.: Rapid resolution of severe neuralgic amyotrophy after treatment with corticosteroids and intravenous immunoglobulin. Muscle Nerve. 2011; 44(2): 304–305. PubMed Abstract | Publisher Full Text\n\nMoriguchi K, Miyamoto K, Takada KM, et al.: Four cases of anti-ganglioside antibody-positive neuralgic amyotrophy with good response to intravenous immunoglobulin infusion therapy. J Neuroimmunol. 2011; 238(1–2): 107–109. PubMed Abstract | Publisher Full Text\n\nNaito K, Fukushima K, Suzuki SM, et al.: Intravenous immunoglobulin (IVIg) with methylprednisolone pulse therapy for motor impairment of neuralgic amyotrophy: clinical observations in 10 cases. Intern Med. 2012; 51(12): 1493–1500. PubMed Abstract | Publisher Full Text\n\nCheung MC, Maguire J, Carey IM, et al.: Review of the neurological manifestations of hepatitis E infection. Ann Hepatol. 2012; 11(5): 618–622. PubMed Abstract"
}
|
[
{
"id": "2574",
"date": "04 Dec 2013",
"name": "Bruce Brew",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case report highlighting HEV as a cause for brachial neuritis. Recognition of this association is clinically important as treatment with corticosteroids should be avoided.",
"responses": []
},
{
"id": "2575",
"date": "20 Dec 2013",
"name": "Thierry Coton",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 about an emerging complication of acute hepatitis E.The following information is missing however:the gender of the patient.if prothrombin time increased during hepatitis.which auto antibodies were screened for.if PCR was realised in stools.the duration of the ribavirine therapy.Additionally in the discussion, the authors must highlight that this case concerned a genotype 3f virus which is predominant in France (Luciano et al. 2012).",
"responses": [
{
"c_id": "659",
"date": "31 Dec 2013",
"name": "Xavier Moisset",
"role": "Author Response",
"response": "We want to thank Dr Coton for the comments and suggestions he made that have helped us to improve the quality of our case report.The gender of the patient (male) was specified in the abstract but not in the core text. It is now corrected. The prothrombin time stayed stable within the normal range throughout the monitoring period. The immunological screening included: antinuclear antibodies, anti-smooth muscles antibodies, anti-mitochondria antibodies, anti LKM antibodies, anti-hepatic cytosol antibodies, complement (C3, C4, CH50), rheumatoid factor, antineutrophil cytoplasmic antibody (ANCA), anti ganglioside antibodies and onconeuronal antibodies (Hu, Ri, Yo, PNMA2, CV2, Amphiphysine). PCR was not initially realized in stools. The only PCR in stools was realized after 3 weeks of Ribavirin treatment and was negative. The ribavirin therapy lasted for 35 days. The treatment was stopped on the basis of negative PCR results in both blood and stools. We now specify in the discussion that this case concerned a genotype 3f virus which is predominant in France."
}
]
},
{
"id": "2868",
"date": "20 Dec 2013",
"name": "José Manuel Echevarrı́a",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describes the association of a convincingly diagnosed case of acute hepatitis E due to HEV genotype 3 infection with the development of severe amyotrophic neuralgia and major dysphagia in the patient. Reports of neurological complications among otherwise healthy patients after acute HEV-3 infection are scarce (I found just six cases from three reports since 2009, five of them reported in 2011), and the manuscript adds significant knowledge to the background.Pathogenic mechanisms underlying neurological complications of hepatitis E are still unclear. Such diseases after viral infection may involve the invasion of the nervous system or may be of a pure, post-infectious, immune-mediated nature. Reports involving immunocompromised patients (Kamar et al., 2011; four patients) found HEV genome in CSF from all of them and detected anti-HEV IgM in one, which confirmed CNS invasion at the time of the onset of the neurological symptoms.Among the six healthy patients mentioned above (Loly et al., 2009; Kamar et al., 2011; Despierres et al., 2011), CSF testing for HEV markers was performed in three cases. One tested positive for both HEV RNA and anti-HEV IgM, and one tested negative for both markers (Despierres et al., 2011). CSF from the remaining patient was tested for viral RNA only and was found negative. It seems likely therefore, that both mechanisms may be involved, in absence of immune impairment.The patient mentioned in the present report tested negative for HEV RNA in CSF, but I understand from the text that anti-HEV testing of CSF was not performed (if the sentence “there was no intrathecal antibody synthesis” refers to immunoglobulins and not to HEV-specific antibody, which should be stated more clearly). It would have provided useful information if the patient’s CSF had been tested for anti-HEV IgG and IgM. The lack of a viral genome at the time of sampling does not exclude CNS invasion, and demonstration of intrathecal synthesis of a specific antibody provides the diagnosis once viral particles are no longer present. As far as I know, nobody has yet communicated results from the evaluation of the intrathecal synthesis of a specific antibody to HEV in these cases (only the presence of anti-HEV IgM has been documented), so I would suggest performing such an evaluation if anti-HEV was detected. From my former experience with the diagnosis of neurological infections caused by varicella-zoster virus, testing for specific IgG antibody optimizes the yield, and the most useful criteria of evaluation is the antibody to albumin index, though several others can be used (see Echevarría et al. 1997).",
"responses": [
{
"c_id": "658",
"date": "31 Dec 2013",
"name": "Xavier Moisset",
"role": "Author Response",
"response": "Dr Echevarrı́a is right; anti-HEV antibody testing of CSF was not performed and this is now clearly specified in our version 2. Indeed, it would be of interest in future cases to test CSF for both RNA and intrathecal synthesis of specific anti-HEV antibodies."
}
]
},
{
"id": "2571",
"date": "23 Dec 2013",
"name": "Michelle Cheung",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a report of an emerging viral infection with well-documented but still uncommon neurological symptoms, and adds to the knowledge of the natural history and potential treatment of this clinical syndrome. It is clearly written and the case is described well.I have the following minor comments:The referenced Cochrane review found no evidence from randomised trials to support any form of treatment for neuralgic amyotrophy, with only one retrospective series reporting a benefit. Therefore it seems an over-statement to say that corticosteroids are a validated treatment for the condition. Are there any cases of prednisolone use specifically for HEV-induced neuralgic amyotrophy? What is the evidence of using ribavirin to treat acute (rather than chronic) HEV, which is a self-limiting infection? The authors state that 'a severely affected subgroup present with signs of liver dysfunction, as seen in HEV' - do you mean that cases associated with HEV infection (and therefore presenting with liver dysfunction) have a more severe clinical course compared to neuralgic amyotrophy associated with other causes? Please justify the conclusion that recognition of HEV infection avoids invasive procedures?",
"responses": [
{
"c_id": "657",
"date": "31 Dec 2013",
"name": "Xavier Moisset",
"role": "Author Response",
"response": "We want to thank Dr Cheung for the comments she made that have helped us to improve the quality of our text. Dr Cheung is right; the Cochrane review identified one open label retrospective series suggesting that prednisone can shorten the duration of the initial pain and leads to earlier recovery in some patients. Thus, the expression “validated treatment” has been replaced by “proposed treatment (with a low level of evidence)”. To our knowledge, there is no description of prednisone use in the specific case of HEV-induced neuralgic amyotrophy. Concerning the use of ribavirin to treat acute HEV, there is a low level of evidence but several authors suggest treating severe acute form in order to preclude the development of acute liver failure (Abbas Z and Afzal R. 2013 [Epub ahead of print] Hepatitis E: when to treat and how to treat). A sentence and this reference are now included in the discussion section. We cannot know if neuralgic amyotrophy secondary to HEV infections are more severe compared with neuralgic amyotrophy associated with other causes. But among the patients with amyotrophic neuralgia, it has been described that a subgroup of patients had a particularly severe clinical course and that these patients had liver dysfunction. Thus, we hypothesized that some of these patients were possibly affected by HEV. Unexplained severe hepatic cytolysis can lead to liver biopsy. HEV infection recognition can avoid this invasive procedure."
}
]
}
] | 1
|
https://f1000research.com/articles/2-259
|
https://f1000research.com/articles/2-287/v1
|
27 Dec 13
|
{
"type": "Review",
"title": "Neurocognitive endophenotypes in CGG KI and Fmr1 KO mouse models of Fragile X-Associated disorders: an analysis of the state of the field",
"authors": [
"Michael R. Hunsaker"
],
"abstract": "It has become increasingly important that the field of behavioral genetics identifies not only the gross behavioral phenotypes associated with a given mutation, but also the behavioral endophenotypes that scale with the dosage of the particular mutation being studied. Over the past few years, studies evaluating the effects of the polymorphic CGG trinucleotide repeat on the FMR1 gene underlying Fragile X-Associated Disorders have reported preliminary evidence for a behavioral endophenotype in human Fragile X Premutation carrier populations as well as the CGG knock-in (KI) mouse model. More recently, the behavioral experiments used to test the CGG KI mouse model have been extended to the Fmr1 knock-out (KO) mouse model. When combined, these data provide compelling evidence for a clear neurocognitive endophenotype in the mouse models of Fragile X-Associated Disorders such that behavioral deficits scale predictably with genetic dosage. Similarly, it appears that the CGG KI mouse effectively models the histopathology in Fragile X-Associated Disorders across CGG repeats well into the full mutation range, resulting in a reliable histopathological endophenotype. These endophenotypes may influence future research directions into treatment strategies for not only Fragile X Syndrome, but also the Fragile X Premutation and Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS).",
"keywords": [
"The FMR1 gene is polymorphic for the number of CGG trinucleotide repeats on the 5´ untranslated region (5´UTR). In the general population there are fewer than 45 of these CGG trinucleotide repeats. This results in what shall be operationally referred to as normal levels of FMR1 messenger RNA (mRNA)",
"and normal levels of the FMR1 protein (FMRP)."
],
"content": "Introduction\n\nThe FMR1 gene is polymorphic for the number of CGG trinucleotide repeats on the 5´ untranslated region (5´UTR). In the general population there are fewer than 45 of these CGG trinucleotide repeats. This results in what shall be operationally referred to as normal levels of FMR1 messenger RNA (mRNA), and normal levels of the FMR1 protein (FMRP).\n\nIn the Fragile X Premutation, there are between 55–200 CGG repeats (individuals with between 45–55 repeats are referred to as carriers of Grey Zone alleles). In the Fragile X Premutation, there are 2–8 fold increases in FMR1 mRNA in peripheral leukocytes and reductions in FMRP expression levels that appear to loosely scale with the CGG trinucleotide repeat length1–6. Carriers of the Fragile X Premutation show increased frequencies of anxiety disorders, neuropsychiatric disorders, and autoimmune as well as other medical co-morbid disorders. Additionally, ~20% of female and ~45% of male Premutation carriers will develop symptoms such as cerebellar gait ataxia, postural sway, intention tremor, Parkinsonism, cognitive decline and dementia, as well as a dysexecutive syndrome during their lifetime. These symptoms have been collectively referred to as Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS). The mechanisms underlying incomplete penetrance of FXTAS in Premutation carriers is an open question currently under investigation7–22.\n\nIn the Fragile X Full Mutation, there are more than 200–230 CGG repeats (often >500 CGG repeats), and in the majority of cases the promoter region of the FMR1 gene becomes hypermethylated, and expression of the FMR1 gene is virtually silenced4,23–26. This results in a virtual absence of FMR1 mRNA and almost no measurable FMRP expression. This leads to phenotypes that include intellectual disability, macroorchidism, and autistic-like features collectively known as Fragile X Syndrome27–29.\n\nBoth the Fragile X Premutation and Full Mutation have associated mouse models that have been developed to study them. Specifically, Rob Willemsen and colleagues in Rotterdam developed a CGG knock-in (KI) mouse model (CGG KI) via homologous recombination (in this case replacing the mouse 5´UTR with a 5´UTR containing 99 CGG repeats of human origin) to model the unstable transmission of CGG repeats across generations30–35. A similar model was developed in 2007 using a CGG-CCG serial ligation method by Karen Usdin and colleagues at NIH (i.e., no human FMR1 DNA was used36). The CGG KI mouse model recapitulates the neuropathological and somatic pathological features associated with the Fragile X Premutation and FXTAS, namely eosinophilic, ubiquitin immuno-positive intranuclear inclusions bodies in neurons and astroglia in the brain, as well as affecting a range of somatic organ systems and the peripheral autonomic and enteric nervous systems. In 1994, a Dutch consortium developed a mouse model wherein the Fmr1 gene was knocked out as a model of Fragile X Syndrome (Fmr1 KO mouse)37. This mouse recapitulates a number of pathological features of the Fragile X Full Mutation, such as macroorchidism and abnormal dendritic arborization in the brain.\n\nUnfortunately, research using these mouse models of Fragile X-Associated Disorders to elucidate gross behavioral phenotypes has proven at best inconsistent. For each of the separate CGG KI mouse lines it was reported that there were very few behavioral phenotypes, and, even when present, the observed effects were rather small. For the Fmr1 KO mouse there has been a slightly greater measure of success in identifying behavioral phenotypes, but the presence and magnitude of any observed effects varies widely across labs, behavioral paradigms, and across background strains38.\n\nBased upon these discrepant and counterintuitive findings (i.e., that of inconsistent or absent phenotypes in mice that are clearly not normal), we used the CGG KI mouse developed by Willemsen and colleagues to develop a battery of behavioral tasks that could identify a neurocognitive endophenotype38–46. We felt that identifying such an endophenotype was possible because the parametric CGG repeat lengths in the CGG KI mouse gave a scalar against which behavioral performance could be associated. Later, an independent group used the paradigms we developed to evaluate behavioral endophenotypes in the Fmr1 KO mouse model47,48. This review will describe the process behind developing a behavioral endophenotyping battery as well as unpacking the resulting behavioral profiles from the experimental process and describe how they inform clinical research into Fragile X-Associated Disorders. Emerging results in the quantity and distribution of neuropathological features will be discussed in the context of human disease, although the present murine models do not entirely recapitulate these findings at present.\n\n\nEndophenotype approach\n\nThere is one clear difference between identifying a behavioral phenotype and identifying a behavioral endophenotype. This difference is that to evaluate a behavioral phenotype, the researcher need only look for a difference in behavior among a homogeneous group of mutant mice relative to a littermate or strain-matched control group. This main effect is then used as evidence for some kind of behavioral impairment. This process is akin to using the same battery of standardized neuropsychological tests to evaluate the behavioral consequences of a number of different genetic disorders and then trying to make inferences about what are the specific profiles of strengths and weaknesses unique to each disorder. In contrast, to evaluate a behavioral endophenotype in the same mice, there is a requirement that any behavioral phenotype predictably scale across some measure. Usually such factors include age, genetic dosage in situations of polymorphic mutations or chromosomal aneuploidy, or some other experimentally controlled factor that is altered parametrically (e.g., stress, environmental toxicant exposure, etc.). This process is similar to the experimental psychology or cognitive neuroscience approaches to studying the behavior of populations carrying genetic mutations. That is, an approach that emphasizes using hypothesis-driven tests that have been designed to evaluate hypothesized effects within the population being studied, irrespective to performance of other populations38,46.\n\nThe importance of finding a behavioral endophenotype is that if there is a predictable relationship among cognitive performance and gene expression, it can be assumed that the genetic mutation alters behavioral output; and subsequently, some sort of relationship between the two exists. Such a finding not only provides a wealth of information that helps the researcher design future experiments, but also data that are useful as outcome measures for studies of intervention that alter or even potentially mitigate some negative impact of the mutation. If there is a more complex relationship wherein age appears to modulate the relationship between the mutation and behavioral output, then those data serve not only as outcome measures, but if well enough understood, could be potentially useful to define risk prodromes to predict future symptomatology or disease progression.\n\nA possible reason for the lack of direct applicability of mouse model research for drug discovery is the unfortunate focus on gross phenotypes that may be either at best secondary to the mutation or result from mouse-unique factors that do not scale evolutionarily. Stated more colloquially, it is much easier to cure disease in mice than to translate the murine research into actually curing human disease. The same general paradigm is prevalent in research into sequelae resultant to neurodevelopmental/neurodegenerative genetic diseases. As a scientific community, we have been able to identify and provide cures for a wide range mouse models of genetic disorders (i.e., Fragile X-Associated Disorders), but to date these ‘cures’ have not proven particularly useful for ameliorating symptoms of human genetic disease: often failing or providing only marginal effects during early phase clinical trials38. Elucidating behavioral or neurocognitive endophenotypes using tasks designed to test specific disease-related hypotheses is one proposed solution to mitigate this lack of efficacy in the mouse model46.\n\nFor these, as well as many other reasons, research into schizophrenia has forced those in the field to change their general approach, and emphasize an endophenotyping approach in the study of prodromal states associated with schizophrenia onset and symptom progression (e.g., focusing research on longitudinal analyses of 22q11.2 deletion populations rather than on de novo schizophrenia cases of unknown or poorly understood genetic origin). By focusing on factors that scale with disease or symptom severity, researchers have been able to understand far more about schizophrenia and what may underlie symptom progression than they would otherwise have been able using a standardized, neuropsychological phenotyping approach38,46.\n\n\nNeurobehavioral endophenotype\n\nThe first step in evaluating the potential for a behavioral phenotype in CGG KI mice was to choose what cognitive domains to test. Unfortunately, at the outset there was a startling lack of data on which to base experimental hypotheses, both from human or mouse research. There were anecdotal data from patients that suggested problems with \"foggy thinking\" in Premutation carriers, which was interpreted by our clinical collaborators as a combination of abnormal hippocampal and parietal cortex function. A separate collaborator of ours that was interested in neurocognition set out to identify any potential prodromal neurocognitive deficits in Fragile X Premutation carriers, focusing primarily on spatiotemporal processing49–51. Using both of these pieces of anecdotal evidence in conjunction with the preliminary data from our collaborators as support for our hypothesis, we set out to evaluate the potential for spatial and temporal processing deficits in CGG KI mice. Similarly, we extended our analysis to visuomotor processing as motor learning and motor performance have a strong dependence on intact spatiotemporal integration40,43.\n\nMore to the point, we examined spatial and temporal processing deficits in CGG KI mice using behavioral tasks designed to test specific hypotheses about the cognitive strengths and weaknesses reported in Fragile X Premutation carriers without FXTAS. These cognitive domains were chosen because a number of neurodevelopmental disorders share fundamental spatiotemporal processing deficits described as a spatiotemporal hypergranularity52. This hypergranularity, or reduced resolution in temporal and spatial attention, impairs spatiotemporal processing that underlies memory formation. What this means is that a greater difference or separation among elements in space or time is required before they can be discriminated by the research subject.\n\nSpatial processing. Using a pair of behavioral tasks designed specifically to evaluate resolution of spatial processing in CGG KI mice (Figure 1A-B), it was demonstrated that CGG KI mice show deficits in processing the specific distances that separate two objects in space using a metric change detection task (also called a coordinate change). These deficits were present in male CGG KI mice as early as 3 months of age, and persisted until at least 12 months of age41,44.\n\nA. Metric or Coordinate change detection task. B. Topological or Categorical change detection task. C. Temporal order for visual objects task. D. Novelty detection for visual objects task. All of these paradigms rely on spontaneous exploration to guide behavior, and thus are not confounded by alterations in affect or the requirement to food deprive animals to provide motivation to perform a task to receive a food or water reward.\n\nPerformance was also tested in a topological spatial memory task that required the mice to remember the relative spatial locations of two dissimilar objects after their positions were transposed (also called a categorical change). Male CGG KI mice did not show deficits at 3 or 6 months of age, but did show deficits when they were 9 and 12 months of age compared to age-matched wildtype littermate controls (Figure 2)41,44.\n\nA. Metric/Coordinate task by repeat length and age. B. Topological/Categorical task by repeat length and age. The red line signifies no response from the animal to spatial change. Data replotted from Hunsaker et al., 2009, 201241,44.\n\nThe results from these spatial experiments suggest that: (1) the resolution of spatial processing in CGG KI mice is reduced at a very young age. This suggests the spatial resolution is due to abnormal development, as this reduced spatial resolution appears to be then be maintained over time, meaning it does not progressively worsen with aging, but does show a relationship with CGG repeat length. (2) General spatial memory as measured by the topological change detection task does show progressive worsening across age as well as showing a relationship with CGG repeat length, with deficits emerging in adults and becoming more profound with advancing age in CGG KI mice.\n\nTemporal processing. To evaluate temporal processing in CGG KI mice, a temporal ordering for visual objects task was used. In this task, mice are presented sequentially with three different pairs of identical objects, and time spent exploring the objects is recorded. The mice are then presented with one object from the first pair and one object from the third pair, and time spent exploring is again recorded. Typically, mice preferentially explore the first over the third object. At least 24 hours later to reduce mnemonic interference, after a different set of objects have been presented, the mice are presented with the first object they were presented with that day as well as a never-before-seen novel object. Mice preferentially explore the novel object over the familiar one. On the novelty detection test the CGG KI mice show more exploration of the novel object as do wildtype littermates, but unlike wildtype mice do not show an object preference in the temporal ordering test (i.e., tested with the first and third presented object). This pattern of results indicates that object recognition is intact, but the processing of temporal order is impaired (Figure 3)42,44.\n\nA. Temporal Ordering for Visual Objects Task in male mice. B. Temporal Ordering for Visual Objects Task in female mice. C. Novelty Detection for Visual Objects Task in male mice. D. Novelty Detection for Visual Objects Task in female mice. Data replotted from Hunsaker et al., 2010, 201242,44.\n\nAn important aspect to these behavioral results is that both male and female CGG KI mice showed deficits. This is important because female Premutation carriers generally show reduced disease severity due to the protective effect of a second, non-mutated FMR1 gene on the inactive X allele (i.e., reduced genetic dosage)56. The presence of temporal processing deficits in both male and female CGG KI mice suggests impairments to these processes are fundamental consequences of the Premutation, since deficits are present and identifiable even in the least affected subgroup within the population. Similar effects are currently being identified and characterized in human Premutation carriers both symptomatic and asymptomatic for motor features associated with FXTAS49–51.\n\nMotor performance. To evaluate potential subclinical gait ataxia or general clumsiness in the CGG KI mice similar to anecdotal reports from our clinical collaborators, a skilled ladder-walking task has been employed. In this test mice are allowed to walk across a series of very thin ladder rungs perpendicular to the direction of travel (similar to walking across a ladder set on the ground). The number of times that the mouse makes an error in foot placement is recorded and is operationally defined as a foot slip. To perform these tasks, the mouse is placed at one end of the apparatus and allowed to cross from one end to the other into a darkened box43.\n\nThe apparatus used in these experiments was modified from previously described ladder rung tasks70–72. The apparatus had black plastic walls separated by approximately 5cm, with 2mm diameter rungs making up the floor. For this initial study, the mice were placed at one end of the apparatus and were allowed to walk back and forth for 2 minutes. The number of foot slips was recorded for the 2 min duration, except when the mouse was turning around. The number of times the mouse traveled from one end of the apparatus to the other was also recorded as a general locomotor measure. On this task, CGG KI mice as young as 2 months of age already showed an increased number of foot slips compared to wildtype litter mate controls (Figure 4)43. Importantly, the CGG KI mice showed more of both forelimb and hindlimb slips than wildtype mice, suggesting both visuospatial and basic motor deficits. That is, forelimb foot slips may suggest visuospatial processing deficits, such as difficulty in planning forepaw placement as well as difficulty updating movements as the step progressed (i.e., as the mouse moved forward the initial planned step has to be modified subtly and an inability to do so results in a foot slip). Hind foot slips however, have less of a visuospatial planning component, but may reflect a subtle motor effect. This is because stepping with the hind limbs has been shown to be more efficient and easier for the mouse to perform cf.43. Difficulty in hind limb stepping may therefore reflect some form of ataxia, and may underlie impairments in rotarod and stationary beam walking reported previously73. Similar to the metric or coordinate task data described above, the lack of an age effect suggests that these motor features are due to abnormal development, not an age-related progressive impairment.\n\nThere were no effects of age present in the data. Data replotted from Hunsaker et al., 201143.\n\nBehavioral analysis. After the initial development of the neurocognitive endphenotype described above in the CGG KI mouse model of the Fragile X Premutation, it became clear that these experiments were begging replication in the Fmr1 KO mouse model of Fragile X Syndrome. We posited two possible outcomes for such a replication and extension study, the first being that the CGG KI mouse and the Fmr1 KO mouse would show completely independent and non-overlapping deficits wherein CGG repeat dosage would be non-predictive of cognitive deficits. The second suggestion was that the CGG KI mouse and the Fmr1 KO mouse may show a predictable relationship with increasing CGG repeat length in CGG KI mice and Fmr1 KO mice showing a linear or otherwise parametric association with each other.\n\nTo test these possibilities, we combined our published CGG KI results44 with the data generated by other labs47,48 wherein they used the battery described above in Figure 1 to evaluate the cognitive deficits in the Fmr1 KO mouse as part of interventional treatment studies. Overall, the data support both of the proposed outcomes. Figure 5 demonstrates that the CG KI mice and Fmr1 KO mice show a relatively linear progression in deficits as a function of CGG repeat length toward the Fragile X Full Mutation range for spatial and temporal processing. However, the data also suggest that the Fmr1 KO mouse demonstrates an unpredicted general memory deficit in the novel object detection task that was not shared with the CGG KI mouse models.\n\nA. Metric or Coordinate task. B. Topological or Categorical task. C. Temporal Ordering for Visual Objects task. D. Novelty Detection for Visual Objects task. Data from Hunsaker et al., 2012; King and Jope, 2013; and Franklin et al., 201344,47,48.\n\nUsing entirely different groups of mice, a group in Rotterdam developed an automated version of the ladder rung task called the Erasmus ladder, upon which the testing and collection of data are fully automated53–55. Similar to the ladder rung apparatus described above, the Erasmus ladder has a series of horizontal ladder-steps that the mouse must traverse in order to enter an escape/start box at the end. The apparatus was designed to use puffs of air to induce the mouse to shuttle between the two boxes by crossing the ladder steps. The system automatically captures step times, foot slips, misplacement of limbs, etc. Cupido53 found that CGG KI mice showed a significantly increased number of foot slips and missteps (Figure 6), although they did not differ from wildtype mice in step time, suggesting intact general motor function. Additionally, the CGG KI mice were able to perform the motor associative learning task as well as wild type litter mate control animals. In contrast, Fmr1 KO mice showed a deficit for the associative learning task, never performing as well as CGG KI and wildtype control animals; however, the number of footslips and missteps did not differ from wildtype controls, suggesting intact visuospatial, visuomotor, and basic motor functions.\n\nNote the dissociation between motor endophenotypes in the CGG KI and Fmr1 KO Mice. All CGG KI mice were from the High CGG repeat group in other figures.\n\nIt has been demonstrated previously that visuomotor function depends on spatiotemporal integration40,43. This means that for an individual to act in space, he must be able to (1) select a goal of action and (2) plan the movements, (3) initiate the movements, (4) modify the ongoing movement using updated spatiotemporal information, and (5) properly terminate the movement as intended. Intact spatiotemporal updating prevents misguided movements, such as reaching for a cup and knocking it off the table because an inability to stop or slow the progress of the hand. This is increasingly important if reaching or acting in space extends to processing angles among stimuli or targets that change over time. This updating process is what may be deficient in Fragile X Premutation carriers and in FXTAS patients, and in the CGG KI mice as they show similar spatiotemporal processing deficits and thus reduced spatial and temporal resolution. This type of spatiotemporal deficit may underlie anecdotal reports of subclinical apraxia and general clumsiness among Premutation carriers.\n\nSimilar to the novel object detection task, it was clear from the data that the Fmr1 KO mice demonstrate a deficit in motor learning that was not present in the CGG KI mouse model. The clear dissociation between motor performance (or visuospatial function) deficits in the CGG KI mouse in the absence of motor learning deficits and the inverse case in the Fmr1 KO mice provides a unique opportunity to evaluate separate components of the motor system in these models. Such data are critical to inform research into the differences between motor phenotypes in Fragile X Premutation carriers with FXTAS and Full Mutation carriers with Fragile X Syndrome.\n\nAnalytical verification. As previously demonstrated44, an unsupervised hierarchal clustering algorithm can correctly sort the CGG KI mouse performance on a number of behavioral tasks into appropriate repeat-length subgroups. To verify that these observations comprised a behavioral endophenotype, a similar clustering analysis was performed on a dataset consisting of the CGG KI mice44 and the Fmr1 KO mice47,48. Only the metric or coordinate task, the topological or categorical task, the temporal ordering and novelty detection are concluded in this analysis as the number of animals in each group were insufficient in the other experiments reported in the review to run this analysis. In all cases, similar to Hunsaker et al. (2012)44, support vector machines (SVM), a supervised machine learning technique, were performed to determine whether patterns in the behavioral data could be interpreted as indications of the expansion of CGG repeat length into the Fragile X Full Mutation. To assess the performance of the SVM classifier, iterative k-fold cross validation (10-fold, 5-fold, 3-fold, and leave-one-out cross validation) methods were used to estimate the accuracy of the classifier that predicts the group classification of a test sample. In the figures, the wildtype mice were removed from the dataset and the analyses were repeated to verify that CGG KI and Fmr1 KO mice could be correctly classified by CGG repeat length (i.e., Low CGG repeat vs. High CGG repeat vs. Fmr1 KO).\n\nFigure 7 is a heatmap representation with all the behavioral tasks included and Figure 8 is a similar heatmap with the novel object detection task removed to evaluate how well the algorithm could sort the Fmr1 KO mice and CGG KI mice without the benefit of the novel object detection task.\n\nNote how the algorithm was capable of separating groups with high levels of accuracy with only a single Fmr1 KO mouse and one High CGG KI mouse being misclassified. The colors along the right hand column of the heatmap correspond to the group colors used in Figure 5.\n\nNote how the algorithm was capable of separating groups with high levels of accuracy, with only a single high CGG KI mouse being misclassified. The colors along the right hand column of the heatmap correspond to the group colors used in Figure 5.\n\n\nEmerging histopathological endophenotype\n\nAlso emerging from studies evaluating histopathological correlates of the different mutations on the FMR1 gene are reports that pathologic features are related to CGG repeat length, albeit in a decidedly nonlinear fashion. Table 1 demonstrates this complicated relationship in human Fragile X Premutation and Full Mutation carriers.\n\nData from Greco et al., 200662; Hunsaker et al., 201143; Pretto et al., 201364; and Tassone et al., 201220.\n\nHuman Premutation carriers. Initially, because of the presumed protective effect of a second FMR1 gene on a non-mutated X chromosome, it was assumed that female Fragile X Premutation carriers would not show similar levels of histopathological features (e.g., intranuclear inclusions) as male Premutation carriers56.\n\nIn 2012, a study was undertaken to specifically determine if this assumption was correct20. Specifically, the same methods as employed earlier57–59 were replicated using sections from female post mortem tissues from Fragile X Premutation carriers. The most intriguing result from this study was that the female Premutation carriers did not in fact have extremely low numbers of intranuclear inclusions in neurons.\n\nThe number of intranuclear inclusions in neurons was virtually identical to that observed in male Premutation carrier cases. However, these female cases demonstrated a clear reduction in the number of intranuclear inclusions in astroglia. This reduction, furthermore, seemed to be inversely related to activation ratio, or directly related to the proportion of premutation FMR1 expressed in brain. This overall effect does suggest an overall reduction in histopathological features in female compared to male Premutation carriers, but not a clear cut one20.\n\nAn additional datum of interest from this study was that one of the female Premutation carrier cases never developed motor features congruent with a FXTAS diagnosis. In fact, this case was included initially in the sample to demonstrate that the presence of intranuclear inclusions is associated with FXTAS. In other words, it was hypothesized that this case would be either free of inclusions or else have at best very low numbers of these pathologic anatomical features. This case also had a very high activation ratio, meaning the mutated FMR1 gene was only expressed in 20% of their cells. Despite these presumed protective factors and lack of motor phenotypes during life, this case showed the highest number of intranuclear inclusions in the study-suggesting that the inclusion bodies appear to be related to the Fragile X Premutation itself, not to the clinical manifestations of genetic disease (e.g., FXTAS).\n\nCGG KI mouse model. In the CGG KI mouse model a parallel experiment was performed with the female Premutation carrier study described above. What was observed was striking: there was a 20–30% reduction in the number of inclusion bodies in neurons of female CGG KI mice when compared to male CGG KI mice with the same CGG repeat length. For this pathology in astroglia, the female CGG KI mice showed a clear and reliable 50% reduction in intranuclear inclusions compared to the male CGG KI mice with the same CGG repeat length60,61.\n\nThese findings suggest there is a clear dosage effect, at least in mice, for the presence of intranuclear inclusions in CGG KI mice. These data strongly suggest that Fmr1 expression levels play an as yet undefined role in inclusion body formation.\n\nHuman Premutation and Full Mutation carriers. In 2011, it was discovered that Fragile X Full Mutation carriers counter intuitively show the hallmark neuropathological feature of The Fragile X Premutation and FXTAS: eosinophilic intranuclear inclusions that stain positive for ubiquitin62,63. To identify how this was possible, given that earlier analyses had suggested that these cases lacked FMR1 mRNA and FMRP expression, further, more sensitive, molecular analyses were performed. These analyses discovered that FMR1 mRNA and FMRP were in fact expressed, just at very low levels that were missed by previous techniques62,63. All said and done, these Fragile X Full Mutation carriers showed approximately 1–2% of the total number of inclusions compared to Fragile X Premutation carriers analyzed in parallel. However, the number of inclusion bodies identified did seem to scale with the rather low expression levels of FMR1 mRNA and FMRP.\n\nSubsequent studies have borne this hypothesis out by evaluating neuropathologic anatomical feature in individuals that have a size mosaic for the Fragile X Premutation and Full Mutation (i.e., they express alleles with Premutation and alleles with Full Mutation length mutations). In one particular case, the expression of histopathological features again scaled with the expression levels of FMR1 mRNA and FMRP in brain. Interestingly, this patient had been diagnosed with Fragile X Syndrome and Parkinsonism during their lifetime, but upon the identification of these neuropathologic features and a re-evaluation of the medical records suggested that this individual had both Fragile X Syndrome as well as FXTAS, despite the fact that this comorbidity had previously been discounted as theoretically impossible64.\n\nCGG KI mouse model. In 2012 it was determined that there was a somewhat linear relationship between CGG repeat length and the quantity of intranuclear inclusions in a number of brain areas in the CGG KI mouse60. The similarity with the human cases carrying the Fragile X Full Mutation described above is that mice carrying a mutation upwards of 350 CGG repeats in length were evaluated. Similar to the cases described above, as CGG KI mice show CGG repeat lengths beyond 230 repeats in length, the number of intranuclear inclusions reduces dramatically. This parallels research done in the same mouse line in Rotterdam73 that suggested Fmr1 mRNA levels and FMRP levels begin to reduce as they approach 200+ CGG repeats.\n\nUnfortunately to date, no matter how long the CGG repeat length becomes in CGG KI mice, the gene does not hypermethylate and silence in the same way as the human FMR1 gene. This means that at present there is no clear and unambiguous comparison between human and mouse Full Mutation alleles, so far as the analysis of histopathological features associated with disease states is concerned.\n\n\nConclusions\n\nOverall, there are two clear conclusions we can draw from the data described in this review. The first is that the behavioral phenotypes of CGG KI mice and Fmr1 KO mice appear to be related in some way. This suggests that there is a behavioral spectrum upon which these two mouse models can be found38. Intriguingly, early models positing a spatial and temporal processing deficit model for Fragile X Syndrome and the Fragile X Premutation now have direct experimental support65. We confirmed this by demonstrating that an unsupervised classifier is capable of using the behavioral data from this rapid battery to identify and sort the CGG KI mice into repeat length groups that are distinct from Fmr1 KO mice, which are reliably sorted as well. These behavioral and analytical results suggest that spatiotemporal processing may be an analytically powerful outcome measure that can be used as an endpoint for interventional, therapeutic, or diagnostic experiments.\n\nHowever, there is also a clear additional behavioral deficit associated with having the Fragile X Full Mutation that is not present in the Fragile X Premutation. This was best observed in the novel object detection task, wherein the CGG KI mice displayed no deficits whatsoever, whereas the Fmr1 KO mice showed a rather profound inability to perform the task. The same type of dissociation exists within the motor domain. CGG KI mice appear to show motor performance deficits, possible from impaired visuomotor function but intact motor learning, whereas the Fmr1 KO mouse shows intact motor function, but impaired motor associative learning. Although these data appear incongruous, the mouse models do actually appropriately model the motor phenotypes associated in the Fragile X-Associated Disorders human population being modeled.\n\nTogether the behavioral results reviewed above demonstrating spatiotemporal and motor phenotypes in CGG KI and Fmr1 KO mice results suggest that the CGG KI and Fmr1 KO mouse models are valid models for evaluating any behavioral effects of the Fragile X Premutation and Full Mutation. This validity stems from the fact that populations of female Premutation carriers demonstrate similar deficits on spatiotemporal processing tasks as the CGG KI mice and Full Mutation carriers demonstrate clear spatiotemporal deficits on standardized testing. Similarly, motor features in female Fragile X Premutation carriers without FXTAS related motor features have been described. Additionally, research involving Premutation carriers are identifying associations between behavioral performance and CGG repeat length, FMR1 mRNA expression levels, and FMRP expression levels; further supporting the use of the associated mouse models for preclinical experimentation.\n\nThe second main conclusion is that the neuropathological features, although complicated, may also be post-mortem endophenotypes of the Fragile X Premutation and Full Mutation. In humans, it appears that there is a complicated but somewhat predictable relationship between expression of the FMR1 gene and the presence of intranuclear inclusions, at least within the range of the Fragile X Premutation. In the Full Mutation, it still appears that there is a relationship between FMR1 expression and pathological features, but this is complicated by the extremely low expression levels and similarly low numbers of intranuclear inclusion bodies20,57–64,66–69.\n\nIn evaluating female and male Fragile X Premutation carriers, it becomes clear that there is an association between gene expression and histopathology-albeit again a complicated one. These results beg for a thorough analysis of cell-type specific FMR1 expression patterns and cell-type specific analyses of pathology.\n\nIntriguingly, although an incomplete model lacking the Fmr1 promoter hypermethylation observed in human Fragile X Full Mutation carriers, the CGG KI mouse appears to recapitulate the results of studies carried out with human tissue. Since the CGG KI mouse model appears to model the histopathological features, it is a fair assumption that the CGG KI mouse should be useful to elucidate the process by which these intranuclear inclusions form in different cell types and potentially preclinically test potential therapies or interventions designed to modulate or manipulate the development of this pathology.\n\nDespite the fact that the CGG KI mouse appears to model the Fragile X Premutation relatively well, there are commonly overlooked weaknesses in the mice used to model the Fragile X Full Mutation that need to be overcome. The first of these difficulties is that at present there are not any good mouse models for FMR1 mutation mosaicism for either size or methylation status. This is important if we are to use the mouse model to adequately model the human condition, given that with increasing study, the genetics underlying Fragile X-Associated Disorders are revealed as more complicated than originally assumed. Additionally, none of the CGG KI mouse models show any evidence for Fmr1 promoter silencing, so none of the mice show the dramatic reduction in gene expression at the 200–230 CGG repeat threshold often described in human studies.\n\nAlthough demonstrating similar pathology such as macroorchidism and altered dendritic development, the Fmr1 KO mouse model does not actually model the genetics of the Fragile X Full Mutation. The Fmr1 KO mouse was developed by inserting a NEO cassette into the first exon of the Fmr1 gene, thus silencing expression. However, although apparently a knockout, it was shown that this original Fmr1 KO mouse does express Fmr1 mRNA and FMRP, albeit nonfunctional and rapidly degraded. This is still the mouse the majority of researchers employ in their research. To eliminate this confound, a second generation of the Fmr1 KO mouse removed the promoter region entirely, thus completely silencing Fmr1 gene expression74. The molecular results of these models are very different from hypermethylation virtually, but not absolutely, silencing transcription with the remaining mRNA and protein being normal in structure and completely functional. To fully model the Fragile X Full Mutation at a molecular level, it is necessary for models that show human-like methylation and expression patterns be developed.\n\nMoving forward, the mouse models associated with Fragile X-Associated Disorders (Fragile X Premutation and Full Mutation) will be an essential tool for informing human disease research as well as for preclinical interventional studies. This utility, however, will only occur so long as care is taken to specifically focus on the similarities with the human populations being studied.\n\nFor studying the development of neuropathological features, these mouse models may serve as testing platforms for early interventional studies. For the Fragile X Premutation, the CGG KI mouse can be used to determine if there is a critical point in development when an intervention may stave off the development of neuropathological features and potential neurodegeneration (i.e., progression toward a neurodegenerative course associated with FXTAS). Similarly, the Fmr1 KO mouse is already in use to determine if early intervention can reliably normalize development and dendritic architecture later in life in the absence of chronic treatment, thus improving outcome.\n\nThe CGG KI mouse model may further be useful to determine what potential factors contribute to the incomplete penetrance of FXTAS. In other words, one can test environmental toxicant exposure, dietary factors, rearing conditions, anxiety, stress, depression, or any number of factors or combinations of factors to determine if there is an environment × gene dosage × age interaction that underlies abnormal neurodevelopment and eventual neurodegeneration. Studies evaluating potential immune mediated pathology in the Fragile X Premutation are already being evaluated75, particularly for any role of pro inflammatory cytokine levels in long term outcome.\n\nFinally, from a purely behavioral standpoint, it is critical that task development continue for these mouse models that explicitly parallel the tasks being developed for humans and the cognitive and functional domains being emphasized in humans. The current paradigm of murine researchers testing compounds with a focus on prepulse inhibition and the water maze is outdated, not translatable to the clinic in any way, and only serves to stagnate the field. There are currently already a number of resources that provide a solution in the form of behavioral assays that can be creatively applied to model human neuropsychological task batteries ranging from those used currently to test Fragile X-Associated Disorders, the 22q11.2 Deletion Syndrome, and even the NIH Toolbox cognitive assessment tools (i.e., http://www.nihtoolbox.org). Only by thoroughly modeling the cognitive assays being used can any data using these models be clearly and validly interpreted38,45,46.\n\nWith any luck, research using the CGG KI and Fmr1 KO mouse models of Fragile X-Associated Disorders will be able to provide critical information to researchers that can be readily translated to clinical applications. The need for rigorous study of these models is evidenced by the increasing frequency of small pharmaceutical companies developing compounds to treat Fragile X-Associated Disorders. However, to date these efforts have been less than successful in identifying clear hypotheses from the mouse models that can be easily translated into the clinic, much less into clinical trials.\n\nResearch into the CGG KI and Fmr1 KO mouse models and into Fragile X-Associated Disorders are at an impasse that only direct basic-clinical science translational research can overcome. It is only through clear communication across levels of science that the basic scientist and clinician can truly work together toward their common goals. It is only through these reciprocal interactions that innovation truly germinates, and the opportune moment to truly begin is now.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe author wishes to acknowledge Dr. Robert F. Berman, Ph.D., Dr. Richard S. Jope, Ph.D., and Dr. Margaret King, Ph.D. for providing data used in the present review.\n\n\nReferences\n\nTassone F, Hagerman RJ, Gane LW, et al.: Strong similarities of the FMR1 mutation in multiple tissues: postmortem studies of a male with a full mutation and a male carrier of a premutation. Am J Med Genet. 1999; 84(3): 240–244. PubMed Abstract | Publisher Full Text\n\nTassone F, Hagerman RJ, Taylor AK, et al.: Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome. Am J Hum Genet. 2000; 66(1): 6–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTassone F, Hagerman RJ, Chamberlain WD, et al.: Transcription of the FMR1 gene in individuals with fragile X syndrome. Am J Med Genet. 2000; 97(3): 195–203. PubMed Abstract | Publisher Full Text\n\nTassone F, Hagerman PJ: Expression of the FMR1 gene. Cytogenet Genome Res. 2003; 100(1–4): 124–128. PubMed Abstract | Publisher Full Text\n\nTassone F, Beilina A, Carosi C, et al.: Elevated FMR1 mRNA in premutation carriers is due to increased transcription. RNA. 2007; 13(4): 555–562. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTassone F, Pan R, Amiri K, et al.: A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations. J Mol Diagn. 2008; 10(1): 43–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHagerman RJ, Leehey M, Heinrichs W, et al.: Intention tremor, parkinsonism, and generalized brain atrophy in male carriers of fragile X. Neurology. 2001; 57(1): 127–130. PubMed Abstract | Publisher Full Text\n\nHagerman RJ, Leavitt BR, Farzin F, et al.: Fragile-X-associated tremor/ataxia syndrome (FXTAS) in females with the FMR1 premutation. Am J Hum Genet. 2004; 74(5): 1051–1056. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJacquemont S, Orrico A, Galli L, et al.: Spastic paraparesis, cerebellar ataxia, and intention tremor: a severe variant of FXTAS? J Med Genet. 2005; 42(2): e14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeehey MA, Munhoz RP, Lang AE, et al.: The fragile X premutation presenting as essential tremor. Arch Neurol. 2003; 60(1): 117–121. PubMed Abstract | Publisher Full Text\n\nLeehey MA, Legg W, Tassone F, et al.: Fibromyalgia in fragile X mental retardation 1 gene premutation carriers. Rheumatology (Oxford). 2011; 50(12): 2233–2236. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeehey MA, Hagerman PJ: Fragile X-associated tremor/ataxia syndrome. Handb Clin Neurol. 2012; 103: 373–386. PubMed Abstract | Publisher Full Text\n\nMoore CJ, Daly EM, Schmitz N, et al.: A neuropsychological investigation of male premutation carriers of fragile X syndrome. Neuropsychologia. 2004; 42(14): 1934–1947. PubMed Abstract | Publisher Full Text\n\nOlichney JM, Chan S, Wong LM, et al.: Abnormal N400 word repetition effects in fragile X-associated tremor/ataxia syndrome. Brain. 2010; 133(pt 5): 1438–1450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaul R, Pessah IN, Gane L, et al.: Early onset of neurological symptoms in fragile X premutation carriers exposed to neurotoxins. Neurotoxicology. 2010; 31(4): 399–402. PubMed Abstract | Publisher Full Text\n\nSchneider A, Ballinger E, Chavez A, et al.: Prepulse inhibition in patients with fragile X-associated tremor ataxia syndrome. Neurobiol Aging. 2012; 33(6): 1045–1053. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeritan AL, Nguyen DV, Farias ST, et al.: Dementia in fragile X-associated tremor/ataxia syndrome (FXTAS): comparison with Alzheimer’s disease. Am J Med Genet B Neuropsychiatr Genet. 2008; 147B(7): 1138–1144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeritan AL, Schneider A, Olichney JM, et al.: Conversion disorder in women with the FMR1 premutation. Am J Med Genet A. 2009; 149A(11): 2501–2506. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSoontarapornchai K, Maselli R, Fenton-Farrell G, et al.: Abnormal nerve conduction features in fragile X premutation carriers. Arch Neurol. 2008; 65(4): 495–498. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTassone F, Greco CM, Hunsaker MR, et al.: Neuropathological, clinical and molecular pathology in female fragile X premutation carriers with and without FXTAS. Genes Brain Behav. 2012; 11(5): 577–585. PubMed Abstract | Publisher Full Text\n\nWirojanan J, Jacquemont S, Diaz R, et al.: The efficacy of melatonin for sleep problems in children with autism, fragile X syndrome, or autism and fragile X syndrome. J Clin Sleep Med. 2009; 5(2): 145–150. PubMed Abstract | Free Full Text\n\nZhang L, Coffey S, Lua LL, et al.: FMR1 premutation in females diagnosed with multiple sclerosis. J Neurol Neurosurg Psychiatry. 2009; 80(7): 812–814. PubMed Abstract | Publisher Full Text\n\nTassone F, Hagerman RJ, Iklé DN, et al.: FMRP expression as a potential prognostic indicator in fragile X syndrome. Am J Med Genet 1999; 84(3): 250–261. PubMed Abstract | Publisher Full Text\n\nTassone F, Hagerman RJ, Loesch DZ, et al.: Fragile X males with unmethylated, full mutation trinucleotide repeat expansions have elevated levels of FMR1 messenger RNA. Am J Med Genet. 2000; 94(3): 232–236. PubMed Abstract | Publisher Full Text\n\nTassone F, Hagerman RJ, Taylor AK, et al.: A majority of fragile X males with methylated, full mutation alleles have significant levels of FMR1 messenger RNA. J Med Genet. 2001; 38(7): 453–456. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTassone F, Hagerman PJ: in The Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) (eds Tassone, F. & Berry-Kravis, E. M.) 67–76 (Springer, New York, 2010).\n\nHagerman PJ, Hagerman RJ: The fragile-X premutation: a maturing perspective. Am J Hum Genet. 2004; 74(5), 805–816. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHagerman PJ: The fragile X prevalence paradox. J Med Genet. 2008; 45(8): 498–499. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHagerman R, Hoem G, Hagerman P: Fragile X and autism: Intertwined at the molecular level leading to targeted treatments. Mol Autism. 2010; 1(1): 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBontekoe CJ, de Graaff E, Nieuwenhuizen IM, et al.: FMR1 premutation allele (CGG)81 is stable in mice. Eur J Hum Genet. 1997; 5(5): 293–298. PubMed Abstract\n\nBrouwer JR, Mientjes EJ, Bakker CE, et al.: Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation. Exp Cell Res. 2007; 313(2): 244–253. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrouwer JR, Severijnen E, de Jong FH, et al.: Altered hypothalamus-pituitary-adrenal gland axis regulation in the expanded CGG-repeat mouse model for fragile X-associated tremor/ataxia syndrome. Psychoneuroendocrinology. 2008; 33(6): 863–873. PubMed Abstract | Publisher Full Text\n\nBrouwer JR, Huizer K, Severijnen LA, et al.: CGG-repeat length and neuropathological and molecular correlates in a mouse model for fragile X-associated tremor/ataxia syndrome. J Neurochem. 2008; 107(6): 1671–1682. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWillemsen R, Hoogeveen-Westerveld M, Reis S, et al.: The FMR1 CGG repeat mouse displays ubiquitin-positive intranuclear neuronal inclusions; implications for the cerebellar tremor/ataxia syndrome. Hum Mol Genet. 2003; 12(9): 949–959. PubMed Abstract | Publisher Full Text\n\nWillemsen R, et al.: in The Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) (eds Tassone, F. & Berry-Kravis, E. M.) 67–76 (Springer, New York, 2010).\n\nEntezam A, Biacsi R, Orrison B, et al.: Regional FMRP deficits and large repeat expansions into the full mutation range in a new Fragile X premutation mouse model. Gene. 2007; 395(1-2): 125–134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFmr1 knockout mice: a model to study fragile X mental retardation. The Dutch-Belgian Fragile X Consortium. Cell. 1994; 78(1): 23–33. PubMed Abstract | Publisher Full Text\n\nHunsaker MR: Comprehensive neurocognitive endophenotyping strategies for mouse models of genetic disorders. Prog Neurobiol. 2012; 96(2): 220–241. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorthwell RM, Hunsaker MR, Willemsen R, et al.: Spatiotemporal processing deficits in female CGG KI mice modeling the fragile X premutation. Behav Brain Res. 2012; 233(1): 29–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDiep AA, Hunsaker MR, Kwock R, et al.: Female CGG knock-in mice modeling the fragile X premutation are impaired on a skilled forelimb reaching task. Neurobiol Learn Mem. 2012; 97(2): 229–234. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunsaker MR, Wenzel HJ, Willemsen R, et al.: Progressive spatial processing deficits in a mouse model of the fragile X premutation. Behav Neurosci. 2009; 123(6): 1315–1324. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunsaker MR, Goodrich-Hunsaker NJ, Willemsen R, et al.: Temporal ordering deficits in female CGG KI mice heterozygous for the fragile X premutation. Behav Brain Res. 2010; 213(2): 263–268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunsaker MR, von Leden RE, Ta BT, et al.: Motor deficits on a ladder rung task in male and female adolescent and adult CGG knock-in mice. Behav Brain Res. 2011; 222(1): 117–121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunsaker MR, Kim K, Willemsen R, et al.: CGG trinucleotide repeat length modulates neural plasticity and spatiotemporal processing in a mouse model of the fragile X premutation. Hippocampus. 2012; 22(12): 2260–2275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunsaker MR, Arque G, Berman RF, et al.: Mouse models of the fragile x premutation and the fragile X associated tremor/ataxia syndrome. Results Probl Cell Differ. 2012; 54: 255–269. PubMed Abstract | Publisher Full Text\n\nHunsaker MR: The importance of considering all attributes of memory in behavioral endophenotyping of mouse models of genetic disease. Behav Neurosci. 2012; 126(3): 371–380. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranklin AV, King MK, Palomo V, et al.: Glycogen Synthase Kinase-3 Inhibitors Reverse Deficits in Long-term Potentiation and Cognition in Fragile X Mice. Biol Psychiatry. 2013. PubMed Abstract | Publisher Full Text\n\nKing MK, Jope RS: Lithium treatment alleviates impaired cognition in a mouse model of fragile X syndrome. Genes Brain Behav. 2013; 12(7): 723–731. PubMed Abstract | Publisher Full Text\n\nGoodrich-Hunsaker NJ, Wong LM, McLennan Y, et al.: Adult Female Fragile X Premutation Carriers Exhibit Age- and CGG Repeat Length-Related Impairments on an Attentionally Based Enumeration Task. Front Hum Neurosci. 2011; 5: 63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodrich-Hunsaker NJ, Wong LM, McLennan Y, et al.: Enhanced manual and oral motor reaction time in young adult female fragile X premutation carriers. J Int Neuropsychol Soc. 2011; 17(4): 746–750. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodrich-Hunsaker NJ, Wong LM, McLennan Y, et al.: Young adult female fragile X premutation carriers show age- and genetically-modulated cognitive impairments. Brain Cogn. 2011; 75(3): 255–260. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimon TJ: A new account of the neurocognitive foundations of impairments in space, time and number processing in children with chromosome 22q11.2 deletion syndrome. Dev Disabil Res Rev. 2011; 14(1): 52–58 X. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCupido A: Detecting Cerebellar Phenotypes with the Erasmus Ladder. Erasmus MC. Dept of Clin Genet Ph.D. 2009; 98. Reference Source\n\nVan Der Giessen RS, Koekkoek SK, van Dorp S, et al.: Role of olivary electrical coupling in cerebellar motor learning. Neuron. 2008; 58(4): 599–612. PubMed Abstract | Publisher Full Text\n\nSaab AS, Neumeyer A, Jahn HM, et al.: Bergmann glial AMPA receptors are required for fine motor coordination. Science. 2012; 337(6095): 749–53. PubMed Abstract | Publisher Full Text\n\nBerry-Kravis E, Potanos K, Weinberg D, et al.: Fragile X-associated tremor/ataxia syndrome in sisters related to X-inactivation. Ann Neurol. 2005; 57(1): 144–147. PubMed Abstract | Publisher Full Text\n\nGreco CM, Hagerman RJ, Tassone F, et al.: Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers. Brain. 2002; 125(pt 8): 1760–1771. PubMed Abstract | Publisher Full Text\n\nGreco CM, Berman RF, Martin RM, et al.: Neuropathology of fragile X-associated tremor/ataxia syndrome (FXTAS). Brain. 2006; 129(pt 1): 243–255. PubMed Abstract | Publisher Full Text\n\nGreco CM, Hunsaker, MR, Berman RF: The Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) (eds Tassone, F. & Berry-Kravis, E. M.) 67–76 (Springer, New York, 2010). Publisher Full Text\n\nSchluter EW, Hunsaker MR, Greco CM, et al.: Distribution and frequency of intranuclear inclusions in female CGG KI mice modeling the fragile X permutation. Brain Res. 2012; 1472: 124–137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWenzel HJ, Hunsaker MR, Greco CM, et al.: Ubiquitin-positive intranuclear inclusions in neuronal and glial cells in a mouse model of the fragile X premutation. Brain Res. 2010; 1318: 155–166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreco CM, Navarro CS, Hunsaker MR, et al.: Neuropathologic features in the hippocampus and cerebellum of three older men with fragile X syndrome. Mol Autism. 2011; 2(1): 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunsaker MR, Greco CM, Tassone F, et al.: Rare intranuclear inclusions in the brains of 3 older adult males with fragile x syndrome: implications for the spectrum of fragile x-associated disorders. J Neuropathol Exp Neurol. 2011; 70(6): 462–469. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPretto DI, Hunsaker MR, Cunningham CL, et al.: Intranuclear inclusions in a fragile X mosaic male. Transl Neurodegener. 2013; 2(1): 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson-Glenberg MC: Fragile X syndrome: Neural network models of sequencing and memory. Cogn Syst Res. 2008; 9(4): 274–292. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreco CM, Soontrapornchai K, Wirojanan J, et al.: Testicular and pituitary inclusion formation in fragile X associated tremor/ataxia syndrome. J Urol. 2007; 177(4): 1434–1437. PubMed Abstract | Publisher Full Text\n\nGreco CM, Tassone F, Garcia-Arocena D, et al.: Clinical and neuropathologic findings in a woman with the FMR1 premutation and multiple sclerosis. Arch Neurol. 2008; 65(8): 1114–1116. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHunsaker MR, Greco CM, Spath MA, et al.: Widespread non-central nervous system organ pathology in fragile X premutation carriers with fragile X-associated tremor/ataxia syndrome and CGG knock-in mice. Acta Neuropathol. 2011; 122(4): 467–479. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTassone F, Hagerman RJ, Garcia-Arocena D, et al.: Intranuclear inclusions in neural cells with premutation alleles in fragile X associated tremor/ataxia syndrome. J Med Genet. 2004; 41(4): e43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhishaw IQ, Travis SG, Koppe SW, et al.: Handshaping in the rat: conserved release and collection vs. flexible manipulation inoverground walking, ladder rung walking, cylinder exploration, and skilledreaching. Behav Brain Res. 2010; 206(1): 21–31. PubMed Abstract | Publisher Full Text\n\nMetz GA, Whishaw IQ: The ladder rung walking task: a scoring system and itspractical application. J Vis Exp. 2009; (28): pii: 1204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrouwer JR, Huizer K, Severijnen LA, et al.: CGG-repeat length and neuropathological and molecular correlates in a mouse model for fragile X-associated tremor/ataxia syndrome. J Neurochem. 2008; 107(6): 1671–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Dam D, Errijgers V, Kooy RF, et al.: Cognitive decline, neuromotor and behavioural disturbances in a mouse modelfor fragile-X-associated tremor/ataxia syndrome (FXTAS). Behav Brain Res. 2005; 162(2): 233–9. PubMed Abstract | Publisher Full Text\n\nMientjes EJ, Nieuwenhuizen I, Kirkpatrick L, et al.: The generation of a conditional Fmr1 knock out mouse model to study Fmrp function in vivo. Neurobiol Dis. 2006; 21(3): 549–55. PubMed Abstract | Publisher Full Text\n\nWinarni TI, Chonchaiya W, Sumekar TA, et al.: Immune-mediated disorders among women carriers of fragile X premutation alleles. Am J Med Genet A. 2012; 158A(10): 2473–81. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3384",
"date": "11 Feb 2014",
"name": "Peter Todd",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Michael Hunsaker covers a large body of literature to which he has significantly contributed related to behavioral assay development in Fragile X spectrum disorder mouse models. The author includes data from several different published reports to analyze and compare behavioral results in CGG KI premutation model mice to similar tests done in Fmr1 KO animals. Interestingly, some though not all of these data follow a trajectory related to CGG repeat length. The author goes on to comment on data comparing inclusion formation in human and mouse models, and address differences between male and female premutation patients and models. It also addresses shortcomings in the current mouse models of Fragile X spectrum disorders. The central conclusion of the paper is that the field needs to work towards development of more patient-applicable endophenotypes to allow for successful translation of basic research discoveries to successful therapeutic development. Overall, the article is well written and the title and abstract are appropriate. Below are a few comments that if addressed would enhance this review and make it more appropriate for indexing. Battery development and implementation: Spatial processing and Figure 1. More explanation of these behavioral tasks would aid reader comprehension. Figure 1 was included to help explain how these tests are performed, but as data are included in the review, it would be useful to have more details on methodology in either the figure legends or in the body of the text. Figures 2-5. A legend explaining that red corresponds to WT, green low CGG, and orange high CGG on these figures should be included either in the figure itself, or in the figure legend. Additionally, it was not clear that the points outside the box plot were outliers and not indicators of significance. More information about how the data are presented would be helpful. It’s also unclear why the individual data points for the WT results are not always included below the box plots. Additionally, were any statistics done on these data? An R2 value for the correlation lines, or an ANOVA for CGG repeat length, or age, would all be appropriate tests to include in these results without having to refer back to the original research manuscripts. Female premutation model mice and Figures 3 and 4: The fact that female CGG KI model mice also show differences in temporal processing with these tests is an interesting observation. Female premutation patients often show less severe symptoms when they have one copy of FMR1 with smaller repeats, making them heterozygous for the pathogenic repeat allele, owing to X-inactivation and mosaic expression. The genotype of the female mice tested in Figures 3, 4, and the text on temporal processing is not indicated, and should be included for clarity. Sex differences and X-inactivation are also mentioned with the histopathological endophenotype discussion, and warrant more explanation for those unfamiliar with the process and impact that X-inactivation skewing can have on this disorder. Additionally, the result in female mice on temporal processing begs the question if female CGG KI mice have been tested in any of the other behavioral tasks discussed in this manuscript. Figure 5: The author mentions that the Fmr1 KO mice is not a perfect model of FXS as Fmr1 expression was eliminated by inserting a neomycin cassette, and not by way of repeat expansion followed by promoter and 5’UTR hypermethylation as in the human disorder. As such, it is probably incorrect to plot the Fmr1 KO data at/beyond the 200 CGG repeat mark. These animals do not have any repeats and as such should be plotted on a separate scale. Analytical verification and Figure 7: Do the two misclassifications noted in Figure 7 correspond to obvious outlier animals? It might be interesting to look back at the data from those animals which the algorithm did not classify with the rest, and comment on why they may have been missed in the analysis. Histopathological endophenotype: The author does a nice job of accumulating histological findings in premutation patients and mouse models. However, the discussion of current hypotheses around the development of inclusion formation and disease pathogenesis is lacking. There are many theories in the FXTAS field that the increase of FMR1 mRNA can cause protein sequestration, and may also trigger production of aberrant protein products which could contribute to inclusion formation. The mechanistic data for both of these hypotheses should be at least mentioned here. Table 1: The percentages reported for frontal cortex and hippocampus are not explained – is this the percentage of inclusions in neuronal/glial cells? Additionally, inclusion of CGG repeat lengths where available should be included in this figure, as the author is commenting on the correlation of repeat length and inclusion formation. Conclusions, Behavioral endophenotype: The comment on associations between CGG length, FMR1 mRNA, and FMRP expression warrants more discussion. Illustrating in some way the differential effects of CGG repeat levels on FMR1 mRNA levels, in contrast to the decrease in FMRP expression with increasing CGG repeats would be helpful (akin to the table generated by (Brouwer et al, 2008). Further, in discussing these endophenotypes in the context of FXS and the Fmr1 KO mouse, there is a difference between no FMRP expression, reduced FMRP expression, and the toxic mRNA gain of function that are all contributing to the unique phenotypes seen in the fragile X spectrum disorders (Ludwig et al., 2014; Pretto et al., 2014) Future directions: The call for more rigorous tests to compare mouse models and human patient phenotypes is indeed warranted. However, in discussing the limitations of the current models, it should be noted that FXTAS and FXS are not unique in the failings of animal models in general. The inability of mice to completely recapitulate human neurological disease is very common, especially in neurodegenerative disease (Parkinson’s disease and Alzheimer’s disease models are both notorious for lacking complete recapitulation of clinical phenotypes). All models (including human iPS cells and simple systems like worms and flies) are fraught with limitations, but there is power in multi-model analysis that one might hope would lead to a greater likelihood for translation from basic science findings to clinical practice. However, leaving the discussion on therapeutic development as disappointing solely because basic research in mouse models has not yet led to successful clinical trials in patients undermines the significant progress made in the field over the past decade.",
"responses": [
{
"c_id": "732",
"date": "12 Mar 2014",
"name": "Michael Hunsaker",
"role": "Author Response",
"response": "Thanks for the great review. I am going to get to work on revisions to all of your comments. I think all of your points are great and I do sometimes have a habit of pointing out limitations rather than celebrate the progress made."
}
]
},
{
"id": "3383",
"date": "26 Feb 2014",
"name": "Anthony Hannan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 on an important topic. Fmr1 knock-out mice have been used as a best approximation of a genetically accurate model of fragile X syndrome, despite the fact that the patients have a tandem repeat expansion rather than a null mutation. The systematic comparison of knock-in and knock-out Fmr1 mice is important not just for our understanding of fragile X syndrome, FXTAS and related disorders, but also for the role of this fascinating tandem repeat polymorphism in healthy brain development and cognitive function. Whilst the maintenance of very long tandem repeat mutations in mouse lines has been a technical challenge (particularly for fragile X syndrome), this can no doubt be overcome so that such tandem repeat disorders are modeled with optimal genetic construct validity. The article summarises and integrates a large body of data. The integration of cognitive endophenotypes with information on cellular and molecular mechanisms is informative. I think it is a useful addition to the literature on tandem repeat polymorphisms and associated disorders, and their effects on the healthy and diseased brain.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-287
|
https://f1000research.com/articles/2-130/v1
|
23 May 13
|
{
"type": "Research Article",
"title": "An a posteriori measure of network modularity",
"authors": [
"Timothée Poisot"
],
"abstract": "Measuring modularity is important to understand the structure of networks, and has an important number of real-world implications. However, several measures exists to assess the modularity, and give both different modularity values and different modules composition. In this article, I propose an a posteriori measure of modularity, which represents the ratio of interactions between members of the same modules vs. members of different modules. I apply this measure to a large dataset of 290 ecological networks, to show that it gives new insights about their modularity.",
"keywords": [
"nodes",
"network",
"modularity",
"interactions"
],
"content": "Introduction\n\nModularity, the fact that groups of nodes within a network interact more frequently with themselves than with other nodes, is an important property of several systems, including genetic1,2, informatics3, ecological4, and socio-economic5 interactions, as well as biogeographic patterns6,7 and disease spread management8. Because of the relevance of modularity for network properties, it is important to assess it correctly. There exists several methods to measure network modularity, some of which rely on the optimization of a given criterion9,10, label propagation11, or combination of these approaches12. These methods return two elements. The first is a value of modularity for the networks, most often within the 0–1 interval. Each method often has a threshold value, above which a network is considered to be modular. Increasing values reflect an increasingly modular structure. The second element is a “community partition”, i.e. the attribution of each node to a module.\n\nRecently, Thébault7 showed that different measures of modularity tailored to presence/absence matrices (i.e. networks in which links have no weight), gave roughly equal estimates of the significance of modularity, but differed in the community partition they returned (i.e. the identity of nodes composing each module varied). In this situation, one might look for a way to choose which community partition should be used. As the criterion that is optimized by each method is different, one possible way to compare the different community partitions is to use an a posteriori measure to quantify modularity, which can be applied to a network regardless of the method used to obtain the community partition.\n\nAn important feature of modular networks is the occurrence of interactions between nodes of different modules. They contribute to the propagation of disturbances4, flow of information13,14, and cross-regulation of biological processes15, inter alia16. In addition to measuring how modular the network is, determining to what extent modules are connected, and to identify nodes and edges responsible for connecting modules, is thus a valuable information. In this article, I propose an a posteriori measure of the proportion of interactions established between modules, i.e. edges connecting different communities. I apply this measure to the community partition identified by the Louvain method on 290 ecological networks, and show that it behaves in a similar way to other modularity measures.\n\n\nThe measure\n\nIn this contribution I define the realized modularity, termed QR. QR measures the extent to which edges, within a network, are established between nodes belonging to the same module. For E edges in a network, if W of them are established between members of the same module, then\n\n\n\nWhen there are no between-module links, then W = E and QR takes the maximal value of 1. When between-module interactions are as numerous as within-modules interactions, then W = E/2, and QR takes the minimal value of 1/2. To express the realized modularity as a value between 0 and 1, it is expressed as:\n\n\n\nThe main advantage of QR is that it is agnostic with regard to the measure used to optimize modularity (and even to the method by which the nodes were assigned to modules, which can be arbitrary), as it acts a posteriori, i.e. after nodes have been attributed to modules. It can therefore be used to select the community detection method maximizing modularity. This measure works on most type of networks, as it makes no difference if links are directional, or if the networks is bipartite/unipartite. An illustration of this measure is given in Figure 1.\n\nNodes of the same modules are identified by different colors. This network has a modularity (Louvain method) of Q = 0.527. Out of the 36 interactions, 31 are established within modules, and 5 between modules. This gives a QR value of 0.86, and Q′R = 0.72.\n\nA python implementation of this measure, using the networkx package, is proposed at https://gist.github.com/tpoisot/4947006. It reads data in the edge list format, and offers additional functions to generate null networks, as detailed in the following section.\n\n\nExample application: realized modularity in ecological networks\n\nIn this section, I analyze the modular structure of a large dataset of 290 ecological networks (187 food webs and 113 host-parasite networks) published in previous meta-analyses17,18. Modularity is an important feature of ecological interaction networks, which is linked to their resilience19,20, stability7, biogeographic structure21, functioning22, and to the evolutionary mechanisms involved in their assembly23. Notably, the occurrence of interactions between and within modules plays a central role in the structure of pollination networks4, and help buffer the effect of species extinctions20.\n\nI used the Louvain method24 to detect modules, due to its rapidity and efficiency on large networks. The Louvain method works in two steps: first it optimizes modularity locally, through clustering of neighboring nodes. These clusters are, in the second step, aggregated together, until modularity ceases to increase. This method is known to give values of modularity comparable to what is found using e.g. simulated annealing, and has been observed to give modules that have a functional relevance. Once the partition is returned by the Louvain method, I recorded its realized modularity Q′R, and its modularity Q (using the Newman and Girvan25 measure).\n\nFor each network, I compared the values of Q and Q′R on the empirical networks to their random estimate using a network null model. The null model is defined as follows. For each node n of the network, I measured its degree dn, its generality (number of successors) gn, and its vulnerability (number of predecessors) vn. In each random network, for each pair of nodes (i, j), the probability that i interacts with j is given by\n\n\n\nand conversely for P(j→i). This null model allowed the generation of pseudo-random networks through a Bernoulli process (in each replicate, the occurrence of a link is randomly determined), with the same connectance, and the same distribution of degrees, generality, and vulnerability, as the original one (these properties are also conserved at the node level). For each of the 289 networks, 1000 pseudo-random replicates are generated. For each of them, the average value of QR and Q′R are estimated along with their 90% confidence interval. When the empirical value lies outside the confidence interval, it can be assumed that the modular structure of the network is different than expected by chance.\n\nThere is a strong, positive relationship between the values of Q′R and the values of Q (Pearsons's product-moment correlation coefficient, as implemented in R 2.1526, ρ = 0.64, 288 d.f., p < 10-6), i.e. networks for which a high modularity is detected tend to have relatively few between-module links (Figure 2). It is worth noting that some Q′R values were negative: in some cases, the best community division resulted in more interactions between than within modules. This result highlights why using an a posteriori measure is useful: other measures of modularity do not reveal the fact that there were more interactions between than within modules. Q and Q′R have different relationships with connectance (Figure 3). Increased connectance values resulted in lower modularity (ρ = –0.61, 288 d.f., p < 10-6), but had no impact on Q′R. This is a desirable property, as it allows easy comparison with the Q′R values of networks with extremely different connectances.\n\nThere exists a strong, positive between the two variables. Worth noting is that fact that, for some networks, the best partition resulted in negative versions of Q′R, i.e. there were more interactions between than within modules. Each dot correspond to a network.\n\nA. Q is negatively affected by connectance, i.e. densely connected networks are more likely not to be modular. B. Q′R is not affected by connectance, allowing to use it to compare different networks. Each dot correspond to a network.\n\nThere is a linear relationship between the deviation from random expectation of Q and Q′R (ρ = 0.78, 288 d.f., p < 10-6 Figure 4). The deviations (respectively ΔQ and ΔQ′R) are calculated as the empirical value, minus the average of the values on the networks generated by the null model. As an example, a ΔQ less than zero indicates that the empirical network is less modular than expected by chance.\n\nNetworks in the red area are detected as being less modular than expected both by Q′R and Q, while networks in the blue area are detected as being more modular. Although the agreement between the two measures is good (see main text for statistics), some networks are detected as having a higher than expected realized modularity Q′R, despite a lower than expected modularity Q. Each dot correspond to a network.\n\nConfidence intervals for the average of the null models were typically very narrow (not represented in the figure to avoid cluttering-see associated dataset), probably owing to the fact that the null model is restrictive on the type of networks which are generated. It is worth noting that for some networks, the diagnostic of the null model analysis conflicted. It is then worth knowing why these networks are less modular but have fewer links between modules, or conversely, than expected. This kind of analyses will help further clarify the importance of species functional role on ecological network modularity.\n\n\nConclusions\n\nThe Q′R measure presented here allows the estimation of the proportion of interactions established between different modules in a network. This measure can be analyzed much in the same way as other measures of modularity, but is applied a posteriori. As such, it can help choosing the “best” community partition according to the property of the network that one wants to maximize. For example, choosing the partition giving the lowest Q′R can help identify which species are more likely to act as connectors between different modules.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nTP is funded by a PBEEE-FQRNT post-doctoral scholarship, and thanks the EEC Canada Research Chair for providing computational support.\n\n\nAcknowledgements\n\nThanks are due to the maintainers and contributors of the free networkx, scipy, and numpy packages used in this project, and to Scott Chamberlain for discussions.\n\n\nReferences\n\nEspinosa-Soto C, Wagner A: Specialization can drive the evolution of modularity. PLoS Comput Biol. 2010; 6(3): e1000719. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBauer-Mehren A, Bundschus M, Rautschka M, et al.: Gene-disease network analysis reveals functional modules in mendelian, complex and environmental diseases. PLoS One. 2011; 6(6): e20284. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFortuna MA, Bonachela JA, Levin SA: Evolution of a modular software network. Proc Natl Acad Sci U S A. 2011; 108(50): 19985–19989. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOlesen JM, Bascompte J, Dupont YL, et al.: The modularity of pollination networks. Proc Natl Acad Sci U S A. 2007; 104(50): 19891–19896. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaavedra S, Stouffer DB, Uzzi B, et al.: Strong contributors to network persistence are the most vulnerable to extinction. Nature. 2011; 478(7368): 233–235. PubMed Abstract | Publisher Full Text\n\nCummings GS, Bodin O, Ernston H, et al.: Network analysis in conservation biogeography: challenges and opportunities. Diversity and Distributions. 2010; 16(3): 414–425. Publisher Full Text\n\nThébault E: Identifying compartments in presence-absence matrices and bipartite networks: insights into modularity measures. J Biogeogr. 2013; 40(4): 759–768. Publisher Full Text\n\nChades I, Martin TG, Nicol S, et al.: General rules for managing and surveying networks of pests, diseases, and endangered species. Proc Natl Acad Sci U S A. 2011; 108(20): 8323–8328. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNewman ME: Modularity and community structure in networks. Proc Natl Acad Sci U S A. 2006; 103(23): 8577–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang XS, Wang RS: Optimization analysis of modularity measures for network community detection. In: The Second International Symposium on Optimization and Systems Biology. Lijiang, China, 2008; 13–20. Reference Source\n\nBarber MJ: Modularity and community detection in bipartite networks. Phys Rev E Stat Nonlin Soft Matter Phys. 2007; 76(6 pt 2): 066102. PubMed Abstract | Publisher Full Text\n\nLiu X, Murata T: Community detection in large-scale bipartite networks. Trans Jpn Soc Artif Intell. 2010; 5(1): 184–192. Publisher Full Text\n\nWiederhold G: Mediators in the architecture of future information systems. IEEE Comput Mag. 1992; 25(3): 38–49. Publisher Full Text\n\nLeskovec J, Lang KJ, Dasgupta A, et al.: Statistical properties of community structure in large social and information networks. In: Proceeding of the 17th international conference on World Wide Web - WWW 08. New York, New York, USA: ACM Press, 2008; 695–704. Publisher Full Text\n\nHartwell LH, Hopfield JJ, Leibler S, et al.: From molecular to modular cell biology. Nature. 1999; 402(6761 Suppl): C47–52. PubMed Abstract | Publisher Full Text\n\nRosvall M, Bergstrom CT: Maps of random walks on complex networks reveal community structure. Proc Natl Acad Sci U S A. 2008; 105(4): 1118–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGravel D, Massol F, Canard E, et al.: Trophic theory of island biogeography. Ecol Lett. 2011; 14(10): 1010–1016. PubMed Abstract | Publisher Full Text\n\nPoisot T, Canard E, Mouillot D, et al.: The dissimilarity of species interaction networks. Ecol Lett. 2012; 15(12): 1353–1361. PubMed Abstract | Publisher Full Text\n\nFortuna MA, Stouffer DB, Olesen JM, et al.: Nestedness versus modularity in ecological networks: two sides of the same coin? J Anim Ecol. 2010; 79(4): 811–817. PubMed Abstract | Publisher Full Text\n\nStouffer DB, Bascompte J: Compartmentalization increases food web persistence. Proc Natl Acad Sci U S A. 2011; 108(9): 3648–3652. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlores CO, Valverde S, Weitz JS: Multi-scale structure and geographic drivers of cross-infection within marine bacteria and phages. ISME J. 2013; 7(3): 520–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThébault E, Loreau M: Food-web constraints on biodiversity-ecosystem functioning relationships. Proc Natl Acad Sci U S A. 2003; 100(25): 14949–14954. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlores CO, Mayer JR, Valverde S, et al.: Statistical structure of host-phage interactions. Proc Natl Acad Sci U S A. 2011; 108(28): E288–297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlondel VD, Guillaume JL, Lambiotte R, et al.: Fast unfolding of communities in large networks. J Stat Mech. 2008; 2008(10): P10008. Publisher Full Text\n\nNewman ME, Girvan M: Finding and evaluating community structure in networks. Phys Rev E Stat Nonlin Soft Matter Phys. 2004; 69(2 pt 2): 026113. Publisher Full Text\n\nR. Development Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria, 2008. Reference Source"
}
|
[
{
"id": "1028",
"date": "26 Jun 2013",
"name": "Carsten F Dormann",
"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 proposed index of modularity is of striking simplicity - and thus likely to be prone to artifacts. In the opening paragraph, Poisot forgot to mention that random networks are also modular. Thus, a Q_R > 0 means, in itself, nothing, as Poisot rightly assumes when employing a null model.The typically log-normal abundance of species in nature will introduce apparent structure into networks, even if the links simply reflect probabilistic interactions (i.e. any species interacts more with a common than a rare species). Thus, without a null model correcting for number of species, for their abundance and for the possibility of random networks also being modular, any index may report only spurious, artefactual results. Poisot uses a null model, but because his example data are binary networks (containing no information about the strength of a link), the best he (or anyone) can do is to use a null model based on degrees, which is only a very poor reflection of the actual abundance. Given that often more than a third of the species in a network are singletons, I believe that their contributions to modularity are overemphasized by any binary measure.Suggested Revisions: 1. Simulate networks (ideally weighted ones) and compare their Q_R values to quantitative null models. How much information does Q_R (and Q) actually contain? 2. Comparison of Q_R not only with Q and connectance but also with other network metrics, such as linkage density or dependence asymmetry (and particularly those with a more or less clear ecological interpretation, such as H2'). The question, again, is: what does Q_R provide in addition to current metrics?3. Gain in ecological knowledge (which follows from 1. and 2.): If there is additional information, what does it mean? Which ecological features (specialization, number of functionally similar species, number of trophic level, number of habitats sampled etc.) contribute to Q_R? (For example along the lines of Pocock et al. 2012, who work on different types of sub networks put together into one large, or Clauset et al. 2008. Are these different sub networks identifiable as modules? If so, what does Q_R stand for?)",
"responses": []
},
{
"id": "1055",
"date": "11 Jul 2013",
"name": "Daniel Carstensen",
"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 aim of the author is interesting and relevant. I am intrigued by the development of a method to quickly evaluate different modularity measures, and an a posteriori method might well be a good solution. Overall the manuscript is generally well written. However, it is not clear to me how much is gained with this approach. Poisot only uses one method to detect the module configuration (Louvain 2008) and one method to calculate the modularity (Newman & Girvan 2004). It would be interesting to explore if the QR differs markedly when applied to the results of different methods. It would also be good to see what existing modularity measures do when optimizing modularity; do they minimize between module links? This is why QR measures and the strong correlation in Figure 2 is not surprising. What is more interesting about Figure 2 is that it shows that below a certain value of Q (~ 0,2 ?) it is not sensible to talk about modularity even if the empirical data is more modular than a random system. In such cases, the presented method seems useful to evaluate results. Other minor revisionsAn earlier reference could be used for the use of modularity in biogeographic networks instead of Cummings et al. 2010 (reference 6). Cummings et al. does not handle modularity. THe author should onsider citing Carstensen & Olesen 2009.In the 'Data and analysis' section the statement starting on line six in this paragraph needs a reference.Null model: What is meant by generality/successors and vulnerability/predecessors?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-130
|
https://f1000research.com/articles/2-284/v1
|
24 Dec 13
|
{
"type": "Case Report",
"title": "Repetitive transcranial magnetic stimulation induced hypomanic symptoms in a woman with a history of electroconvulsive therapy induced mania: a case report",
"authors": [
"Noah S. Philip",
"S. Louisa Carpenter",
"S. Louisa Carpenter"
],
"abstract": "Repetitive transcranial magnetic stimulation (rTMS) is a comparatively novel option for the treatment of major depressive disorder (MDD) and other psychiatric illnesses. Previous research has shown rTMS to be safe and without significant side effects compared to pharmacologic options. However, rare cases of rTMS-induced mania have been reported. This case report describes such an affective switch in a 52 year old female veteran with treatment-resistant MDD and a history of electroconvulsive therapy (ECT)-induced mania. Six treatments of rTMS were administered at 5 Hz for a total of 3000 pulses per day, when the patient began to display multiple hypomanic symptoms. These symptoms decreased after the termination of treatment and abated within a couple of days. In conclusion, caution should be used when administering rTMS to patients with a history of ECT-induced mania.",
"keywords": [
"rTMS induced hypomanic symptoms",
"hypomania",
"rTMS",
"case report"
],
"content": "Introduction\n\nRepetitive transcranial magnetic stimulation (rTMS) is a US FDA-cleared neuromodulatory treatment for major depressive disorder (MDD). rTMS targets the dorsolateral prefrontal cortex, a brain region associated with atypical activation patterns in mood disorders1. Both high (≥10 Hz, considered more excitatory) and lower (5 Hz) frequency rTMS has been shown to be efficacious in reducing depressive symptoms and is generally safe to use in MDD and other populations2–5. However, rare cases of rTMS-induced mania and hypomania have been reported in bipolar subjects6,7. The following case report documents 5 Hz rTMS-induced hypomanic symptoms in a patient with MDD but without a definitive history of bipolar disorder. Written informed consent for publication of their clinical details was obtained from the patient.\n\n\nCase report\n\nMs. A, a 52 year-old Caucasian veteran with MDD and post-traumatic stress disorder (PTSD), was referred to the rTMS clinic at her local Veterans Affairs hospital, due to continued depressive symptoms despite multiple unsuccessful prior pharmacologic interventions, in October of 2013. In her current major depressive episode, Ms. A had been treated with citalopram (40mg daily) and diazepam (10mg daily), with limited efficacy. On evaluation, Ms. A endorsed active depressive symptoms, including hypersomnia, intense feelings of sadness, weight loss, difficulty with decision-making, anhedonia, psychomotor retardation, anergia, and passive thoughts of death, without outright suicidal ideation. On measures of symptom severity, Ms. A reported scores consistent with a severe symptom burden on the Quick Inventory of Depressive symptoms, self-report (QIDS-SR = 19)8, the Patient Health Questionnaire 9 (PHQ9 = 16)9, and PTSD Checklist (PCL = 62)10.\n\nRegarding her past psychiatric history, Ms. A’s depressive and PTSD symptoms began during her childhood, when she experienced an extended period of reported sexual abuse from close family members and friends during the ages of 3–17. Her MDD and PTSD symptoms were exacerbated when she was sexually assaulted during military service at the age of 22. She still experiences active PTSD symptoms, including avoidance, hypervigilance, and marked response to triggers, on a daily basis. She reported a history of alcohol abuse but no substance abuse.\n\nShe had received multiple past interventions for her MDD and PTSD with limited efficacy or significant side effects. Previously trialled antidepressant included mono- and combination therapies of citalopram (up to 60mg daily), escitalopram (10mg daily), fluoxetine (up to 40mg daily), paroxetine (up to 20mg daily), sertraline (up to 50mg daily), bupropion (up to 450mg daily), mirtazapine (30mg at bedtime), nortriptyline (up to 40mg daily), duloxetine (20mg daily), and venlafaxine (up to 150mg daily). Previous augmentation regimens included treatment with lithium (up to 600mg daily), lamotrigine (up to 150mg daily), quetiapine (up to 200mg), risperidone (up to 1mg daily), olanzapine up to 15mg daily) and aripiprazole (up to 10mg daily). Additionally prazosin (up to 1mg daily), had been used for PTSD symptoms. She began a course of electroconvulsive therapy (ECT) 10 years ago, which was discontinued after 14 treatments due to an induced manic episode and subsequent hospitalization with pharmacotherapy for mood stabilization. Manic symptoms resolved after the termination of ECT treatment and since that time there have been no hypomanic or manic symptoms. She had not been on any pharmacotherapy for mood stability since 2003.\n\nPast medical history included gastroesophageal reflux/Barrett’s esophagus, Graves’ disease and subsequent iatrogenic hypothyroidism, and chronic myelopathy. Associated current medications were levothyroxine 0.088mg, simvastatin 40mg, and sucralfate 500mg; all had been stable for >4 months prior to consultation. Results from a recent laboratory screening were within normal limits; most recent thyroid stimulating hormone level was 3.9 μIUnits/ml (within normal range). The physical and neurologic exams were consistent with her past medical history and revealed no acute changes.\n\nAfter signing informed consent, rTMS was administered at 5 Hz for 4 sec, with an inter-train interval of 12 sec, over the left dorsolateral prefrontal cortex at 120% of the motor threshold for a total of 3000 pulses per session, delivered five days a week. This parameter choice reflected the current literature in addition to our previous clinical experience in which patients with significant anxiety often are unable to tolerate 10 Hz. After her fifth treatment Ms. A reported improved MDD and PTSD symptoms, with scores decreasing to 9, 11 and 43 for the QIDS, PHQ9 and PCL, respectively. After the sixth treatment, Ms. A arrived at the clinic displaying multiple hypomanic symptoms, including inflated self-esteem/grandiosity, hypermotoric behavior, pressured speech, and distractibility. She reported an increase in goal-directed behavior, namely shopping, and reduced sleep to 3–4 hours. She denied psychotic symptoms and risk-taking behaviors. She reported her “thoughts were racing” and she felt like \"this could get worse and [she] would be in trouble.\" A diagnosis of rTMS-induced hypomanic symptoms was made. Treatment was suspended and symptoms were monitored. Hypomanic symptoms began decreasing in severity over the following 24 hours and continued to normalize over the next couple of days. A week post-termination of treatment, she reported stable mood and regular sleep of 6–8 hours per night for the previous 5 days, which was maintained at 2-weeks. Her scores on rating scales at one and two-weeks post-treatment reflected relatively retained improvements in mood and PTSD symptoms (QIDS-SR = 10; PHQ9 = 10; PCL = 50, and QIDS-SR = 10; PHQ9 = 11; PCL = 52 for one and two weeks, respectively). At one month post-treatment, there had been no evidence of hypomania since cessation of rTMS.\n\n\nDiscussion\n\nThe strong temporal relationship between the presentation of symptoms and the course of treatment, as well as the patient’s history of ECT-induced mania, suggest that rTMS triggered Ms. A’s hypomanic symptoms. However, compared to previously reported cases6,7, the patient demonstrated no consistent symptoms of bipolar disorder prior to rTMS therapy.\n\nThe use of 5 Hz stimulation, compared to 10 Hz should also be noted. 5 Hz rTMS has been safely used in bipolar patients taking mood stabilizers3,5,11. Lower frequency rTMS has been associated with an accelerated antidepressant effect as an add-on treatment to medication in MDD subjects compared to sham2. Nonetheless, singular cases of rTMS-induced hypomania in bipolar patients have been reported for both 5 and 10 Hz parameters6,7.\n\nWe hypothesize that the increased treatment parameters (3000 pulses per day versus 1600 pulses per day in past 5 Hz literature)3,5,11 and the aforementioned accelerating effect of rTMS treatment on antidepressants might have triggered the patient’s affective switch. Her history of ECT-induced mania, but not antidepressant-induced switching, suggests a mood regulatory system prone to severe shifts during neuromodulatory intervention. Unlike subjects in previous studies, Ms. A was not taking mood stabilizers that may act as a protective factor3,5. The presented case report therefore indicates that caution should be used when treating patients with rTMS who have a history of ECT-induced manic symptoms. Further study of patients with a history of affective switch with multiple neuromodulatory interventions is warranted, to better characterize a potentially at-risk population.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nNSP was the clinician overseeing the patient’s treatment. He revised the manuscript for editorial, medical, and intellectual content. SLC was the TMS technician and wrote the first draft of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors have no conflicts of interest to disclose; NSP has received research support from Neuronetics, Inc., and NeoSync, Inc. through contracts with Butler Hospital.\n\n\nGrant information\n\n\n\n\nReferences\n\nYe T, Peng J, Nie B, et al.: Altered functional connectivity of the dorsolateral prefrontal cortex in first-episode patients with major depressive disorder. Eur J Radiol. 2012; 81(12): 4035–40. PubMed Abstract | Publisher Full Text\n\nRumi DO, Gattaz WF, Rigonatti SP, et al.: Transcranial magnetic stimulation accelerates the antidepressant effect of amitriptyline in severe depression: a double-blind placebo-controlled study. Biol Psychiatry. 2005; 57: 162–166. PubMed Abstract | Publisher Full Text\n\nNahas Z, Kozel FA, Li X, et al.: Left prefrontal transcranial magnetic stimulation (TMS) treatment of depression in bipolar affective disorder: a pilot study of acute safety and efficacy. Bipolar Disord. 2003; 5: 40–47. PubMed Abstract | Publisher Full Text\n\nCarpenter LL, Janicak PG, Aaronson ST, et al.: Transcranial magnetic stimulation (TMS) for major depression: a multisite, naturalistic, observational study of acute treatment outcomes in clinical practice. Depress Anxiety. 2012; 29(7): 587–96. PubMed Abstract | Publisher Full Text\n\nGeorge MS, Nahas Z, Molloy M, et al.: A controlled trial of daily left prefrontal cortex TMS for treating depression. Biol Psychiatry. 2000; 48(10): 962–70. PubMed Abstract | Publisher Full Text\n\nHuang CC, Su TP, Shan IK: A case report of repetitive transcranial magnetic stimulation-induced mania. Bipolar Disord. 2004; 6(5): 444–5. PubMed Abstract | Publisher Full Text\n\nDolberg OT, Schreiber S, Grunhaus L: Transcranial magnetic stimulation-induced switch into mania: a report of two cases. Biol Psychiatry. 2001; 49(5): 468–70. PubMed Abstract | Publisher Full Text\n\nRush AJ, Bernstein IH, Trivedi MH, et al.: An evaluation of the Quick Inventory of Depressive Symptomatology and the Hamilton Rating Scale for Depression: A sequenced treatment alternatives to relieve depression trial report. Biol Psychiatry. 2006; 59(6): 493–501. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKroenke K, Spitzer RL, Williams JB: The PHQ-9: validity of a brief depression severity measure. J Gen Intern Med. 2001; 16(9): 606–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeathers F, Litz B, Herman D, et al.: The PTSD Checklist (PCL): reliability, validity, and diagnostic utility. Paper presented at the Annual Convention of the International Society for Traumatic Stress Studies, San Antonio, TX. October 1993. Reference Source\n\nSu TP, Huang CC, Wei IH: Add-on rTMS for medication-resistant depression: a randomized, double-blind, sham-controlled trial in Chinese patients. J Clin Psychiatry. 2005; 66(7): 930–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2892",
"date": "06 Jan 2014",
"name": "Anne Weigand",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting case of rTMS-induced hypomanic symptoms in a 52 year old woman with treatment-resistant MDD and PTSD. Importantly, the patient had a history of a manic episode induced by a course of ECT 10 years ago. No hypomanic or manic symptoms have been reported after ECT termination and the patient had not been taking mood stabilizers since 2003.To date, rare cases of affective switches triggered by rTMS have been reported in bipolar subjects. This important case report indicates that further caution is needed when treating patients with rTMS who have a history of ECT-induced mania.",
"responses": []
},
{
"id": "3381",
"date": "18 Feb 2014",
"name": "Martijn Arns",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 study of rTMS-induced hypomanic symptoms. It is important that such cases and information be published and made public, since it might also provide us more information about the working mechanism of rTMS, and in this case suggests some overlap between ECT and rTMS. The case is well written and clearly presented.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-284
|
https://f1000research.com/articles/2-283/v1
|
23 Dec 13
|
{
"type": "Case Report",
"title": "A case report of simultaneous PML-IRIS during corticosteroids tapering in a patient with an anti-synthetase syndrome",
"authors": [
"Guillaume Martin-Blondel",
"David Brassat",
"Hervé Dumas",
"Emmanuelle Uro-Coste",
"Daniel Adoue",
"Hans Lassmann",
"Michel Clanet",
"David Brassat",
"Hervé Dumas",
"Emmanuelle Uro-Coste",
"Daniel Adoue",
"Hans Lassmann",
"Michel Clanet"
],
"abstract": "We report a case of simultaneous progressive multifocal leukoencephalopathy-associated immune reconstitution inflammatory syndrome (PML-IRIS) during corticosteroid tapering in a patient with an anti-synthetase syndrome. We describe the challenges associated with the diagnosis and the management of this emerging inflammatory neurological condition in this immunocompromised patient with a severe rheumatic disease. We highlight that, in the setting of IRIS, the low-level of the JC virus viral load requires a sensitive PCR assay before excluding PML.",
"keywords": [
"JC virus",
"Progressive multifocal leukoencephalopathy",
"Immune reconstitution inflammatory syndrome",
"Antisynthetase syndrome",
"Corticosteroids."
],
"content": "Introduction\n\nProgressive multifocal leukoencephalopathy (PML) is a devastating disease due to reactivation of the Polyomavirus JC virus (JCV) in immunocompromised patients1. PML has been associated with immune reconstitution inflammatory syndrome (IRIS) during immune recovery of HIV-infected patients treated by antiretroviral therapy, or in non HIV-infected patients after the withdrawal of therapeutic monoclonal antibodies2,3. Here we describe a case of simultaneous PML-IRIS during corticosteroids tapering in a patient with an antisynthetase syndrome (ASS).\n\n\nCase report\n\nA 62-year-old right-handed French Caucasian woman was diagnosed in October 2007 for an ASS and treated by corticosteroids (methylprednisolone 40 mg/week and prednisolone 20 mg/day) and mycophenolate mofetil (3 g/day). In March 2010 white blood cell count showed profound lymphopenia (486/mm3, normal range 1,500 to 4,000/mm3, Figure 1). Because the ASS was controlled, methylprednisolone was stopped and prednisolone was progressively tapered to 7.5 mg/day. In May 2010 she presented progressive cognitive impairment, followed by a brisk worsening in July 2010 with dizziness and falls. At admission on July 8th 2010 neurological examination revealed mental slowness, attention and memory troubles, and paresis of the left lower limb. Brain axial T2-WI MRI revealed confluent subcortical white matter hyperintensities of the right frontal and parietal region (Figure 2A). Axial T1-WI MRI displayed hypointensities with multiple foci of gadolinium enhancement (Figure 2B). Prednisolone and mycophenolate mofetil were stopped. The differential diagnoses were viral encephalitis, tuberculosis, cerebral lymphoma, paraneoplastic disorder and CNS involvement of a connective tissue disorder. General examination did not demonstrate any activity of the ASS. Blood cell count showed 750 lymphocytes/mm3 (Figure 1). Cerebrospinal fluid (CSF) examination on day 2 was normal, and in-house PCR (Herpesviridae, enterovirus, JCV, BK virus, Toxoplasma gondii and Mycobacterium tuberculosis), and serologies (HIV, Borrelia and syphilis), were negative, as well as direct staining and cultures for bacteria and fungi. Blood immunophenotyping showed 673 CD4+ T cells/mm3 (normal range 500–1,500), 82 CD8+ T cells/mm3 (normal range 250–950) and 37 CD19+ B cells/mm3 (normal range 100–600) (Figure 1). The level of anti-Jo1 antibodies previously detected (Nov 2009, 7.4 AI (normal range 0–0.9) was decreasing (5.9 AI) and a screening for anti-neutrophil cytoplasmic and onconeuronal antibodies was negative. A computed tomography scan showed steady lung interstitial infiltrates, and no evidence for sarcoidosis, tuberculosis or cancer. A stereotactic brain biopsy of the right parietal lobe was performed on July 16th 2010. Neuropathological examination showed demyelinated lesions with axonal loss and a severe inflammatory reaction with a vasculitic component and endothelial damage (Figure 2D–E). Perivascular and parenchymal inflammatory infiltrates showed a pronounced CD3+ T cell infiltrate (Figure 2F) composed mostly by CD4+ T cells, in association with a few CD68+ macrophages/microglial cells and CD138+ plasma cells. Anti-Simian virus 40 (SV40) immunohistochemistry (cross-reacting with the JCV) was positive, and a second aliquot of the CSF taken on day 2, sent to Dr. Major’s laboratory at the NIH, was positive for JCV by Real-time TaqMan PCR at a low level (23 copies/ml)4, both firmly establishing the diagnosis of PML. A diagnosis of simultaneous PML-IRIS with vasculitis was made. As in the meantime her neurological status had stabilized, the patient did not receive corticosteroids. When followed up in November 2010, mental slowness and paresis of the left lower limb had completely recovered, and repeated MRI showed improvement of previous lesions. In parallel, blood immunophenotyping showed partial normalization (Figure 1). However, concomitantly she presented with a severe flare of the ASS, with myositis, polyarthritis and active interstitial pneumonitis. A one-week course of oral corticosteroids was initiated together with monthly intravenous polyclonal immunoglobulin therapy (IVIg). By December 2012 the ASS was considered under control with IVIg alone. The patient was fully independent without any neurological abnormalities, while MRI showed sequellar lesions (Figure 2C).\n\nArrows underneath the graph represent treatment periods. Single arrow heads represent treatments that were begun or stopped outside of the time period represented on the graph. ASS: Anti-synthetase syndrome; MMF: mycophenolate mofetil.\n\nA–B: Brain MRI at the onset of the simultaneous PML-IRIS showing white matter hyperintensities of the right frontal and parietal region (A, axial T2-WI) with multiple foci of gadolinium enhancement (B, axial T1-WI). C: Brain MRI nine months after the onset of PML-IRIS showing sequellar lesions (axial T2-WI). D–E: Histopathology showing severe perivascular infiltrates (D, H&E × 200) with a vasculitic component (E, H&E × 630, star: fibrin deposit, arrow: neutrophils and serohematic material, arrowhead: lymphocytes infiltrating vessel wall). F: Immunohistochemistry analysis (×400) showing perivascular inflammatory infiltrates positive for CD3 antibody (clone F7.2.38, DAKO).\n\n\nDiscussion\n\nThe diagnosis of PML in this immunocompromised patient is firmly established by the detection of JCV DNA in the CSF and of viral proteins on the brain biopsy sample, and by the exclusion of alternative infections or tumors1.\n\nHowever, this PML case is associated with very unusual inflammatory features, as attested by contrast enhancement on brain MRI and T cell infiltrates with a vasculitic component on brain biopsy. Because the level of immunosuppression was recently alleviated in this patient, as suggested by the increase of the blood lymphocyte count at admission, we believe that this patient developed a simultaneous PML-IRIS. The development of neurologic abnormalities due to an unusual inflammatory form of PML in the setting of immune recovery is consistent with the definition of simultaneous IRIS5. IRIS results from the restoration of an antimicrobial immune response that causes disproportionate tissue damage in infected organs6. In this case, the corticosteroid tapering, by restoring partially immune surveillance, might have unleashed the T-cell mediated immune response underlying PML-IRIS. The subsequent control of the viral replication might explain the low-level of the CSF viral load in this patient, highlighting that a sensitive PCR assay is required to exclude PML in the setting of IRIS.\n\nBrain infiltrates were mainly composed of CD4+ T cells, which is another unusual feature of this case. Indeed, a clear dominance of CD8+ T cells in infiltrates has been observed in natalizumab-associated PML-IRIS in patients with multiple sclerosis (MS)7, and in PML-IRIS in HIV-infected patients8. Nevertheless, a recent case report suggested a central role for CD4+ T cells in natalizumab-associated PML-IRIS in a patient with MS9. The fact that lymphopenia in our patient mainly relies on CD8+ T cells, and not on CD4+ T cells, conversely to the situation in HIV-infected patients, might in part explain this phenomenon.\n\nDespite severe neurological deterioration, this PML correlates with favorable outcome without corticosteroid treatment. The inflammatory reaction associated with IRIS is often self-limited and does not seem to alter survival of patients with PML10. A better control of viral replication might also have contributed to this positive outcome. Because corticosteroids have a profound impact on the JCV-specific T-cell response, they should be reserved for life-threatening PML-IRIS11. Finally when the ASS relapsed, the IVIg therapy was a suitable way to manage the risks of immunosuppression12.\n\nIn conclusion, PML-IRIS might occur in patients with rheumatic diseases not receiving therapeutic monoclonal antibodies when immunosuppression is alleviated. PML presentation is unusual in this setting, and diagnosis requires a sensitive PCR assay, and/or brain biopsy.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nG. Martin-Blondel: Drafting/revising the manuscript for content, including medical writing for content, Study concept or design, acquisition of data, analysis and interpretation of data, study coordination. D. Brassat: Drafting/revising the manuscript for content, acquisition of data, analysis and interpretation of data. H. Dumas: Revising the manuscript for content, neuroradiological analysis. E. Uro-Coste: Revising the manuscript for content, pathological analysis. D. Adoue: Revising the manuscript for content, acquisition of data. H. Lassmann: Revising the manuscript for content, pathological analysis. M. Clanet: Drafting/revising the manuscript for content, acquisition of data, analysis and interpretation of data.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Dr. Camille Laurent who carried out in part the brain histological analysis, and Dr. Eugene O. Major for performing the JCV PCR assay and for reading the manuscript.\n\n\nReferences\n\nTan CS, Koralnik IJ: Progressive multifocal leukoencephalopathy and other disorders caused by JC virus: clinical features and pathogenesis. Lancet Neurol. 2010; 9(4): 425–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan IL, McArthur JC, Clifford DB, et al.: Immune reconstitution inflammatory syndrome in natalizumab-associated PML. Neurology. 2011; 77(11): 1061–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan K, Roda R, Ostrow L, et al.: PML-IRIS in patients with HIV infection: clinical manifestations and treatment with steroids. Neurology. 2009; 72(17): 1458–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRyschkewitsch C, Jensen P, Hou J, et al.: Comparison of PCR-southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid. J Virol Methods. 2004; 121(2): 217–21. PubMed Abstract | Publisher Full Text\n\nJohnson T, Nath A: Neurological complications of immune reconstitution in HIV-infected populations. Ann N Y Acad Sci. 2010; 1184: 106–20. PubMed Abstract | Publisher Full Text\n\nMartin-Blondel G, Delobel P, Blancher A, et al.: Pathogenesis of the immune reconstitution inflammatory syndrome affecting the central nervous system in patients infected with HIV. Brain. 2011; 134(pt 4): 928–46. PubMed Abstract | Publisher Full Text\n\nMetz I, Radue EW, Oterino A, et al.: Pathology of immune reconstitution inflammatory syndrome in multiple sclerosis with natalizumab-associated progressive multifocal leukoencephalopathy. Acta Neuropathol. 2012; 123(2): 235–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartin-Blondel G, Bauer J, Cuvinciuc V, et al.: In situ evidence of JC virus control by CD8+ T cells in PML-IRIS during HIV infection. Neurology. 2013; 81(11): 964–970. PubMed Abstract | Publisher Full Text\n\nAly L, Yousef S, Schippling S, et al.: Central role of JC virus-specific CD4+ lymphocytes in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome. Brain. 2011; 134(pt 9): 2687–702. PubMed Abstract | Publisher Full Text\n\nHarrison DM, Newsome SD, Skolasky RL, et al.: Immune reconstitution is not a prognostic factor in progressive multifocal leukoencephalopathy. Journal of Neuroimmunology. 2011; 238(1–2): 81–6. PubMed Abstract | Publisher Full Text\n\nAntoniol C, Jilek S, Schluep M, et al.: Impairment of JCV-specific T-cell response by corticotherapy: effect on PML-IRIS management?. Neurology. 2012; 79(23): 2258–64. PubMed Abstract | Publisher Full Text\n\nRiminton DS, Hartung HP, Reddel SW: Managing the risks of immunosuppression. Curr Opin Neurol. 2011; 24(3): 217–23. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3404",
"date": "10 Feb 2014",
"name": "Gabriele Arendt",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an article on a patient with an antisynthetase syndrome developing progressive multifocal leucoencephalopathy = PML-IRIS under cortisone tapering. The case is interesting, well presented and important, because it shows that IRIS-PML might occur also when a relatively mild immunosuppression is reduced.",
"responses": []
},
{
"id": "3910",
"date": "03 Mar 2014",
"name": "Joseph Berger",
"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 fascinating patient with ASS and PML-IRIS. The latter appeared to develop concomitant with tapering of corticosteroid therapy. I would suggest the following to improve the manuscript:The focus of the paper should be expanded or modified. The occurrence of PML-IRIS at the time of the initial diagnosis of PML is a well-described phenomenon in patients being treated with natalizumab. In fact, as many as 40% of the them have IRIS concomitant with their PML. This literature should be cited. More importantly, this appears to be the first case of ASS and PML. There is at least one case report of polymyositis and PML, but I could not find another of ASS and PML and I think that is probably more important than the concomitant appearance of PML and PML-IRIS. The figures should include some histopathological sections to demonstrate the demyelination and the SV40 staining. Were bizarre astrocytes and enlarged oligodendroglial nuclei not observed? If so, it should be described. Does ASS ever occur with vasculitis or brain disease? A discussion of this aspect of the differential diagnosis would be helpful to the reader. The authors reserve comment on the specific clinical features of the rheumatological condition to the very end of their case description. I think it would be more useful to incorporate it earlier in their description. When did it develop, what were the clinical features, etc? Do the authors think that the mycophenolate mofetil contributed to the PML? After all, it carries a black box warning in the U.S. for PML. Change the wording from \"mainly relies on CD8\" to \"mainly characterized by CD8\" or something similar. This patient had resolution of the IRIS in the absence of corticosteroid treatment. This is another important point as the neurological community has widely embraced employment of steroids at the outset of the disorder. A discussion of this dilemma can be found in Berger, JR (2009).",
"responses": []
},
{
"id": "3911",
"date": "07 Mar 2014",
"name": "Xin Dang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presence of JCV genomic DNA in CSF is the key evidence for making a diagnosis of PML. However, from time to time in our research, CSF samples from diagnosed PML patients could be JCV negative in conventional PCR; only the most sensitive real time PCR can detect the extremely low JC viral load in these CSF samples. For this reason, I suspect that some PML patients may be misdiagnosed. The remarkable work of Dr. Martin-Blondel's group will encourage other clinicians to look into a possible PML diagnosis in some neural ataxia cases when occurring in immunosuppressed patients.I have two suggestions that will improve this work:Based on my own experience, JC viral load may increase to a higher level after the PML lesions become visible. If post PML diagnosis CSF samples are available, the data of the JC viral replication kinetic in this patient will help readers gain a better understanding of how JCV replicates in this PML-IRIS patient. This patient also shows cognitive dysfunction, which I suspect may be the symptom of a newly defined JCV associated cerebellar disease called JCV Granule Cell Neuronopathy (JCVGCN). Unfortunately, the MRI of the cerebellum is not available in this paper and the authors didn't look into the presence of GCN type JCV strains. However this won't hurt the integrity of this paper.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-283
|
https://f1000research.com/articles/2-158/v1
|
16 Jul 13
|
{
"type": "Research Article",
"title": "Adaptability of the nociceptive withdrawal reflex",
"authors": [
"Nathan Eckert",
"Zachary A Riley",
"Nathan Eckert"
],
"abstract": "The nociceptive withdrawal reflex is a protective mechanism to mediate interactions within a potentially dangerous environment. The reflex is formed by action-based sensory encoding during the early post-natal developmental period, and it is unknown if the protective motor function of the nociceptive withdrawal reflex in the human upper-limb is adaptable based on the configuration of the arm or if it can be modified by short-term practice of a similar or opposing motor action. In the present study, nociceptive withdrawal reflexes were evoked by a brief train of electrical stimuli applied to digit II, 1) in five different static arm positions and, 2) before and after motor practice that was opposite (EXT) or similar (FLEX) to the stereotyped withdrawal response, in 10 individuals. Withdrawal responses were quantified by the electromyography (EMG) reflex response in several upper limb muscles, and by the forces and moments recorded at the wrist. EMG onset latencies and response amplitudes were not significantly different across the arm positions or between the EXT and FLEX practice conditions, and the general direction of the withdrawal response was similar across arm positions. In addition, the force vectors were not different after practice in either the practice condition or between EXT and FLEX conditions. We conclude the withdrawal response is insensitive to changes in elbow or shoulder joint angles as well as remaining resistant to short-term adaptations from the practice of motor actions, resulting in a generalized limb withdrawal in each case. It is further hypothesized that the multisensory feedback is weighted differently in each arm position, but integrated to achieve a similar withdrawal response to safeguard against erroneous motor responses that could cause further harm. The results remain consistent with the concept that nociceptive withdrawal reflexes are shaped through long-term and not short-term action based sensory encoding.",
"keywords": [
"reflex",
"nociception",
"withdrawal response",
"electromyography",
"sensory encoding"
],
"content": "Introduction\n\nNoxious electrical stimulation of the digits in humans produces a coordinated reflex response that results in the withdrawal of the limb, akin to removing the hand from touching a hot stove1,2. This reflexive action, known as the nociceptive withdrawal reflex3, is an essential protective mechanism for interactions between parts of the body and the environment, allowing for quick removal from noxious stimuli. The relatively short-latency reflex responses occur as a result of transmission from Aδ afferent fibers to the motor neuron pools of several muscles4. Previously, it was suggested that stimulation of these afferent fibers consistently produces excitatory postsynaptic potentials in flexor motor neurons and inhibitory postsynaptic potentials in extensor motor neurons of the cat hindlimb5. However, fixed nociceptive input would limit the protective capabilities of the nervous system to only producing flexion at individual joints (e.g. elbow, shoulder), and a summary of more recent work has dispelled this hypothesis (see review by Clarke and Harris (2004)6).\n\nTo initiate reflexive motor actions such as withdrawing the whole limb, nociceptive afferent input would need to be distributed to the motor neuron pools of muscles across several joints to coordinate the movement. In the hindlimb of rats activation of afferent neurons with receptive fields in a specific area of skin coordinates the activity of one or more muscles best suited to remove it from the noxious stimuli3,7. Specifically, the pairing of afferent neurons and resulting muscle activity was designated as reflex \"modules\"3,7,8. Reflex modules are not limited to only activating the synergist muscles required to withdraw the limb, but can also inhibit muscles that would oppose it. Thus, organizing nociceptive sensory-motor interactions into reflex modules would result in a more efficient, coordinated limb withdrawal.\n\nIt has been suggested that the reflex modules are shaped by use, or action-based sensory encoding9,10. As a purely protective mechanism, the nociceptive withdrawal reflex adapts or develops according to the repetitive motor actions performed and the environment in which there are constant sensory-motor interactions. Since most of these adaptations transpire early in post-natal development11–15 it is assumed that they shape the functional sensory-motor behaviors required later in maturity14. However, it is unknown how reflex modules are influenced by short-term action-based motor practice or use after development has ceased. Features such as habituation and dishabituation (wind-up), as well as human correlates of long-term potentiation (LTP) and long-term depression (LTD), have been well described in relation to short-term synaptic plasticity in nociceptive pathways2,16–19. However, it is yet to be determined how short-term synaptic plasticity of nociceptive pathways controls the activation of reflex modules translating to the appropriate motor actions. Furthermore, the evidence presented for the activation of specific reflex modules has been demonstrated only by stimulating different receptive fields3,7.\n\nThe purpose of the present study is to test the adaptability of the nociceptive withdrawal reflex through two different tasks. First, the study seeks to determine if different nociceptive reflex modules are activated in the upper-limb of unimpaired humans by stimulating the same receptive field with the arm in different static arm positions by independently changing either the elbow or shoulder joint angles. We hypothesized that the static muscle length changes would alter group II afferent input that has been shown to have a strong excitatory input to interneurons regulating the withdrawal response20–22. Specifically, if there was modular organization in the spinal cord, we expected to observe different patterns of muscle activity (latencies, amplitude), and subsequent endpoint forces, evoked by stimulation in the various positions. Secondly, the study seeks to determine whether the protective motor function of the nociceptive withdrawal reflex in the upper-limb could be modified by short-term practice of a similar or opposing motor action voluntarily triggered by a non-noxious stimuli.\n\n\nExperimental procedure\n\nTen healthy adults (7 males: 29.9 ± 7.5 yrs; range: 21–42 yrs) participated in the study. None of the subjects reported any neurological disorders or other upper limb musculoskeletal impairments. Each subject provided informed consent prior to participating. The protocol was approved by the Indiana University Institutional Review Board (Study # 1105005484) and was performed in accordance with the Declaration of Helsinki.\n\nNociceptive withdrawal reflexes were examined in five static conditions (see Figure 1A–C) where the arm was fully supported in front of the body in a general position similar to reaching to a stove top or opening a cabinet door. The five conditions were achieved by independently manipulating the elbow or shoulder joint angles in the transverse plane:\n\n1) With the elbow flexed and the shoulder in a neutral position (ELBFLEX; θ = 75° and 95°, respectively);\n\n2) Elbow extended and shoulder neutral (ELBEXT; θ = 115° and 95°, respectively);\n\n3) Elbow and shoulder both neutral (NEUT; θ = 95° for both);\n\n4) Shoulder flexed and elbow neutral (SHLFLEX; θ = 80° and 95°, respectively);\n\n5) Shoulder extended and elbow neutral (SHLEXT; θ = 120° and 95°, respectively).\n\nA) Sagittal view of the subject with the wrist secured to the force transducer and the orientation of the forces recorded for all of the experiments. B) When independently changing elbow angle (a. ELBFLEX; b. NEUT; c. ELBEXT) while keeping the same shoulder angle (θ = 95°); and C) When independently changing shoulder angle (a. SHLFLEX; b. NEUT; c. SHLEXT) while keeping the same elbow angle (θ = 95°). Note that the dashed line (b.) in both B and C is the same NEUT condition and was only tested once during the trials and also again after all of the joint positions had been completed. D) Schematic showing the joint configuration (τ = elbow ~95°, shoulder ~95°) for both EXT and FLEX practice protocols. E) Representative data from one subject demonstrating the magnitude and direction of the endpoint force vector from the nociceptive withdrawal response, after practice, from the individual trials (gray-thin lines) and the average responses (black-thick lines) in the (+/-)Y and +X directions. The average responses from the EXT and FLEX trials are also superimposed on panel D.\n\nWhen the subject was fixed in each position shoulder abduction was ~75° and the only attachment point was at the wrist where it was affixed to the force transducer. This allowed the subject to completely relax the limb and did not require any stabilizing muscle activation from the shoulder or surrounding muscles. Each subject completed the five static conditions in a single experimental session, though the order of conditions was randomized for each subject. To examine if there was habituation in the withdrawal response the NEUT condition was repeated at the end of every experiment.\n\nNociceptive withdrawal reflexes were also examined in the right upper-limb of the subjects before and after two practice conditions performed on separate days. In this task the subject was in the aforementioned NEUT position for all of the trials. For the extension practice condition (EXT) the subject was asked to produce a submaximal, voluntary extension of their arm, as quickly as possible, against the force transducer upon receiving non-painful electrical stimulation of digit II (index finger). As the subject was secured at the wrist, this was an isometric contraction. This was repeated for 200 trials. The conditioning stimulations were delivered randomly with a period of 5–15s between stimuli in order to eliminate any fatiguing influence. The second condition, flexion practice (FLEX), was performed on a different day and required the subject to isometrically flex the arm at the elbow as quickly as possible. The experimental setup and endpoint force vectors generated by one subject in each practice condition are displayed in Figure 1D&E. Nociceptive withdrawal reflexes were assessed before and after the completion of the 200 trials in each condition. Shoulder and elbow flexion were kept at a constant ~95° and shoulder abduction was maintained at ~75° during each of the testing sessions. There was a minimum of two weeks between testing the two conditions and the order in which they were performed was randomized.\n\nFor all testing the subjects were comfortably seated in an upright position with only the right wrist secured to a six degree-of-freedom force transducer (75E20; JR-3, Woodland, CA), which kept them in a static position with each arm configuration. The JR-3 force transducer continuously sampled endpoint forces (Fx, Fy, Fz) and moments (Mx, My, Mz) during the experiments at 200 Hz. There were constant fluctuations in the gravitational force and moment (Fz, Mz) with the position of the limb in the two tasks, so this axis of force was excluded from further analysis.\n\nFor both the joint position task and the conditioning task noxious stimulation was applied using a Grass S88X stimulator (Grass Technologies, Astro-Med, USA) connected in-series with a Digitimer DS7AH constant current electrical stimulator (Digitimer LTD, UK). Short trains of electrical stimuli were delivered to digit II using stainless steel ring electrodes secured to the medial and proximal phalanges, while the subject was at rest. Each stimulus train consisted of 10 pulses (200 μs duration) delivered at 300 Hz, which was consistent with previous research evoking withdrawal reflexes in the upper limb at rest2. Electrical current from the stimulator was slowly increased to determine perceptual threshold at the beginning of the experiment. Then it was increased in ~2 mA steps (relative to perceptual threshold) until the subject reported that further increases were intolerable. This resulted in a stimulus intensity of between 30–50 mA for all subjects, which was consistent with other studies in the upper limb1,23,24. The stimulus trains used to elicit the reflex response during the experimental protocols were delivered at random intervals between 5 and 15s. Eight stimulus trains were delivered in all experiments where the nociceptive withdrawal reflex was examined. Stimulation intensity remained constant across all conditions for a given subject within each day of testing.\n\nIn the conditioning task non-noxious stimulation for the 200 practice trials was performed at an intensity of 5–15 mA (1.5× perceptual threshold). The same stimulation trains were delivered during the practice trials, only at a much lower intensity that was not sufficient to evoke a noticeable short-latency reflex in any of the muscles. Stimulus trains were delivered randomly between 5 and 15s for the practice trials as well. Five minutes of rest was given between the initial nociceptive withdrawal reflex testing and the practice trials, and between the practice trials and nociceptive withdrawal post-testing.\n\nSurface electromyography (EMG) signals were recorded the same in both tasks, with single differential bar electrodes (Delsys Inc, MA, USA). The signals were amplified and conditioned using a 16-channel Bagnoli EMG System (Delsys) with high- and low-pass cut-off frequencies of 20 Hz and 1,000 Hz, respectively, before being stored at a final gain of 1,000 Hz with Spike2 software (CED, Cambridge, UK). Surface EMG activity was recorded from the abductor pollicus brevis (APB), brachioradialis (BRD), biceps brachii long head (BBL), triceps brachii lateral head (TRI), anterior deltoid (AD), and posterior deltoid (PD) muscles in the right upper limb. The right (ESR) and left erector spinae (ESL) were also recorded to monitor postural responses. The skin overlying each muscle was cleaned prior to affixing the electrode over the individual muscle belly. A single reference electrode was placed over the acromion process.\n\nForces and moments were considered relative to subject coordinates with the +X direction pointing to the right of the subject, the –Y direction pointing towards the subject, and the +Z direction pointing upwards. Analysis of the endpoint forces and moments were confined to the horizontal (X-Y) plane. For all of the nociceptive withdrawal reflex trials the resultant two-dimensional (X-Y) endpoint force vectors were computed from the local peak force up to 200ms after stimulation. EMG for each muscle was processed by first removing the DC offset; then rectifying the signal. The latencies for withdrawal reflex onsets were designated when the reflex response exceeded a threshold of three standard deviations (SDs) above the mean EMG amplitude during a 100ms pre-stimulus baseline period (-100ms-stimulation). Withdrawal reflex offsets were determined based on previous observations of voluntary withdrawal latencies, such that the response reported was limited to the nociceptive spinal pathways25. Withdrawal reflex responses in the upper-limb muscles were quantified by calculating the mean EMG for a time window between onset and offset latencies. The analyses were performed on each individual stimulation trial and the resulting values were averaged across the 8-stimuli for statistical comparisons. In addition, resultant two-dimensional (X-Y) endpoint force vectors were computed from the local peak force up to 350ms for the 200 FLEX and EXT practice trials.\n\nIn the joint position task analysis of variance (ANOVA) was used to compare the direction and magnitude of the reflex endpoint force vectors across all arm positions. Arm position was considered as an independent factor and subject as a random factor for all multiple comparisons. Mean EMG reflex responses and onset latencies for each muscle were compared with separate ANOVAs across all positions. Bonferroni adjustments were made to correct for multiple comparisons in any additional post-hoc analysis. The forces, moments, EMG response amplitudes, and EMG onset latencies were compared between the initial and follow-up NEUT conditions, examining habituation, with independent t-tests.\n\nThe conditioning task was examined using paired t-tests to compare the direction and magnitude of the endpoint force vectors, peak forces and moments, and mean EMG reflex responses and onset latencies before and after each set of practice trials. To enable comparisons between the EXT and FLEX conditions that were tested on different days the data was expressed as a percent change from pre-post practice (i2-i1/i1*100) within each condition. The percent change in the direction and magnitude of the endpoint force vectors, peak forces and moments, and mean EMG reflex responses were then compared between EXT and FLEX conditions with independent t-tests. Onset latencies were compared between EXT and FLEX conditions as the absolute change from pre-post (ms). All data processing and statistical analyses were performed in MATLAB (Mathworks, MA, USA). Results were considered significant if p < 0.05.\n\n\nResults\n\nThe average withdrawal response evoked in the NEUT position for one subject is presented in Figure 2. Specifically, the withdrawal responses from all muscles except the erector spinae are displayed, along with the corresponding X- and Y-endpoint force traces. There were never any clear responses in the erector spinae, so that data is not presented. There were no differences in any of the EMG or force variables between the initial and follow-up NEUT position testing, suggesting there was minimal habituation to the stimulus, or at the least that the stimulation and recording conditions did not change. Onset latencies for the withdrawal responses were not significantly different across the arm positions for any of the muscles recorded. In addition, the amplitude of the withdrawal EMG response was not different across positions for any of the muscles shown in the grouped data.\n\nThe average onset time is indicated by the dashed line with the arrow. The DC offset was removed and the signals were rectified prior to averaging for each channel. The time window is from the stimulation onset-200ms.\n\nThe individual endpoint forces (Fx, Fy) and moments (Mx, My) were not different across positions. When the endpoint force vectors were calculated from the peak Fx (+x) and Fy (-y) magnitudes (posterior-lateral direction, see Figure 3), significant differences were present in the direction of withdrawal between the SHLFLEX and SHLEXT positions (p < 0.05). In addition, the magnitude of the endpoint force vector was significantly greater in the ELBEXT position than in the ELBFLEX or SHLFLEX positions (p = 0.025).\n\nA) Representative figure showing the arm in the NEUT position and the corresponding group force vector response to stimulation. B) Average force vector for all subjects in each position. The dashed line is the NEUT position. The lines with arrows are for the independent manipulation of the elbow angle, while the lines with circles correspond to changing shoulder angle. The same data from panel B is displayed in panels C and D, separated for the elbow and shoulder positions, respectively.\n\nThe average withdrawal reflex response before and after EXT and FLEX practice is shown for the PD muscle and the endpoint force vectors for one subject in Figure 4. The only significant group difference observed in reflex onset latency after EXT practice was in the PD muscle where the response was observed at 78 ± 11ms before practice and 84 ± 18ms after EXT practice (p = 0.001). In the FLEX condition the reflex onset latency was significantly delayed after practice in the BRD (86 ± 12ms–94 ± 18ms, p = 0.012), but no significant differences were noted for any other muscle in the FLEX condition. Furthermore, there were no significant differences in the pre-post absolute change in reflex latency (ms) between the EXT and FLEX conditions. Table 1 displays the variability in onset latency changes pre-post practice for each of the conditions. In addition, no differences were present in the EMG reflex response amplitudes for any of the muscles before and after practice, for either EXT or FLEX conditions. Similarly, there were no differences between the EXT and FLEX practice conditions, with the exception of the AD muscle that showed a significantly greater decrease in the EMG reflex response in the FLEX condition (p = 0.037).\n\nThe muscles listed are the biceps brachii long head (BBL), triceps brachii (TRI), brachioradialis (BRD), posterior deltoid (PD), anterior deltoid (AD), abductor pollicis brevis (APB), right erector spinae (ESR), and left erector spinae (ESL).\n\nThe individual endpoint peak forces and moments were not significantly different before and after either EXT or FLEX practice with the exception of the My-moment (pitch), which was significantly lower after EXT practice (p = 0.007). There were no differences in the direction or magnitude of the endpoint force vectors calculated from the peak Fx (+x) and Fy (-y)magnitudes (posterior-lateral direction) pre-post practice within either condition, or between EXT and FLEX conditions (Figure 5).\n\nThe inset on each panel shows the average for all subjects. No significant differences were noted.\n\n\nDiscussion\n\nThe purpose of the present study was to examine the adaptability of the nociceptive withdrawal reflex across different tasks. First, changing the elbow or shoulder joint angles independently resulted in a consistent nociceptive withdrawal response to the stimulation of the same receptive field on digit II. This conclusion is based on the pattern of withdrawal reflex activity in the upper-limb muscles being similar, regardless of the position of the limb. Some statistical differences were observed in the withdrawal force vectors, however, these differences did not correspond to changes in withdrawal reflex muscle activity across positions, suggesting they were due to altered limb biomechanics. More importantly, the direction of the withdrawal force response was relatively constant no matter the configuration of the upper-limb (see Figure 3). This was even the case when re-testing the NEUT position at the end of the experiment, where adjustments in limb position and repeated noxious stimuli did not show habituation in the nociceptive reflex, which has been observed previously2. Second, the nociceptive withdrawal response was resistant to change, at least to the short-term motor practice performed. While there were a few significant differences observed in individual EMG and force variables, the main observation was that the reflex responses were not consistently modified by either EXT or FLEX practice. If the nociceptive withdrawal reflexes in the upper limb are considered merely a protective mechanism then it would be expected that the response would be robust. These results lead us to conclude that the action-based shaping of nociceptive reflex modules in the spinal cord help to prevent erroneous adaptations that would potentially compromise safety.\n\nMuch of the work examining the organization of nociceptive withdrawal reflexes has demonstrated distinct excitatory and inhibitory nociceptive reflex responses depending on the receptive fields stimulated (see review by Clarke and Harris (2004)6). In the present study the withdrawal reflexes only elicited consistent excitatory responses in the upper-limb muscles regardless of the joint angle that was altered. This result is incongruent with the notion that the synergist muscles removing the limb should be excited while the opposing muscles should be inhibited3. It is difficult to ascertain the benefit of co-activating muscles throughout the entire upper limb in response to noxious stimulation, particularly if it slows down the physical withdrawal of the limb. Co-activation of the muscles throughout the upper limb in response to nociceptive stimulation could be specific to the spinal motor neurons being relatively quiescent during these trials, as reported previously26. There is also the potential that EMG cross-talk between opposing muscles (e.g. biceps brachii and triceps brachii) could have obscured smaller changes such as suppression, in particular with the muscle already quiescent27. Alternatively, there are distinct excitatory and inhibitory responses in upper limb muscles during movement, even demonstrating dependence on the phase of movement26,28. It has been suggested that tactile afferent input is organized by the actions the system performs routinely; for example, reaching and withdrawing the limb voluntarily10. If this nociceptive organization is linked to development13, or is action-based, it can be reasoned that the nociceptive withdrawal response at rest would be much less finely-tuned or that the synaptic input would be largely dispersed in the spinal cord. Specific to the present study, this rationale would also explain the similar directions of withdrawal for each of the arm configurations.\n\nThe original hypothesis that the nociceptive withdrawal reflex response would vary with the joint position was based on the expectation that each joint position would have an optimal withdrawal motor response and that the static muscle length changes around the elbow or shoulder joints would alter group II afferent input to interneurons that help to gate the withdrawal response20,21. It appears this assumption was too generalized since it is known that group II afferents from secondary muscle spindle endings have a relatively constant discharge rate and low discharge rate variability in static postures29–31; and thus the difference in group II afferent feedback may not have been sufficient to modulate nociceptive input. It has been suggested that the nociceptive withdrawal response arises from the integration of multiple sensory feedback sources, and that there is a complex sensorimotor transformation mediating the appropriate motor response8,10,15. In this case the group II afferent feedback alone would just be part of a larger, more complex set of sensory inputs that might be weighted differently across individual joints and arm positions. This would be feasible since there is a large amount of redundancy in multi-joint control of the upper limb and it has been reported that weighted sensory feedback in the upper limb improves control of the limb32. The five positions examined in the present study only encompassed a small area of the workspace of the upper limb, even confined to a single plane, so in this context very little weighting of sensory feedback would even be required to produce the same withdrawal response.\n\nNociceptive input is widely distributed to motorneurons innervating muscles across several joints and with different primary movements8, and because of this it was originally hypothesized that short-term motor practice would modulate the withdrawal reflex response. However, the results of the present study suggest that the motor practice the subjects performed was unable to alter this distribution of input. Presumably, this was because the practice was non-specific to the nociceptive withdrawal reflex, meaning that the subjects were practicing a voluntary motor task. However, the results of this study have caused us to re-examine the actual benefit of short-term plasticity in the nociceptive withdrawal reflex. Assuming the withdrawal reflex is organized with reflex modules, it is necessary for the reflex modules encoding nociceptive input to be developed over time and based on the motor actions commonly performed by the individual3,7,8,10,14,15,33,34. This being the case, short-term adaptations would likely compromise the built-in protective capabilities of the nociceptive system. This is not to suggest that habituation and dishabituation (wind-up) of the nociceptive pain pathways does not occur2,16–19, but rather that the musculotopic organization underlying the activation of specific reflex modules9, ultimately producing the motor action, is relatively insensitive to short-term practice. This remains in agreement with the demonstrated results, however the task utilized in the current study may fail to elicit plasticity within the withdrawal reflex.\n\nThe results presented suggest potential environmental factors that must be taken into account. The timeline for the purported action-based shaping of reflex modules remains unclear. As mentioned previously, the model of action-based sensory encoding or somatosensory imprinting has been described in early post-natal development13,15, where spinal organization can be determined by simple tactile feedback from spontaneous muscle twitches during sleep35,36. However, the mature nociceptive system is much less flexible, presumably because withdrawal receptive fields have already been optimized to eliminate or depress erroneous connections and strengthen appropriate connections11,12. Consequently, to override, or adapt the adult nociceptive reflex modules to new biomechanical, anatomical, or action-based constraints there would need to be a much greater amount of practice. It has been suggested that the long-term plasticity occurs as a result of a greater transfer of information between primary motor cortex (M1)-basal ganglia-cerebellum37. Alternatively, the initial phase of adaptation can occur in as little as 10–30 minutes of practice, and can be driven by the disfacilitation of intracortical circuits in M138,39. Consequently, the short-term motor practice in the current study could have been specifically targeting the primary motor cortex and was not sufficient to cause adaptations in cerebellar circuits and pathways mediated through the cerebellum.\n\nThe original purpose of the present study was to examine the adaptability of the nociceptive withdrawal reflex across different tasks. The manipulation of joint position and reflex conditioning would activate specific reflex modules that would result in an efficient, coordinated limb withdrawal distinctly different from the control. The present results do not directly support the notion that different reflex modules were activated by independently changing elbow or shoulder joint angles. However, this does not suggest that there is no modular organization in the spinal cord for nociceptive input; rather it could just provide additional evidence that the reflex modules are specific to the receptive field activated. Instead, we hypothesize that the current data implies there is an additional layer of complexity to the sensorimotor transformation of nociception, meaning that the convergence of weighted multisensory input in the spinal cord was integrated to activate the same reflex module(s) for each arm position. In addition, since there appears to be a specific reflex encoder for each muscle in the dorsal horn of the spinal cord34, the integration of multisensory input had to be similar for each muscle to result in the same pattern of activation. Chang et al.40 recently demonstrated that whole limb kinematics are preferentially preserved over individual joint kinematics following peripheral nerve injury in the hindlimb of cats, and a similar compensatory mechanism may be responsible for the preservation of the withdrawal force direction despite altering individual joint angles. Thus, while there appears to be very complex processing of nociceptive input through interneuronal spinal circuits, the limb is consistently withdrawn in a general posterior direction towards the body. This could be a natural consequence of development15,35, or could be specific to non weight-bearing muscles that receive fewer cross-spinal inputs. Though it has been demonstrated repeatedly that the nociceptive withdrawal system is not hardwired, as was first believed5, the results of the present study imply that the nociceptive reflex modules are also resistant to short-term adaptations from practicing motor actions. This is consistent with the protective function of the nociceptive withdrawal reflex, as it guards the body from erroneous responses that could cause further harm. The data collected in the current study demonstrates a level of complexity within the nociceptive withdrawal reflex that was previously unclear. The results suggest that the motor response of the nociceptive withdrawal reflex remains fixed, which is in agreement with the long-term development of nociceptive reflexes, and regardless of the specific mechanisms involved, the nociceptive withdrawal response in the upper limb serves as an efficient means of protecting the hand.",
"appendix": "Author contributions\n\nBoth authors were equally involved in the experimental design, data collection, data analysis, manuscript preparation, and manuscript revisions.\n\n\nCompeting interests\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\nDewald JP, Beer RF, Given JD, et al:Reorganization of flexion reflexes in the upper extremity of hemiparetic subjects. Muscle Nerve. (1999); 22: 1209–1221.\n\nFloeter MK, Gerloff C, Kouri J, et al:Cutaneous withdrawal reflexes of the upper extremity. Muscle Nerve. (1998); 21: 591–598.\n\nSchouenborg J, Kalliomaki J: Functional organization of the nociceptive withdrawal reflexes. I. Activation of hindlimb muscles in the rat. Exp Brain Res. (1990); 83: 67–78.\n\nSchomburg ED, Steffens H, Marx B: Nociceptive reflexes evoked by TTX-resistant C-fibre afferents and their sensitivity to opioids in the cat. Acta Physiol Pharmacol Bulg. (2001); 26: 193–196.\n\nEccles JC, Lundberg A: Synaptic actions in motoneurones by afferents which may evoke the flexion reflex. Arch Ital Biol. (1959); 97: 199–221.\n\nClarke RW, Harris J: The organization of motor responses to noxious stimuli. Brain Res Brain Res Rev. (2004); 46: 163–172.\n\nSchouenborg J, Holmberg H, Weng HR: Functional organization of the nociceptive withdrawal reflexes. II. Changes of excitability and receptive fields after spinalization in the rat. Exp Brain Res. (1992); 90: 469–478.\n\nSchouenborg J: Modular organisation and spinal somatosensory imprinting. Brain Res Brain Res Rev. (2002); 40: 80–91.\n\nLevinsson A, Holmberg H, Broman J, et al:Spinal sensorimotor transformation: relation between cutaneous somatotopy and a reflex network. J Neurosci. (2002); 22: 8170–8182.\n\nSchouenborg J: Action-based sensory encoding in spinal sensorimotor circuits. Brain Res Rev. (2008); 57: 111–117.\n\nHolmberg H, Schouenborg J: Developmental adaptation of withdrawal reflexes to early alteration of peripheral innervation in the rat. J Physiol. (1996); 495(Pt 2): 399–409.\n\nHolmberg H, Schouenborg J, Yu YB, et al:Developmental adaptation of rat nociceptive withdrawal reflexes after neonatal tendon transfer. J Neurosci. (1997); 17: 2071–2078.\n\nLevinsson A, Luo XL, Holmberg H, et al:Developmental tuning in a spinal nociceptive system: effects of neonatal spinalization. J Neurosci. (1999); 19: 10397–10403.\n\nSchouenborg J: Learning in sensorimotor circuits. Curr Opin Neurobiol. (2004); 14: 693–697.\n\nWaldenstrom A, Thelin J, Thimansson E, et al:Developmental learning in a pain-related system: evidence for a cross-modality mechanism. J Neurosci. (2003); 23: 7719–7725.\n\nArendt-Nielsen L, Brennum J, Sindrup S, et al:Electrophysiological and psychophysical quantification of temporal summation in the human nociceptive system. Eur J Appl Physiol Occup Physiol. (1994); 68: 266–273.\n\nArendt-Nielsen L, Sonnenborg FA, Andersen OK: Facilitation of the withdrawal reflex by repeated transcutaneous electrical stimulation: an experimental study on central integration in humans. Eur J Appl Physiol. (2000); 81: 165–173.\n\nHagbarth KE: Spinal withdrawal reflexes in the human lower limbs. J Neurol Neurosurg Psychiatry. (1960); 23: 222–227.\n\nKlein T, Magerl W, Hopf HC, et al:Perceptual correlates of nociceptive long-term potentiation and long-term depression in humans. J Neurosci. (2004); 24: 964–971.\n\nAlstermark B, Lundberg A, Pinter M, et al:Subpopulations and functions of long C3-C5 propriospinal neurones. Brain Res. (1987); 404: 395–400.\n\nAlstermark B, Lundberg A, Pinter M, et al:Long C3-C5 propriospinal neurones in the cat. Brain Res. (1987); 404: 382–388.\n\nCrowe A, Matthews PB: Further studies of static and dynamic fusimotor fibres. J Physiol. (1964); 174: 132–151.\n\nKofler M: Functional organization of exteroceptive inhibition following nociceptive electrical fingertip stimulation in humans. Clin Neurophysiol. (2003); 114: 973–980.\n\nKofler M, Stetkarova I, Wissel J: Nociceptive EMG suppression in triceps brachii muscle in humans. Clin Neurophysiol. (2004); 115: 1052–1056.\n\nRiley ZA, Krepkovich ET, Mayland EC, et al:Flexion-withdrawal reflexes in the upper-limb adapt to the position of the limb. Palo Alto, CA 2009.\n\nSerrao M, Pierelli F, Don R, et al:Kinematic and electromyographic study of the nociceptive withdrawal reflex in the upper limbs during rest and movement. J Neurosci. (2006); 26: 3505–3513.\n\nKofler M, Fuhr P, Leis AA, et al:Modulation of upper extremity motor evoked potentials by cutaneous afferents in humans. Clin Neurophysiol. (2001); 112: 1053–1063.\n\nDon R, Pierelli F, Ranavolo A, et al:Modulation of spinal inhibitory reflex responses to cutaneous nociceptive stimuli during upper limb movement. Eur J Neurosci. (2008); 28: 559–568.\n\nBurke D, Skuse NF, Stuart DG: The regularity of muscle spindle discharge in man. J Physiol. (1979); 291: 277–290.\n\nMatthews PB: Evidence that the secondary as well as the primary endings of the muscle spindles may be responsible for the tonic stretch reflex of the decerebrate cat. J Physiol. (1969); 204: 365–393.\n\nMatthews PB: A possible function for the secondary ending of the muscle spindle. J Physiol. (1969); 201: 102P–103P.\n\nBunderson NE, Ting LH, Burkholder TJ: Asymmetric interjoint feedback contributes to postural control of redundant multi-link systems. J Neural Eng. (2007); 4: 234–245.\n\nSchouenborg J, Weng HR: Sensorimotor transformation in a spinal motor system. Exp Brain Res. (1994); 100: 170–174.\n\nSchouenborg J, Weng HR, Kalliomaki J, et al:A survey of spinal dorsal horn neurones encoding the spatial organization of withdrawal reflexes in the rat. Exp Brain Res. (1995); 106: 19–27.\n\nPetersson P, Granmo M, Schouenborg J: Properties of an adult spinal sensorimotor circuit shaped through early postnatal experience. J Neurophysiol. (2004); 92: 280–288.\n\nPetersson P, Waldenstrom A, Fahraeus C, et al:Spontaneous muscle twitches during sleep guide spinal self-organization. Nature. (2003); 424: 72–75.\n\nPascual-Leone A, Nguyet D, Cohen LG, et al:Modulation of muscle responses evoked by transcranial magnetic stimulation during the acquisition of new fine motor skills. J Neurophysiol. (1995); 74: 1037–1045.\n\nClassen J, Liepert J, Wise SP, et al:Rapid plasticity of human cortical movement representation induced by practice. J Neurophysiol. (1998); 79: 1117–1123.\n\nRosenkranz K, Kacar A, Rothwell JC: Differential modulation of motor cortical plasticity and excitability in early and late phases of human motor learning. J Neurosci. (2007); 27: 12058–12066.\n\nChang YH, Auyang AG, Scholz JP, et al:Whole limb kinematics are preferentially conserved over individual joint kinematics after peripheral nerve injury. J Exp Biol. (2009); 212: 3511–3521."
}
|
[
{
"id": "2598",
"date": "02 Dec 2013",
"name": "Hong-You Ge",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of the article “Adaptability of the nociceptive withdrawal reflex” is not so specific to the current study. The title of the article should be confined to the main research results of the study, i.e., the nociceptive withdrawal reflex responses in the upper limb are resistant to the change of joint position and short term motor practice.The abstract represents a suitable summary of the work. The experimental design and methods are appropriate and the discussion is well balanced. The conclusion is justified on the basis of the results of the study.",
"responses": []
},
{
"id": "1087",
"date": "10 Dec 2013",
"name": "Brach Poston",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 purpose of the present study was to examine the adaptability of the nociceptive withdrawal reflex in different task conditions after motor practice. Specifically, this was accomplished by applying electrical stimulation to the second digit in a total of five different arm positions (shoulder and elbow joint angle combinations), both before and after motor practice performed either in the opposite or same direction as the stereotyped withdrawal response.The primary dependent measures of interest were the force responses recorded at the wrist and the EMG reflex responses obtained from six muscles of the upper limb. The main findings were:The amplitude and onset latencies of the EMG reflex responses were similar in all of the arm configurations for every muscle studied.There were minimal differences in the direction of the withdrawal force response for the different arm configurations. A single session of motor practice in either the opposite or same direction as the stereotyped withdrawal response did not change the pattern of the EMG or Force responses.The authors concluded that the nociceptive withdrawal response is resistant to short-term motor practice. Thus, if it is even possible to significantly modify the withdrawal reflex response, it would likely take extensive motor practice over a long period of time, which makes intuitive sense as it would not be beneficial for protective reflex responses to be modified by short-term perturbations. Therefore, it seems that the author’s interpretation of their findings is appropriate. Overall, the manuscript is very well-written, the data appear to have been collected carefully, and there are no major flaws or concerns, although the authors could probably have shortened the Introduction and Discussion sections.The topic addressed seems to be relatively novel as I don’t believe any previous studies in humans have addressed the degree to which the nociceptive withdrawal reflex can be modified in these task conditions with motor practice. Therefore, the current work makes a significant contribution to the current body of literature on the topic.To the credit of the authors, they presented a balanced and justified interpretation of the results even though some of the findings did not match their original hypotheses. In addition, they pointed out any limitations of their study, which will have to be addressed in future research.Finally, the focus of the research seems to be appropriate for F1000 Research and of interest to many readers of the journal.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-158
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https://f1000research.com/articles/2-279/v1
|
18 Dec 13
|
{
"type": "Research Article",
"title": "Prmt7 is dispensable in tissue culture models for adipogenic differentiation",
"authors": [
"Yu-Jie Hu",
"Saïd Sif",
"Anthony N. Imbalzano",
"Yu-Jie Hu",
"Saïd Sif"
],
"abstract": "Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.",
"keywords": [
"Research over the past 15 years has demonstrated the importance of protein arginine methylation in various biological processes including transcriptional regulation",
"DNA repair",
"RNA processing",
"and signal transduction1–5. The fact that hundreds of cellular proteins have been identified as the targets of protein arginine methylation supports the idea that arginine methylation regulates diverse cellular processes6. Arginine methylation is catalyzed by protein arginine methyltransferases (Prmts) that transfer a methyl group from S-adenosyl methionine (AdeMet) to protein substrates. To date",
"nine protein arginine methyltransferases have been identified in the mammalian genome and have been classified as type I",
"type II and type III enzymes by the activity of producing ω-NG",
"NG-asymmetric dimethylarginine (ADMA)",
"ω-NG",
"NG-symmetric dimethylarginine (SDMA)",
"and ω-NG-monomethylarginine (MMA)",
"respectively2",
"4",
"7."
],
"content": "Introduction\n\nResearch over the past 15 years has demonstrated the importance of protein arginine methylation in various biological processes including transcriptional regulation, DNA repair, RNA processing, and signal transduction1–5. The fact that hundreds of cellular proteins have been identified as the targets of protein arginine methylation supports the idea that arginine methylation regulates diverse cellular processes6. Arginine methylation is catalyzed by protein arginine methyltransferases (Prmts) that transfer a methyl group from S-adenosyl methionine (AdeMet) to protein substrates. To date, nine protein arginine methyltransferases have been identified in the mammalian genome and have been classified as type I, type II and type III enzymes by the activity of producing ω-NG,NG-asymmetric dimethylarginine (ADMA), ω-NG,NG-symmetric dimethylarginine (SDMA), and ω-NG-monomethylarginine (MMA), respectively2,4,7.\n\nAmong the family of Prmts, Prmt7 is unique in that it possesses two AdoMet-binding domains, which may have resulted from a gene duplication event8. As for many of the Prmts, histones are among the identified substrate molecules, suggesting a functional role for Prmt7 in regulating gene expression as a chromatin modifier. Initial work indicated that H2A and H4 were symmetrically dimethylated by Prmt7 in vitro9, whereas several studies have reported that Prmt7 only produces MMA on histones and other substrates8,10,11. A recent study indicated that Prmt7 symmetrically dimethylates H4R3 and H2AR3 in a manner that contributes to the repression of expression of genes involved in DNA repair12. Another study revealed that Prmt7-mediated H4R3 symmetric dimethylation antagonizes MLL4-catalyzed H3K4 methylation on neuron-specific gene promoters during differentiation13, suggesting that Prmt7 might negatively regulate tissue differentiation by its chromatin-modifying activity. Aside from the role in neuronal differentiation, the regulatory function of Prmt7 in the differentiation of other lineages has not been reported.\n\nAdipocyte differentiation is one of the most intensively studied differentiation processes. Both human and mouse mesenchymal stem cells (MSCs) that reside in fat pads and bone marrow undergo lineage commitment and terminal differentiation to become mature adipocytes14–16. The adipogenic differentiation process is controlled by a number of tissue-specific transcription factors, such as the CCAAT/enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptor gamma (PPARγ), and numerous chromatin remodeling and modifying enzymes including the ATP-dependent SWI/SNF complex and Prmts17–20. It has been shown that Prmt5 interacts with Brg1-based SWI/SNF complex21 and facilitates the binding of Brg1 to the PPARγ and to PPARγ target promoters to facilitate the activation of adipogenic genes19. Furthermore, Prmt4, which also has been shown to interact with Brg1-based SWI/SNF complex22, acts as a co-activator of PPARγ to promote adipocyte gene expression20. Prmt7 had been recently shown to interact with Brg1-based SWI/SNF complex12, but whether Prmt7 has functional roles in adipocyte differentiation remains unclear.\n\nIn the present study, we examined the role of Prmt7 in adipocyte differentiation in mouse C3H10T1/2 embryonic mesenchymal cells and in C/EBPα-reprogrammed murine NIH3T3 fibroblasts. By knocking down and over-expressing Prmt7, we showed that Prmt7 has no effect on lipid accumulation and adipogenic gene expression in differentiating cells. Based on the results, we concluded that Prmt7 is not required for differentiation in tissue culture models of adipogenesis.\n\n\nMethods\n\nMouse C3H10T1/2 and NIH3T3 cells were obtained from the ATCC. C3H10T1/2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Sigma) and 100U/ml of penicillin/streptomycin (Invitrogen). NIH3T3 cells were maintained in DMEM high glucose with 10% calf serum (Sigma). 293T and BOSC23 cells were obtained from S.N. Jones (UMass Medical School) and R.E. Kingston (Massachusetts General Hospital), respectively, and were grown in the same medium as C3H10T1/2 cells. For adipogenic differentiation, two-day postconfluent cells were differentiated with DMEM medium containing 10% FCS, 10μg/ml insulin, 0.5mM 3-isobutyl-1-methyxanthine, 1μM dexamethasone, and 10μM troglitazone (Sigma). After 48 hours incubation, media on the differentiating cells was replaced with media containing 5μg/ml insulin. Subsequently, the media was changed every other day until harvest. To evaluate cell proliferation, 1×105 cells were seeded in 6-well plates (Corning Inc.), and the number of viable cells was counted under a microscope (CK2, Olympus) each day from day 1 to day 4 with a hemocytometer (Hausser Scientific).\n\npENTR/pTER+ vector and pLentiX2 Dest vector were gifts from Dr. Eric Campeau (UMass Medical School). The preparation of lentiviral small hairpin RNA (shRNA) constructs was done as previously described12,23. Briefly, shPrmt7-1, shPrmt7-2 and scrambled control oligonucleotides were cloned into a pENTR/pTER+ vector. These constructs were individually incubated with the pLentiX2 DEST vector and LR clonase II enzyme mix (Invitrogen) to generate pLentiX2 DEST/ shPrmt7-1, pLentiX2 DEST/shPrmt7-2 and pLentiX2 DEST/shCtrl constructs. These lentiviral constructs were amplified in Stbl3 competent cells (Invitrogen) for generating lentiviruses. The pBABE puromycin empty vector (pBABE vector), FLAG-tagged PRMT7 construct (pBABE PRMT7) and pBABE CEBPα construct were previously described12,24 and were individually amplified in TOP10 competent cells (Invitrogen) for generating retroviral DNA as previously described12,24.\n\nThe preparation of viruses was performed as previously described23,25. Briefly, for lentiviruses, the packaging vectors pLP1, pLP2, pVSVG (Invitrogen) and pLentiX2 DEST/shRNA constructs were co-transfected into 293T cells with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. BOSC23 cells were used for pBABE-based retrovirus production. After 48 hours incubation, the supernatant was harvested and filtered through 0.45μm syringe filter (Millipore). For viral infection, 1ml of the filtered supernatant and 4μg/ml of polybrene (Sigma) were used to infect one million cells. After 48 hours incubation, virus infected cells were selected in 2.5μg/ml puromycin (Invitrogen).\n\nCells were washed twice with cold PBS and were harvested in RIPA buffer (50mM Tris-HCl pH7.4, 150mM NaCl, 1mM EDTA, 1% Nonidet P-40 (Thermo Scientific) and 0.25% sodium deoxycholate) supplemented with protease inhibitor cocktail (Roche). The samples were sonicated at high intensity setting for 3 minutes with 30sec on/off cycle in a Bioruptor (UCD-200, Diagenode). After quantifying the protein concentration by means of a Bio-Rad protein assay, the protein samples were then mixed with 4× SDS loading buffer (240mM Tris-HCl pH6.8, 8% SDS, 40% glycerol, 0.01% bromophenol blue and 10% β-mercaptoethanol) and boiled at 95°C for 10min. 30μg protein samples were separated on 10% SDS-PAGE and transferred onto PVDF membrane (Bio-Rad). The blots were blocked overnight in 3% non-fat milk (Essential Everyday). The next day, proteins were detected using specific antibodies (1:1000 dilution) and HRP-conjugated secondary antibodies (1:2000 dilution). The rabbit polyclonal antibodies against human PRMT7 (sc-98882) and rat C/EBPα (sc-61) were purchased from Santa Cruz Biotechnology. The mouse monoclonal antibody against mouse PPARγ (sc-7273) and the goat polyclonal antibody against human PRMT5 (sc-22132) were also purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-PI3K (ABS233) antibody was from Millipore. The secondary antibodies (NA9340 and NA9310) were purchased from GE Healthcare Life Sciences. The blots were developed on X-ray films with ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences). The signal intensity was quantified by ImageJ.\n\nTotal RNA was isolated from samples using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was prepared from 1μg of total RNA by Superscript III reverse transcriptase kit (Invitrogen). Quantitative PCR was performed on StepOne Plus real-time PCR machine with Fast SYBR Green Master mix (Applied Biosystems). The specific primers for gene expression analysis were:\n\nFasn forward 5′-CGTGTTGGCCTACACCCAGAGCT-3′;\n\nFasn reverse 5′-GGCAGCAGGGCCTCCAGCACCTT-3′;\n\nAdipoQ forward 5′-CAGTGGATCTGACGACACCA-3′;\n\nAdipoQ reverse 5′-CGAATGGGTACATTGGGAAC-3′;\n\nFabp4 forward 5′-GCGTGGAATTCGATGAAATCA-3′;\n\nFabp4 reverse 5′-CCCGCCATCTAGGGTTATGA-3′;\n\n5S rRNA forward 5′-GTCTACGGACATACCACCCTG-3′;\n\n5S rRNA reverse 5′-TACAGCACCCGGTATTCCCAG-3′.\n\nRelative expression levels were determined by the comparative Ct method26.\n\nDifferentiating cells were washed once with PBS and fixed in 10% phosphate-buffered formalin (Fisher Scientific) overnight. The next day, the fixed cells were washed with 60% isopropanol and air-dried completely. The cells were then stained with 60% Oil Red O (AMRESCO) for 10 minutes and washed repeatedly with tap water to remove excess stain. To quantify staining, Oil Red O was extracted from the cells with 100% isopropanol, and the optical density was measured at 500nm (OD500).\n\n\nResults\n\nThe C3H10T1/2 cell line was established from C3H mouse embryos and has served as a faithful cell culture model for mesenchymal lineage differentiation27–29. C3H10T1/2 cells can be differentiated into mature adipocytes by treating the confluent cells with a cocktail that contains insulin, dexamethasone, 3-isobutyl-1-methyxanthine (IBMX) and PPARγ ligands30. Using this model, we first examined Prmt7 protein levels during adipogenic differentiation by Western blot analysis. We found that Prmt7 protein levels are relatively constant from the onset of differentiation (day 0) through the day 6 post-differentiation (Figure 1A and Figure 1B) (Dataset 1). We concluded that Prmt7 protein levels were not altered in differentiating C3H10T1/2 cells.\n\n(A) Western blot analysis of the protein extracts from day 0 to day 6 post-differentiated C3H10T1/2 cells. Duplicate blots were probed with anti-Prmt7 antibody and with anti-PI3K antibody as a loading control. (B) The quantification of (A) by ImageJ. The levels of Prmt7 were normalized to PI3K loading control and presented as the relative expression levels to the day 0 sample (day 0=1). The data represent the average of two independent experiments (n=2).\n\nTo study the function of Prmt7, we used viral vectors to knock down or over-express Prmt7 in C3H10T1/2 cells. Two lentiviral constructs (pLentiX2 DEST/shPrmt7-1 and pLentiX2 DEST/shPrmt7-2) that encode shRNAs against Prmt7 mRNA were used for knocking down endogenous Prmt7 in proliferating C3H10T1/2 cells. A pBABE retroviral construct (pBABE-PRMT7) encoding FLAG-tagged PRMT7 was used to over-express PRMT7. The virus-infected cells were selected with puromycin and the levels of Prmt7 in the selected cells were examined by Western blot analysis (Figure 2A). Endogenous Prmt7 levels were reduced 10-fold or more in the knockdown cells compared to the scrambled shRNA control cells (Figure 2B). Prmt7 levels were increased more than 5-fold in the FLAG-tagged PRMT7 over-expression cells compared to the pBABE empty vector control (Figure 2B) (Dataset 2). Since Prmt5 is the major type II arginine methyltransferase and is also associated with SWI/SNF complexes21, we examined Prmt5 protein levels in our samples. We observed no changes in Prmt5 levels in Prmt7 knockdown and over-expression C3H10T1/2 cells (Figure 2A).\n\n(A) A representative Western blot analysis from proliferating C3H10T1/2 cells with Prmt7 knockdown and over-expression. Endogenous Prmt7 was specifically depleted by two different lentiviral shRNA constructs (shPrmt7-1 and shPrmt7-2). The scrambled shRNA lentiviral construct (shCtrl) was used as a control. The pBABE retroviral construct encoding FLAG-tagged PRMT7 (pBABE - PRMT7;12) was used to ectopically express PRMT7, and the pBABE empty vector (pBABE vector) was used as a control. Duplicate blots were probed with anti-Prmt7 and anti-Prmt5 antibodies and with anti-PI3K antibody as a loading control. (B) The quantification of Prmt7 in (A) by ImageJ. The levels of Prmt7 were normalized to the PI3K loading control and are presented as expression levels relative to the scrambled shRNA control or pBABE empty vector control. The data represent the average of two independent experiments (n=2).\n\nBoth Prmt7 and Prmt5 exhibit type II arginine methyltransferase activity. However, unlike Prmt5, Prmt7 has no effect on cell proliferation in NIH3T3 fibroblasts12. We measured the cell proliferation rate of Prmt7 knockdown and over-expression C3H10T1/2 cells, and found that neither the reduction of Prmt7 nor the over-expression of Prmt7 affected the proliferation of C3H10T1/2 cells (Figure 3) (Dataset 3). This result is consistent with the results from the previous study on NIH3T3 fibroblasts12.\n\nThe growth rates of control (shCtrl and pBABE vector), Prmt7 knockdown (shPrmt7-1 and shPrmt7-2) and Prmt7 over-expression (pBABE PRMT7) C3H10T1/2 cells were measured by seeding 1×105 cells in 6-well plates, and the viable cells were counted each day for 4 days after seeding. The data represent the average of two independent experiments (n=2) counted in duplicate. Error bars show the standard deviation.\n\nTo determine whether Prmt7 affects adipogenesis, the Prmt7 knockdown and over-expression C3H10T1/2 cells were grown to confluence and treated with the differentiation cocktail. At day 6 post-differentiation, the accumulation of intracellular neutral lipids was measured by Oil Red O staining. The Oil Red O staining showed similar levels of lipid accumulation in Prmt7 knockdown as well as Prmt7 over-expression cells as compared to the control cells. (Figure 4A and Figure 4B) (Dataset 4). This result suggests that the changes in Prmt7 levels did not affect lipid accumulation in differentiating C3H10T1/2 cells.\n\n(A) Representative Oil-Red O staining images of day 6 post-differentiated C3H10T1/2 cells in which Prmt7 was either knocked down (shPrmt7-1 and shPrmt7-2) or over-expressed (pBABE PRMT7). The Prmt7 knockdown and over-expression C3H10T1/2 cells were grown to confluence and differentiated 48 h later. At day 6 post-differentiation, the cells were fixed with 10% formalin and stained with Oil-Red O. (B) The quantification of (A). The Oil-Red O stain was extracted with 100% isopropanol and the optical density at 500nm (OD500) was determined. The data represent the average of two independent experiments (n=2) assayed in duplicate. Error bars show standard deviation. (C) A representative Western blot analysis of endogenous Prmt7, PPARγ and C/EBPα expression in day 6 post-differentiated C3H10T1/2 cells. The levels of PI3K are presented as a loading control. (D) Gene expression analysis on day 6 post-differentiated C3H10T1/2 cells. The mRNA levels of fatty acid synthase (Fasn), adiponectin (AdipoQ) and fatty acid binding protein 4 (Fabp4) were measured by RT-qPCR. The individual mRNA levels were normalized to 5S rRNA. The normalized expression levels of the control cells in one of the experiments were set as 1. The data are presented as the average of relative expression levels from two independent experiments (n=2) assayed in duplicate. Error bars show standard deviation.\n\nPPARγ and C/EBPα are the key transcription factors for adipogenic differentiation and for the maintenance of the adipocyte phenotype31–34. We examined the protein levels of PPARγ and C/EBPα in day 6 post-differentiation cells by Western blot analysis and found no significant difference in either Prmt7 knockdown or Prmt7 over-expression cells compared to the corresponding controls (Figure 4C). In addition, to rule out the possibility that Prmt7 functions as a cofactor of PPARγ and C/EBPα, we measured the mRNA expression levels of PPARγ and C/EBPα target genes in day 6 post-differentiation samples by real-time quantitative PCR (Figure 4D) (Dataset 5). We found that Prmt7 had no significant impact on fatty acid synthase (Fasn), adiponectin (AdipoQ), and fatty acid binding protein 4 (Fabp4) gene expression in the differentiating cells. These results suggest that Prmt7 is dispensable for adipogenic gene expression.\n\nPrevious studies had shown that ectopic expression of PPARγ or C/EBPα alone in non-adipogenic NIH3T3 cell line is able to reprogram NIH3T3 fibroblasts into adipocyte-like cells33,34. To test if Prmt7 is required for the reprogramming of NIH3T3 fibroblasts, we first knocked down and over-expressed Prmt7 in NIH3T3 fibroblasts by using the same viral constructs that we used in C3H10T1/2 cells, and confirmed the knockdown and over-expression of Prmt7 by Western blot analysis (Figure 5A). These cells were further infected with retroviruses encoding C/EBPα at 70% confluence. After the cells reached confluence, the differentiation cocktail was added to stimulate adipogenic differentiation. At day 6 post-differentiation, the accumulated lipid was evaluated by Oil Red O staining (Figure 5B). We found that neither knockdown nor over-expression of Prmt7 caused a significant difference in Oil Red O staining in C/EBPα-reprogrammed NIH3T3 fibroblasts, which is consistent with the results in C3H10T1/2 cells.\n\n(A) Western blot analysis on Prmt7 knockdown and over-expression NIH3T3 fibroblasts. Endogenous Prmt7 was specifically depleted by the lentiviral shRNA constructs (shPrmt7-1 and shPrmt7-2). The scrambled shRNA lentiviral construct (shCtrl) was used as a control. The pBABE retroviral construct encoding FLAG-tagged PRMT7 (pBABE PRMT7) was used to ectopically express PRMT7 and the pBABE empty vector was used as a control. The blot was probed with anti-Prmt7 antibody, and the PI3K levels are presented as a loading control. (B) Oil-Red O staining images of C/EBPα-reprogrammed NIH3T3 fibroblasts at day 6 post-differentiation. Prmt7 knockdown and over-expression NIH3T3 fibroblasts were infected with retroviruses encoding C/EBPα at 70% confluence. Two day post-confluent cells were differentiated. At day 6 post-differentiation, the cells were fixed with 10% formalin and stained with Oil-Red O.\n\n\n\n\nDiscussion\n\nChanges in gene expression during cell differentiation require alterations in higher-order chromatin organization as well as in local chromatin structure. Cells possess histone modifying enzymes and ATP-dependent chromatin remodeling enzymes to facilitate chromatin changes. The interplay between these two families of enzymes has been shown to be crucial for both transcription activation and repression18,21,35. Prmt7 was identified as a histone arginine methylating enzyme9–11 and was shown to associate with Brg1-based SWI/SNF ATP-dependent chromatin remodeling complex12. These findings led us to investigate the possible roles of Prmt7 in adipogenic differentiation, which is a process that requires the function of Brg1-based SWI/SNF complex24. Our data clearly showed that Prmt7 levels were significantly changed in the knockdown or over-expression cells, but manipulation of Prmt7 levels did not cause a differentiation deficiency. It is established that Brg1-based SWI/SNF complex is recruited to the adipogenic promoters upon differentiation24. However, whether Prmt7 associates with Brg1-based SWI/SNF complex at adipogenic promoters is still unknown. Since Prmt7 has no effect on adipogenic gene expression, we expect that Prmt7 is not recruited to adipogenic promoters. Alternatively, even if there is binding, the function of Prmt7 is dispensable at these loci.\n\nFunctional redundancy within the Prmt family has not been characterized. Prmt7 was classified as a type II and a type III arginine methyltransferase by characterization of its in vitro catalytic activity8–11. Whether other Prmts functionally compensate for Prmt7 is still unknown. The predominant type II arginine methyltransferase Prmt5 catalyzes the formation of MMA and SDMA in a nonprocessive fashion36,37. Type I arginine methyltransferases also produce MMA38–40. It is possible that Prmt5 or type I Prmts partially or fully compensate for the loss of Prmt7 in the cells. Our data showed that Prmt7 knockdown or over-expression has no effect on Prmt5 protein levels in C3H10T1/2 cells. This observation is consistent with the results from the previous study in HeLa cells41. However, we still cannot rule out the possibility that Prmt5 compensates for Prmt7 enzyme activity, even though Prmt5 protein levels remain constant. Further investigation is needed to address the possible crosstalk between Prmt5 and Prmt7 in chromatin regulation.\n\nTo our knowledge, Prmt7 knock-out or transgenic mice have not been reported. Whether changes in Prmt7 levels cause any developmental deficiencies in vivo remains unknown. However, several studies using cell lines or tissues have revealed regulatory roles for Prmt7 in tissue-specific gene expression. For example, PRMT7 negatively regulates neuronal differentiation of a human embryonal carcinoma cell line by repressing the expression of differentiation-specific genes13. In mouse germ cells, Prmt7 was recruited to the imprinting control region through physical interaction with CTCFL, a testis-specific nuclear protein, and repressed imprinted gene expression42. Furthermore, mouse embryonic stem cells and germ cells have relative high levels of Prmt7 compared with mouse embryonic fibroblasts43,44. This evidence suggests that Prmt7 might have important functions in the maintenance of stem cell pluripotency and that the down-regulation of Prmt7 might be required for early cell fate determination. Taken together, these data suggest that Prmt7 might have a role during early development.",
"appendix": "Author contributions\n\n\n\nANI and SS conceived the study. Y-JH, SS and ANI designed the experiments and reviewed the data. Y-JH and ANI wrote the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by NIH grants DK084278 to ANI and SS and GM56244 to ANI. ANI is a member of the UMass Medical School Diabetes and Endocrine Research Center, supported by the National Institutes of Health grant DK32520.\n\n\nAcknowledgements\n\nWe thank AR Barutcu and KM Imbalzano for comments on the manuscript.\n\n\nReferences\n\nBedford MT, Richard S: Arginine methylation an emerging regulator of protein function. Mol Cell. 2005; 18(3): 263–272. PubMed Abstract | Publisher Full Text\n\nBedford MT, Clarke SG: Protein arginine methylation in mammals: who, what, and why. Mol Cell. 2009; 33(1): 1–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee YH, Stallcup MR: Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation. Mol Endocrinol. 2009; 23(4): 425–433. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDi Lorenzo A, Bedford MT: Histone arginine methylation. FEBS Lett. 2011; 585(13): 2024–2031. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang Y, Bedford MT: Protein arginine methyltransferases and cancer. Nat Rev Cancer. 2013; 13(1): 37–50. PubMed Abstract | Publisher Full Text\n\nBoisvert FM, Cote J, Boulanger MC, et al.: A proteomic analysis of arginine-methylated protein complexes. Mol Cell Proteomics. 2003; 2(12): 1319–1330. PubMed Abstract | Publisher Full Text\n\nKarkhanis V, Hu YJ, Baiocchi RA, et al.: Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem Sci. 2011; 36(12): 633–641. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiranda TB, Miranda M, Frankel A, et al.: PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity. J Biol Chem. 2004; 279(22): 22902–22907. PubMed Abstract | Publisher Full Text\n\nLee JH, Cook JR, Yang ZH, et al.: PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine. J Biol Chem. 2005; 280(5): 3656–3664. PubMed Abstract | Publisher Full Text\n\nZurita-Lopez CI, Sandberg T, Kelly R, et al.: Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues. J Biol Chem. 2012; 287(11): 7859–7870. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeng Y, Maity R, Whitelegge JP, et al.: Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets R-X-R Sites in Lysine and Arginine-Rich Regions. J Biol Chem. 2013; (in press). PubMed Abstract | Publisher Full Text\n\nKarkhanis V, Wang L, Tae S, et al.: Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1. J Biol Chem. 2012; 287(35): 29801–29814. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDhar SS, Lee SH, Kan PY, et al.: Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4. Genes Dev. 2012; 26(24): 2749–2762. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang W, Zeve D, Suh JM, et al.: White fat progenitor cells reside in the adipose vasculature. Science. 2008; 322(5901): 583–586. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodeheffer MS, Birsoy K, Friedman JM: Identification of white adipocyte progenitor cells in vivo. Cell. 2008; 135(2): 240–249. PubMed Abstract | Publisher Full Text\n\nGesta S, Tseng YH, Kahn CR: Developmental origin of fat: tracking obesity to its source. Cell. 2007; 131(2): 242–256. PubMed Abstract | Publisher Full Text\n\nFarmer SR: Transcriptional control of adipocyte formation. Cell Metab. 2006; 4(4): 263–273. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde la Serna IL, Ohkawa Y, Imbalzano AN: Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers. Nat Rev Genet. 2006; 7(6): 461–473. PubMed Abstract | Publisher Full Text\n\nLeBlanc SE, Konda S, Wu Q, et al.: Protein arginine methyltransferase 5 (Prmt5) promotes gene expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and its target genes during adipogenesis. Mol Endocrinol. 2012; 26(4): 583–597. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYadav N, Cheng D, Richard S, et al.: CARM1 promotes adipocyte differentiation by coactivating PPARgamma. EMBO Rep. 2008; 9(2): 193–198. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPal S, Yun R, Datta A, et al.: mSin3A/histone deacetylase 2- and PRMT5-containing Brg1 complex is involved in transcriptional repression of the Myc target gene cad. Mol Cell Biol. 2003; 23(21): 7475–7487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu W, Cho H, Kadam S, et al.: A methylation-mediator complex in hormone signaling. Genes Dev. 2004; 18(2): 144–156. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCampeau E, Ruhl VE, Rodier F, et al.: A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS One. 2009; 4(8): e6529. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalma N, Xiao H, Mueller E, et al.: Temporal recruitment of transcription factors and SWI/SNF chromatin-remodeling enzymes during adipogenic induction of the peroxisome proliferator-activated receptor gamma nuclear hormone receptor. Mol Cell Biol. 2004; 24(11): 4651–4663. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgenstern JP, Land H: Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990; 18(12): 3587–3596. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong ML, Medrano JF: Real-time PCR for mRNA quantitation. Biotechniques. 2005; 39(1): 75–85. PubMed Abstract\n\nTaylor SM, Jones PA: Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell. 1979; 17(4): 771–779. PubMed Abstract | Publisher Full Text\n\nKonieczny SF, Emerson CP Jr: 5-Azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell. 1984; 38(3): 791–800. PubMed Abstract | Publisher Full Text\n\nTang QQ, Otto TC, Lane MD: Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci U S A. 2004; 101(26): 9607–9611. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCho YC, Zheng W, Yamamoto M, et al.: Differentiation of pluripotent C3H10T1/2 cells rapidly elevates CYP1B1 through a novel process that overcomes a loss of Ah Receptor. Arch Biochem Biophys. 2005; 439(2): 139–153. PubMed Abstract | Publisher Full Text\n\nLefterova MI, Zhang Y, Steger DJ, et al.: PPARgamma and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale. Genes Dev. 2008; 22(21): 2941–2952. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmidt SF, Jorgensen M, Chen Y, et al.: Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites. BMC Genomics. 2011; 12: 152. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreytag SO, Paielli DL, Gilbert JD: Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev. 1994; 8(14): 1654–1663. PubMed Abstract | Publisher Full Text\n\nTontonoz P, Hu E, Spiegelman BM: Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. 1994; 79(7): 1147–1156. PubMed Abstract | Publisher Full Text\n\nMusri MM, Gomis R, Parrizas M: Chromatin and chromatin-modifying proteins in adipogenesis. Biochem Cell Biol. 2007; 85(4): 397–410. PubMed Abstract | Publisher Full Text\n\nBranscombe TL, Frankel A, Lee JH, et al.: PRMT5 (Janus kinase-binding protein 1) catalyzes the formation of symmetric dimethylarginine residues in proteins. J Biol Chem. 2001; 276(35): 32971–32976. PubMed Abstract | Publisher Full Text\n\nWang M, Xu RM, Thompson PR: Substrate specificity, processivity, and kinetic mechanism of protein arginine methyltransferase 5. Biochemistry. 2013; 52(32): 5430–5440. PubMed Abstract | Publisher Full Text\n\nLin WJ, Gary JD, Yang MC, et al.: The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase. J Biol Chem. 1996; 271(25): 15034–15044. PubMed Abstract | Publisher Full Text\n\nOsborne TC, Obianyo O, Zhang X, et al.: Protein arginine methyltransferase 1: positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis. Biochemistry. 2007; 46(46): 13370–13381. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrankel A, Yadav N, Lee J, et al.: The novel human protein arginine N-methyltransferase PRMT6 is a nuclear enzyme displaying unique substrate specificity. J Biol Chem. 2002; 277(5): 3537–3543. PubMed Abstract | Publisher Full Text\n\nGonsalvez GB, Tian L, Ospina JK, et al.: Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins. J Cell Biol. 2007; 178(5): 733–740. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJelinic P, Stehle JC, Shaw P: The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation. PLoS Biol. 2006; 4(11): e355. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuhr N, Carapito C, Schaeffer C, et al.: Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells. Electrophoresis. 2008; 29(11): 2381–2390. PubMed Abstract | Publisher Full Text\n\nGirardot M, Hirasawa R, Kacem S, et al.: PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome. Nucleic Acids Res. 2013; (in press). PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3357",
"date": "27 Jan 2014",
"name": "Jeffrey M. Gimble",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 designed, well conducted, and well described study from a leading lab in the evaluation of histone mediated transcriptional regulation of adipogenesis. The authors provide compelling evidence that Prmt-7 is not directly involved in adipogenic regulation using loss of function/gain of function approaches in two independent pre-adipocyte models in vitro. The authors have evaluated their findings objectively and placed it appropriately in the context of the existing literature. The manuscript and figures are of top quality and require no further modification. The findings provide information that will benefit others in the scientific community by documenting the absence of a direct relationship between Prmt-7 and adipogenic mechanisms.",
"responses": []
},
{
"id": "3483",
"date": "13 Feb 2014",
"name": "Yaacov Barak",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 manuscript, Hu et al. performed a series of high-quality, well-designed and properly controlled experiments in search of potential functions of the protein arginine methylase Prmt7 in adipogenesis of C3H10T1/2 cell cultures. The motivation for the experiment was the group’s previous work that identified central functions of the closely related Prmt5 in the process, which led them to hypothesize guilt-by-homology. Moreover, hints of developmental functions of Prmt7 in other systems, such as neuronal differentiation, warranted testing a broader effect on differentiation in other system. The conclusion that Prmt7 has no bearing on adipogenesis is fully justified by the clean data, which showed no effect in both loss and gain-of-function approaches. It is reassuring that the experiment was conducted by the same group that identified the roles of Prmt5 in the process, and thus the negative data are likely not due to experimental system variability. In the Discussion, the authors allow that this lack of effect may be the result of compensation by redundant methylases; this is probably an overly cautious, “just-in-case” argument, considering that even in most cases of true redundancy there are at least some quantitative effects, which is not the case here whatsoever. It is more likely that arginine methylases are target-specific and have a clear partition of labor among them. With heaps of negative data buried in the archives of many laboratories which never see the light of day, the team has to be commended for publishing their own negative data to seal the issue and allow other research groups that might be drawn to the question to steer away and focus on more productive hypotheses.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-279
|
https://f1000research.com/articles/2-211/v1
|
10 Oct 13
|
{
"type": "Short Research Article",
"title": "Protein structure quality assessment based on the distance profiles of consecutive backbone Cα atoms",
"authors": [
"Sandeep Chakraborty",
"Ravindra Venkatramani",
"Basuthkar J. Rao",
"Bjarni Asgeirsson",
"Abhaya M. Dandekar",
"Ravindra Venkatramani",
"Basuthkar J. Rao",
"Bjarni Asgeirsson",
"Abhaya M. Dandekar"
],
"abstract": "Predicting the three dimensional native state structure of a protein from its primary sequence is an unsolved grand challenge in molecular biology. Two main computational approaches have evolved to obtain the structure from the protein sequence - ab initio/de novo methods and template-based modeling - both of which typically generate multiple possible native state structures. Model quality assessment programs (MQAP) validate these predicted structures in order to identify the correct native state structure. Here, we propose a MQAP for assessing the quality of protein structures based on the distances of consecutive Cα atoms. We hypothesize that the root-mean-square deviation of the distance of consecutive Cα (RDCC) atoms from the ideal value of 3.8 Å, derived from a statistical analysis of high quality protein structures (top100H database), is minimized in native structures. Based on tests with the top100H set, we propose a RDCC cutoff value of 0.012 Å, above which a structure can be filtered out as a non-native structure. We applied the RDCC discriminator on decoy sets from the Decoys 'R' Us database to show that the native structures in all decoy sets tested have RDCC below the 0.012 Å cutoff. While most decoy sets were either indistinguishable using this discriminator or had very few violations, all the decoy structures in the fisa decoy set were discriminated by applying the RDCC criterion. This highlights the physical non-viability of the fisa decoy set, and possible issues in benchmarking other methods using this set. The source code and manual is made available at https://github.com/sanchak/mqap and permanently available on 10.5281/zenodo.7134.",
"keywords": [
"Computational biology",
"protein structure prediction",
"Model quality assessment programs",
"Boltzmann distribution",
"Annnsen's thermodynamic hypothesis",
"statistical potentials",
"protein backbone",
"decoy sets"
],
"content": "Introduction\n\nThe structure of a protein is a veritable source of information about its physiological relevance in the cellular context1. In spite of rapid technical advances in crystallization techniques, the number of protein sequences known far exceeds the known structures. There are essentially two different computational approaches to predict protein structures from its primary sequence: 1) Template based methods (TBM) which are based on features obtained from the database of known protein structures2–4 and 2) ab initio or de novo methods which are based on the intrinsic laws governing atomic interactions and are applicable in the absence of a template structure with significant sequence homology5,6. While at present TBM methods fare much better than the de novo approaches, the requirement of a known template protein can sometimes be a constraining factor. Both these methods typically generate multiple possibilities for the native structure of a given sequence. Selecting the best candidate from the set of putative structures is an essential aspect that is performed by model quality assessment programs (MQAP).\n\nMQAPs can be classified as energy based, consensus based or knowledge based. The refinement of structures based on modeling of atomic interactions in energy based methods, such as molecular dynamic simulations, are subject to limited sampling of possible conformations due to large run times, and force field inaccuracies due to the approximations involved in describing the dynamics of large multi-atomic systems7–10. Consensus methods are based on the principle that sub-structures of the native structure are likely to feature frequently in a set of near-native structures11–14. These methods are currently the best performing amongst MQAPs13, but are prone to be computationally intensive due structure-to-structure comparison of all models14, and are of limited use when the number of possible structures is small15. Knowledge based methods rely on the assignment of an empirical potential (also known as statistical potential) from the frequency of residue contacts in the known structures of native proteins16,17. In statistical physics, for a system in thermodynamic equilibrium, the accessible states are populated with a frequency which depends on the free energy of the state and is given by the Boltzmann distribution. The Boltzmann hypothesis states that if the database of known native protein structures is assumed to be a statistical system in thermodynamic equilibrium, specific structural features would be populated based on the free energy of the protein conformational state. Sippl argued using a converse logic that the frequencies of occurrence of structural features such as interatomic distances in the database of known protein structures could determine a free energy (potential of mean force) for a given protein conformation, and thus be used to discriminate the native structure18,19. A crucial aspect in applying statistical potentials is the proper characterization of the reference state20. The application of such empirical energy functions to predict and assess protein structures, while quite popular, are vigorously debated21,22, and several approaches for using statistical potentials for protein structure prediction been described to date20,23–26.\n\nHere, we propose a new statistical potential based MQAP for assessing the quality of protein structures based on the distances of consecutive Cα atoms - Protein structure quality assessing based on Distance profile of backbone atoms (PROQUAD). We first propose a statistical potential based on the distance of consecutive Cα distances. In a set of high quality protein structures (top100H27), we demonstrate that the distance between consecutive Cα atoms are distributed normally with a mean of 3.8 Å and standard deviation of 0.04 Å. Based on this observation, the reference state for our statistical potential calculations is defined as one where all consecutive Cα atoms are 3.8 Å apart. We propose a scoring function which measures the deviation of consecutive Cα atoms from 3.8 Å, and hypothesize that this score is minimized in native structures. Based on the top100H database, we chose a cutoff of 0.012 Å for this scoring function to identify non-native states. We show that all the decoy structures from the fisa decoy set taken from the Decoys 'R' Us database28 are distinguished using this discriminator. It has been previously proposed that native structures have constrained interatomic distances29. Interatomic distances, and other metrics, have been combined in several such methods - Molprobity (http://molprobity.biochem.duke.edu/), PROSA (https://prosa.services.came.sbg.ac.at/prosa.php) and the WHATIF server (http://swift.cmbi.ru.nl/whatif)30–32. These identify possible anomalies in a given protein structure. While Molprobity and WHATIF identified steric clashes in the decoy structure in fisa, distance checks between consecutive Cα are not part of checks in these methods, and they failed to detect the consecutive Cα atoms anomaly in the fisa decoy set.\n\nThus, we propose a simple and fast discriminator for protein structure quality based on the distance profiles of consecutive backbone Cα atoms that identifies decoy structures that are physically nonviable.\n\n\nResults and discussion\n\nThe frequency distribution of the distance of consecutive Cα atoms in ~100 proteins in the top100H database (a database consisting of high quality structures)27 shows that the distance between consecutive Cα atoms are distributed normally with a mean of 3.8 Å and standard deviation of 0.04 Å (Figure 1a). Out of 16,162 pairs of consecutive Cα atom distances, 14,281 (88%) were spaced 3.8 Å apart, 1297 (8%) were spaced 3.9 Å apart and 553 (3%) were spaced 3.7 Å apart. Only 31 (0.1%) pairs of consecutive Cα atom distances had values different than these (highest being 4 Å and the lowest being 2.9 Å). It would be interesting to correlate these distance deviant residue pairs to structural or functional aspects of the protein - It is well worth examining every outlier and either correcting it if possible, giving up gracefully if it really cant be improved (more often true at low resolution), or celebrating the significance of why it is being held in an unfavorable conformation33.\n\n(a) Probability distribution (P(x)) for the distance of consecutive Cα in ~100 proteins in the top100H database. (b) RDCC in ~100 high quality structures from the top100H database. (c) Variation in specificity based on the cutoff value. We choose 0.012 Å as the cutoff for filtering out non-native structures.\n\nFigure 1b plots the root-mean-square deviation of the distance of consecutive Cα (RDCC) for these ~100 proteins. All structures in the top100H database have low RDCC values, barring three proteins (PDBids: 2ER7, 1XSO and 4PTP), which had multiple conformations for some residues, and were excluded from the processing. This validates our hypothesis that RDCC is minimized in native structures. Hence, structures that have a RDCC value more than a user specified threshold can be pruned out as structures with low quality or non-native structures.\n\nWe evaluated the results using the measures of specificity (the ability of a test to identify negative results) which is defined as:\n\n\n\n(TN = true negatives, FP = false positives). The specificity variation with the cutoff chosen is shown in Figure 1c. We choose 0.012 Å as the cutoff value for RDCC, which has a specificity of 1. We have applied this cutoff on decoy sets from the Decoys 'R' Us database28. The first protein (the native structure) in all decoy sets has RDCC below the 0.012 Å cutoff (Figure 1c). Figure 2 shows the RDCC for the hg_structral and fisa decoy sets from the Decoys 'R' Us database. All 500 decoy structures in each protein structure in the fisa decoy set are discriminated by applying the RDCC criterion. Figure 3 shows the superimposition of the native structure and the first decoy structure (AXPROA00-MIN) for a protein (PDBid: 1FC2) taken from the fisa decoy set. The distance between Ile12/Cα and Leu13/Cα atoms is 3.8 Å and 4.1 Å in the native and the decoy structures, respectively. According to our hypothesis, a 4.1 Å distance between consecutive Cα atoms is typically unfeasible in protein structures, and their occurrence should be relatively rare. The presence of such deviations throughout the protein structures categorizes it as a non-native structure. MolProbity30 and ProSA31 are two programs used as a pre-processing step for structures used in CASP34. MolProbity was able to discriminate the decoy structure (AXPROA00-MIN) from the native structure (PDBid: 1FC2) using a metric called the ClashScore (the number of serious steric overlaps) and the Cβ deviations35. PROSA was unable to discriminate between the decoy and the native structures, reporting equivalent Zscores of -4.12 and -5.28, respectively. The WHATIF server report also reports steric clashes in the decoy structures (Data File 1). None of the above mentioned methods use a metric similar to the RDCC proposed in this paper, and thus did not report the abnormal distance between consecutive Cα atoms in the decoy structure.\n\nThe hg_structal and misfold decoy sets are indistinguishable using the distance discriminator, unlike the fisa decoy set. We have shown ~25 decoy structures from the fisa set, but the values apply to all the decoys (more than 500). The first protein (the native structure) in each set has RDCC below the 0.012 Å cutoff.\n\nThe native structure is in red, and the decoy structure is in green. The structures are superimposed using MUSTANG53. The distance between Ile12/Cα and Leu13/Cα atoms is 3.8 Å and 4.1 Å in the native and the decoy structures, respectively.\n\nThe hg_structal and misfold decoy sets are indistinguishable using this distance discriminator, while only a few decoy structures failed in the 4state_reduced decoy set. This relationship between RDCC and proteins structure quality is therefore not an equivalence relationship. In propositional calculus, a relationship is equivalent if 'A' implies 'B' and 'B' implies 'A'. A high RDCC implies a low quality structure, but a low quality structure does not necessitate a high RDCC. We therefore suggest the usage of the RDCC measure as a first pass to rule out the non-native contacts prior to applying other discriminators.\n\nThe model quality assessment program (MQAP) used to choose the best structure from the multiple closely related structures generated by structure prediction programs is of critical importance. We have in the past used electrostatic congruence to detect a promiscuous serine protease scaffold in alkaline phos-phatases36 and a phosphoinositide-specific phospholipase C from Bacillus cereus37, and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio alkaline phosphatase38,39. However, continuum models40 that compute potential differences and pKa values from charge interactions in proteins41 are sensitive to the spatial arrangement of the atoms in the structure. Thus, an incorrect model will generate an inaccurate electrostatic profile of the peptide42. It is thus possible to functionally characterize a protein from its sequence by applying such in silico tools subsequent to the protein structure prediction and MQAPs tools43.\n\nThe estimation of the model quality by MQAPs is achieved by formalizing a scoring function44, referred to as a knowledge-based or statistical potential, constructed from the database of known structures, assuming that the distribution of the structural features obtained from these structures follows the Boltzmann distribution20,23,24,26. The validity of statistical potential and the method to choose a proper reference frame in such models are still widely debated21,22. Methods that use consensus values from numerous models outperform other MQAP methods11–14, and are 'very useful for structural meta-predictors'45. It has been shown that many of the MQAP programs perform considerably better when different statistical metrics are combined46–48. The state of the art methods for predicting structures49 and MQAPs34,45,50 are evaluated by researchers every two years.\n\nHere, we propose a discriminator (RDCC) based on the distance of consecutive Cα atoms in the peptide structure. The discriminator is independent of the database of structures51, and is thus an absolute discriminator. Our proposed RDCC criterion is satisfied in high quality protein structures taken from the top100H database. As a specific application, we show that all decoy structures in the fisa decoy set from the Decoys 'R' Us database Cα atoms do not satisfy this criterion. It has been previously shown that the fisa decoy set violates the van der Waals term52. We propose a fast complementary method to identify this transgression. It is also an interesting fact that most consensus methods will fare poorly in the fisa decoy set, since the majority of sub-structures are incorrect in all the decoy sets. Therefore, the fisa decoy set consists of physically nonviable structures and one should exercise caution when benchmarking other methods using this decoy set.\n\n\n\n\nMaterials and methods\n\nThe set of proteins Φproteins consists of the native structure P1 and M-1 decoys structures (Equation 2). We ignore the first x=IgnoreNTerm and last y=IgnoreCTerm pairs of residues in the protein structure to exclude the terminals (Equation 3). For every consecutive pair of residues in the structure we calculate the distance between the consecutive Cα atoms (Resn(Cα) and Resn+1(Cα)), and its deviation from the ideal value of 3.8 Å. The square of the summation of these deviations is then normalized based on the number of pairs processed, and results in the CADistScore. We hypothesize that CADistScoreP1 is minimum in a native structure (Equation 4). Algorithm 1 shows the pseudocode for the function that generates the CADistScore.\n\n\n\n\n\n\n\nInput: P1 : Protein under consideration\n\nInput: IgnoreNTerm: Ignore this number of residues in the N Terminal\n\nInput: IgnoreCTerm: Ignore this number of residues in the C Terminal\n\nOutput: CADistScore: Score indicating deviation of successive Cα atoms from 3.8 Å\n\nbegin\n\nCADistScore = 0 ; NumberCompared = 0 ; N = NumberOfResidues(P 1);\n\nfor p ← IgnoreNTerm to N – IgnoreCTerm do\n\nq = p + 1 ;\n\nCADist = Distance(p, q, Cα, Cα)\n\nNumberCompared = NumberCompared + 1 ;\n\ndiff = absolute(CADist – 3.8 Å) ;\n\nCADistScore = CADistScore + diff * diff;\n\nend\n\n/* Normalize */\n\nCADistScore = sqrt(CADistScore/(NumberCompared * NumberCompared));\n\nreturn (CADistScore);\n\nend\n\nIn order to validate our hypothesis on known structures, we applied our discriminator to the top100H database (a database consisting of high quality structures)27 - http://kinemage.biochem.duke.edu/databases/top100.php. In order to benchmark model quality assessment programs, we used decoy sets from the Decoys 'R' Us database28 - http://dd.compbio.washington.edu/. Each set has several structures that are supposed to be ranked worse than the native structure.\n\nStructural superimposition has been done using MUSTANG53. Protein structures were rendered by Py-Mol (http://www.pymol.org/). The source code and manual is made available at https://github.com/sanchak/mqap and permanently available from http://dx.doi.org/10.5281/zenodo.7134.",
"appendix": "Author contributions\n\n\n\nConceived and performed the experiments: SC. Analyzed the data, and improved experiments: SC BA AMD BJR RV. Wrote the manuscript: SC BA AMD BJR RV.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nBJ and RV acknowledge financial support from Tata Institute of Fundamental Research (Department of Atomic Energy). Additionally, BJR is thankful to the Department of Science and Technology for the JC Bose Award Grant. BA extends gratitude to the University of Iceland Research Found for supporting the project financially. AMD wishes to acknowledge grant #12-0130-SA from California Department of Food and Agriculture CDFA PD/GWSS Board.\n\n\nReferences\n\nWise EL, Rayment I: Understanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes. Acc Chem Res. 2004; 37(3): 149–158. PubMed Abstract | Publisher Full Text\n\nSoding J: Protein homology detection by HMM-HMM comparison. Bioinformatics. 2005; 21(7): 951–960. PubMed Abstract | Publisher Full Text\n\nPeng J, Xu J: RaptorX: exploiting structure information for protein alignment by statistical inference. Proteins. 2011; 79(Suppl 10): 161–171. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Y: Template-based modeling and free modeling by I-TASSER in CASP7. Proteins. 2007; 69(Suppl 8): 108–117. PubMed Abstract | Publisher Full Text\n\nWu S, Skolnick J, Zhang Y: Ab initio modeling of small proteins by iterative TASSER simulations. BMC Biol. 2007; 5: 17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRohl CA, Strauss CE, Misura KM, et al.: Protein structure prediction using Rosetta. Methods Enzymol. 2004; 383: 66–93. PubMed Abstract | Publisher Full Text\n\nChen J, Brooks CL: Can molecular dynamics simulations provide high-resolution refinement of protein structure? Proteins. 2007; 67(4): 922–930. PubMed Abstract | Publisher Full Text\n\nZhu J, Fan H, Periole X, et al.: Refining homology models by combining replica- exchange molecular dynamics and statistical potentials. Proteins. 2008; 72(4): 1171–1188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaval A, Piana S, Eastwood MP, et al.: Refinement of protein structure homology models via long, all-atom molecular dynamics simulations. Proteins. 2012; 80(8): 2071–2079. PubMed Abstract | Publisher Full Text\n\nLee MR, Tsai J, Baker D, et al.: Molecular dynamics in the endgame of protein structure prediction. J Mol Biol. 2001; 313(2): 417–430. PubMed Abstract | Publisher Full Text\n\nGinalski K, Elofsson A, Fischer D, et al.: 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics. 2003; 19(8): 1015–1018. PubMed Abstract | Publisher Full Text\n\nTerashi G, Oosawa M, Nakamura Y, et al.: United3D: a protein model quality assessment program that uses two consensus based methods. Chem Pharm Bull (Tokyo). 2012; 60(11): 1359–1365. PubMed Abstract | Publisher Full Text\n\nWallner B, Elofsson A: Prediction of global and local model quality in CASP7 using Pcons and ProQ. Proteins. 2007; 69(Suppl 8): 184–193. PubMed Abstract | Publisher Full Text\n\nAdamczak R, Pillardy J, Vallat BK, et al.: Fast geometric consensus approach for protein model quality assessment. J Comput Biol. 2011; 18(12): 1807–1818. PubMed Abstract | Publisher Full Text\n\nMcGuffin LJ: Benchmarking consensus model quality assessment for protein fold recognition. BMC Bioinformatics. 2007; 8: 345. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTanaka S, Scheraga HA: Model of protein folding: inclusion of short-, medium-, and long-range interactions. Proc Natl Acad Sci U S A. 1975; 72(10): 3802–3806. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiyazawa S, Jernigan RL: Estimation of effective interresidue contact energies from protein crystal structures: quasi-chemical approximation. Macromolecules. 1985; 18(3): 534–552. Publisher Full Text\n\nSippl MJ: Calculation of conformational ensembles from potentials of mean force. An approach to the knowledge-based prediction of local structures in globular proteins. J Mol Biol. 1990; 213(4): 859–883. PubMed Abstract | Publisher Full Text\n\nSippl MJ: Knowledge-based potentials for proteins. Curr Opin Struct Biol. 1995; 5(2): 229–235. PubMed Abstract | Publisher Full Text\n\nZhou H, Zhou Y: Distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction. Protein Sci. 2002; 11(11): 2714–2726. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThomas PD, Dill KA: Statistical potentials extracted from protein structures: how accurate are they? J Mol Biol. 1996; 257(2): 457–469. PubMed Abstract | Publisher Full Text\n\nHamelryck T, Borg M, Paluszewski M, et al.: Potentials of mean force for protein structure prediction vindicated, formalized and generalized. PLoS ONE. 2010; 5(11): e13714. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSamudrala R, Moult J: An all-atom distance-dependent conditional probability discriminatory function for protein structure prediction. J Mol Biol. 1998; 275(5): 895–916. PubMed Abstract | Publisher Full Text\n\nShen MY, Sali A: Statistical potential for assessment and prediction of protein structures. Protein Sci. 2006; 15(11): 2507–2524. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRajgaria R, McAllister SR, Floudas CA: A novel high resolution Calpha-Calpha distance dependent force field based on a high quality decoy set. Proteins. 2006; 65(3): 726–741. PubMed Abstract | Publisher Full Text\n\nLu H, Skolnick J: A distance-dependent atomic knowledge-based potential for improved protein structure selection. Proteins. 2001; 44(3): 223–232. PubMed Abstract | Publisher Full Text\n\nWord JM, Lovell SC, LaBean TH, et al.: Visualizing and quantifying molecular goodness-of-fit: small-probe contact dots with explicit hydrogen atoms. J Mol Biol. 1999; 285(4): 1711–1733. PubMed Abstract | Publisher Full Text\n\nSamudrala R, Levitt M: Decoys 'R' Us: a database of incorrect conformations to improve protein structure prediction. Protein Sci. 2000; 9(7): 1399–1401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEngh RA, Huber R: Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallographica Section. 1991; A47: 392–400. Publisher Full Text\n\nChen VB, Arendall WB, Headd JJ, et al.: MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 1): 12–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWiederstein M, Sippl MJ: ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 2007; 35(Web Server issue): W407–410. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVriend G: WHAT IF: a molecular modeling and drug design program. J Mol Graph. 1990; 8(1): 52–56. PubMed Abstract | Publisher Full Text\n\nRichardson J, Richardson D: The zen of model anomalies – correct most of them. Treasure the meaningful valid few. Live serenely with the rest! Advancing Methods for Biomolecular Crystallography. , Springer Netherlands: 2013; 1–10. Publisher Full Text\n\nKryshtafovych A, Monastyrskyy B, Fidelis K: Casp prediction center infrastructure and evaluation measures in casp10 and casp roll. Proteins. 2013. PubMed Abstract | Publisher Full Text\n\nLovell SC, Davis IW, Arendall WB, et al.: Structure validation by Calpha geometry: phi, psi and Cbeta deviation. Proteins. 2003; 50(3): 437–450. PubMed Abstract | Publisher Full Text\n\nChakraborty S, Minda R, Salaye L, et al.: Active site detection by spatial conformity and electrostatic analysis–unravelling a proteolytic function in shrimp alkaline phosphatase. PLoS ONE. 2011; 6(12): e28470. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRendon-Ramirez A, Shukla M, Oda M, et al.: A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold. PLoS ONE. 2013; 8(8): e70923. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHelland R, Larsen RL, Asgeirsson B: The 1.4 Å crystal structure of the large and cold-active Vibrio. sp. alkaline phosphatase. Biochim Biophys Acta. 2009; 1794(2): 297–308. PubMed Abstract | Publisher Full Text\n\nChakraborty S, Asgeirsson B, Minda R, et al.: Inhibition of a cold-active alkaline phosphatase by imipenem revealed by in silico modeling of metallo-β-lactamase active sites. FEBS Lett. 2012; 586(20): 3710–3715. PubMed Abstract | Publisher Full Text\n\nHonig B, Nicholls A: Classical electrostatics in biology and chemistry. Science. 1995; 268(5214): 1144–1149. PubMed Abstract | Publisher Full Text\n\nBashford D, Karplus M: pKa's of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry. 1990; 29(44): 10219–10225. PubMed Abstract | Publisher Full Text\n\nBaker NA, Sept D, Joseph S, et al.: Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A. 2001; 98(18): 10037–10041. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChakraborty S, Rao BJ: A measure of the promiscuity of proteins and characteristics of residues in the vicinity of the catalytic site that regulate promiscuity. PLoS ONE. 2012; 7(2): e32011. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen MY, Sali A: Statistical potential for assessment and prediction of protein structures. Protein Sci. 2006; 15(11): 2507–2524. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKryshtafovych A, Fidelis K, Tramontano A: Evaluation of model quality predictions in CASP9. Proteins. 2011; 79(Suppl 10): 91–106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBenkert P, Tosatto SC, Schomburg D: QMEAN: A comprehensive scoring function for model quality assessment. Proteins. 2008; 71(1): 261–277. PubMed Abstract | Publisher Full Text\n\nTosatto SC: The victor/FRST function for model quality estimation. J Comput Biol. 2005; 12(10): 1316–1327. PubMed Abstract | Publisher Full Text\n\nBagaria A, Jaravine V, Huang YJ, et al.: Protein structure validation by generalized linear model root-mean-square deviation prediction. Protein Sci. 2012; 21(2): 229–238. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoult J: A decade of CASP: progress, bottlenecks and prognosis in protein structure prediction. Curr Opin Struct Biol. 2005; 15(3): 285–289. PubMed Abstract | Publisher Full Text\n\nKryshtafovych A, Barbato A, Fidelis K, et al.: Assessment of the assessment: Evaluation of the model quality estimates in CASP10. Proteins. 2013. PubMed Abstract | Publisher Full Text\n\nBenkert P, Biasini M, Schwede T: Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics. 2011; 27(3): 343–350. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHandl J, Knowles J, Lovell SC: Artefacts and biases affecting the evaluation of scoring functions on decoy sets for protein structure prediction. Bioinformatics. 2009; 25(10): 1271–1279. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKonagurthu AS, Whisstock JC, Stuckey PJ, et al.: MUSTANG: a multiple structural alignment algorithm. Proteins. 2006; 64(3): 559–574. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2091",
"date": "30 Oct 2013",
"name": "Bairong Shen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 an absolute discriminator to identify non-native protein structures based on backbone Cα atom distance. Interestingly the authors found that most methods performed poorly in the fisa decoy set from the Decoys “R” Us database and remind researchers to be cautious of using this decoy set since most of the sub-structures in the fisa decoy set are incorrect. This work provides a simple and fast assessment for protein structure quality.",
"responses": [
{
"c_id": "624",
"date": "18 Nov 2013",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Shen,Thank you for taking the time to review our manuscript. We appreciate your encouraging comments. We have revised our manuscript with some additional results from the CASP8 I-TASSER decoy set, and also cited some recent manuscripts which had been published since the version 1 went online. We hope you find the revised version improved.Warm regards,Sandeep"
}
]
},
{
"id": "2086",
"date": "06 Nov 2013",
"name": "Simon Lovell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nice, straightforward analysis of features of protein decoy sets. The authors find that a simple, novel measure of protein geometry is sufficient to distinguish native structures from decoys in the popular fisa decoy set. A rational response to this work would be to include measures of geometry such as that which are used here in the generation of the decoys. This would make the measure less useful, but would improve decoy sets.",
"responses": [
{
"c_id": "623",
"date": "18 Nov 2013",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Lovell,Thank you for your encouraging comments on our manuscript. We took a lot of inspiration from your work \"Artefacts and biases affecting the evaluation of scoring functions on decoy sets for protein structure prediction. Bioinformatics. 2009\" (ref 52), while writing our manuscript.Additionally, we have revised our manuscript adding some additional results we have obtained on the CASP8 I-TASSER decoy set.Warm regards,Sandeep"
}
]
}
] | 1
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https://f1000research.com/articles/2-211
|
https://f1000research.com/articles/2-277/v1
|
17 Dec 13
|
{
"type": "Case Report",
"title": "Endovascular treatment of chronic mesenteric ischemia in an adolescent male: case report",
"authors": [
"Sadiq Muhammed Al-Hammash",
"Abd El-Salam Dawood Al-Ethawi",
"Kasim Abbas Ismail",
"Sadiq Muhammed Al-Hammash",
"Kasim Abbas Ismail"
],
"abstract": "Chronic mesenteric ischemia (CMI) is a condition characterized by inadequate blood flow to the bowel resulting from stenosis of one or more of the three mesenteric arteries. Fibromuscular dysplasia is the most common cause in children and young adults; whereas atherosclerosis is the most common cause in the elderly. Treatment is mandatory in symptomatic patients, because it may lead to malnutrition, bowel infarction or even death.Herein, we present the case of a 14-year old male, diagnosed with CMI who underwent percutaneous balloon angioplasty (PTA) followed by stent placement with immediate positive results.",
"keywords": [
"fibromuscular dysplasia",
"mesenteric ischemia",
"endovascular procedures",
"percutaneous transluminal angioplasty"
],
"content": "Introduction\n\nChronic mesenteric ischemia (CMI) is a condition characterized by inadequate blood flow to the bowel resulting from stenosis of one or more of the three mesenteric arteries: the celiac artery (CA), the superior mesenteric artery (SMA), and the inferior mesenteric artery (IMA). Many case-series include patients with ischemic bowel due to stenosis of a single artery1–3.\n\nFibromuscular dysplasia (FMD) is a rare disorder of medium-sized arteries with no signs of inflammation or atherosclerosis4. It is the most common cause of stenosis in children and young adults. The reported worldwide incidence is around 1%5.\n\nRenal arteries (60–75%) and cerebral arteries (25–30%) are the most commonly involved vasculatures; followed by the mesenteric, iliac, and subclavian arteries5.\n\nIn those older than 60 years atherosclerotic plaques are the most common cause of stenosis of the mesenteric arteries, with females affected three times more often than males6.\n\nSymptomatic untreated bowel ischemia may cause malnutrition or acute bowel ischemia with infarction –a complication that is associated with a bad prognosis6. A fatal outcome has been reported in some cases7. Therefore, treatment is mandatory in symptomatic cases.\n\nThe gold standard therapeutic option is surgical (either a bypass or endarterectomy) with a combined morbidity of 15–47% and a mortality of 0–17%8–12. Stenoses of the mesenteric vessels are focal, and found at the ostium or proximal part of the artery – two features that make them accessible to transluminal therapy. Endovascular treatment of bowel ischemia was first described in 198013 and subsequently good results were found in two small studies14,15. Encouraged by these results, several centers, including ours, started using transluminal angioplastic techniques as the first line therapy for mesenteric ischemia.\n\nHerein, we present the case of a 14-year old Arabic Muslim male from the center of Iraq who was diagnosed with chronic mesenteric ischemia and who underwent a percutaneous balloon angioplasty (PTA) followed by stent placement with immediate positive results.\n\n\nCase report\n\nThe condition started in infancy when the child had recurrent attacks of respiratory tract infections and exhibited a failure to thrive. He was diagnosed at that time as a case of moderate-sized patent ductus arteriosus (PDA) and small restrictive perimembranous ventricular septal defect (VSD). The patient had been prescribed anti- heart failure medications (in the form of digoxin oral drops (10 mcg/kg/day in two divided doses, furosemide 1 mg/kg bid by mouth (PO), and captopril 0.5 mg/kg by mouth per day (QD PO)) and the patient had frequent follow-up visits. His PDA had been closed via a transcatheter approach using an Amplatzer® ductal occluder device at the age of 8 months and the VSD closed spontaneously as suggested by clinical follow-up exams and confirmed by echocardiography. The child’s medication was stopped at the age of 2 years and he was thriving well.\n\nOne year ago (at the age of 13), the patient started to develop post-prandial nausea and abdominal pain necessitating a complete work-up including investigations to exclude peptic ulcer disease (PUD) as a cause of these symptoms.\n\nThe patient was referred back to our center by his pediatrician to exclude any complicating event associated with his ex-PDA and VSD. An initial clinical examination was unremarkable. A screening echocardiography was performed that showed turbulence in the abdominal aorta near the origin of the celiac artery (CA), which raised the possibility of chronic mesenteric ischemia as the cause of his symptoms.\n\nCatheterization was done and a 6 F short sheath inserted in the right femoral artery. A non-selective abdominal aortography followed by selective angiography using a 6 F Judkins Right® (JR) catheter confirmed the presence of a narrowing at the ostial and proximal segments of the celiac artery. The length of the narrowed segment, the diameter of the stenosed segment and the nearby normal segment were measured, expert consultation was done, and the patient was scheduled for subsequent therapeutic catheterization including percutaneous balloon angioplasty (PTA) with or without stent placement (Figure 1A).\n\nA. Non-selective angiography (lateral view) showing ostial and proximal segment stenosis of the celiac artery. (The inset shows a close-up view of the stenosis; arrow). B. PTA balloon inflated showing “waist” formation (arrow). C. Residual stenosis (arrow) after initial PTA. D. Stent in-situ with no residual stenosis (arrow).\n\nSix weeks later (after being prepared with aspirin 100 mg QD PO for seven days and clopidogrel 75 mg QD PO for three days, a 6 F short sheath introducer was inserted in the right femoral artery and another 6 F hydrophilic short sheath introducer was inserted in the right radial artery after confirming palmar arch patency using modified Allen's test16 and skin infiltration with 1.5 ml lidocaine as a local anaesthetic. Heparin 5000 IU was given intravenously immediately after inserting the radial sheath and 200 mcg nitroglycerine was administered via the sheath. The femoral sheath was used for monitoring, and non-selective angiography (for proper localization of the balloon), while the radial sheath was used to deliver the balloon (with or without the stent) into the celiac artery. A 6 French guiding catheter was passed over a 0.030\" exchange guide wire (GW) reaching up to the ostium of the CA from the radial artery. It was then replaced with a 0.014\" soft-tipped GW, which was passed a few centimeters within the CA. A 4 mm diameter percutaneous coronary angioplasty (PTCA) balloon was passed over the 0.014' GW and dilatation of the ostial and proximal segment of the CA was done (reaching nominal pressure and kept inflated for 30 seconds) (Figure 1B and C). A residual stenosis of about 40% was noticed after the balloon angioplasty, necessitating the use of a Promus® drug-eluting stent (PROMUS Element™ Plus; Everolimus-Eluting Platinum Chromium Coronary Stent System), premounted on a balloon, measuring 4 mm in diameter and 12 mm in length. The balloon was inflated just-below burst pressure and kept inflated for 30 seconds. An angiography conducted after this procedure, showed a diameter equal to the nearby normal segment (Figure 1D).\n\nThe radial artery sheath was removed immediately after completing the procedure and hemostasis was secured using a TR band® (compression strap) and a femoral artery sheath, removed after 60 minutes.\n\nThere were no immediate post-operative complications. The patient was discharged on the second day after the operation and was instructed to take both aspirin 100 mg/day QD PO and clopidogrel 75 mg/day QD PO for the following six months followed by use of aspirin alone for life. In the subsequent follow-up visits conducted one- and six-months after the procedure, there was neither post-prandial abdominal pain, nor nausea and the patient gained 7 kilograms weight over 6 months.\n\n\nDiscussion\n\nUntreated chronic mesenteric ischemia (CMI) can lead to symptoms of post-prandial abdominal pain, malnutrition, or even death. Therefore, a symptomatic patient needs treatment. The conventional treatment is surgical via a bypass surgery with or without endarterectomy6. Transluminal endovascular intervention, first described in 198013 and the good results obtained by two small studies encouraged many centers- including ours- to adopt the endovascular treatment as the first line therapy.\n\nThe aims of therapy are to: 1) achieve symptomatic resolution, 2) prevent bowel infarction and resultant morbidity and mortality and 3) prevent and treat malnutrition. Some groups also recommend prophylactic revascularization even in asymptomatic patients who are due for an operation in the aorta (e.g. aneurysm, coarctation, etc.) or before being scheduled for major abdominal surgery that might jeopardize the collateral circulation6.\n\nThe patient had a moderate–sized PDA and small restrictive VSD diagnosed in infancy. The former was closed by a transcatheter approach and the latter closed spontaneously. Up to the time of writing this article, the authors were unable to find a study showing an association between these two defects and mesenteric ischemia due to celiac artery stenosis. As such these findings appear coincidental.\n\nTwo catheterization sessions were needed in our patient because expert consultation and support was needed. However, if the patient is well-prepared in terms of antiplatelet medications (seven days of aspirin and three days of clopidogrel in our center); equipment and expert consultation is available, one catheterization session might suffice for confirming the diagnosis, planning, measuring and proceeding with angioplasty with or without stent placement.\n\nSince the initial PTA result was unsatisfactory with 40% residual stenosis, a drug-eluting covered stent (PROMUS Element™ Plus; Everolimus-Eluting Platinum Chromium Coronary Stent System) with a length ensuring 1 mm protrusion in the aortic lumen and 1–2 mm coverage of the nearby normal-width segment was inserted. The diameter chosen depends on the diameter of the nearby normal segment (we added 1 mm to the diameter of the normal segment).\n\nSome centers also recommend using stents in the following situations: 1) lesions located at the ostium or eccentric lesions, 2) History of dissection after a previous angioplasty and 3) Chronic stenosis17.\n\nSince the take-off of the celiac artery has an acute angle to the aortic wall, the superior approach (i.e. radial) provides a smoother curve and easier access of this artery. For that reason we chose the right radial artery after using a modified Allen test16 to confirm patency of the palmar arch.\n\nThe use of vasodilators (nitroglycerine) given in the sheath is necessary to prevent radial artery vasospasm.\n\n\nConclusion\n\nSymptomatic CIM can be found in the pediatric age group. We contribute this case report to the growing body of information regarding the role and effectiveness of endovascular treatment of this condition. Further research is needed to elucidate the “long-term” efficacy and safety of this therapeutic option.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient's parents.",
"appendix": "Author contributions\n\n\n\nSadiq, Abd El-Salam, and Kasim conceived the study. Sadiq prepared the patient for the procedure in addition to Kamal and Amer. Sadiq and Abd El-Salam conducted the procedure and Kasim monitored its technical aspects. Abd El-Salam wrote the first draft of the manuscript. All authors had been involved in revising the manuscript and agreed to its final contents.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors acknowledge Dr. Kamal Ali and Dr. Amer Na'aes for their medical care for the patient pre- and postoperatively and for technical support in preparing the images displayed in this article.\n\n\nReferences\n\nSharafuddin MJ, Olson CH, Sun S, et al.: Endovascular treatment of celiac and mesenteric arteries stenoses: application and results. J Vasc Surg. 2003; 38(4): 692–8. PubMed Abstract | Publisher Full Text\n\nMaspes F, Mazzetti di Pietralata G, Gandini R, et al.: Percutaneous transluminal angioplasty in the treatment of chronic mesenteric ischemia: results and 3 years of follow-up in 23 patients. Abdom Imaging 1998; 23(4): 358–63. PubMed Abstract | Publisher Full Text\n\nSteinmetz E, Tatou E, Favier-Blavoux C, et al.: Endovascular treatment as first choice in chronic intestinal ischemia. Ann Vasc Surg. 2002; 16(6): 693–9. PubMed Abstract | Publisher Full Text\n\nHarrison EG Jr, McCormack LJ: Pathologic classification of renal arterial disease in renovascular hypertension. Mayo Clin Proc. 1971; 46(3): 161–7. PubMed Abstract\n\nSlovut DP, Olin JW: Fibromuscular dysplasia. N Engl J Med. 2004; 350(18): 1862–71. PubMed Abstract | Publisher Full Text\n\nLoffroy R, Steinmetz E, Guiu B, et al.: Role of endovascular therapy in chronic mesenteric ischemia. Can J Gastroenterol. 2009; 23(5): 365–73. PubMed Abstract | Free Full Text\n\nGuill CK, Benavides DC, Rees C, et al.: Fatal mesenteric fibromuscular dysplasia: a case report and review of literature. Arch Intern Med. 2004; 164(10): 1148–53. PubMed Abstract | Publisher Full Text\n\nAbuRahma AF, Stone PA, Bates MC, et al.: Angioplasty/stenting of the superior mesenteric artery and celiac trunk: early and late outcomes. J Endovasc Ther. 2003; 10(6): 1046–53. PubMed Abstract | Publisher Full Text\n\nKasirajan K, O'Hara PJ, Gray BH, et al.: Chronic mesenteric ischemia: open surgery versus percutaneous angioplasty and stenting. J Vasc Surg. 2001; 33(1): 63–71. PubMed Abstract | Publisher Full Text\n\nRose SC, Quigley TM, Raker EJ: Revascularisation for chronic mesenteric ischemia: comparison of operative arterial bypass grafting and percutaneous transluminal angioplasty. J Vasc Interv Radiol. 1995; 6(3): 339–49. PubMed Abstract | Publisher Full Text\n\nCunningham CG, Reilly LM, Rapp JH, et al.: Chronic visceral ischemia: Three decades of progress. Ann Surg. 1991; 214(3): 276–88. PubMed Abstract | Free Full Text\n\nMateo RB, O'Hara PJ, Hertzer NR, et al.: Elective surgical treatment of symptomatic chronic mesenteric occlusive disease: early results and late outcomes. J Vasc Surg. 1999; 29(5): 821–31. PubMed Abstract | Publisher Full Text\n\nFurrer J, Grüntzig A, Kugelmeier J, et al.: Treatment of abdominal angina with percutaneous dilatation of an arteria mesenterica superior stenosis. Preliminary communication. Cardiovasc Intervent Radiol. 1980; 3(1): 43–4. PubMed Abstract | Publisher Full Text\n\nMatsumoto AH, Tegtmeyer CJ, Fitzcharles EK, et al.: Percutaneous transluminal angioplasty of visceral arterial stenoses: results and long-term clinical follow-up. J Vasc Interv Radiol. 1995; 6(2): 165–74. PubMed Abstract | Publisher Full Text\n\nAllen RC, Martin GH, Rees CR, et al.: Mesenteric angioplasty in the treatment of chronic intestinal ischemia. J Vasc Surg. 1996; 24(3): 415–21. PubMed Abstract | Publisher Full Text\n\nBarone JE, Madlinger RV: Should an Allen test be performed before radial artery cannulation? J Trauma. 2006; 61(2): 468–70. PubMed Abstract | Publisher Full Text\n\nKougias P, El Sayed HF, Zhou W, et al.: Management of chronic mesenteric ischemia: The role of endovascular therapy. J Endovasc Ther. 2007; 14(3): 395–405. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "4553",
"date": "25 Apr 2014",
"name": "Madathipat Unnikrishnan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an excellent and well-written case of a 14 year old boy who reported with features of mesenteric angina due to ostioproximal stenosis of the coeliac trunk managed by percutaneous angioplasty combined with stenting. Technical success was followed by clinical relief of symptoms along with considerable weight gain for the boy as expected.However, I wish to place a few of my thoughts and suggestions regarding this case scenario: The last line of the introduction (just prior to Case report) is redundant. Isolated coeliac artery stenosis is rarely the cause for mesenteric angina in the presence of a full calibre superior mesenteric artery. Also ostioproximal lesion is not typical of FMD which usually involves the body of an artery (Renal /internal carotid). In this 14 year old boy, having had congenital cardiac (VSD and PDA)lesions, rarely found coeliac artery syndrome might have been the cause that had given rise to the symptoms described.I congratulate the authors and suggest they also discuss the possibility or otherwise of coeliac artery syndrome in discussion which would make this a tidy and valuable contribution.",
"responses": [
{
"c_id": "795",
"date": "28 Apr 2014",
"name": "Madathipat Unnikrishnan",
"role": "Reviewer Response",
"response": "In continuation to my referee report, in the Conclusion the authors should change 'CIM' to 'CMI'"
}
]
},
{
"id": "4650",
"date": "13 May 2014",
"name": "Lefeng Qu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reported a case of a 14-year old male, diagnosed with CMI who underwent PTA followed by stent placement with immediate positive results.The history was reported clearly, and the procedure was described in detail. The authors also pointed out their experiences concerning the tips of the procedure, such as choosing the right radial artery as the approach to overcome the acute angle of the celiac artery, and the use of nitroglycerine to prevent radial artery vasospasm.Generally, this is an interesting article, and has certain merits for indexing.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-277
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https://f1000research.com/articles/2-276/v1
|
17 Dec 13
|
{
"type": "Opinion Article",
"title": "Creating Minimum Harm Practice (MiHaP): a concept for continuous improvement",
"authors": [
"Ranjit Singh"
],
"abstract": "The author asks for the attention of leaders and all other stakeholders to calls of the World Health Organization (WHO), the Institute of Medicine (IOM), and the UK National Health Service (NHS) to promote continuous learning to reduce harm to patients. This paper presents a concept for structured bottom-up methodology that enables and empowers all stakeholders to identify, prioritize, and address safety challenges. This methodology takes advantage of the memory of the experiences of all persons involved in providing care. It respects and responds to the uniqueness of each setting by empowering and motivating all team members to commit to harm reduction. It is based on previously published work on “Best Practices Research (BPR)” and on “Systematic Appraisal of Risk and Its Management for Error Reduction (SARAIMER)”. The latter approach, has been shown by the author (with Agency for Healthcare Research and Quality (AHRQ) support), to reduce adverse events and their severity through empowerment, ownership and work satisfaction. The author puts forward a strategy for leaders to implement, in response to national and international calls for Better health, Better care, and Better value (the 3B’s of healthcare) in the US Patient Protection and Affordable Care Act. This is designed to enable and implement “A promise to learn- a commitment to act”. AHRQ has recently published “A Toolkit for Rapid-Cycle Patient Safety and Quality Improvement” that includes an adapted version of SARAIMER.",
"keywords": [
"The September 2012 Institute of Medicine (IOM) report1 called for higher quality at lower cost through leadership that fosters continuous learning. It makes ten important recommendations (e.g. “involve patients and families in decisions regarding health and health care",
"tailored to fit their preferences” and “continuously improve health care operations to reduce waste",
"streamline care delivery",
"and focus on activities that improve patient health”). The IOM very rightly asserts that there can be no quality without safety."
],
"content": "Introduction\n\nThe September 2012 Institute of Medicine (IOM) report1 called for higher quality at lower cost through leadership that fosters continuous learning. It makes ten important recommendations (e.g. “involve patients and families in decisions regarding health and health care, tailored to fit their preferences” and “continuously improve health care operations to reduce waste, streamline care delivery, and focus on activities that improve patient health”). The IOM very rightly asserts that there can be no quality without safety.\n\nAn August 2013 report2 to the British Prime Minister by Britain’s National Advisory Group on the Safety of Patients in England, draws attention to the fact that the current state of patient safety is in “Crisis”. It calls for “A promise to learn- a commitment to act” for improving the safety of patients in England. This report identifies fifteen problems including the use of “quantitative targets” without caution. Based on these, it makes ten recommendations (e.g. “The NHS should continually and forever reduce patient harm by embracing whole heartedly an ethic of learning” and “All leaders concerned with NHS healthcare-political, regulatory, governance, executive, clinical and advocacy – should place quality of care in general, and patient safety in particular, at the top of their priorities for investment, inquiry, improvement, regular reporting, encouragement and support”). Both reports make the vital statement that the power of bottom-up commitment for continuous learning for quality improvement is much greater than that of the top-down efforts that are based on rules, standards and enforcement. Bottom-up initiatives and acts will help fulfill the mission of the US Patient Protection and Affordable Care Act. We need to develop a facility that enables and helps implementation of “A promise to learn- a commitment to act”2. Providers are frustrated with the top-down management methods that are by their very nature mechanistic (Taylorism), as against humanistic (bottom-up) methods. Top-down methods include: external audits, practice profiles, quality and safety indicators, and trigger tools. These tools do not adequately recognize the problems involved with defining and quantifying harm3.\n\nOrganizational changes required to reduce the huge global burden of harm in healthcare should take advantage of recommendations in these two reports, supported by previously published work by Mold and Gregory4 on “Best Practice Research (BPR)” and Singh et al. on “Systematic Appraisal of Risk and Its Management for Error Reduction (SARAIMER)”5–7. The Agency for Healthcare Research and Quality (AHRQ) has recently published “A Toolkit for Rapid-Cycle Patient Safety and Quality Improvement” that includes an adapted version of SARAIMER8.\n\nThis paper presents a concept that embeds the BPR in SARAIMER for creating a sustainable and affordable methodology for continuously improving safety-based quality of care efficiently and effectively. The guiding principle in healthcare is that improvement science is not about developing a theory begging for application but rather is about understanding societal needs and wants, and engineering means of meeting them in an efficient manner that is sustainable in the never ending journey toward excellence.\n\n\nWhat is Best Practice Research (BPR) method in healthcare?\n\nThis is a bottom-up systematic study that identifies, narrates, combines and disseminates effective and efficient management strategies designed by and for practicing providers. The primary objective is to create a list of desirable (BEST) qualities followed by prioritizing them with stipulated minimum standards for each. This approach can help fill the gap (in knowledge of desirable qualities and of their prioritization) often left in medical school and residency training programs9. This approach to research captures the collective experiences and wisdom of providers working in their unique settings. Development of BPR was motivated by frustration with the top-down management methods.\n\nIn general, the BPR method includes:\n\nDevelopment of a conceptual model. This essentially captures all the contributing parts of the process, including an appropriate process flow diagram.\n\nDefinition of “BEST” method. This creates a list of desirable qualities, prioritizing them and setting a minimum standard for each.\n\nEvaluation of the potential methods for each process.\n\nCombining best processes.\n\nTesting of the combined method.\n\nPut forward by Mold and Gregory, this method invokes improvement science10. Their goal is to find a solution for best practice. They provide examples in their 2003 paper4. Their records showed positive responses from all participants in their research. Lessons learnt from their approach can build a strong house of quality on the triad of Structure-Process-Outcome put forward by Donabedian11. This triad led the IOM in 2001 to form the list of six components of quality care namely: efficient, effective, timely, equitable, safe, and patient centered.\n\n\nWhat is Systematic Appraisal of Risk And Its Management for Error Reduction (SARAIMER)?\n\n(a): Introduction: Despite numerous national and international calls for improvements in safety, healthcare progress has been painfully slow. In the US up to 200,000 avoidable deaths per year occur in primary and outpatient settings alone12,13. In the US alone nearly eleven million patients are harmed every year due to avoidable errors14,15. The associated costs are estimated to be $1 trillion/year out of a total healthcare annual budget of $3.2 trillion. Reduction of these is an ethical, societal, and fiscal imperative that demands priority from all stakeholders. Recently, various accreditation bodies have started to pay greater attention to the reduction of this huge burden of harm to patients. The US Accreditation Council of Graduate Medical Education (ACGME) has recently called for implementation of a “Next GME Accreditation System”16. This current paper draws particular attention to “System-Based Practice” and “Practice-Based Learning and improvement”. These two competencies have, hitherto, received little attention despite their importance.\n\nThe most commonly used methods of measuring harm in healthcare, are based on error reports, external audits and profiles, quality and safety indicators, and trigger tools. The Health Affairs issue of April 2011 published a review showing that, for a specific setting and time, the total number of adverse events measured with voluntary reporting, AHRQ Safety Indicators and global trigger tool17, were 4, 35 and 354 respectively in a total of 795 patient records.These different measures of the same reality obtained with different methods of observation testify to the fact that what we measure in healthcare is a function of how and why we measure. A statement that is often made in medical literature is: “if you cannot measure it you cannot improve it”. This is attractive and relevant in the engineering and manufacturing domains. In the science of healthcare, however, unconditional reliance on these so-called measures narrows the range and depth of our progress. Science of improvement10 is a function of the complex Donabedian Triad of Structure-Process-Outcome11. The prevalent reductionist approaches are a barrier to advances in outcome improvements such as patient harm reduction. It is relevant to recall the observation made by the Nobel Laureate Prigogine that “science is an expression of contemporary culture”.\n\nThese thought processes led the author and his team to develop SARAIMER, which captures the memory18,19 of providers and patients in each unique setting and empowers, enables, and motivates them at the point of care to make sustainable continuing improvements.\n\n(b) Description of SARAIMER: This systems approach to practice-based learning and improvement is built on three considerations and five principles as illustrated in Figure 1. SARAIMER, adapted from Failure Mode and Effects Analysis (FMEA), is designed to foster mutual respect, trust, understanding, collaboration, cooperation, and work and patient satisfaction. The approach has been demonstrated5,6, with AHRQ support, to reduce adverse events and their severity through empowerment and ownership.\n\nThis approach loops the science of observed systems with that of observing them resulting in a cyclic methodology as illustrated in Figure 2. In the concept proposed by the author the third stage in SARAIMER is enriched with BPR input. All the SARAIMER stages are briefly described5,6 below:\n\nStage (1) Assess baseline safety with annonymous on-line survey: The first step in this adapted FMEA process is to begin to understand the system of care in the setting. This is done by first identifying the various entities in the practice (such as the patient, provider, nurse, and chart), listing the main interactions between them, and then portraying them in a diagram. Memory of staff about hazards in each entity is captured with a specially designed instrument.\n\nStage (2) Identify the most significant system problems using hazard analysis for identification of the most hazardous failure modes and prioritization: The goal of the analysis is to rank the failure modes from the survey according to the size of their effects. This is followed by a web-based consensus forming process (Delphi Technique) the results of which are presented to staff in a graphic format. This results in team decisions regarding which hazards to address.\n\nStage (3) Establish team based feasible solutions to prioritized hazards: In light of the resources available and the capabilities of the unique setting, the teams develop solutions to address the prioritized vulnerabilities. These solutions are informed by: (a) established safety principles, (b) safety strategies and (c) desirable equipment safety design features. BPR-identified effective and efficient management strategies are invoked at this stage to enrich feasible solutions.\n\nStage (4) Implementation of team based improvement interventions and tracking their effects: The staff are helped to form implementation teams with clear allocation of responsibilities and time schedules. Along with this, the outcomes of the interventions are tracked with the aid of the software written for this purpose.\n\nIn his 2012 papers5,6 the author provides details of methodology of SARAIMER interventionin various offices. This intervention (without BPR enrichment) resulted in significant reductions in frequency and severity of harm to patients. Participants, including all staff, in every practice expressed great enthusiasm, and recommended wider dissemination of his team-based methodology because it encouraged and enabled them to continuously learn to reduce harm with greater work satisfaction.\n\n(c) Minimum Harm Practice (MiHaP) (an acronym of May I Have Your Attention Please!) by embedding BPR, and safety principles and strategies in SARAIMER: At philosophical and improvement science levels, the development process for MiHaP must use a system engineering approach. Medical practices operate in fast changing, complex circumstances. They need to be able to adapt quickly, and to find order where there is disorder. Order helps to create continually learning practice.\n\nIt was in 1946 that T.S. Eliot asked: “Where is the wisdom we have lost in knowledge?, where is the knowledge we have lost in information?” Even today, we compartmentalize information and knowledge into different fields of science, such as physics, biology, mathematics, etc. In other words we tend to fragment our information and knowledge, thus retarding our progress to wisdom. The fragmentation in the US healthcare ‘non-system’ compounds the complexity. To bring about order we must avoid reductionism by adopting systems thinking so as to integrate information and knowledge, leading to wisdom that enables ethical advancements. This thought is expressed in Figure 3.\n\nAt a practical level, MiHaP should help form best medical practice built on Donabedian’s house of quality triad with attention to a sound foundation and lateral supports (flying buttresses) as shown in Figure 4. To make this house a continuously learning practice it should take advantage of the September 2012 IOM report recommendations: provide real-time access to knowledge by digitally capturing care experiences (e.g. BPR), engaging empowered patients, providing transparency with incentives aligned for value, and provide leadership that cultivates a culture of learning in system-based practice and practice-based learning and improvement. This is illustrated in Figure 5.\n\nAs described earlier, SARAIMER presents an operational framework in which can be embedded, as shown in lower part of Figure 2, not only the safety principles and strategies (including equipment features) but also effective and efficient best practice management strategies designed by and for providers and disseminated through Practice Based Research Networks (PBRNs). The overall objective of BPR has to be the delivery of safe, effective, efficient, equitable, timely and patient-centered care, as called for by IOM. These have to be supported by a caring organization that pays attention to continuity of care20.\n\nFor harm reduction MiHaP has to be armed with sharp awareness of and attention to the following systemic threats of the process of care:\n\nVariability from patient to patient\n\nInconsistency in the standards of care\n\nPoor interfacing (e.g. transition between settings)\n\nLack of error-preventing barriers\n\nLack of initiative to handle the unforeseen\n\nUse of inappropriate time constraints\n\nUse of a hierarchical culture in the system and\n\nHuman fallibility – to err is human\n\nAs stated earlier, there are national and international calls for Better health, Better care, and Better value (3B’s of Healthcare). In response to the 2012 IOM report and the Britain’s National Advisory Group, the author of this paper proposes a concept for MiHaP portrayed in Figure 6. The details in this figure are a reflection of the complexity of care. SARAIMER is designed to inculcate order in this complexity by fostering mutual respect, trust, understanding, collaboration and cooperation as well as worker and patient satisfaction. The bottom part of this figure emphasizes the business case for improving safety, focusing on the costs of harm to patients. This figure captures the primary driving forces in best practice.\n\n\nConcluding remarks\n\nA safe organization is a cost effective quality organization. A concept for a structured bottom-up methodology is put forward that motivates, engages, enables and empowers all stake holders to identify, prioritize, and address safety challenges. This methodology takes advantage of the memory of all team members involved in providing care. It respects and responds to the uniqueness of each setting by empowering and motivating all to a never ending commitment to harm reduction. A strategy is put forward for leaders to implement, in response to national and international calls for Better health, Better care, and Better value (3B’s of Healthcare). The author’s contention is that incorporation of BPR in SARAIMER will accelerate the journey to the desired outcome (reduced harm). This strategy is designed to enable and implement “A promise to learn- a commitment to act”2. It is useful to add that AHRQ has recently published “A Toolkit for Rapid-Cycle Patient Safety and Quality Improvement” that is based on an adapted paper version of SARAIMER.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author is grateful to AHRQ for research grants (Nos. R21 HS014867 and R18 HS017020) and its support.\n\n\nAcknowledgements\n\nWork associated with the aforementioned grants by all the team members, particularly by Diana Anderson, Elizabeth McLean-Plunkett, Don McLean, Ron Brooks, Chester Fox, Renee Kee, Nik Satchidanand, Angela Bosinski, John Taylor, Thomas Rosenthal and Gurdev Singh, is gratefully acknowledged.\n\n\nReferences\n\nIOM. Best Care at Lower Cost: The path to continuously Learning Health Care in America. September 2012. Reference Source\n\nNational Advisory Group on the Safety of Patients in England. A promise to learn – a commitment to act. August 2013. National Health Service, UK. Reference Source\n\nBates DW, Cohen M, Leape LL: Reducing the frequency of errors in medicine using information technology. J Amer Med Inform Assoc. 2001; 8(4): 299–308. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMold JW, Gregory ME: Best Practice Research. Fam Med. 2003; 35(2): 131–4. PubMed Abstract\n\nSingh R, Anderson DR, McLean-Plunkett EA, et al.: IT-enabled systems engineering approach to monitoring and reducing ADEs. Am J Manag Care. 2012; 18(3): 169–175. PubMed Abstract\n\nSingh R, Anderson DR, McLean-Plunkett EA, et al.: Effects of self-empowered teams on rates of adverse drug events in primary care. Int J Fam Med. 2012; 2012: Article ID 374639, 6 pages. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh R, Hickner J, Mold J, et al.: \"Chance Favors Only the Prepared Mind\": Preparing Minds to Systematically Reduce Hazards in the Testing Process in Primary Care. J Patient Saf. JPS-11-22R.\n\nAHRQ. Improving Your Office Testing Process: A Toolkit for Rapid-Cycle Patient Safety and Quality Improvement. August 2013. Agency for Healthcare Research and Quality, Rockville, MD. Reference Source\n\nSingh R, Singh A, Fish R, et al.: A patient safety objective structured clinical examination. J Patient Saf. 2009; 5(2): 55–60. PubMed Abstract | Publisher Full Text\n\nBerwick DM: The Science of Improvement. JAMA. 2008;299(10): 1182–1184. PubMed Abstract | Publisher Full Text\n\nDonabedian A: Evaluating the quality of medical care. 1966. Milbank Q. 2005; 83(4): 691–729. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeingart SN, McL Wilson R, Gibberd RW, et al.: Epidemiology of medical error. West J Med. 2000; 172(6): 390–393. PubMed Abstract | Free Full Text\n\nStarfield B: Is US health really the best in the world? JAMA. 2000; 284(4): 483–485. PubMed Abstract | Publisher Full Text\n\nConsumer Report. New hospital-safety plan leaves patients in the dark. Apr 12, 2011. Reference Source\n\nMakary M: Unaccountable: What Hospitals Won’t Tell You and How Transparency Can Revolutionize Health Care. 2012: Bloomsbury Press, New York. Reference Source\n\nNasca TJ, Philibert I, Brigham T, et al.: The next GME accreditation system--rationale and benefits. N Engl J Med. 2012; 366(11): 1051–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClassen DC, Resar R, Griffin F, et al.: Global trigger tool’ shows that adverse events in hospitals may be ten times greater than previously measured. Health Aff (Millwood). 2011; 30(4): 581–589. PubMed Abstract | Publisher Full Text\n\nDonaldson L: Measuring the unmeasurable. Liam Donaldson. Measuring the unmeasurable. Reference Source\n\nDonaldson L: An organisation with a memory. Clin Med. 2002; 2(5): 452–7. PubMed Abstract | Publisher Full Text\n\nSingh R, Roberts AC, Singh A, et al.: Improving transitions in inpatient and outpatient care using a paper or web-based journal. JRSM Short Rep. 2011; 2(2): 6. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2808",
"date": "21 Jan 2014",
"name": "John Martin Hickner",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a U.S. family physician, and I have been aware of Dr. Singh's important work in safety and quality for nearly a decade. I am also aware of the \"Best Practices\" model for improvement championed by Mold and others. Both of these approaches are pragmatic and build on the knowledge of those at the front line, much like the Toyota model has done for industry. This manuscript is a well written summary of these two improvement methods, firmly grounded in the context of calls for healthcare improvement in the U.S. and the U.K.There is no question that improvement in healthcare will come from both top down and bottom up approaches. So far, the balance of improvement efforts have been top down. To the extent that practitioners and their support staff can become engaged in improvement efforts, the pace will accelerate. Organizations that are able to harness the knowledge and spirit of their employees are likely to forge ahead of the rest in quality and safety. We are already seeing this in the U.S.",
"responses": []
},
{
"id": "3472",
"date": "05 Feb 2014",
"name": "Christian Gericke",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 conceptual paper, proposing a bottom-up methodology for quality and safety projects in healthcare that has real potential for widespread use throughout healthcare systems.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-276
|
https://f1000research.com/articles/2-275/v1
|
17 Dec 13
|
{
"type": "Case Report",
"title": "Massive Solitary Fibrous Tumor (SFT) of the infratemporal and pterygomaxillary fossa treated by combined endoscopic approach",
"authors": [
"François Cloutier",
"Geneviève Lapointe",
"Sylvie Nadeau",
"Geneviève Lapointe",
"Sylvie Nadeau"
],
"abstract": "Solitary fibrous tumors (SFTs) were first described as spindle-cell tumors originating from the pleura. Until recently, there was some debate with regards to the name and origin of these tumors and the distinctions with the term haemangiopericytomas (HPCs), a rare type of vascular tumor.Morphological, immunohistochemical and clinical features of HPCs were not specific for one entity. With the exception of myopericytoma, infantile myofibromatosis and HPC-like lesions of the sinonasal tract showing myoid differentiation, all other HPC-like lesions are best considered as subtypes of SFT. Due to their mesenchymal origin, we are now aware that SFTs may involve several extrapleural sites including soft tissues or meninges. When SFTs involve the skull base and show malignant histological characteristics, they can be an important challenge for the surgical team.We report a case of a 54-year-old man complaining of poor vision and facial pain that had worsened over the last year. Computed tomography and magnetic resonance imaging indicated a large mass, involving the right infratemporal and pterygomaxillary fossa. A biopsy proved positive for a solitary fibrous tumor with malignant features. Surgery was performed using a combined approach of frontotemporal craniotomy and nasal endoscopy. The subtotal resection, conducted due to nodularity on the dura mater, was a success and was complemented by postoperative radiation therapy. Follow-up MRIs showed no recurrence of the tumor.To the authors’ knowledge, this is the first reported case of a massive SFT involving this region, treated with minimal invasive surgery without any facial osteotomies.",
"keywords": [
"Solitary Fibrous Tumor",
"Haemangiopericytoma",
"pterygomaxillary and infratemporal fossa",
"neoplasms"
],
"content": "Introduction\n\nSolitary fibrous tumors (SFTs) were first described by Klempere and Rabin7 in 1931 as spindle-cell tumors originating from the pleura. Until recently, there was some debate with regards to the name and origin of these tumors11. The term haemangiopericytomas (HPCs) was used by Stout and Murray10 in 1942 when they described a type of vascular tumor derived from the capillary pericytes, previously described by Zimmermann in 1923.\n\nHowever, over the years, the use of the term HPC has raised many issues. Morphological, immunohistochemical and clinical features of HPCs are not specific for one entity3,8. With the exception of myopericytoma, infantile myofibromatosis and HPC-like lesions of the sinonasal tract showing myoid differentiation, all other HPC-like lesions are best considered as subtypes of SFT3. Due to their mesenchymal origin5, we now are aware that SFTs may involve several extrapleural sites including soft tissues or meninges5,8.\n\nOnly a few cases of SFT have been described in the literature involving the skull base9,14, but a massive malignant SFT of the soft tissue involving the infratemporal and the pterygomaxillary fossa is extremely rare and represents a multidisciplinary clinical and surgical challenge. The vast extent of the tumor and its localisation near vital structures makes it an important challenge for the surgical team, especially when the tumor shows malignant histological characteristics. Classical transfacial surgical approaches may leave unsightly scars with high morbidity. However, a combined approach with nasoendoscopy provides excellent access for tumor resection without any concerns over the aesthetic result.\n\nTo our knowledge, this is the first case reported of a massive SFT involving this region, treated with minimal invasive surgery without any facial osteotomies.\n\n\nCase report\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.\n\nEarly December 2010, a 54-year-old caucasian man was referred to our otolaryngologist head and neck surgery department. He presented no relevant personal or family history. He had noticed progressive loss of vision and facial pain over the last year. The ophthalmologist, who saw him for decreased vision in the right eye, found a mass on the axial CT that was causing his compressive optic neuropathy.\n\nThe clinical examination revealed subtle proptosis of the right eye. Using flexible nasolaryngoscopy, we saw small bulging in the nasal cavity on the right side. All cranial nerves were intact and no lymph node enlargement was found. The patient was not complaining of epistaxis or nasal obstruction.\n\nThe first computer tomography scan with intravenous iodine contrast, requested by the ophthalmologist, indicated a lesion in the right pterygomaxillary fossa eroding the base of the skull to the temporal lobe and to the right cavernous sinus. There was also an extension in the orbital apex and erosion of the pterygoid process with bulging of the posterior wall of the right nostril. The mass was visible with contrast gadolinium enhancement on the MRI. Its dimensions were 4.6cm anterioposterior × 3.3cm transverse × 4.7cm cranio-caudal (Figure 1–Figure 3).\n\nShortly after the MRI, a transmaxillary endoscopic biopsy was performed. The result of the frozen tissue examination was compatible with a diagnosis of neuroendocrine carcinoma or hemangiopericytoma. The final pathology indicated a solitary fibrous tumor and this was confirmed by an expert otolaryngologist pathologist in Toronto. There was hypercellularity and a mitotic index of 4 mitoses per field, a sign of a highly aggressive tumor8 (Figure 4).\n\nThe case was discussed at the multidisciplinary oncology clinic of our otolaryngology department. With neurosurgical and radio-oncology advice, it was decided to schedule surgery. A preoperative angiography was made with embolization of the tumoral branches by the right internal maxillary artery. It was made the day before surgery to minimize the risks of severe intraoperative bleeding.\n\nThe surgery began with the removal of the inferior and superior right turbinate. After that, we removed the median portion of the maxillary sinus. The tumor was bulging at this location and increased at the level of the sphenoid sinus.\n\nTo attain greater exposure, a Caldwell-Luc incision was made with minimal osteotomy of the anterior maxillary sinus. Using endoscopy, we witnessed the fibrous tumor covering the complete posterior wall of the maxillary sinus.\n\nLaterally, the resection was made until we saw the fatty tissue of the infratemporal fossa. The vidian nerve had to be removed.\n\nWe excised the tumor with the capsule as much as possible. We had to terminate the surgery with a subtotal resection (STR), because it was noted that some nodularity persisted on the dura mater on the lateral-superior side of the tumor (Figure 3a,b). It was left there, because the sealing at this location would make it very difficult to reach and remove. In addition, it was considered a benign tumor with aggressive potential. As a result, radiotherapy was determined to be necessary, in any case.\n\nThe neurosurgeon performed frontotemporal craniotomy with right infratemporal fossa exploration. The debulking was made using ultrasonic suction-irrigation (Cavitron).\n\nOne month later, the patient started external beam radiation (60 Gy by 30 fractions) of the paranasal sinus with infra temporal fossa and pterygomaxillary fossa.\n\nOn monthly follow-up, it appeared that the scars were healing well. There were no problems with the sinuses and no further complaints of facial pain. However, there is no improvement of the visual acuity in the patient’s right eye. Subsequent MRI follow-up showed a continual small asymptomatic residual tumor, but no signs of recurrence more than one year later.\n\n\nDiscussion\n\nSFTs are rare spindle-cell neoplasms of variable histological grades. Despite some confusion in the past regarding HPC and SFT, the relationship between the two is now more obvious. It is also known that SFT of the soft tissue can occur in the head and neck region and throughout the body4,6,12.\n\nMost cases of soft tissue SFTs occur in the early fifth decade of life8 with no sex predilection2,3. Its occurrence is less than 2% of all soft tissue tumors8.\n\nSkull base SFTs may include a wide variety of symptoms, although they are usually asymptomatic on presentation13. The symptoms manifest most frequently as a slowly expanding painless mass. Decreased vision, nasal obstruction, local pain, recurrent sinusitis, epistaxis, headaches, dural erosion, cerebrospinal fluid rhinorrhea, anosmia and lower cranial nerve palsies may all be part of the patient symptomatology.\n\nSFTs from any site are usually benign and surgical resection alone is curative4,11. However, malignancy is possible, although the criteria remain imprecise. It may be suspected with the radiology exam (mass >10cm) or by the presence of metastasis13. The presence of infiltrative margins with surrounding tissues, high mitotic count (≥4 mitoses per 10 high-power high fields) of cellular pleomorphism and tumor necrosis also suggests malignancy4,8.\n\nThe main treatment is surgical for benign and malignant SFTs. There is little evidence (principally from case reports) from adjuvant radiotherapy and none supporting the use of chemotherapy. But when histopathology suggests malignancy or when there are positive surgical resection margins, radiotherapy must be discussed, as for other sarcomas1,8.\n\nIn conclusion, we successfully managed a case of massive solitary fibrous tumor of the soft tissue, involving the infratemporal and pterygomaxillary fossa. With the combination of conventional frontotemporal craniotomy with sinus surgery endoscopy, we were capable of removing most of the tumor while preserving vital structures close to the tumor and without the need for facial osteotomies, leaving the patient without any undesirable scars. With the addition of adjuvant radiotherapy treatment, we were able to achieve no sign of recurrence at the one-year follow-up.\n\nSFT is very uncommon but should be part of the differential diagnosis in patients with a skull base lesion. Diagnosis is difficult but pathologists should be aware of the classical finding of this disease consisting of spindled cells in a disorganized pattern, with alternating hypocellular and hypercellular areas separated by hyalinized collagen and branching HPC-like vessels. Also the immunophenotyping staining positive for the presence of CD34 and Bcl-2 can be useful. The surgical approach needs to weigh risks and benefits of subtotal vs. total resection because of the surrounding vital structures.",
"appendix": "Author contributions\n\n\n\nFrançois Cloutier: first author, acquisition of data, study design, participated in drafting the article and revising it critically for important intellectual content.\n\nGave final approval of the version to be submitted.\n\nGeneviève Lapointe: Second author, substantial contribution to conception and design of this study.\n\nparticipated in drafting the article and revising it critically for important intellectual content, gave final approval of the version to be submitted and any revised version.\n\nSylvie Nadeau: Third author, substantial contribution to conception and design, participated in drafting the article and revising it critically for important intellectual content, and gave final approval of the version to be submitted and any revised version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank the entire otolaryngology and neurosurgery department from the Hôpital de l’Enfant-Jesus.\n\n\nReferences\n\nBowe SN, Wakely PE Jr, Ozer E: Head and neck solitary fibrous tumors: Diagnostic and therapeutic challenges. Laryngoscope. 2012; 122(8): 1748–1755. PubMed Abstract | Publisher Full Text\n\nEngland DM, Hochholzer L, McCarthy MJ: Localized benign and malignant fibrous tumors of the pleura. A clinicopathologic review of 223 cases. Am J Surg Pathol. 1989; 13(8): 640–658. PubMed Abstract\n\nGengler C, Guillou L: Solitary fibrous tumour and haemangiopericytoma: evolution of a concept. Histopathology. 2006; 48(1): 63–74. PubMed Abstract | Publisher Full Text\n\nGold JS, Antonescu CR, Hajdu C, et al.: Clinicopathologic correlates of solitary fibrous tumors. Cancer. 2002; 94(4): 1057–1068. PubMed Abstract | Publisher Full Text\n\nJanjua A, Sklar M, Macmillan C, et al.: Endoscopic resection of solitary fibrous tumors of the nose and paranasal sinuses. Skull Base. 2011; 21(2): 129–134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhalifa MA, Montgomery EA, Azumi N, et al.: Solitary fibrous tumors: a series of lesions, some in unusual sites. South Med J. 1997; 90(8): 793–799. PubMed Abstract | Publisher Full Text\n\nKlemperer P, Rabin C: Primary neoplasms of the pleura: a report of five cases. Arch Pathol. 1931; 11: 385–412.\n\nPenel N, Amela EY, Decanter G, et al.: Solitary fibrous tumors and so-called hemangiopericytoma. Sarcoma. 2012; 2012: 690251. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRansom ER, Judy KD, Bigelow DC: Concurrent Cochlear Implantation with Resection of Skull Base Hemangiopericytoma following Sudden Deafness in an Only Hearing Ear. Skull Base. 2010; 20(4): 279–284. PubMed Abstract | Free Full Text\n\nStout AP, Murray MR: Hemangiopericytoma: A Vascular Tumor Featuring Zimmermann's Pericytes. Ann Surg. 1942; 116(1): 26–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuster S, Nascimento AG, Miettinen M, et al.: Solitary fibrous tumors of soft tissue. A clinicopathologic and immunohistochemical study of 12 cases. Am J Surg Pathol. 1995; 19(11): 1257–1266. PubMed Abstract | Publisher Full Text\n\nVallat-Decouvelaere AV, Dry SM, Fletcher CD: Atypical and malignant solitary fibrous tumors in extrathoracic locations: evidence of their comparability to intra-thoracic tumors. Am J Surg Pathol. 1998; 22(12): 1501–1511. PubMed Abstract\n\nWignall OJ, Moskovic EC, Thway K, et al.: Solitary fibrous tumors of the soft tissues: review of the imaging and clinical features with histopathologic correlation. AJR Am J Roentgenol. 2010; 195(1): W55–62. PubMed Abstract | Publisher Full Text\n\nZeitler DM, Kanowitz SJ, Har-El G: Malignant solitary fibrous tumor of the nasal cavity. Skull Base. 2007; 17(4): 239–246. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2799",
"date": "13 Jan 2014",
"name": "Masafumi Fukuda",
"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 rare case report of solitary fibrous tumor (SFT) occurred in the infratemporal and pterygomaxillary fossa treated by combined approach of frontotemporal craniotomy and nasal endoscopy. However, the points that the authors emphasized are not obvious. Do the authors want to stress the regions occurring the tumors, pathological findings, or the surgical approach? In the discussion , only the general considerations of SFT were described. Are there differences between the previous reports and the case regarding initial symptoms, radiological findings, surgical approach, or final outcome? The authors should show the tables including the information previously reported about SFT in the skull base. Minor revisionsThe authors used “minimum invasive surgery” in some parts. I think that craniotomy in addition to endonasal approach was not minimally invasive. The term “combined surgery” or “combined approach” is better. In the 4th paragraph of the introduction, the authors state“without any facial osteotomies”. However, in the 3rd paragraph of \"Treatment\", the authors described that “a Cadwell-Luc incision was made with minimal osteotomy the anterior maxillary sinus.” Are these inconsistent? In \"Treatments\", the authors should describe the findings with respect to tumor characteristics, such as soft or hard, massive bleeding or not, massive invasion to the adjacent bone structures or not. In the discussion, the first paragraph the authors have already said in the introduction. This paragraph should be deleted. In the 6th paragraph of the discussion, the authors said that “With the addition of adjuvant radiotherapy treatment, we were able to achieve no sign of recurrence at the one-year follow-up”. SFT has slow growth rate and long-term follow-up is required, such as 10 or 20 years. The sentence should be deleted or re-wrote.",
"responses": []
},
{
"id": "3993",
"date": "10 Mar 2014",
"name": "Giulio Cantu",
"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 write that the nasal endoscopic approach is better that the classical transfacial approach because this later may leave “unsightly scars with high morbidity”. The emphasis in this sentence is probably excessive. The scars may sometimes be “unsightly” (although a good skin closure makes the scar quite invisible) but I never saw “high morbidity” for the scar per se.The authors write that the inferior portion of the tumor was capsulated whilst it was not the case of the portion adherent to the dura. The reported MRI images confirm this data. So, I believe that this tumor was resectable with the only infratemporal intracranial approach. Moreover, I believe that it was indicated a dural resection in order to achieve a complete radicality. I agree that the repair of the dura in that region is not easy but it is possible without CSF leakage (very rare in my experience, especially filling the dead space with the rotation of the temporal muscle or with a free flap). It is worthy of consideration that the postoperative radiotherapy is not a guarantee of success in case of macroscopic residual tumor, mainly when one is dealing with low grade tumors that often have a low radiosensitivity.The reported sentence “With the addition of adjuvant radiotherapy treatment, we were able to achieve no sign of recurrence at one-year follow-up” has a very low oncologic meaning. I have seen relapses of solitary fibrous tumor after 12 years of the resection.Finally, the postoperative figure 3A and 3B show a very enlarged liquor space: Why?",
"responses": []
},
{
"id": "4772",
"date": "14 May 2014",
"name": "Rakesh K Chandra",
"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 that is well illustrated and has some instructive elements. My only criticism is that it is not clear why a craniotomy was done if this was a benign (albeit aggressive) tumor and a subtotal resection followed by XRT was done anyhow. Perhaps the authors could discuss their surgical decision-making in this regard. The surgical defect in the postoperative MRI also appears to involve areas that were not previously involved radiologcially.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-275
|
https://f1000research.com/articles/2-273/v1
|
16 Dec 13
|
{
"type": "Systematic Review",
"title": "Economic evaluations of peritoneal dialysis and hemodialysis: 2004-2012",
"authors": [
"Frank Xiaoqing Liu",
"Tiffany P. Quock",
"John Burkart",
"Les L. Noe",
"Gary Inglese",
"Tiffany P. Quock",
"John Burkart",
"Les L. Noe",
"Gary Inglese"
],
"abstract": "Background: End-stage renal disease (ESRD) is a debilitating condition resulting in death unless treated. Treatment options include conservative care, transplantation, and dialysis. Major alternative dialysis modalities include peritoneal dialysis (PD) and in-center hemodialysis (ICHD), which have been shown to produce similar outcomes and survival. The need to provide dialysis treatment for patients with ESRD represents a significant financial challenge for global health care systems. Changes in clinically-appropriate dialysis delivery leading to more efficient use of resources would increase health systems’ ability to meet that challenge. The purpose of this paper is to evaluate the economic literature of PD and ICHD within the context of continued economic uncertainty and pressure on healthcare resource use.Methods: A systematic literature search was conducted for studies published between 2004 and 2012. Articles are included if they were original research studies in English which reported costs and/or cost effectiveness associated with PD and ICHD.Results: Twenty-four articles are included in our review, six of which are cost effectiveness studies comparing PD and ICHD.Conclusions: Our findings echo those of prior published reviews, showing that PD is significantly cost-saving compared to ICHD therapy in most developed countries and some developing countries. Increasing the use of clinically-appropriate PD would substantially reduce healthcare costs.",
"keywords": [
"End-stage renal disease (ESRD) is a debilitating condition resulting in death unless treated. Kidney transplantation",
"dialysis",
"and conservative therapy supported by palliative care are the three primary treatment options for patients with ESRD1",
"2. Although kidney transplantation is the most cost-effective therapy",
"the supply of donor kidneys is limited3. In addition",
"patients who are eligible for and choose kidney transplantation usually have to wait months to years for a kidney to become available. During this waiting period",
"patients are treated with dialysis. The two main types of dialysis are hemodialysis (HD) and peritoneal dialysis (PD). In HD",
"the removal of wastes and excess fluid is accomplished by circulating blood outside the body through an external filter (dialyzer). HD is usually performed in a hospital or clinic (in-center HD or ICHD)",
"but can also be performed at home (HHD). Conventional HD is typically performed three times per week",
"with each treatment lasting 3 to 5 hours. In PD",
"the removal of wastes and excess fluid is accomplished inside the body",
"in the abdominal or peritoneal cavity. A dialysis solution is placed in the peritoneal cavity",
"which allows wastes to move from the blood to this solution. After a period of time",
"the solutions are drained from the body4. Unlike HD5",
"PD is usually a continuous therapy",
"in which dialysis solutions are repeatedly drained from and instilled into the peritoneal cavity so there is a possibility of dialysate dwell (and hence solute removal) up to 24 hours/day 7 days a week4. However",
"some patients are treated with intermittent PD",
"which is either performed manually (the patient uses gravity to fill and drain the abdominal cavity) through continuous ambulatory PD (CAPD) treatment",
"or with a cycler (a machine that automatically fills and drains the abdominal cavity",
"usually while the patient sleeps)",
"in automated PD (APD) treatment6."
],
"content": "Introduction\n\nEnd-stage renal disease (ESRD) is a debilitating condition resulting in death unless treated. Kidney transplantation, dialysis, and conservative therapy supported by palliative care are the three primary treatment options for patients with ESRD1,2. Although kidney transplantation is the most cost-effective therapy, the supply of donor kidneys is limited3. In addition, patients who are eligible for and choose kidney transplantation usually have to wait months to years for a kidney to become available. During this waiting period, patients are treated with dialysis. The two main types of dialysis are hemodialysis (HD) and peritoneal dialysis (PD). In HD, the removal of wastes and excess fluid is accomplished by circulating blood outside the body through an external filter (dialyzer). HD is usually performed in a hospital or clinic (in-center HD or ICHD), but can also be performed at home (HHD). Conventional HD is typically performed three times per week, with each treatment lasting 3 to 5 hours. In PD, the removal of wastes and excess fluid is accomplished inside the body, in the abdominal or peritoneal cavity. A dialysis solution is placed in the peritoneal cavity, which allows wastes to move from the blood to this solution. After a period of time, the solutions are drained from the body4. Unlike HD5, PD is usually a continuous therapy, in which dialysis solutions are repeatedly drained from and instilled into the peritoneal cavity so there is a possibility of dialysate dwell (and hence solute removal) up to 24 hours/day 7 days a week4. However, some patients are treated with intermittent PD, which is either performed manually (the patient uses gravity to fill and drain the abdominal cavity) through continuous ambulatory PD (CAPD) treatment, or with a cycler (a machine that automatically fills and drains the abdominal cavity, usually while the patient sleeps), in automated PD (APD) treatment6.\n\nThe prevalence of ESRD (estimated to be 2.8 million globally at the end of 20117) and, consequently, the demand for dialysis, continues to increase due to an aging population, extended life expectancies, the increased prevalence of precipitating conditions such as diabetes6. Worldwide, hemodialysis continues to be the most common mode of therapy for individuals with ESRD6 and the need to provide dialysis treatments for a large and growing patient population places a heavy economic burden on the global health care system8. In the United States, (US), for example, 1.3% of Medicare patients with ESRD accounted for 7.5% of Medicare spending in 20105.\n\nIn response to such pressures, initiatives aimed specifically at controlling dialysis-related costs have already been implemented in many countries, including Australia, Portugal, Thailand, United Kingdom (UK) and US9–14. For example, the Australian government implemented a range of national health reforms in 2012, including a national efficient price (NEP) for hospital services, which was based on the average costs of best practice pathways14. Similarly, Portugal implemented a bundled payment program for dialysis, bundling dialysis services and products into one rate11. In addition, the Centers for Medicare and Medicaid Services (CMS) in the US enacted an expanded bundled prospective payment system (PPS) for dialysis services in order to control for the unpredictable and escalating costs associated with the provision of dialysis, and to combine pharmaceuticals and lab services that were previously charged separately13. These and other initiatives are similar in striving for maintaining healthcare costs and identifying interventions that are less resource intensive and/or cost-effective. Given this changing economic environment, where payers and providers around the world are facing pressure to control healthcare costs while improving patient outcomes, we conducted a systematic review of economic evaluations of PD and ICHD published since 2004. Our purpose was to assess whether the recent literature continues to support the use of PD to be a less resource intensive therapy compared with HD from payer, provider and societal perspectives15.\n\n\nMethods\n\nWe conducted a systematic literature search for peer-reviewed economic evaluations of dialysis modalities published between January 1, 2004 and March 31, 2012 in the second quarter of 2012. To ensure that we captured articles that might not have been included in the database for the prior literature synthesis which assessed literature from 1996–2006, our literature search included an overlap of three years of inclusion (2004–2012) with the prior literature synthesis. We applied our search strategies in PubMed; EMBASE; Cochrane Collaboration; Database of Abstracts of Reviews of Effects; UK National Health System Economic Evaluations Database; and Health Technology Assessment Database. Medicine indexing terms for dialysis therapies, including renal replacement therapy, peritoneal dialysis, renal dialysis, and hemodialysis units, hospital that linked to the subheading economics, and free-text search terms, including hemodialysis, peritoneal dialysis, renal dialysis, continuous ambulatory peritoneal dialysis, conventional hemodialysis, automated peritoneal dialysis, cost, costs, expenditures, cost analysis, cost effectiveness, cost benefit analysis, pharmacoeconomics, health economics, and economics, were used in the database search.\n\nOur search identified 498 publications (Figure 1). Two investigators (FXL and TPQ) screened 40% of all the found abstracts independently based on our research objective. With 96% of the selected articles matched, one investigator (TPQ) continued the remaining selection. Articles were excluded (n=444) if they were of the following publication types: Editorial, Letter, Case Report, Comment, Interview, Lecture, News, Newspaper Article, or Patient Education Handout, or if the title/abstract did not suggest that a comparison was made between the costs of at least one sub-modality of PD to at least one sub-modality of ICHD. Articles were included (n=54) if they were original research studies with text in English, and if they compared costs and/or cost effectiveness associated with PD and ICHD or subtypes of PD and ICHD. To be comprehensive, additional articles (n=15) were identified through a review of references from the selected papers. These 69 articles were subjected to full-text review and further inclusion criteria were applied. Studies that did not focus on an economic evaluation of dialysis, or did not differentiate between the costs of PD and ICHD (or their subtypes) were excluded, as were review articles with no original data. Twenty-four articles (18 cost studies and 6 cost-effectiveness studies) met all of these inclusion criteria. Relevant study data were manually extracted (TPQ) and cross-checked for accuracy (FXL).\n\n\nResults\n\nThe 24 articles included in our review provide economic and cost-utility data from 13 high-income (Australia, Austria, Finland, France, Greece, Italy, Japan, New Zealand, Singapore, Spain, Switzerland, UK, US), three upper-middle income (Chile, Malaysia, Mexico), and six lower-middle income (China, Columbia, India, Romania, Thailand, Turkey) countries. Designations of high, upper-middle, and lower-middle income countries are based on World Bank definitions whereby economies are divided according to the 2011 gross national income (GNI) per capita, which is calculated using the World Bank Atlas method. Defined groups are: low income, $1,025 or less; lower-middle income, $1,026–$4,035; upper-middle income, $4,036–$12,475; and high income, $12,476 or more16. Table 1 outlines the costs of ICHD and PD found in 19 countries from our 18 cost studies.\n\nCross-country comparisons of economic data are challenging to perform well under the best of circumstances, but perhaps especially in the case of dialysis, where ICHD-related costs are comprised primarily of labor and capital expenses, whereas PD-related costs are associated primarily with disposables, such as dialysate solutions and supplies. In this review, the usual concerns were compounded by the variability of the data compared within individual studies and by incomplete transparency within individual reports. Cost perspectives, where specified, ranged from patients, service providers, and payers to government health systems. With different perspectives, different costs are considered relevant and different methods of apportioning direct and overhead expenses may influence the reported results17. The currency years reported in these studies, where specified, spanned more than a decade (1997 to 2010), demanding significant assumptions to adjust for price inflation, international currency valuations, and purchasing power parity in order to arrive at a common-currency metric. Furthermore, differences in provider payment and healthcare financing systems, variation in rates of HD versus PD use affecting economies of scale across different countries, and potential differences in treatment settings intensify the challenges for cross-country comparisons of these studies. Data sources were similarly varied, including literature-derived secondary sources, hospital records, insurance information, disease registries, government publications, expert opinions, surveys, and semi-structured interviews. Finally, the 19 countries from which dialysis costs were reported are at different levels of economic development. As such, they have widely varying infrastructures available to support dialysis services, as well as different barriers to the use of each dialysis modality18,19. Each of these factors further magnify the danger of cross-country comparisons20. Nevertheless, the data shown in Table 1 remain valuable insofar as they highlight the magnitude of dialysis costs and the relative (not absolute) cost differences between the dialysis modalities across countries. Ratios of costs for ICHD compared to PD are included in Table 1 to assist with this exercise. Ratios less than 1.00 (parity) denote lower cost of ICHD compared to PD.\n\nNote: medical direct costs (dialysis, laboratory, and other directly related costs); total medical (medical direct and non-medical spending, including transportation) costs; total medical costs plus indirect treatment costs (such as loss of productivity); \"-\" not available\n\nBased on World Bank definitions for classifying economies: low income, $1,205 or less; lower-middle income, $1,026–$4,035; upper-middle income, $4,036-$12,475; and high income, $12,476 or more\n\na Small sample size of 29 patients and is insufficient to assess costs\n\nb For Satellite HD: weighted calculation=(129*52*3*0.83+207*52*3*0.14)/0.97\n\nc For Hospital HD: weighted calculation=(155*52*3*0.83+232*52*3*0.14+258*52*3*0.02)\n\n§PD: peritoneal dialysis; HD: hemodialysis; ICHD: in-center hemodialysis; CAPD: continuous ambulatory peritoneal dialysis; APD: automated peritoneal dialysis; cost ratios are calculated by dividing highest value with lowest value\n\nThe findings from our review demonstrate that ICHD costs, whether they are medical direct-, total medical-, or total medical plus indirect costs, are generally (but not always) higher than PD costs. A 2009 Finnish study21 of the total medical costs (payer perspective) of PD and ICHD reported that the cost of ICHD tended to be about 25%–38% higher than the cost of PD (ICHD:PD ratio = 1.25-1.38). In contrast, a 2007 study evaluating the direct costs of PD and ICHD (payer perspective) in Japan22, found that the cost of ICHD tended to be about 86% of the cost of PD (ICHD:PD ratio = 0.86). Nevertheless, data from 82% (9/11) of the high-income, 50% (1/2) of the upper-middle income, and 83% (5/6) of the lower-middle income countries represented in our review show the cost of ICHD to be more than that of PD. Figure 2 arrays the country-specific cost ratios for ICHD compared to PD.\n\nSix of the 24 studies in our review were cost-effectiveness or cost-utility studies; that is, they considered the outcomes of each dialysis modality in terms of patient life-years, and/or healthy life-years (typically measured as quality-adjusted life years) in addition to costs. Despite wide variability in methodologies, outcome measures, perspectives, time horizons, costs, and analytic approaches used in these six studies, each came to a similar conclusion: the cost-effectiveness of PD is equivalent to (1 study), or better than (5 studies) the cost-effectiveness of ICHD (Table 2).\n\n*LY = life years. QALY = quality-adjusted life years\n\nThe 24 economic analyses reviewed in the context of this study were of variable quality. This is a particular concern because the individuals who need to use these analyses to guide decisions may not be experts at evaluating them23. To document the quality differences between studies in the context of this review, we characterized the reports using criteria adapted from the Quality of Health Economics Studies (QHES) instrument23, which was specifically developed to emphasize appropriate methods; and valid, transparent, and comprehensive results reporting in health economic analyses23.\n\nCharacteristics of high-quality studies not only clearly specified the objective, perspective, and data source, but also included a detailed description of the study methodologies; total direct, non-direct, indirect, and intangible medical costs; and addressed both limitations and biases. While the majority of cost-effectiveness studies clearly described study objective, perspective, and study estimates (83%–100%), fewer studies addressed the validity of outcome measures and biases (67%). Similarly, cost studies frequently reported study objective and direct medical costs, but none of the cost studies included intangible costs and only a few studies (17%) included indirect costs. Intangible costs may be difficult to assess well in the context of dialysis due to the lack of a firm value and the challenge in quantifying pain and suffering within a heterogeneous population.\n\n\nDiscussion\n\nWe conducted a systematic search for peer-reviewed economic evaluations of dialysis modalities published between January 2004 and March 2012, with the goal of evaluating the economic literature within the context of recent global dialysis payment policies encouraging greater use of PD, while building on an earlier systematic review of the literature15 that was published between 1996 and 2006. The results of our review reiterate that ICHD is generally more costly, and less cost-effective than PD in the majority of studies and countries that examined costs and/or cost effectiveness associated with the different dialysis modalities. These results are consistent with previously published systematic reviews of the literature15,24. In fact, there is a growing body of evidence suggesting greater use of PD or the use of PD as first treatment modality for appropriate ESRD patients25–27. Our findings show that existing reimbursement incentives for delivering dialysis therapies have implications for both clinical practice and health policy. The burden of rising healthcare costs globally, coupled with competing economic priorities, highlight the importance of establishing policies that encourage the use of PD over ICHD, whenever feasible and clinically appropriate.\n\nThere are exceptions where PD has been reported to be more costly than ICHD; these are noted in Table 1. The reasons behind these exceptions are myriad, but chief among them may be methodological differences between studies, as well as variations in practice patterns, cost estimations, and the inclusion/exclusion of specific cost elements. For example, a study conducted in Spain28 included staff wages for direct PD-related medical costs, whereas ICHD costs excluded these costs in the calculation of direct medical costs. In effect, this particular study reported PD to be more costly than ICHD.\n\nMost of the studies in our review consider only direct medical and non-medical costs in their analyses. Direct medical costs include physician and other staff costs (fees; salaries); capital costs of hemodialysis machines and PD cyclers; costs associated with establishing and maintaining vascular access; ongoing use of dialysis-related supplies such as solutions, medications, and tubing; as well as costs associated with radiology, laboratory and hospitalization. Direct non-medical costs include overheads for administering and maintaining buildings and facilities but they may also include patients’ travel to a dialysis center or physician’s office. It is important to keep in mind that PD and ICHD rely on very different direct cost components. The direct cost of ICHD is driven primarily by the cost of staff, buildings, infrastructure, and capital equipment (i.e., fixed costs), whereas the direct cost of PD is driven primarily by the supply costs of dialysis solutions, tubing and other disposables (i.e., variable costs). Thus, it is not enough to compare dialysis modalities based on differences in one or two cost components. Rather, cost comparisons of PD and ICHD must be based on the total costs of therapy, as direct medical costs alone do not have the same relevance from all economic perspectives. For instance, in the developing world where labor is relatively inexpensive and the cost of imported dialysis solutions and supplies is high, it is easy to perceive PD as being more expensive than ICHD29. The reverse may be true in developed economies where labor is relatively more costly than disposable supplies.\n\nIndirect costs, or productivity losses of patients and caregivers were rarely included in the studies we reviewed, even in the four studies focusing on six countries for which the stated perspective of the analysis was societal30–33. Of these, the most comprehensive array of indirect costs is represented in a Thai study that considers all resources used by dialysis patients and/or caregivers, including capital, labor and materials costs of health-care providers, as well as the real and opportunity costs associated with patient treatment time, informal patient care, and sick leave31. Of course, the inclusion of indirect and/or productivity costs will substantially increase the reported cost of any dialysis modality, but these costs have a particular impact on the cost of ICHD which requires regular, lengthy, dialysis treatments that are provided outside of patients’ homes.\n\nIntangible costs denote those consequences of an intervention that are particularly challenging to measure, such as the value of improved health per se, or the pain, suffering, and impairment in quality of life associated with treatment. Only five30,31,34–36 of the 24 studies included in our review considered intangible costs, all of them via the utility scores used to arrive at quality-adjusted life years.\n\nOur review has several limitations. First, our review focused on economic evidence comparing PD and HD; articles comparing the clinical and patient outcomes associated with PD and HD were not included in this review. Second, we included all articles we found that compared the costs and/or cost effectiveness of PD vs. ICHD. These articles are of varying quality and our report did not differentiate the quality of these findings. As such, it is difficult to make generalizations about the published literature of dialysis economics owing to the limited number of studies available and differences among them in terms of study methods, geography, scope, and quality. Some studies rely on decades-old data that may not be relevant to present-day economies; other studies fail to indicate the currency-year used in analysis. Between-country differences in economic development, practice patterns, reimbursement structures, provider payment, healthcare system financing, and social norms often make it difficult to interpret the findings from one country in the context of another country. In addition, the proportion of patients on HD vs. PD and the treatment settings (provider demographics such as freestanding/satellite vs. hospital-based dialysis clinics) vary across countries. Even studies that report ‘direct costs’ may include/exclude different components of direct cost and, thus, may not necessarily produce comparable results. Further, some authors clearly specify the perspective taken in their analyses (e.g., societal, payer, provider); in other papers the perspective is only implied. Reported study methods, too, may be insufficiently transparent to evaluate study quality; for example, the calculations used to derive costs or the elements of costs (e.g., frequency of dialysis, prescription drug use) are often lacking or unclear. Data sources may vary among studies as well; data sources may include disease registries; hospital, insurer, or government databases; secondary literature; government publications; dialysis product distributors; dialysis center data; and/or individual patient interviews. Just one of these between-study differences introduces uncertainty when drawing comparisons among study results. Because published studies of the economics of dialysis often vary on many of these parameters, specific, robust between-study comparisons cannot be made.\n\nDespite the methodological differences and variable quality across the studies within our review, ICHD clearly emerged as a more expensive modality than PD in most of the 19 countries represented in our review. These results are consistent with earlier systematic reviews of the dialysis literature15,37. Similarly, the cost-effectiveness studies presented in Table 2 begin to show a nascent picture of cost-savings related to PD; the limited number of cost-effectiveness studies and the methodological differences between them (e.g., differences in the variables considered within each modelled analysis) preclude the drawing of specific conclusions.\n\n\nConclusion\n\nThe magnitude of costs associated with providing dialysis therapies globally is large and growing. Our findings echo those of prior published reviews and add to the growing body of evidence finding PD is significantly cost-saving compared to ICHD therapy in most developed countries and some developing countries. Additionally, evidence continues to suggest that PD is a cost-effective strategy relative to ICHD. Therefore, it appears that increasing the use of clinically-appropriate PD would substantially reduce healthcare costs and help health systems meet ever-tightening budget constraints.",
"appendix": "Author contributions\n\n\n\nFXL and TQ conceived, designed, and implemented the review. JB contributed to the data extraction of the study from reviewed papers and provided expertise in nephrology. FXL, TQ, and GI prepared the first draft of the manuscript. LN contributed to the design and preparation of the manuscript. All authors were involved in reviewing and revising the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nFrank Xiaoqing Liu is an employee and stockholder of Baxter Healthcare Corporation, Deerfield, IL USA; At the time this research was conducted, Tiffany Quock and Les Noe were employees of ICON, which received research funding from Baxter Healthcare Corporation for the systematic review. Tiffany is now at Covance Market Access Services Inc. and Les is now at InVentiv Health Clinical; John Burkart is faculty at Wake Forest University, Winston-Salem, NC, USA and has been an ad hoc consultant for Baxter Healthcare Corporation; Gary Inglese was an employee of Baxter Healthcare Corporation at the time of this manuscript development and is now at Hollister Incorporated.\n\n\nGrant information\n\nThe study was supported by funding from Baxter Healthcare Corporation, Deerfield, IL 60015 USA. The publication of the study results was not contingent on the sponsor's approval or censorship of the manuscript.\n\n\nAcknowledgements\n\nThe authors acknowledge Nancy Neil, Ph.D. from Chordata Consulting, LLC and University of Washington, Seattle, WA, USA, for her help on the preparation of the manuscript and Kathryn Webb, MS, for her help on the literature search.\n\n\nReferences\n\nFassett RG, Robertson IK, Mace R, et al.: Palliative care in end-stage kidney disease. Nephrology (Carlton). 2011; 16(1): 4–12. PubMed Abstract | Publisher Full Text\n\nJassal SV, Kelman EE, Watson D: Non-dialysis care: an important component of care for elderly individuals with advanced stages of chronic kidney disease. Nephron Clin Pract. 2011; 119(suppl 1): c5–c9. PubMed Abstract | Publisher Full Text\n\nAbecassis M, Bartlett ST, Collins AJ, et al.: Kidney transplantation as primary therapy for end-stage renal disease: a National Kidney Foundation/Kidney Disease Outcomes Quality Initiative (NKF/KDOQITM) conference. Clin J Am Soc Nephrol. 2008; 3(2): 471–480. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRenal Resource Center 2010. An Introduction to Peritoneal Dialysis. Reference Source.\n\nGhaffari A, Kalantar-Zadeh K, Lee J, et al.: PD First: Peritoneal Dialysis as the Default Transition to Dialysis Therapy. Semin Dial. 2013; 26(6): 706–713. PubMed Abstract | Publisher Full Text\n\nU.S. Renal Data System. USRDS 2012 Annual Data Report: Atlas of Chronic Kidney Disease and End-Stage Renal Disease in the United States. Bethesda, MD: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases. 2012.\n\nFresenius Medical Care. ESRD Patients in 2011. Reference Source.\n\nZelmer JL: The economic burden of end-stage renal disease in Canada. Kidney Int. 2007; 72(9): 1122–1129. PubMed Abstract | Publisher Full Text\n\nPraditpornsilpa K, Lekhyananda S, Premasathian N, et al.: Prevalence trend of renal replacement therapy in Thailand: impact of health economics policy. J Med Assoc Thai. 2011; 94(suppl 4): s1–s6. PubMed Abstract\n\nSirivongs DKV, Wangsiripaisal A, et al.: Fact about PD First Policy in Thailand. Vol ISBN 978-616-223-129-2. Nonthaburi Thailand: Sahamitrprinting & publishing.co, Ltd.\n\nRocha MJ, Ferreira S, Martins LS, et al.: Cost analysis of renal replacement therapy by transplant in a system of bundled payment of dialysis. Clin Transplant. 2012; 26(4): 529–531. PubMed Abstract | Publisher Full Text\n\nIglehart JK: Bundled Payment for ESRD - including ESAs in Medicare’s Dialysis Package. N Engl J Med. 2011; 364(7): 593–595. PubMed Abstract | Publisher Full Text\n\nGolper TA, Guest S, Glickman JD, et al.: Home dialysis in the new USA bundled payment plan: implications and impact. Perit Dial Int. 2011; 31(1): 12–16. PubMed Abstract | Publisher Full Text\n\nCouncil of Australian Governments. National Healthcare Agreement. 2011. Australia. 2011; Reference Source.\n\nJust PM, Riella MC, Tschosik EA, et al.: Economic evaluations of dialysis treatment modalities. Health Policy. 2008; 86(2–3): 163–180. PubMed Abstract | Publisher Full Text\n\nThe world bank. how we classify countries. 2013. Reference Source.\n\nMoore R, Marriott N: Cost and price in the NHS: the importance of monetary value in the decision-making framework--the case of purchasing renal replacement therapy. Health Serv Manage Res. 1999; 12(1): 1–14. PubMed Abstract\n\nNissenson A, Prichard SS, Cheng IK, et al.: Non-medical factors that impact on ESRD modality selection. Kidney Int Suppl. 1993; 40: s120–127. PubMed Abstract\n\nLittle J, Irwin A, Marshall T, et al.: Predicting a patient's choice of dialysis modality: experience in a United Kingdom renal department. Am J Kidney Dis. 2001; 37(5): 981–986. PubMed Abstract | Publisher Full Text\n\nWordsworth S, Ludbrook A: Comparing costing results in across country economic evaluations: the use of technology specific purchasing power parities. Health Econ. 2005; 14(1): 93–99. PubMed Abstract | Publisher Full Text\n\nHallinen T, Soini EJ, Martikainen JA, et al.: Costs and quality of life effects of the first year of renal replacement therapy in one finnish treatment centre. J Med Econ. 2009; 12(2): 136–140. PubMed Abstract | Publisher Full Text\n\nFukuhara S, Yamazaki C, Hayashino Y, et al.: The organization and financing of end-stage renal disease treatment in Japan. Int J Health Care Finance Econ. 2007; 7(2–3): 217–231. PubMed Abstract | Publisher Full Text\n\nOfman JJ, Sullivan SD, Neumann PJ, et al.: Examining the value and quality of health economic analyses: implications of utilizing the QHES. J Manag Care Pharm. 2003; 9(1): 53–61. PubMed Abstract\n\nMacLeod A, Grant A, Donaldson C, et al.: Effectiveness and efficiency of methods of dialysis therapy for end-stage renal disease: systematic reviews. Health Technol Assess. 1998; 2(5): 1–166. PubMed Abstract | Publisher Full Text\n\nBurkart J: The future of peritoneal dialysis in the united states: optimizing its use. Clin J Am Soc Nephrol. 2009; 4(suppl 1): s125–131. PubMed Abstract | Publisher Full Text\n\nChaudhary K, Sangha H, Khanna R: Peritoneal dialysis first: rationale. Clin J Am Soc Nephrol. 2011; 6(2): 447–456. PubMed Abstract | Publisher Full Text\n\nJose MD, Johnson DW, Mudge DW, et al.: Peritoneal dialysis practice in Australia and New Zealand: a call to action. Nephrology (Carlton). 2011; 16(1): 19–29. PubMed Abstract | Publisher Full Text\n\nLamas Barreiro J, Alonso Suarez M, Saavedra Alonso JA, et al.: Costs and added value of haemodialysis and peritoneal dialysis outsourcing agreements. Nefrologia. 2011; 31(6): 656–663. PubMed Abstract | Publisher Full Text\n\nChugh KS, Jha V, Chugh S: Economics of dialysis and renal transplantation in the developing world. Transplant Proc. 1999; 31(8): 3275–3277. PubMed Abstract | Publisher Full Text\n\nKontodimopoulos N, Niakas D: A socio-economic comparison of hemodialysis and peritoneal dialysis in greece. Int J Healthcare Technology and Management. 2005; 6(3): 296–306. Publisher Full Text\n\nTeerawattananon Y, Mugford M, Tangcharoensathien V: Economic evaluation of palliative management versus peritoneal dialysis and hemodialysis for end-stage renal disease: evidence for coverage decisions in Thailand. Value Health. 2007; 10(1): 61–72. PubMed Abstract | Publisher Full Text\n\nNeil N, Walker DR, Sesso R, et al.: Gaining efficiencies: resources and demand for dialysis around the globe. Value Health. 2009; 12(1): 73–79. PubMed Abstract | Publisher Full Text\n\nPacheco A, Saffie A, Torres R, et al.: Cost/Utility study of peritoneal dialysis and hemodialysis in Chile. Perit Dial Int. 2007; 27(3): 359–363. PubMed Abstract\n\nHaller M, Gutjahr G, Kramar R, et al.: Cost-effectiveness analysis of renal replacement therapy in Austria. Nephrol Dial Transplant. 2011; 26(9): 2988–2995. PubMed Abstract | Publisher Full Text\n\nHoward K, Salkeld G, White S, et al.: The cost-effectiveness of increasing kidney transplantation and home-based dialysis. Nephrology (Carlton). 2009; 14(1): 123–132. PubMed Abstract | Publisher Full Text\n\nKontodimopoulos N, Niakas D: An estimate of lifelong costs and QALYs in renal replacement therapy based on patients' life expectancy. Health Policy. 2008; 86(1): 85–96. PubMed Abstract | Publisher Full Text\n\nMenzin J, Lines LM, Weiner DE, et al.: A review of the costs and cost effectiveness of interventions in chronic kidney disease: implications for policy. Pharmacoeconomics. 2011; 29(10): 839–861. PubMed Abstract | Publisher Full Text\n\nBenain JP, Faller B, Briat C, et al.: [Cost of dialysis in France]. Nephrol Ther. 2007; 3(3): 96–106. PubMed Abstract | Publisher Full Text\n\nDurand-zaleski I, Combe C, Lang P: International Study of Health Care Organization and Financing for end-stage renal disease in France. Int J Health Care Finance Econ. 2007; 7(2–3): 171–183. PubMed Abstract | Publisher Full Text\n\nPontoriero G, Pozzoni P, Vecchio LD, et al.: International study of health care organization and financing for renal replacement therapy in Italy: an evolving reality. Int J Health Care Finance Econ. 2007; 7(2–3): 201–215. PubMed Abstract | Publisher Full Text\n\nAshton T, Marshall MR: The organization and financing of dialysis and kidney transplantation services in New Zealand. Int J Health Care Finance Econ. 2007; 7(4): 233–252. PubMed Abstract | Publisher Full Text\n\nVilla G, Rodriguez-carmona A, Fernandez-ortiz l, et al.: Cost analysis of the Spanish renal replacement therapy programme. Nephrol Dial Transplant. 2011; 26(11): 3709–3714. PubMed Abstract | Publisher Full Text\n\nSandoz MS, Ess SM, Keusch GW, et al.: Prevalence and direct medical costs of end-stage renal disease in patients with type 2 diabetes mellitus in Switzerland for 2001. Swiss Med Wkly. 2004; 134(31–32): 448–458. PubMed Abstract\n\nBaboolal K, McEwan P, Sondhi S, et al.: The cost of renal dialysis in a UK setting--a multicentre study. Nephrol Dial Transplant. 2008; 23(6): 1982–1989. PubMed Abstract | Publisher Full Text\n\nBerger A, Edelsberg J, Lnglese GW, et al.: Cost comparison of peritoneal dialysis versus hemodialysis in end-stage renal disease. Am J Manag Care. 2009; 15(8): 509–518. PubMed Abstract\n\nNeil N, Guest S, Wong L, et al.: The financial implications for Medicare of greater use of peritoneal dialysis. Clin Ther. 2009; 31(4): 880–888. PubMed Abstract | Publisher Full Text\n\nShih YC, Guo A, Just PM, et al.: Impact of initial dialysis modality and modality switches on Medicare expenditures of end-stage renal disease patients. Kidney Int. 2005; 68(1): 319–329. PubMed Abstract | Publisher Full Text\n\nJeloka TK, Upase S, Chitikeshi S, et al.: Monthly cost of three exchanges a day peritoneal dialysis is same as of thrice a week hemodialysis in self-paying Indian patients. Indian J Nephrol. 2012; 22(1): 39–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErek E, Sever MS, Akoglu E, et al.: Cost of renal replacement therapy in Turkey. Nephrology (Carlton). 2004; 9(1): 33–38. PubMed Abstract | Publisher Full Text\n\nSalonen T, Reina T, Oksa H, et al.: Alternative strategies to evaluate the cost-effectiveness of peritoneal dialysis and hemodialysis. Int Urol Nephrol. 2007; 39(1): 289–298. PubMed Abstract | Publisher Full Text\n\nHooi IS, Lim TO, Goh A, et al.: Economic evaluation of centre haemodialysis and continuous ambulatory peritoneal dialysis in Ministry of Health hospitals, Malaysia. Nephrology (Carlton). 2005; 10(1): 25–32. PubMed Abstract | Publisher Full Text"
}
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[
{
"id": "2781",
"date": "07 Feb 2014",
"name": "Scott Klarenbach",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have conducted a systematic review of the cost of provision of peritoneal dialysis versus hemodialysis. They have considered a more recent era than previous reviews, evaluating studies published between 2004 and 2012. Using a well described search strategy and inclusion and exclusion criteria, they provided a narrative review of the 24 cost and cost-effectiveness studies. In tabular format they provided a brief description of each study, and for costing studies provided a ratio of the costs of hemodialysis versus PD. The results of each country were categorized by national income status.The results of the 24 studies are congruent with previous reviews suggesting that compared to in center hemodialysis, peritoneal dialysis is less costly to provide. The limitations of the studies including differences in perspective, cost categories, and quality are well described. This is a generally well-written and well conducted study and is an important addition to the literature as healthcare systems strive for economic sustainability.There is a conflict of interest as Baxter is a major supplier of PD equipment and supplies, and many of the author are or were employees / stockholders. This however is clearly outlined in the competing interests section. There does not appear to be any major discernible bias imparted by this.I have some suggestions to improve clarity and organization of the paper:Major commentsIn several places including the abstract and final conclusion it is stated that increase in the use of clinically appropriate PD would reduce healthcare costs. More precise language should be used to indicate that PD should be used instead of hemodialysis (technical efficiency). This is simply to highlight the fact that some health care systems may not provide dialysis therapy at all, or provide dialysis for only a small proportion of the population. This analysis does not compare the cost cost-effectiveness of PD versus conservative management. It might be prudent to make this explicit at some point in the paper, perhaps the discussion. There are several sentences or paragraphs in the results section which I think would be better suited to the methods section. First is the discussion of how income status of each of the countries included in the review were determined. This is in the first paragraph of the results section. The first paragraph of column 2 on page 4 discusses how the ratio of HD to PD costs are determined and I would suggest they should also be in the method section. There are also several sections in the results paragraphs that are more of an interpretation of the results than simply stating the results of their systematic review. For example page 4 first column third paragraph describe the challenges of cross-country comparison but these first few sentences do not describe any results. This would be better suited to the discussion section. I would suggest that the authors go through the results section and move interpretive statements to the discussion. Further, statements around limitations would also be better suited to the discussion. While I agree with the conclusion that increasing the proportion of patients treated with dialysis therapy to PD would lower health care costs, the feasible proportion of patients suitable for peritoneal analysis is controversial. However it is clear that at least in several jurisdictions such as Canada and United States, there are segments of the patient population that would not be suitable for PD, recognizing that PD is generally thought to be significantly underutilized. Exploration of this in the discussion would be useful to place the results in context.Other commentsPage 3 column 2 methods section: The prior literature synthesis from 1996 to 2006 should be referenced. Page 7 column 1 discusses “reimbursement incentives”. Reimbursement implies payment for delivery of a service versus actual cost. These are two different concepts. As this article is looking at cost rather than the effect of reimbursement practices or incentives, I would suggest that this terminology be removed. A recent paper has been published that attempts to look at reasons why PD costs may be higher in some low or middle income countries. This paper suggested that lack of local manufacturing of PD supplies, a low number of PD patients, and high import tariffs may make PD less attractive compared to hemodialysis. It may be worthwhile to incorporate this into the discussion (Karopadi et al., 2013).",
"responses": []
},
{
"id": "5023",
"date": "25 Jun 2014",
"name": "Allen R. Nissenson",
"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 systematic review of the comparative cost of dialysis modalities. In some ways it is a self-fulfilling prophecy- led by Baxter, clearly with a strong proprietary interest in seeing expansion of peritoneal dialysis. Having said that, however, the authors do a reasonable job of articulating the limitations of the current analysis. There are a number of issues, however, that need to be addressed for the conclusions to be supportable:The authors need to more clearly distinguish between “cost” and “payment”. Although this is complex and may be difficult with the papers that were available for analysis, this is key if the purpose is to inform policy makers or clinicians in various countries about the relative value provided by ICHD vs. PD. For example, despite being “less costly” than ICHD, PD may not be viable in a country where the actual payment is less than the actual cost of delivering the modality. It is not clear how the comparison of total costs is accounted for vs. the costs of the modality per se. It would be much more useful to provide information on the distribution of costs by category and how these differ by modality and country. At the end of the day it is the total costs of care that matter- one might envision a country where there is little payment for in-patient care. Any differences in hospitalization rates might not be reflected in the data presented but may be important in determining which modality is most cost effective. In addition to the comment above, the authors very glibly state that clinical outcomes for ICHD and PD are the same. This may not be the same in all countries, however. For example, if peritonitis rates are high and hospitalizations are frequent for infection or catheter malfunctions the total costs of PD may be significantly higher than ICHD. The paragraph in the Introduction about the changing reimbursement systems is too simplistic and rapidly becoming outdated. The authors need to contextualize this work by clearly discussing the need to examine total costs of care and the overall value of competing modalities (quality/cost). The tables would be easier to interpret if the currency were normalized. I understand the complexity of this, but just showing the comparison between ICHD and PD within a country and not among countries seems to be a missed opportunity.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-273
|
https://f1000research.com/articles/2-92/v1
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21 Mar 13
|
{
"type": "Clinical Practice Article",
"title": "A case series discussing the anaesthetic management of pregnant patients with brain tumours",
"authors": [
"Alaa A Abd-Elsayed",
"Jose Díaz-Gómez",
"Gene H Barnett",
"Andrea Kurz",
"Maria Inton-Santos",
"Sabri Barsoum",
"Rafi Avitsian",
"Zeyd Ebrahim",
"Vesna Jevtovic-Todorovic",
"Ehab Farag",
"Alaa A Abd-Elsayed",
"Jose Díaz-Gómez",
"Gene H Barnett",
"Andrea Kurz",
"Maria Inton-Santos",
"Sabri Barsoum",
"Rafi Avitsian",
"Zeyd Ebrahim",
"Vesna Jevtovic-Todorovic"
],
"abstract": "Pregnancy may aggravate the natural history of an intracranial tumour, and may even unmask a previously unknown diagnosis. Here we present a series of seven patients who had brain tumours during pregnancy. The aim of this case series is to characterize the current perioperative management and to suggest evidence based guidelines for the anaesthetic management of pregnant females with brain tumours. This is a retrospective study. Information on pregnant patients diagnosed with brain tumours that underwent caesarean section (CS) and/or brain tumour resection from May 2003 through June 2008 was obtained from the Department of General Anaesthesia and the Rose Ella Burkhardt Brain Tumour & Neuro-Oncology Centre (BBTC) at the Cleveland Clinic, OH, USA. The mean age was 34.5 years (range 29-40 years old). Six patients had glioma, two of whom had concomitant craniotomy and CS. Six cases had the tumour in the frontal lobe. Four cases were operated on under general anaesthesia and three underwent awake craniotomy. The neonatal outcomes of the six patients with elective or emergent delivery were six viable infants with normal Apgar scores. Pregnancy was terminated in the 7th patient. In conclusion, management of brain tumours in pregnant women is mainly reliant on case reports and the doctor’s personal experience. Therefore, close communication between the neurosurgeon, neuroanaesthetist, obstetrician and the patient is crucial. General anaesthesia, propofol, dexmedetomidine and remifentanil were used in our study and were safe. Although this may not agree with previous studies, desflurane and isoflurane were used in our patients with no detectable complications.",
"keywords": [
"pregnancy",
"brain tumours",
"propofol",
"dexmedetomidine",
"remifentanil",
"anaesthetic"
],
"content": "Introduction\n\nPregnancy may increase the growth of a previously existing intracranial tumour, and can even unmask a previously undiscovered tumour. A previous study that included 8 patients who had been diagnosed antenatally with a malignant brain tumour stated that all had severe neurological manifestations, and six of them had a severe neurological event that lead to premature termination of the pregnancy1. It was suggested that immunological tolerance and steroid mediated growth led to this exacerbation during pregnancy2.\n\nIn a population based study; Haas et al. reported that the number of meningiomas, acoustic neuromas, and primary malignant intracranial neoplasms diagnosed during pregnancy was less than expected with the ratio of observed/expected tumours associated with pregnancy to be 0.383.\n\nIn 1988, Simon4 postulated a theory to predict the prevalence of brain tumours in pregnant patients by using the intersection of the probability of being pregnant at any given time with the probability of having a brain tumour at a specific age and sex. Based on this theory the author calculated that in the USA there are about 89 pregnant women per year that also have brain tumours.\n\nBrain tumours in pregnant patients impose a unique risk to both the foetus and mother. There are no previous studies that proposed any guidelines for the anaesthetic management of pregnant patients with brain tumours.\n\nThe aim of this case series is to characterize the current perioperative management of pregnant patients with brain tumours and to suggest guidelines for the proper anaesthetic management.\n\n\nMethods\n\nInformation on pregnant patients diagnosed with brain tumours that underwent CS and/or brain tumour resection from May 2003 to June 2008 was obtained from the Department of General Anaesthesia and the Rose Ella Burkhardt Brain Tumour & Neuro-Oncology Centre (BBTC) IRB-approved databases at the Cleveland Clinic in OH, USA. Patients were managed by the Departments of Neurosurgery, Obstetrics and Gynaecology, Anaesthesiology and BBTC. We used the Anaesthesia Record Keeping System (ARKS) to obtain the electronic record of the anaesthetic management. Additional data from the patients’ electronic and paper charts were used to complete the pre- and post-operative patient information.\n\n\nResults\n\nFive pregnant patients presented with brain tumours during their pregnancy. An additional two patients had their diagnosis of brain tumours made in the immediate postpartum period. Diagnoses (Table 1) included meningioma (1 patient) and glioma (6 patients). The mean age was 34.5 years (range 29–40 years) and parity was 0 (2 patients), 1 (1 patient), and >2 (4 patients). More than half of the patients (57%) underwent CS with craniotomy performed, on average, 45 days after the CS (range: 2–90 days). One case was diagnosed with a brain tumour at the 6th week of gestation and she had a craniotomy during her pregnancy (Table 2). All our patients were managed by general anaesthesia or monitored anaesthesia care (MAC). Inhalational anaesthetic agents (isoflurane and desflurane) were used under 1-minimal alveolar concentration for the maintenance of anaesthesia. Four drugs were used in our patients for both induction and maintenance of anaesthesia; propofol in 3 patients, remifentanyl in 3 patients, dexmeditomidine in 2 patients and alfentanyl in one patient. Foetal heart rate monitoring was applied in one patient receiving MAC for an “awake” craniotomy. Rapid sequence induction was not universally applied. Four cases had general anaesthesia and used rocuronium as a muscle relaxant to facilitate endotracheal intubation. There were no major intraoperative events (Table 1 and Table 3). The neonatal outcomes of the six patients with elective or emergent delivery were six viable infants with normal Apgar scores. Pregnancy was terminated in the 7th patient. There was neither operative mortality nor significant sustained morbidity in this series. Only one patient suffered a pulmonary embolus in the postoperative period.\n\n*FHR: Foetal Heart Rate.\n\n*ICP: Intra-Cranial Pressure.\n\n*EBL: Estimated Blood Loss.\n\n** ETCO2: End Tidal CO2.\n\n\nDiscussion\n\nBrain tumours tend to increase in size during pregnancy due to several factors such as fluid retention, increased blood volume and hormonal changes and therefore may be diagnosed earlier5. The decision to proceed with neurosurgery during pregnancy depends on the site, size, type of tumour, neurological signs and symptoms, age of the foetus, and the patient’s wishes6,7.\n\nThere are no guidelines for the management of intracranial tumours in pregnant women. A possible algorithm to follow is shown in Figure 1 (modified from Tewari et al.1).\n\nCorticosteroids have been recommended as they are safe in pregnancy, promote foetal lung maturity and reduce cerebral oedemas1.\n\nDuring the first and early second trimesters, if the patient is stable, it is acceptable to permit pregnancy to proceed into the early second trimester and surgery can then be performed at this time. It is also possible to administer radiotherapy, radio-surgery and image guided surgery beyond the first trimester. If the patient is unstable, undergoing an urgent neurosurgery is recommended1.\n\nAt the end of the second trimester; in stable patients, proceed with pregnancy with close observation. But if the patient has a worsening neurological status; radiotherapy can be used to delay surgery. If the patient is unstable and shows symptoms of impending herniation, it is recommended to use general anaesthesia to deliver the baby by CS which is followed by surgical decompression1.\n\nAt term;. in a stable patient, induction of vaginal delivery is permitted8. A shortened second stage can be achieved with epidural anaesthesia9. CS should only be performed for accepted indications as it has been shown that CS does not seem to provide any advantage over vaginal delivery in protecting against increased intracranial pressure. In unstable patients, perform, as above, CS under general anaesthesia, followed by surgical decompression1.\n\nMannitol and hypocapnia were avoided in our patients to prevent foetal dehydration and cerebral ischemia/hypoxia, respectively10.\n\nGeneral anaesthesia is safe to be used in patients with intracranial tumours. Tracheal intubation is very important as it allows maternal hyperventilation thereby controlling raised intracranial pressure11. Patients should be pre-medicated with ranitidine 50 mg I.V. to protect the patient against possible vomiting and aspiration.\n\nPropofol was used in six of our patients without producing any side effects. The main side effect is that it has a relaxing effect on the gravid uterus12. It is still controversial whether its use is safe with newborns. Bacon et al. did not report any adverse effects of propofol in newborns after emergency CS13 while another study reported seizure, ataxia, and hallucinations after prolonged propofol anaesthesia for more than 6 hours14.\n\nMeanwhile, isoflurane is known to produce many adverse effects on the foetus15,16. It was used in 3 of our patients but our records did not show any adverse effects. Desflurane was used in one of our patients with no complications. But the neurotoxicity of desflurane and sevoflurane is still a controversial issue17,18.\n\nRemifentanil was used in 3 of our patients without producing any adverse effects; this may be explained by the fact that it has a unique metabolism by plasma and tissue esterases and a context-sensitive half-life of 3 to 4 min, independent of the duration of infusion19. One concern is that the transfer of opioids, such as remifentanil, across the placenta may lead to neonatal depression. However, remifentanil can be metabolized and redistributed to both the mother and the foetus rapidly20. Remifentanil has opioid properties that allow both control of the intraoperative stress response and a more rapid recovery compared to other opioids. Because of its metabolism and short duration of action, remifentanil is therefore considered to be safe and effective for general anaesthesia for emergency CS in patients with neurological risk factors21.\n\nClinically relevant concentrations of remifentanil induce rapid, persistent increases in NMDA-induced ion currents. Since NMDA-receptor blockade during a critical stage in brain development leads to depression of neuronal activity and as such is known to initiate the apoptotic cell death cascade in immature neurons22, we suggest that remifentanil may be safe for the developing brain. In addition, remifentanil is known to offer a neuron-protective effect in cases of opioid induced hyperalgesia or tolerance23. Dexmedetomidine was used in 2 of our patients and its use is recommended in pregnant patients24.\n\nIn the following paragraphs we will discuss drugs that were not used in our study but have been investigated before.\n\nThere are no human trials examining the effects of nitrous oxide on neuronal structure and neurocognitive performance in young children. Some case studies showed that the exposure of neonates to nitrous oxide in utero during the third trimester or during CS can result in transient neurological sequelae25.\n\nAlthough sevoflurane is one of the most prevalent volatile anaesthetics, a recent study has suggested that it can cause epileptic seizure activity, neurotoxicity, and both acute and chronic impairment in synaptic plasticity in neonatal rats26.\n\nOxytocin has been used in patients with intracranial tumours without any adverse effects. Ergotamine can cause hypertensive responses, which may increase the intracranial cranial pressure and can lead to haemorrhage. It should be avoided in pregnant women with brain tumours27.\n\nDexamethasone has been traditionally used to reduce brain oedema. It is safe to use it in an acute setting but its chronic use may be harmful to the foetus as it may cause hypoadrenalism. Weighting the risks and benefits for treating seizures with anticonvulsants; it is recommended to use them in this setting to avoid seizures that may lead to maternal and foetal hypoxia and acidosis28.\n\nSeveral studies investigated the mechanism of anaesthesia-induced neurotoxicity. Previous reports suggested depression of neuronal activity due to anaesthesia induced GABA A receptor activation and NMDA receptor blockade during a critical stage in brain development20. Several adjuvants, such as estradiol, pilocarpine, melatonin and dexmedetomidine, have been identified in animal studies to ameliorate anaesthesia induced neurodegeneration29–31. It is still controversial whether etomidate is neurotoxic or not. There is evidence that the rarely used anaesthetic, xenon, in clinical doses does not have neurodegenerative effects and may be neuroprotective17,18.\n\nA recent study showed that the administration of lithium significantly increased the activation of a neuroprotective pathway in the hippocampus. Further studies and human trials are necessary to fully investigate the beneficial effects of lithium in the anaesthetic management of pregnant patients with brain tumours32.\n\n\nConclusion\n\nManagement of brain tumours in pregnant women is mainly reliant on case reports and the doctor’s personal experience. Therefore, close communication between the neurosurgeon, neuroanaesthetist, obstetrician and the patient is crucial. General anaesthesia, propofol, dexmedetomidine and remifentanil were used in our study and were safe. Although this may not agree with previous studies, desflurane and isoflurane were used in our patients with no detectable complications.",
"appendix": "Author contributions\n\n\n\nAlaa A Abd-Elsayed: designed, analyzed, interpreted data and wrote the manuscript. Jose Diaz Gomez: designed, analyzed and interpreted data. Gene H Barnett: designed the study and drafted the article. Andrea Kurz: interpreted data and drafted the article. Maria Inton-Santos: interpreted data and drafted the article. Sabri Barsoum: designed the study and drafted the article. Rafi Avistian: interpreted data and drafted the article. Zeyd Ebrahim: designed the study and drafted the article. Vesna J Todorovic: designed the study and drafted the article. Ehab Farag: substantial contribution to conception and design, acquisition, analysis and interpretation of data. All authors approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nAll authors declare that there are 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\nTewari KS, Cappuccini F, Asrat T, et al.: Obstetric emergencies precipitated by malignant brain tumors. Am J Obstet Gynecol. 2000; 182(5): 1215–21. PubMed Abstract | Publisher Full Text\n\nGoldberg M, Rappaport ZH: Neurosurgical, obstetric and endocrine aspects of meningioma during pregnancy. Isr J Med Sci. 1987; 23(7): 825–8. PubMed Abstract\n\nHaas JF, Janisch W, Staneczek W: Newly diagnosed primary intracranial neoplasms in pregnant women: a population-based assessment. J Neurol Neurosurg Psychiatry. 1986; 49(8): 874–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimon RH: Brain tumors in pregnancy. Semin Neurol. 1988; 8(3): 214–21. PubMed Abstract | Publisher Full Text\n\nWlody D: Neurosurgery in the pregnant patient. Newfield P, Cottrell J, editors. Philadelphia: WW Lippincott; 1999.\n\nChang L, Looi-Lyons L, Bartosik L, et al.: Anesthesia for cesarean section in two patients with brain tumours. Can J Anaesth. 1999; 46(1): 61–5. PubMed Abstract | Publisher Full Text\n\nKorula G, Farling P: Anesthetic management for a combined Cesarean section and posterior fossa craniectomy. J Neurosurg Anesthesiol. 1998; 10(1): 30–3. PubMed Abstract | Publisher Full Text\n\nBayram Cirak N, Memduh K, Kadir T: Neurosurgical procedures in pregnancy. Acta Chir Brasil. 2003; 18: 5–9. Publisher Full Text\n\nGrocott HP, Mutch WA: Epidural anesthesia and acutely increased intracranial pressure. Lumbar epidural space hydrodynamics in a porcine model. Anesthesiology. 1996; 85(5): 1086–91. PubMed Abstract | Publisher Full Text\n\nCottrell JE, Smith DS: Anesthesia and neurosurgery, 4th Edn.St Louis: Mosby. BJA. 2002; 89(1): 189. Publisher Full Text\n\nGiannini A, Bricchi M: Posterior fossa surgery in the sitting position in a pregnant patient with cerebellopontine angle meningioma. Br J Anaesth. 1999; 82(6): 941–4. PubMed Abstract | Publisher Full Text\n\nLee TL, Adaikan PG, Lau LC, et al.: Effects of propofol on gravid human uterine muscle. J Anesthesia. 1997; 11(1): 71–4. Publisher Full Text\n\nBacon RC, Razis PA: The effect of propofol sedation in pregnancy on neonatal condition. Anaesthesia. 1994; 49(12): 1058–60. PubMed Abstract | Publisher Full Text\n\nBendiksen A, Larsen LM: Convulsions, ataxia and hallucinations following propofol. Acta Anaesthesiol Scand. 1998; 42(6): 739–41. PubMed Abstract | Publisher Full Text\n\nHughes J, Leach HJ, Choonara I: Hallucinations on withdrawal of isoflurane used as sedation. Acta Paediatr. 1993; 82(10): 885–6. PubMed Abstract | Publisher Full Text\n\nArnold JH, Truog RD, Rice SA: Prolonged administration of isoflurane to pediatric patients during mechanical ventilation. Anesth Analg. 1993; 76(3): 520–6. PubMed Abstract\n\nZhao M, Joo DT: Enhancement of spinal N-methyl-D-aspartate receptor function by remifentanil action at delta-opioid receptors as a mechanism for acute opioid-induced hyperalgesia or tolerance. Anesthesiology. 2008; 109(2): 308–17. PubMed Abstract | Publisher Full Text\n\nEishima K: The effects of obstetric conditions on neonatal behaviour in Japanese infants. Early Hum Dev. 1992; 28(3): 253–63. PubMed Abstract | Publisher Full Text\n\nJevtovic-Todorovic V, Hartman RE, Izumi Y, et al.: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003; 23(3): 876–82. PubMed Abstract\n\nLoop T, Priebe HJ: Recovery after anesthesia with remifentanil combined with propofol, desflurane, or sevoflurane for otorhinolaryngeal surgery. Anesth Analg. 2000; 91(1): 123–9. PubMed Abstract | Publisher Full Text\n\nMichelsen LG, Hug CC Jr: The pharmacokinetics of remifentanil. J Clin Anesth. 1996; 8(8): 679–82. PubMed Abstract\n\nRoss AK, Davis PJ, Dear Gd GL, et al.: Pharmacokinetics of remifentanil in anesthetized pediatric patients undergoing elective surgery or diagnostic procedures. Anesth Analg. 2001; 93(6): 1393–401, table of contents. PubMed Abstract | Publisher Full Text\n\nJevtovic-Todorovic V: General anesthetics and the developing brain: friends or foes? J Neurosurg Anesthesiol. 2005; 17(4): 204–6. PubMed Abstract | Publisher Full Text\n\nSouza KM, Anzoategui LC, Pedroso WC, et al.: [Dexmedetomidine in general anesthesia for surgical treatment of cerebral aneurysm in pregnant patient with specific hypertensive disease of pregnancy: case report.]. Rev Bras Anestesiol. 2005; 55(2): 212–6. PubMed Abstract | Publisher Full Text\n\nEdwards DA, Shah HP, Cao W, et al.: Bumetanide alleviates epileptogenic and neurotoxic effects of sevoflurane in neonatal rat brain. Anesthesiology. 2010; 112(3): 567–75. PubMed Abstract | Publisher Full Text\n\nFredriksson A, Archer T, Alm H, et al.: Neurofunctional deficits and potentiated apoptosis by neonatal NMDA antagonist administration. Behav Brain Res. 2004; 153(2): 367–76. PubMed Abstract | Publisher Full Text\n\nTodd MM, Warner DS, Sokoll MD, et al.: A prospective, comparative trial of three anesthetics for elective supratentorial craniotomy. Propofol/fentanyl, isoflurane/nitrous oxide, and fentanyl/nitrous oxide. Anesthesiology. 1993; 78(6): 1005–20. PubMed Abstract\n\nMcCarroll CP, Paxton LD, Elliott P, et al.: Use of remifentanil in a patient with peripartum cardiomyopathy requiring Caesarean section. Br J Anaesth. 2001; 86(1): 135–8. PubMed Abstract | Publisher Full Text\n\nGerstner T, Demirakca S, Demirakca T, et al.: Relevance of phenobarbitalinduced neuronal apoptosis. Psychomotor development of 8 to 14 year olds after neonatal phenobarbital treatment. Monatsschr Kinderheilkund. 2005; 163: 1174–81.\n\nOlney J, Wang H, Qin Y, et al.: Pilocarpine pretreatment reduces neuroapoptosis induced by midazolam or isoflurane in infant mouse brain. Atlanta, GA2006 Contract No. Program No. 286.15.\n\nYon JH, Carter LB, Reiter RJ, et al.: Melatonin reduces the severity of anesthesia-induced apoptotic neurodegeneration in the developing rat brain. Neurobiol Dis. 2006; 21(3): 522–30. PubMed Abstract | Publisher Full Text\n\nTajes M, Yeste-Velasco M, Zhu X, et al.: Activation of Akt by lithium: pro-survival pathways in aging. Mech Ageing Dev. 2009; 130(4): 253–61. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1393",
"date": "27 Aug 2013",
"name": "Michael James Paech",
"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\nThere is not much information about modern management of cerebral tumours during pregnancy, so this paper is of some interest despite the small number of cases. Abstract This draws a conclusion and makes general recommendations– yet there was no related discussion within the Discussion section of the body of the paper. It is inappropriate anyway to draw conclusions based on a small retrospective case series, so this needs revision. After describing the cases and the drugs used, I suggest the author indicates what follows next in the paper ie. a more detailed description of cases and discussion of the drugs used for general anaesthesia. Introduction This reads well and is informative. I prefer the use of “fetus” and there is a good linguistic argument to be made for this spelling. Guidelines should be evidence-based, or in the absence of reasonable evidence at least consensus based, from a group of experts. I don’t think it is reasonable to suggest guidelines should come from ‘studies’ or be based on single institution experience with 7 cases – perhaps the authors should suggest a plan of obstetric and neurosurgical management. Results “Four patients (57%) underwent …” Please replace “case” with “patient’. Remifentanil and alfentanil are misspelt in several places. Table 2 legend - change to “Intracranial pressure”. Correct Table 1 heading to “Postoperative events”. Delete “Only” from the last sentence.\n\nNeonatal condition would be better placed in Table 2, because Table 1 describes several patients in whom tumour resection was much later than delivery, and Table 2 has the obstetric data. The GA technique for tumour resection is mentioned but not the GA technique for CS, which becomes a source of confusion when you later discuss drugs and their effects on the developing brain etc. Can this be added? Discussion A comment should be made about the exclusive use of GA. Regional block may be appropriate (and preferred by the patient) for CS if there is no evidence of raised intracranial pressure or recent seizures – in this series all patients had increased ICP or seizures. A comment about the location of fetal monitoring might also be useful. Correct to “cerebral oedema” and “epidural analgesia” in Management Issues. Preoperative seizure control and the implications of anticonvulsant therapy warrant a mention.‘Motherhood’ statements such as “general anaesthesia is safe to be used with intracranial tumours” should be removed. The discussion of propofol should be re-focused and expanded a little – this is now widely considered suitable for induction in pregnancy, and the main issues are the dose-response curve compared with thiopentone and the neonatal outcome, rather than uterine relaxation. The potential neurotoxicity is not mentioned, yet later the volatiles are implicated and other drugs considered. This area of science is very interesting but no clinical conclusions have been drawn as yet, and I think it has been over-emphasised throughout the author's Discussion, to the detriment of clinical studies and information about the implications of drugs during neurosurgery. In relation to animal neurotoxicity studies, I suggest only drawing comparisons between various drugs and balance these with clinical information. The evidence condemning isoflurane is not supported by the references, which are paediatric case reports rather than fetal exposure. Few would consider isoflurane unacceptable, despite not being ideal; nor would desflurane or sevoflurane be avoided. References after 17 are muddled and do not apply to the text. This needs full review. In addition there are some odd choices of reference – e.g. the case report by McCarroll on remifentanil and peripartum cardiomyopathy is used to justify a comment about anticonvulsants, and ergometrine is discussed using a reference about craniotomy (or – all perhaps the McCarroll case? – but this would still be inappropriate).Again, suggesting that remifentanil is safe for the developing brain is premature if based only on mechanistic action considerations. Likewise use of dexmedetomidine, quoting a single case report! Please discuss mechanisms but don’t generalise. It would make more sense to discuss other drugs, some of which were surely used in the patients, in a more logical sequence (no information provided about GA technique for CS, but presumably oxytocin must have been used, possibly also nitrous oxide?). A restructure of the Discussion section with different headings should be considered.Please do not draw conclusions about safety based on a 7 patient case series.",
"responses": [
{
"c_id": "644",
"date": "11 Dec 2013",
"name": "Alaa Abd-Elsayed",
"role": "Author Response",
"response": "\"After describing the cases and the drugs used, I suggest the author indicates what follows next in the paper\"We have changed the conclusion as advised by the referee. \"I prefer the use of “fetus” and there is a good linguistic argument to be made for this spelling\"The spelling of \"fetus\" was suggested by the journal. \"I don’t think it is reasonable to suggest guidelines should come from ‘studies’ or be based on single institution experience with 7 cases\"We have discussed the evidence from the literature. Having 7 cases from one institute with pregnant patients with brain tumors is a reasonable number, and we are not aware of any other institutions who have published in this topic. It is a rare condition and not that common. \"Neonatal condition would be better placed in Table 2\"Table 2 is already busy, but we will be happy to do this if okay with the journal format. \"The GA technique for tumour resection is mentioned but not the GA technique for CS\"We think this is not relevant to the topic we wanted to discuss. We have corrected the manuscript by changing the number of patients to the pregnant patients who had the tumor resection during pregnancy, to make sure it is relevant to the discussion of the effects of variable anesthetics on the developing fetus. \"A comment should be made about the exclusive use of GA\"We just described what was done, multiple anesthesiologists managed our patients and each did it according to his beliefs and practice. We did not state we did the best management, as there are not clear guidelines for the management. And this is the message - we need to have some guidelines for managing these patients. \"A comment about the location of fetal monitoring might also be useful\"It is always done using external monitoring. \"Correct to “cerebral oedema” and “epidural analgesia” in Management Issues\"We are not sure to what correction the referee refers. \"Preoperative seizure control and the implications of anticonvulsant therapy warrant a mention\"These are already mentioned in the discussion section. \"The discussion of propofol should be re-focused and expanded a little – this is now widely considered suitable for induction in pregnancy, and the main issues are the dose-response curve compared with thiopentone and the neonatal outcome, rather than uterine relaxation\"We have modified the conclusion. Thiopentone is not widely used in our insitute. \"In relation to animal neurotoxicity studies, I suggest only drawing comparisons between various drugs and balance these with clinical information\"This has been mentioned in our discussion. \"...suggesting that remifentanil is safe for the developing brain is premature if based only on mechanistic action considerations. Likewise use of dexmedetomidine, quoting a single case report! Please discuss mechanisms but don’t generalise.\"There has not been much published on these topics, and we quoted the case reports as this is what has been published. \"Please do not draw conclusions about safety based on a 7 patient case series\"We have changed the conclusion. We have addressed and corrected the wordings advised by the referee. The issue with the references has also been checked."
}
]
},
{
"id": "1900",
"date": "07 Oct 2013",
"name": "Hiroyuki Sumikura",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 read this report with interest, as I have just experienced a case of cesarean section with a brain tumor. I wondered if spinal anesthesia was contraindicated for these patients, hence, if the author could add some discussion about this topic it would be greatly appreciated.",
"responses": [
{
"c_id": "645",
"date": "11 Dec 2013",
"name": "Alaa Abd-Elsayed",
"role": "Author Response",
"response": "This is a very good point mentioned by the reviewer, we just think it is out of the scope of this article, and is a topic with a lot of controversy that really needs a full another manuscript to discuss. We focused on the good drugs and agents to use and not the technique of management."
}
]
},
{
"id": "1899",
"date": "24 Oct 2013",
"name": "Carolyn F Weiniger",
"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 case series reports brain tumors and cesarean delivery (CD) over a five year period in Cleveland Clinics, Ohio. The stated aim was to elucidate management suggestions from previously performed cases in their institution.The case series includes seven patients; 5 with preCD and 2 with postCD diagnosis. The results state that \"over half patients underwent CD with craniotomy up to 45 days post CD\". Only one woman had craniotomy during pregnancy. Tables 1 and 2 somewhat clarify this confusing information. The authors report in the results that one patient had tumor diagnosed during pregnancy and had peripartum craniotomy, yet table 2 reports three such cases.Table 1 reports the anesthesia drugs used for craniotomy surgery in the seven patients. Since only 3 patients had craniotomy with CD, it is not clear why Apgar scores are reported here for the patients who had post CD craniotomy.Three patients, including one at 22 weeks gestation and two postCD had awake craniotomys. The authors report that fetal monitoring was used in only one of the awake craniotomy patients. It is my understanding that the other two had already delivered at time of craniotomy. The stated study aim was to provide management suggestions for anesthetic management of pregnant patients with brain tumors. I assumed this meant management for the CD. There are few details regarding the CD surgery, and four patients undergoing craniotomy were no longer pregnant at time of the craniotomy. Therefore table 3 also doesn't make sense- it is titled \"management of pregnant patients for brain tumor resection\" - again, four patients are no longer pregnant.The authors present an algorithm for delivery of women with brain tumors. I could not find in their results any case of VD, all seemed to have CD. I wonder why the two postpartum diagnosed patients had CD? Their tumor was discovered 1 and 3 weeks postpartum.The authors mention that VD is a delivery option, but their study does not bring new evidence for this. The algorithm combines information from other studies and is quite clear, but I wonder if their series adds to our knowledge. For example, stating that propofol was used in six patients and is controversial with newborns is not relevant for the four women who had craniotomy postpartum. Same goes for isoflurane and later - dexmetotomidine. The literature review of pharmacological agents is nicely presented. However again the conclusion reporting safety of des/iso is not particularly relevant, as some women were not pregnant when the drugs were given.",
"responses": [
{
"c_id": "646",
"date": "11 Dec 2013",
"name": "Alaa Abd-Elsayed",
"role": "Author Response",
"response": "We would like to thank the reviewer for her valid points.\"The authors report in the results that one patient had tumor diagnosed during pregnancy and had peripartum craniotomy, yet table 2 reports three such cases\"We have changed this to two patients, as the third case had both managed together. \"Table 1 reports the anesthesia drugs used for craniotomy surgery in the seven patients. Since only 3 patients had craniotomy with CD, it is not clear why Apgar scores are reported here for the patients who had post CD craniotomy.\"We wanted to show the outcomes of all pregnancies regardless of the final management, and we think it is important to mention the fetal outcome. \"There are few details regarding the CD surgery, and four patients undergoing craniotomy were no longer pregnant at time of the craniotomy. Therefore table 3 also doesn't make sense- it is titled \"management of pregnant patients for brain tumor resection\" - again, four patients are no longer pregnant\"We agree with the reviewer - we still think it is important to have this table, but maybe we can change the title to \"Intraoperative management\". \"The authors present an algorithm for delivery of women with brain tumors. I could not find in their results any case of VD, all seemed to have CD. I wonder why the two postpartum diagnosed patients had CD?\"That is a very good point. We wanted to include the algorithm as part of our discussion, as it serves very well to help people managing their cases with the same disease. Our article mainly carries the message of what we did and what can be done better. \"The authors mention that VD is a delivery option, but their study does not bring new evidence for this. The algorithm combines information from other studies and is quite clear, but I wonder if their series adds to our knowledge. For example, stating that propofol was used in six patients and is controversial with newborns is not relevant for the four women who had craniotomy postpartum. Same goes for isoflurane and later - dexmetotomidine. The literature review of pharmacological agents is nicely presented. However again the conclusion reporting safety of des/iso is not particularly relevant, as some women were not pregnant when the drugs were given.\"We agree with the reviewer, and have changed this in the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/2-92
|
https://f1000research.com/articles/2-174/v1
|
14 Aug 13
|
{
"type": "Short Research Article",
"title": "Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells ",
"authors": [
"Anchit Khanna"
],
"abstract": "Background: Protein phosphatase 2A inhibition is one of the pre-requisites for human cell transformation. Previously, we have identified an endogenous inhibitor of PP2A, CIP2A (Cancerous Inhibitor of Protein Phosphatase 2A) in human fibrosarcoma cells (HT1080) using tandem affinity purification. CIP2A over expression has been demonstrated in almost every tumour type studied so far. However, our understanding on the mechanisms regulating CIP2A expression in human cancers, especially in sarcomas, is still emerging. Methods: Human fibrosarcoma (HT1080) cells were treated with small molecule inhibitors against the three major signalling pathways, namely p38, MEK and JNK pathways to identify the pathway regulating CIP2A expression in the sarcoma cells. This was followed by verification of the results using small interfering RNAs (siRNA) for the kinases. Results: In line with previous observations, small molecule inhibitor for MEK pathway (PD98059) decreased CIP2A mRNA and protein expression. Interestingly, small molecule inhibitor for the JNK pathway, SP600125 decreased mRNA and protein levels of CIP2A oncoprotein with negligible effect of SB203580 (p38 kinase) inhibitor on CIP2A expression in HT1080 cells. However, siRNAs specific to either JNK1 or JNK2 kinases did not result in decrease in CIP2A expression. Contrarily, two different CIP2A siRNAs, which were used as positive controls, decreased JNK2 expression in HT1080 cells. Conclusion: Although it is well established that SP600125 inhibits JNK kinases, it has also been shown to inhibit a spectra of other kinases. SP600125 inhibits CIP2A protein expression both in time and concentration dependent manner. However, depletion of both JNK1 and JNK2 kinases using specific siRNAs fails to decrease CIP2A protein expression levels, thereby indicating the need to verify the results obtained by treatment with small molecular inhibitors of kinases by independent approaches like two different target specific siRNAs. Finally, fortuitously we identify JNK2 as a CIP2A downstream target in HT1080 cells.",
"keywords": [
"Fibrosarcoma",
"HT1080",
"SP600125",
"CIP2A",
"JNK2",
"siRNA",
"PP2A"
],
"content": "Introduction\n\nIt has been recently established that regardless of phenotypic variability between different cancer types, perturbation of a limited number of genetic elements is sufficient to induce transformation in different human cell types1. Experimentally, it was demonstrated that activation of RAS and telomerase (TERT), along with inactivation of the tumour suppressor proteins P53 and Retinoblastoma protein (RB) can immortalize a variety of human cell types, which can subsequently transform to a tumourigenic state in response to inhibition of protein phosphatase 2A (PP2A)1,2. Various independent studies have shown that inhibition of PP2A activity is a pre-requisite for human cell transformation1,3–5. Therefore, understanding the mechanisms by which PP2A is inhibited in cancer cells is vital for developing new anti-cancer therapies.\n\nCancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a recently identified oncogene, which has been demonstrated to inhibit the endogenous tumour suppressive activity of PP2A in cancer cells6. Several layers of evidence, both from us and others, have shown CIP2A to be required for malignant cell growth and in vivo tumour formation7–12. In addition, the prognostic role of CIP2A has been demonstrated in several human tumours8,11–13. Moreover, since CIP2A overexpression has been observed at a high frequency in most human cancers studied so far6–9,13–20, identification of mechanisms regulating its expression in human cancers becomes important to address.\n\nAlthough several transcription factors like MYC8, ETS117, E2F19 and ATF219 have been identified as positive regulators of CIP2A in various carcinomas, factors influencing CIP2A expression in non-hematopoietic mesenchymal cells or sarcomas, are yet to be discovered. Notably, CIP2A amplification has been observed in soft-tissue sarcomas21. In addition, since CIP2A was identified using HT1080 (human fibrosarcoma cell line) cell extracts6 this cell line was selected to dissect the mechanisms for high CIP2A expression in sarcomas. Since p38, ERK and JNK signalling pathways are commonly perturbed in cancers, we assessed the role of these pathways in CIP2A expression in HT1080 cells. To this end, respective small molecule kinase inhibitors, namely SB203580 (p38 pathway inhibitor), PD98059 (MEK pathway inhibitor) and SP600125 (JNK pathway inhibitor) were used to inhibit signalling through these pathways in HT1080 cells.\n\n\nMaterial and methods\n\nSP600125 was purchased from Calbiochem (Cat No. - 420119, Merck-Millipore CAS 129-56-6, San Diego, CA) and stocked as a 20 mM solution in DMSO. PD98059 was purchased from Calbiochem (Cat No. - 513000, Merck-Millipore, San Diego, CA) and stocked as 40 mM stock in DMSO. SB203580 was purchased from Calbiochem (Cat No. - 559389, Merck-Millipore, San Diego, CA) and stocked as 20 mM.\n\nThe siRNAs to inhibit CIP2A expression were obtained from Eurofins MWG operon (Ebersberg, Germany). Either of the following double-stranded oligonucleotides was transiently transfected into HT1080 cell line as CIP2A siRNAs: CIP2A.1, 5´-CUGUGGUUGUGUUUGCACUTT-3´, and CIP2A.2, 5´-ACCAUUGAUAUCCUUAGAATT-3´. As a control, a scrambled siRNA with the sequence 5´-UAACAAUGAGAGCACGGCTT-3´ was used instead. HP-validated siRNAs for human JNK1 and JNK2 were purchased from Qiagen Sciences (Germantown, MD). Either of the following oligonucleotides were transiently transfected into HT1080 at 30%–50% confluency in a six-well plate were transfected with the siRNA in antibiotic free medium using RNAiMAX Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.\n\nProteins were extracted in hot Laemmli sample buffer and subjected to immunoblot analysis. Thirty micrograms of total protein extracts was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Helsinki, Finland) and transferred to nitrocellulose membranes (Thermo Scientific Pierce Protein Biology Products, Rockford, USA). Membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS; 20 mM Trizma Base and 150 mM NaCl dissolved in distilled water and adjusted with HCl to pH 7.5) containing 0.1%-NP40 (Igepal Ca-630; Sigma-Aldrich)8. Nitrocellulose membranes (Thermo Scientific Pierce Protein Biology Products, Rockford, USA) were incubated with antibodies to JNK1 (Cat. No. sc-1648: 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), JNK2 (Cat. No. sc-827: 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) in 5% milk in TBS-NP40 (Igepal Ca-630; Sigma-Aldrich) at 4°C overnight, with a 1:5000 dilution of the rabbit polyclonal anti-CIP2A antibody8 at 4°C overnight, or with a 1:1000 dilution of goat polyclonal anti-β-Actin antibody (Cat. No. sc-47778, Santa Cruz Biotechnology) at room temperature for 1 hour. The proteins were visualized by enhanced chemiluminescence (ECL) with Proteome Grasp ECL Kit (Thermo Scientific Pierce Protein Biology Products, Rockford, USA).\n\nHT1080 cells originally were obtained from ATCC and were cultured in DMEM (Gibco) supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin (Bio-Whittaker Europe, Verviers, Belgium).\n\nTotal mRNA was extracted from cells using the RNeasy kit (Qiagen, Valencia, CA) and converted to cDNA by using the M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant cDNA synthesis kit (Promega Corporation, Madison, WI). cDNAs were subjected to quantitative real-time polymerase chain reaction (PCR) by using the Light Cycler (Roche Diagnostics, Mannheim, Germany) and SYBR Green PCR Master Mix kit (Roche Diagnostics). Primer sequences (Sigma-Proligo, St Louis, MO) used for PCR of CIP2A were as follows: CIP2A forward, 5´-CTGGTGAGATAATCAGCAATTT-3´ and CIP2A reverse, 5´-CGAAACATTCATCAGACTTTTCA-3´. Transcript levels were normalized to levels of TATA-binding protein (TBP) or β-Actin expression, which were determined by PCR of the same samples using the following primers: TBP forward, 5´-GAATATAATCCCAAGCGGTTTG-3´, and TBP reverse, 5´-ACTTCACATCACAGCTCCCC-3´; Actin forward, 5´-CGAGCACAGAGCCTCGCCTTTGC-3´, and Actin reverse: 5´-CATAGGAATCCTTC TGACCCATG-3´.\n\nCancer cell line encyclopaedia (http://www.broadinstitute.org/software/cprg/?q=node/11) was used to get the expression levels of JNK1 and JNK2 in HT1080 cells22. This is a resource which provides analysis and visualization of DNA copy number, mRNA expression, mutation data and more, for 1000 cancer cell lines.\n\nStudent T-test was used obtain the statistical significance value using Graph Pad software.\n\n\nResults\n\nTo determine the oncogenic signalling pathways that may be involved in regulating CIP2A expression in human fibrosarcoma, HT1080 cells were treated with small molecule inhibitors for the p38 (SB203580; 20 µM), JNK (SP600125; 10 µM) and ERK (PD98058; 20 µM) signalling pathways. As previously observed in gastric cancer cells17, PD98058 reduced CIP2A mRNA and protein expression in HT1080 cells (Figure 1A and B). Importantly, while SB203580 showed negligible effect, SP600125 potently inhibited CIP2A mRNA and protein expression in HT1080 (Figure 1A and B) cells. Furthermore, inhibition of CIP2A protein expression by SP600125 was observed to be both time and concentration dependent (Figure 1C and D).\n\nA. qRT-PCR showing the effect of small molecule inhibitors against the p38 (SB203580), MEK-ERK (PD98059) and JNK (SP600125) pathways on CIP2A mRNA expression (12h timepoint; Shown is mean values ± S.D., of representative results from three independent experiments. Student T-test was used obtain the statistical significance value) B. Western blot showing the effect of small molecule inhibitors against the p38 (SB203580), MEK-ERK (PD98059) and JNK (SP600125) pathways on CIP2A protein expression (24h timepoint). Shown is the representative picture of two independent experiments. C and D. Effect of small molecule inhibitors against JNK (SP600125) pathway on CIP2A protein expression in concentration (C) and time (D) dependent manner. Shown is the representative picture of two independent experiments.\n\nSP600125-mediated inhibition of CIP2A expression suggested the involvement of the c-Jun N-Terminal Kinases (JNKs) in regulation of CIP2A expression in HT1080 cells. Since the JNK3 isoform is well known to be neural-specific23, specific validated siRNAs against JNK1 and JNK2 isoforms were transfected into HT1080 cells and CIP2A expression estimated. Although, both JNK1 and JNK2 siRNAs reduced their target proteins (JNK1 and JNK2 respectively), there was no change in CIP2A protein expression with either of them individually (Figure 2A and B) or in combination (Figure 2C). Since the JNK2 isoform is expressed more than 10-fold higher than the JNK1 isoform in the HT108022 (Figure 2D) cell line we transfected two validated and specific siRNAs against JNK2. In addition, we also transfected the HT1080 cells with two different CIP2A siRNA as positive controls. In line with our previous observation (Figure 2B and C), the two different siRNAs for JNK2 knocked out JNK2 expression (Figure 2E), while CIP2A expression remained unaltered. Surprisingly, two different CIP2A siRNA, which worked well as positive controls, efficiently decreased JNK2 expression (Figure 2E).\n\nA, B, and C. Western blots showing the effect of JNK1 (A), JNK2 (B) and combination of both JNK1 and JNK2 (C) siRNAs on CIP2A protein expression, 72h post-transfection. Shown is a representative result from two independent experiments. D. mRNA expression of JNK1 and JNK2 in HT1080 cell line from the cancer cell encyclopaedia study. E. Western blot showing the effect of two different siRNAs specific for JNK2 and CIP2A proteins and their protein expression levels 72h post-transfection. The numbers below the blot are the quantified values for CIP2A and JNK2 protein levels normalized to Actin protein levels, relative to the levels in Scrambled (control) transfected cells. Shown is a representative result of two independent experiments.\n\n\nDiscussion\n\nEven though small molecule inhibitors are an emerging therapeutic option against cancers, the specificity issues limit their potential to be used in clinics. They have been extensively used to study various cell signalling pathways. In particular SP600125 has been used to study the effect of c-Jun N-Terminal Kinases (JNKs) in various processes24–26. Our results suggest that even though we were able to see decrease in CIP2A expression in HT1080 cells on treatment with SP600125 (Figure 1), at doses used previously to inhibit c-Jun NH2-terminal kinase (JNKs) activity27,28, we were not able to validate the findings using two different siRNAs specific to the JNK2 kinase (Figure 2E). Interestingly, a decade ago a previous study emphatically demonstrated the effect of SP600125 on different kinases29. The study revealed that SP600125, although a JNK inhibitor, could inhibit the activity of several other kinases29. In fact, SP600125 was demonstrated to inhibit kinases like CK1, DYRK1 and AMPK even more effectively than JNK kinase itself29 (Supplementary Table 1).\n\nSurprisingly, two different CIP2A siRNAs used as positive controls, decreased JNK2 expression levels in HT1080. This has also been observed in a separate study in an epithelial origin cell line, HeLa20. Interestingly, JNK2 has been shown to regulate CIP2A expression via ATF2 transcription factor in mouse embryo fibroblasts (MEFs)19 in the pre-transformed stage. Since we observe the vice versa in fully transformed HT1080 cells, it can suggest that there may be a molecular switch between JNK2 and CIP2A which may have a possible role in the RAS-transformation of mesenchymal cells. Nevertheless, the functional consequence of CIP2A-mediated JNK2 expression in mesenchymal cells would require further exploration.\n\nAltogether, our study highlights the need for the validation of results obtained by small molecule treatments with independent approaches like two or more target specific siRNAs, shRNAs or use of inducible systems like RNAi or Tamoxifen/Tetracycline-induced overexpression systems8.",
"appendix": "Competing interests\n\n\n\nNo competing interests to declare.\n\n\nGrant information\n\nThe work was funded by Academy of Finland through the Tampere Graduate Program in Biomedicine and Biotechnology.\n\n\nAcknowledgements\n\nProfessor Jukka Westermarck is acknowledged for his guidance.\n\n\nSupplementary table\n\n\n\n\nReferences\n\nZhao JJ, Roberts TM, Hahn WC: Functional genetics and experimental models of human cancer. Trends Mol Med. 2004; 10(7): 344–50. PubMed Abstract | Publisher Full Text\n\nMumby M: PP2A: Unveiling a Reluctant Tumor Suppressor. Cell. 2007; 130(1): 21–4. PubMed Abstract | Publisher Full Text\n\nWestermarck J, Hahn WC: Multiple pathways regulated by the tumor suppressor PP2A in transformation. Trends Mol Med. 2008; 14(4): 152–60. PubMed Abstract | Publisher Full Text\n\nHahn WC, Dessain SK, Brooks MW, et al.: Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol Cell Biol. 2002; 22(7): 2111–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHahn WC, Weinberg RA: Rules for making human tumor cells. N Engl J Med. 2002; 347(20): 1593–603. PubMed Abstract | Publisher Full Text\n\nJunttila MR, Puustinen P, Niemela M, et al.: CIP2A inhibits PP2A in human malignancies. Cell. 2007; 130(1): 51–62. PubMed Abstract | Publisher Full Text\n\nCome C, Laine A, Chanrion M, et al.: CIP2A is associated with human breast cancer aggressivity. Clin Cancer Res. 2009; 15(16): 5092–100. PubMed Abstract | Publisher Full Text\n\nKhanna A, Bockelman C, Hemmes A, et al.: MYC-dependent regulation and prognostic role of CIP2A in gastric cancer. J Natl Cancer Inst. 2009; 101(11): 793–805. PubMed Abstract | Publisher Full Text\n\nLaine A, Sihto H, Come C, et al.: Senescence sensitivity of breast cancer cells is defined by positive feedback loop between CIP2A and E2F1. Cancer Discov. 2013; 3(2): 182–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen KF, Liu CY, Lin YC, et al.: CIP2A mediates effects of bortezomib on phospho-Akt and apoptosis in hepatocellular carcinoma cells. Oncogene. 2010; 29(47): 6257–66. PubMed Abstract | Publisher Full Text\n\nDong QZ, Wang Y, Dong XJ, et al.: CIP2A is overexpressed in non-small cell lung cancer and correlates with poor prognosis. Ann Surg Oncol. 2011; 18(3): 857–65. PubMed Abstract | Publisher Full Text\n\nHe H, Wu G, Li W, et al.: CIP2A Is Highly Expressed in Hepatocellular Carcinoma and Predicts Poor Prognosis. Diagn Mol Pathol. 2012; 21(3): 143–9. PubMed Abstract | Publisher Full Text\n\nBockelman C, Lassus H, Hemmes A, et al.: Prognostic role of CIP2A expression in serous ovarian cancer. Br J Cancer. 2011; 105(7): 989–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBockelman C, Hagstrom J, Makinen LK, et al.: High CIP2A immunoreactivity is an independent prognostic indicator in early-stage tongue cancer. Br J Cancer. 2011; 104(12): 1890–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBockelman C, Koskensalo S, Hagstrom J, et al.: CIP2A overexpression is associated with c-Myc expression in colorectal cancer. Cancer Biol Ther. 2012; 13(5): 289–95. PubMed Abstract | Publisher Full Text\n\nKhanna A, Böckelman C, Laine A, et al.: 859 Constitutive DNA-damage signaling promotes cancer cell proliferation through Chk1-CIP2A pathway. Ejc Supplements. 2010; 8(5): 217. Publisher Full Text\n\nKhanna A, Okkeri J, Bilgen T, et al.: ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in human cancer cells. PloS One. 2011; 6(3): e17979. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhanna A, Okkeri J, Westermarck J: KIAA1524. Atlas of Genetics and Cytogenetics in Oncology and Haematology. 2010; 14(10): 976–978. Publisher Full Text\n\nMathiasen DP, Egebjerg C, Andersen SH, et al.: Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation. Oncogene. 2012; 31(3): 390–401. PubMed Abstract | Publisher Full Text\n\nNiemela M, Kauko O, Sihto H, et al.: CIP2A signature reveals the MYC dependency of CIP2A-regulated phenotypes and its clinical association with breast cancer subtypes. Oncogene. 2012; 31(39): 4266–78. PubMed Abstract | Publisher Full Text\n\nBarretina J, Taylor BS, Banerji S, et al.: Subtype-specific genomic alterations define new targets for soft-tissue sarcoma therapy. Nat Genet. 2010; 42(8): 715–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarretina J, Caponigro G, Stransky N, et al.: The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012; 483(7391): 603–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNamgung U, Xia Z: Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase. J Neurosci. 2000; 20(17): 6442–51. PubMed Abstract\n\nGuillemot L, Citi S: Cingulin regulates claudin-2 expression and cell proliferation through the small GTPase RhoA. Mol Biol Cell. 2006; 17(8): 3569–77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Y, Gorelik GJ, Strickland FM, et al.: Decreased ERK and JNK signaling contribute to gene overexpression in \"senescent\" CD4+CD28- T cells through epigenetic mechanisms. J Leukoc Biol. 2010; 87(1): 137–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCao Y, Xia DS, Qi SR, et al.: Epiregulin can promote proliferation of stem cells from the dental apical papilla via MEK/Erk and JNK signalling pathways. Cell Prolif. 2013; 46(4): 447–56. PubMed Abstract | Publisher Full Text\n\nMartinez K, Kennedy A, McIntosh MK: JNK inhibition by SP600125 attenuates trans-10, cis-12 conjugated linoleic acid-mediated regulation of inflammatory and lipogenic gene expression. Lipids. 2011; 46(10): 885–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang G, Yue Z, Talloczy Z, et al.: Autophagy induced by Alexander disease-mutant GFAP accumulation is regulated by p38/MAPK and mTOR signaling pathways. Hum Mol Genet. 2008; 17(11): 1540–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBain J, McLauchlan H, Elliott M, et al.: The specificities of protein kinase inhibitors: an update. Biochem J. 2003; 371(Pt 1): 199–204. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1558",
"date": "20 Aug 2013",
"name": "Kristopher Clark",
"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\nInhibition of the protein phosphatase PP2A contributes to the acquisition of a cancerous phenotype in cells. One mechanism by which the inhibition of PP2A can be achieved is through the up-regulation of endogenous inhibitors of PP2A such as CIP2A. However, the signaling pathways regulating CIP2A expression in cancer cells remain poorly defined. In this article, Anchit Khanna has studied the role of the different mitogen-activated protein (MAP) kinase cascades in regulating the expression of CIP2A in the HT1080 human fibrosarcoma cell line. Treatment of HT1080 cells with the pharmacological inhibitor SP600125 led to a drop in the mRNA and protein expression of CIP2A in HT1080 cells. SP600125 has often been used to implicate the protein kinases of the JNK family in the cellular process under study, but as pointed out by the author, this compound has many off-target effects. Anchit Khanna therefore attempted to validate the results using siRNA mediated knock-down of JNK1 and JNK2 expression in HT1080 cells. Unfortunately, CIP2A expression was normal in the cells in which JNK1 and/or JNK2 were depleted using siRNA oligos. The author concluded that the effects of SP600125 on CIP2A expression were not due to JNK1 or JNK2 inhibition, and stated the importance of validating experimental results using independent methods. The data as presented does not confirm or refute a role for the JNKs in regulating CIP2A expression in HT1080 cells, as alternative explanations for the data remain possible. For instance, the knock-down of JNK1 or JNK2 may not be sufficiently effective, leaving residual JNK1/2 catalytic activity in cells which drives the expression of CIP2A. Indeed, the data presented in figure 2A show that JNK1 expression was suppressed by only 50% and the author failed to show the level of JNK1 expression in the experiment depicted in figure 2C. In addition to blotting for the respective kinase, it would have also been informative in both the inhibitor and RNAi studies to measure the phosphorylation of a substrate such as c-Jun to verify that these treatments indeed blocked JNK function in HT1080 cells. It is also good practice to use structurally-unrelated protein kinase inhibitors to reduce the chance of off-target effects contributing to the phenotype under study. Since SP600125 entered the market, much improved JNK inhibitors have been developed such as JNK-in-8 (Zhang et al. 2012 Chem Biol). It would be worthwhile to test the effect of such compounds on CIP2A expression, which could help establish whether a role for JNKs exist in this pathway. The author is correct in stating that one must validate results using different approaches. I was therefore very surprised to read that the author concluded that CIP2A is involved in regulating the expression of JNK2 in HT1080 using a single approach. RNAi technology, like other methodologies, is fraught with off-target problems. It is essential to validate the results of these experiments by re-expressing the protein using an RNAi-resistant cDNA of CIP2A. This control experiment would strongly support the author’s conclusion that CIP2A controls JNK2 expression in HT1080 cells. I acknowledge that these experiments are challenging and the author may be able to provide supporting evidence using alternative approaches to the one used in this article.Although the experiments are well performed, the study is incomplete and does not allow one to make a strong case for or against involvement of the JNKs in regulating CIP2A expression. Based on this assessment, I have scored this paper ‘Approved with Reservations’. Additional Minor Points: 1. The author omitted to indicate the statistical significance of the data in Figure 1A. 2. In a single author paper, one should not use the terms ‘we’ and ‘us’ rather ‘I’ and ‘me.’",
"responses": [
{
"c_id": "643",
"date": "11 Dec 2013",
"name": "Anchit Khanna",
"role": "Author Response",
"response": "''The data as presented does not confirm or refute a role for the JNKs in regulating CIP2A expression in HT1080 cells, as alternative explanations for the data remain possible. For instance, the knock-down of JNK1 or JNK2 may not be sufficiently effective, leaving residual JNK1/2 catalytic activity in cells which drives the expression of CIP2A.\"I thank Dr. Clark for his prompt and valuable comments on the manuscript. However, I would like to highlight Figures 2B, C and E wherein almost 100% knockdown of JNK2 kinase was achieved. In fact, two different siRNAs for JNK2 isoform, which is the isoform that is majorly expressed in HT1080 (Fig. 2D) cells, completely abolished JNK2 expression leaving little room for the existence of residual JNK2 activity in these cells for maintaining CIP2A levels. Additionally, 10 fold higher expression of JNK2 isoform in comparison to the JNK1 isoform in Ht1080 cells (Fig 2D), suggests it to be more relevant functionally. \"...the data presented in figure 2A show that JNK1 expression was suppressed by only 50% and the author failed to show the level of JNK1 expression in the experiment depicted in figure 2C.\"I thank Dr. Clark for this relevant observation. I have now attached the JNK1 levels from the experiment shown in Fig 2C as supplementary figure 1A with quantifications. As shown in the figure, despite complete knockdown of JNK1 in these cells, CIP2A levels remain almost unaltered, thereby re-emphasizing the fact that JNK1 does not regulate CIP2A expression in HT1080 cells. Complete knockdown of JNK1 also rules out the possibility of residual JNK1 activity driving CIP2A expression and the use of c-Jun as marker of JNK function in this experiment. With reference to SP600125, the concentrations used in Figure 1 have been demonstrated previously in several studies to inhibit JNK activity (p-Jun levels). Few examples of the studies that have used similar concentrations of SP600125 to inhibit JNK activity are Yue et al., 2011 (used 20uM of SP600125), Duan et al., 2011 (5uM of SP600125 in HT1080 cells), and Kong et al., 2013 (10uM of SP600125). \"It is also good practice to use structurally-unrelated protein kinase inhibitors to reduce the chance of off-target effects contributing to the phenotype under study\"I thank Dr. Clark for sharing his expertise in the field and totally agree that structurally–unrelated kinase inhibitors have decreased off target effects and their use would definitely further confirm the negative findings. However, the main aim of this study is to highlight the un-specificity of SP600125 compound which has been used extensively as specific JNK inhibitor. In addition, using two different siRNAs (to overcome off-target effects due to one siRNA) that knock down JNK2 expression almost completely (Fig 2E) without changing the levels of CIP2A significantly strongly suggest that JNK2 does not regulate CIP2A expression in HT1080 cells. \"The author is correct in stating that one must validate results using different approaches. I was therefore very surprised to read that the author concluded that CIP2A is involved in regulating the expression of JNK2 in HT1080 using a single approach. RNAi technology, like other methodologies, is fraught with off-target problems. It is essential to validate the results of these experiments by re-expressing the protein using an RNAi-resistant cDNA of CIP2A. This control experiment would strongly support the author’s conclusion that CIP2A controls JNK2 expression in HT1080 cells. I acknowledge that these experiments are challenging and the author may be able to provide supporting evidence using alternative approaches to the one used in this article.\"I thank Dr. Clark for highlighting the issue of siRNA specificity and totally agree that there are off-target effects that have to be ruled out with this approach. It is for this reason most journals and the general scientific community requests the use of two independent siRNAs for verifying the effects due to depletion of the target gene. Accordingly, as shown in Fig 2E, there have been two independent siRNAs against both JNK2 and CIP2A used which not only almost completely deplete their target proteins but also rule out the element of un-specificity which usually arises by the use of a single siRNA against the target protein. Additionally, an independent study (Niemelä et al., 2012) using microarray approach identified JNK2 as downstream target of CIP2A in HeLa cells, thereby lending further confidence to these findings. Moreover, as mentioned in the manuscript this finding surfaced just by chance as CIP2A siRNAs were being used as positive controls in the experiment. Additionally, I agree and have now further highlighted in the manuscript as well that CIP2A–mediated regulation of JNK2 would need further exploration. Finally, I would like to emphasize that the main aim of this study is to highlight that SP600125, concentrations at which it has been demonstrated to inhibit JNK activity, may also inhibit other kinases as highlighted by an elegant study previously (kinases that it inhibits have been listed in supplementary table 1). Interestingly, we recently identified CHK1 kinase (which is almost as sensitive as JNK to SP600125) as a positive regulator of CIP2A expression (Khanna et al., 2013). Therefore, to explore the possible role of CHK1 and other SP600125 sensitive kinases in regulating CIP2A expression, we did a pilot screen and transfected HT1080 cells with 2 different siRNAs against CHK1 (Kinase almost as sensitive to SP600125 as JNK according to previously published study) and with siRNAs against CDK2 and DYRK1 (two other kinases that are more sensitive to SP600125 than JNK2). As shown in supplementary figure 1B, two different siRNAs against CHK1 decreased CIP2A expression as observed previously, while CDK2 and DYRK1 didn’t change CIP2A expression. This suggests that most likely the SP600125 kinase regulating CIP2A expression in HT1080 is CHK1, as observed in other cancers cells like prostate, breast, gastric and cervical cancers (Khanna et al., 2013). \"Although the experiments are well performed, the study is incomplete and does not allow one to make a strong case for or against involvement of the JNKs in regulating CIP2A expression.\"I thank Dr. Clark for his positive and encouraging comments and hope that the my response, new data and changes in the text mitigate his previous concerns and are enough to conclude that there are un-specificity issues that may exist with SP600125, the small molecule inhibitor of JNK2 kinase as highlighted by the study. Additional Minor Points:The revised figure with the p-values using student t-test is now attached and is in accordance to the figure legend. I thank Dr. Clark for bringing this to my notice and have revised this in the modified manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/2-174
|
https://f1000research.com/articles/2-219/v1
|
17 Oct 13
|
{
"type": "Research Article",
"title": "Does excitatory fronto-extracephalic tDCS lead to improved working memory performance?",
"authors": [
"Níall Lally",
"Camilla L. Nord",
"Vincent Walsh",
"Jonathan P. Roiser",
"Camilla L. Nord",
"Vincent Walsh",
"Jonathan P. Roiser"
],
"abstract": "Evidence suggests that excitatory transcranial direct current stimulation (tDCS) may improve performance on a wide variety of cognitive tasks. Due to the non-invasive and inexpensive nature of the method, harnessing its potential could be particularly useful for the treatment of neuropsychiatric illnesses involving cognitive dysfunction. However, questions remain regarding the efficacious stimulation parameters. Here, using a double-blind between-subjects design, we explored whether 1 mA excitatory (anodal) left dorsolateral prefrontal cortex stimulation with a contralateral extracephalic reference electrode, leads to enhanced working memory performance across two days, relative to sham stimulation. Participants performed the 3-back, a test of working memory, at baseline, and during and immediately following stimulation on two days, separated by 24-48 hours. Active stimulation did not significantly enhance performance versus sham over the course of the experiment. However, exploratory comparisons did reveal a significant effect of stimulation group on performance during the first stimulation phase only, with active stimulation recipients performing better than sham. While these results do not support the hypothesis that dorsolateral prefrontal cortex tDCS boosts working memory, they raise the possibility that its effects may be greatest during early learning stages.",
"keywords": [
"Transcranial direct current stimulation (tDCS) has been utilised as a non-invasive brain stimulation methodology to improve performance on a variety of cognitive tasks in healthy volunteers",
"including decision-making1",
"planning2 and working memory3",
"4. Due to the minimal risk profile",
"arising as function of the very low current delivered to the scalp",
"and the relatively inexpensive nature of the device",
"it has high potential as a clinical tool. Tentative evidence thus far suggests tDCS may be effective in ameliorating neurological and psychiatric illnesses associated with cognitive deficits. Prominent amongst these are recent developments in the study of the treatment of addiction",
"depression",
"schizophrenia and stroke5. However",
"questions remain over stimulation condition blinding6",
"optimal stimulation frequency7 and appropriate electrode placement8 as these parameters can strongly influence neuronal activity and the interpretation of effects on cognitive performance. Questions have also been raised about behavioural and stimulation controls9."
],
"content": "Introduction\n\nTranscranial direct current stimulation (tDCS) has been utilised as a non-invasive brain stimulation methodology to improve performance on a variety of cognitive tasks in healthy volunteers, including decision-making1, planning2 and working memory3,4. Due to the minimal risk profile, arising as function of the very low current delivered to the scalp, and the relatively inexpensive nature of the device, it has high potential as a clinical tool. Tentative evidence thus far suggests tDCS may be effective in ameliorating neurological and psychiatric illnesses associated with cognitive deficits. Prominent amongst these are recent developments in the study of the treatment of addiction, depression, schizophrenia and stroke5. However, questions remain over stimulation condition blinding6, optimal stimulation frequency7 and appropriate electrode placement8 as these parameters can strongly influence neuronal activity and the interpretation of effects on cognitive performance. Questions have also been raised about behavioural and stimulation controls9.\n\nMany studies have reported enhanced task performance in healthy volunteers following application of the excitatory anodal electrode to a location of the scalp corresponding to a task-relevant region of the brain, including: the temporal parietal junction to enhance social cognition10; inferior frontal cortex to enhance target detection11,12; Wernicke’s area to enhance visual picture naming13; and dorsolateral prefrontal cortex (DLPFC) to enhance decision making1,14 and working memory3,4,7. The application of the excitatory tDCS electrode to the scalp is thought to cause an increase in neuronal excitability in the stimulated area by altering the resting potential15. To complete the electrical circuit, the reference or inhibitory cathodal electrode must be placed somewhere on the head or body being stimulated.\n\nThe majority of studies exploring cognitive enhancement using tDCS have targeted DLPFC as their region of excitation while the inhibitory electrode has typically been placed on the contralateral supra-orbit (or DLPFC). For example, in a single blind investigation using this electrode montage, Ohn et al.4 found that 30 minutes of 1 mA tDCS while participants performed the n-back task led to significant improvements in task performance over sham during stimulation only. However, placing the reference electrode on the scalp introduces a potential confound in the interpretation of any resulting behavioural effects: these could arise as a result of excitation, inhibition, or a combination of the two electrodes. The location of the reference electrode, whether extra or intra-cephalic, has been show to play a prominent role in the efficacy of the excitatory electrode16.\n\nThe n-back17 is a cognitive task commonly used to assess aspects of executive function, and is thought to engage working memory in particular. Although criticisms have been made with respect to its construct validity18, the n-back has frequently been used in the context of functional neuroimaging experiments in both healthy volunteers and patients with psychiatric and neurological illnesses. The results of these studies consistently implicate a network of brain regions including parietal cortex and DLPFC, which are engaged with increasing load during n-back performance19. Importantly, altered DLPFC function is associated with several psychiatric conditions20–22. For example, research using the n-back has identified that the DLPFC is hyperactive in patients diagnosed with major depressive disorder (MDD)23,24, suggesting that MDD patients may need to use greater resources to achieve the same level of performance. Similar findings have been reported in schizophrenia25.\n\nWorking memory dysfunction, and executive function deficits more broadly, have been found across a number of psychopathologies, including attention deficit disorder, autistic spectrum disorder, traumatic brain injury, Alzheimer’s disease, schizophrenia and depression26–28. Indeed, executive function performance has been identified as a tractable endophenotype to explore across neuropsychiatric illnesses due to its prevalence, particularly in depression and schizophrenia29,30. Thus, there is significant potential for non-invasive brain stimulation techniques such as tDCS to be applied clinically to ameliorate cognitive dysfunction. However, in order to establish whether tDCS has the potential to improve clinical conditions through modulatory effects on executive function, it is pertinent to first establish the effects of specific stimulation parameters in healthy volunteers.\n\nA recent single-blind within-subjects investigation by Zaehle et al.31 demonstrated that 15 minutes of 1 mA excitatory tDCS applied to left DLPFC during rest, with the inhibitory electrode placed ipsilaterally at the mastoid, resulted in enhanced post-stimulation performance on the 2-back task in comparison with cathodal, but not sham, stimulation. The position of the reference electrode can however affect the underlying cortical excitability16. Importantly, Zaehle et al.31 utilized a fronto-extracephalic montage, which attenuates interpretational difficulties; but it remains unknown whether contralateral or ipsilateral positioned reference electrodes or bifrontal montages are superior in the stimulation of DLPFC. Furthermore, it remains to be determined whether stimulation during a task or while at rest is more beneficial. Andrews et al.3 found tentative evidence to suggest that DLPFC tDCS applied concurrently with a cognitive task may provide more robust effects on subsequent working memory performance that stimulation during rest.\n\nHere, we sought to build on prior research3,4,31 by conducting a double-blind between-subjects experiment to examine whether excitatory DLPFC tDCS applied across two days would lead to enhancement of n-back performance during and post-stimulation. Specifically, we assessed whether excitatory fronto-extracephalic DLPFC tDCS, with the reference on the contralateral cheek, could improve performance on the 3-back in healthy volunteers across two stimulation days. We hypothesized that those receiving active stimulation would have greater task performance improvement, relative to baseline, in comparison with sham stimulation recipients.\n\n\nMethods\n\nTwenty-one (14 females, M = 23.09 years, SD = 3.95) right-handed participants were recruited from the Psychology subject pool at University College London, UK. Participants self-reported no history of mental or neurological illness, current psychiatric medication use, no prior or current participation in another brain stimulation experiment within the previous 24 hours and had normal or corrected to normal vision. There were no significant age (t(19) = 0.211, P = 0.835) or gender (X2(1) = 1.527, P = 0.361) differences between the two stimulation groups (active and sham). All participants provided written informed consent and were compensated for their time. The study was approved by the UCL ethics committee.\n\nTask. The n-back (Figure 1) consisted of a continuous sequence of 300 (150 for baseline) centrally presented consonants (500 ms) interleaved with fixation crosses (1500 ms). Participants were instructed to respond to every appearance of a letter, pressing the ‘H’ key only when the letter onscreen matched the letter 3-back, and pressing the ‘F’ key for all other instances. It is thought that this version of the n-back may afford increased sensitivity to working memory performance than versions that focus solely on hits31,32. Matches (one-fifth of all stimuli) and non-matches to 3-back stimuli were randomized in order but their ratio was fixed throughout the experiment. The task was coded in Matlab (release 2008b for Windows; Mathworks, Natick, MA, USA) using the Cogent Toolbox (http://www.vislab.ucl.ac.uk/cogent_2000.php) and is available for free online (available at https://sites.google.com/site/nialllally/home/code/ and https://github.com/nialllally/nback_dprime/blob/master/NBack.zip). The code is also permanently available at 10.5281/zenodo.7148.\n\nStimuli (consonants) were presented centrally for 500 ms and followed by a fixation cross for 1500 ms. Participants were instructed to respond to every stimulus, indicating whether the stimulus matched the letter 3-back (‘H’) or not (‘F’).\n\ntDCS was administered continuously at 1 mA using the Neuroconn DC-Stimulator (Neuroconn, Germany) via a pair of rubber electrodes (7 cm × 5 cm) housed in small synthetic sponges dampened with salt water to increase conductivity. The excitatory (anodal) electrode was placed over F3 (Figure 2A), corresponding to the left DLPFC, while the reference (cathodal) electrode was placed on the contralateral cheek. F3 was located using a 10–20 electroencephalography cap and demarcated using a removable marker. Left DLPFC was chosen as the anodal electrode position as this region has been consistently implicated in working memory paradigms19. Additionally as the task involved processing static letters, the left side of the brain was considered most appropriate33. Once the area was located, the electrodes were fastened in position using two headbands (a polyester hairband across the forehead and a rubber band beneath the jaw and around the circumference of the head; Figure 2B).\n\nBefore arrival, participants were randomized to one of two brain stimulation conditions using Matlab, active (N=10) or sham (N=11). Specific codes were selected from the tDCS device manual by an independent researcher not involved with the study and were assigned to each condition and randomized to each participant. Importantly, utilizing the ‘study mode’ of the device allowed the stimulation-administering researchers to remain blinded to the condition participants were in as the readout on the stimulation apparatus was identical for both active and sham stimulation. The administered current was applied for 10 minutes with an additional 15-second fade-in and fade-out ramping period to minimize discomfort and facilitate participant blinding. Sham stimulation was limited to small pulses of 100–200 μA every 400–550 ms between a 15-second fade-in and fade-out voltage ramp34.\n\nDuring the baseline session on day 1 (D1), participants were first trained on the n-back with a brief exposure to 1, 2 and finally 3-back. Thereafter, participants completed a 5-minute version of the 3-back, which served as a baseline pre-stimulation measure of performance. Immediately after, tDCS was administered for 10 minutes while participants performed the 3-back task (D1 tDCS; Figure 2C). Following this, participants completed a further 10-minute session of the 3-back (D1 post-tDCS) without stimulation. Participants were instructed that continuation to day 2 was dependent on task performance but were not given feedback until the end of day 1. Continuation to day 2 was dependent on above chance performance, which was any positive d' value:\n\nd' = Z(hit rate) - Z(false alarm rate) (1)\n\nwhere hit and false alarm rate are the number of correct or incorrect ‘H’ responses, respectively, divided by the total number of opportunities (1/5 or 4/5 of total stimulus letters) and Z is the inverse of the cumulative Gaussian distribution. Participants received £10 for their participation on day 1 irrespective of task performance. Day 2 (24–48 hours later) consisted of one 10-minute task run with stimulation (D2 tDCS) and one post-stimulation (D2 post-tDCS). Participants were told that performing better than the test phase of day 1 would result in a bonus of £10 on top of the £5 basic payment on day 2.\n\nA, B) The excitatory anodal electrode (red) was positioned using a 10–20 standard electroencephalography electrode cap under F3, which corresponds to dorsolateral prefrontal cortex. The inhibitory cathodal electrode (blue) was positioned on the contralateral cheek. C) Timeline of events in the study. Participants performed the 3-back on five separate occasions, once at baseline on day 1, twice during and twice following stimulation on both days.\n\nTo assess the effect of active stimulation versus sham over time, we conducted a linear mixed model in SPSS, version 21 (IBM Corp New York 2012). The dependent variable was d'. Follow up models also were conducted using hit rate, correct rejection rate (1 - false alarm rate) and reaction time for both of these variables. The four testing sessions after baseline (D1 tDCS, D1 post-tDCS, D2 tDCS, D2 post-tDCS) were entered as a fixed effect of time; tDCS and sham stimulation were entered as a fixed effect of group; and their interaction (time-by-group) was also entered as a fixed effect. Participant number was entered as a random effect and baseline performance was entered as a covariate. A heterogeneous first order autoregressive covariance structure was employed. Bonferonni corrected tests between the groups at each time point were conducted using linear contrasts to assess between-group differences. Follow up assessments of significant points were assessed using a general linear model with baseline performance entered as a covariate. Performance differences at baseline were assessed using an independent sample t-test. Based on our sample size we had 80% power to detect a large effect size (d=1.3) at P = 0.05 (two-tailed) between the stimulation groups.\n\n\nResults\n\nOne participant (sham group) scored a negative d' value for day 1 and did not participate in session 2, but their data from day 1 were included in the linear mixed model. Additionally, the testing computer malfunctioned during the day 1 post-tDCS assessment for 1 participant (active group), approximately 40% through the task. These data were included in the model, and the participant completed a further post-tDCS test, which was used only to determine progress to day 2; these data were not included in the model. There was no significant difference in d' performance between the groups at baseline (t(19) = 1.044, P = 0.309; Figure 3). As expected, there was a significant main effect of time (F(3,36) = 7.669, P < 0.001) on d' performance, reflecting improvement across both groups with increasing exposure to the task. However, contrary to our hypothesis, no main effect of stimulation group was identified (F(1,16) = 2.228, P = 0.155) and there was no group × time interaction (F(3,36) = 1.339, P = 0.277).\n\nExploratory Bonferonni corrected pairwise comparisons were carried out to assess group performance differences at the four post-baseline time points. A significant difference between active sham stimulation was identified at the day 1 tDCS time point (F(1,13.373) = 10.747, P = 0.006; controlling for baseline performance and Bonferonni corrected for multiple comparisons), indicating a large effect size (Cohen’s d = 1.427, r2 = 0.337). No other stimulation group differences in d' were found at other time points (all F < 1.2, P > 0.3; see Table 1 for group performance across sessions and task components). Further analyses of performance during stimulation during day 1 (including baseline as a covariate) revealed that both the hit rate (F(1,18) = 4.454, P = 0.049, ηp2 = 0.198) and correct rejection rate (F(1,18) = 3.680, P = 0.071, ηp2 = .170) of active stimulation recipients were significantly, or at trend level, better than sham. However, no significant reaction time differences were found for this time point for either hits (F(1,18) = 0.010, P = 0.923, ηp2 = 0.001) or correct rejections (F(1,18) = 0.202, P = 0.659, ηp2 = 0.011).\n\nThe active stimulation group always performed better than the sham group but only statistically significantly so during stimulation on day 1 (D1 tDCS), denoted by an asterisk (*). Baseline performance did not differ between the groups but was included in the model as a covariate. Error bars represent ±1 standard error of the mean.\n\nD1 = day 1, HR = hit rate, CRR = correct rejection rate, RT = reaction time, CR = correct rejection.\n\n\n\n\nDiscussion\n\nDeficits in executive function, including working memory, have been implicated in many neurological and psychiatric conditions and have also been targeted as potentially tractable endophenotypes in both schizophrenia35 and depression29. Although tDCS has been suggested as a therapeutic tool for many cognitive and neurological impairments, very few tDCS studies have conducted double-blind assessments in either clinical or non-clinical populations. Furthermore, the specific ameliorative stimulation parameters, such as amplitude, frequency and electrode positioning, are largely undefined. Importantly, few electrode montages have been tested1,31,36,37, fewer studies yet have applied tDCS for greater than one session38–40 and many of the variables in the available parameter space have not been subject to systematic manipulation9. Contrary to our hypothesis, no effect of tDCS stimulation was identified in this study. However, exploratory tests did suggest that active stimulation was associated with enhanced performance relative to sham stimulation during the first stimulation period on day 1 only. Our results may therefore indicate that the performance enhancement effects of excitatory tDCS may be limited to earlier stages of learning40. They also suggest that reports of improvements after one session of tDCS – the most common report in enhancement studies – may not translate to continual improvement with additional stimulation.\n\nOur results do not support the hypothesis that excitatory tDCS applied to DLPFC results in post-stimulation improvement on the n-back task across multiple days. This result is consistent with some previous research; in comparison with sham stimulation, neither Zaehle et al.31 nor Ohn et al.4 demonstrated significant performance enhancements on the n-back task immediately following excitatory DLPFC tDCS. Nevertheless, we did find evidence for a specific improvement in performance during stimulation on day 1 only, an outcome consistent with results from Ohn et al.4 and others41. Andrews et al.3 found that DLPFC excitatory tDCS applied during a working memory task (n-back) led to significant improvements in post-stimulation performance in comparison with baseline on an alternative working memory task (digit span forward but not backward). The improvements found3 were not present for either sham stimulation in conjunction with task performance or stimulation without task performance. Behavioural data were not reported for the task during stimulation and an intracephalic reference electrode was used, limiting direct comparison with the present study. Furthermore, Hoy et al.7 found that 1 mA excitatory tDCS applied to DLPFC at rest resulted in an enhancement in 2-back reaction times 40 minutes post-stimulation, but found no improvement in accuracy. However, other reports have found evidence for more enduring cognitive enhancement following tDCS2,39 (but see Walsh9).\n\nThis discrepancy between results may reflect the different tasks, electrode montages, stimulation parameters, sample sizes and study designs used. For example, it is possible that the payment schemes that served as a performance motivator here limited the potential to observe performance enhancing effects of tDCS. As there was no monetary motivation during the stimulation phase on day 1, participants may not have exerted themselves fully and thus the effects of stimulation may have had greater leverage; while on day 2, participants in both groups may have reached a level whereby any potential for further enhancement of performance through tDCS was limited. Whilst the sample size used here is low for a between-subjects study, few tDCS studies have thus far been conducted using large sample sizes, and future studies should address the issue of stimulation parameter optimization using large sample sizes. Nevertheless, our results suggest that tDCS may be particularly sensitive to earlier stages of learning40.\n\nIn theory, the beneficial effects of tDCS may be most pronounced in poorer performers. Indeed, there is some evidence that tDCS may be particularly useful as a cognitive enhancer with lower performing individuals42. As the population utilised here primarily comprised students from University College London, between-group differences arising as a function of tDCS may have been attenuated due to high initial baseline ability. Finally, evidence suggests that individual differences in genotype may play a large part in susceptibility to the plasticity enhancing capabilities of tDCS. Fritsch et al.43 found that tDCS was more efficacious in both mice and humans possessing the homozygous Val/Val genotype of the brain-derived neurotrophic factor polymorphism (rs6265), than Met carriers, though we did not have a sufficiently large sample to explore such moderators in the current study.\n\nThe results of this experiment require careful replication and extension to validate the potential role for tDCS in executive function enhancement. In particular, the evaluation of result specificity represents a prominent hole in the current literature. Few studies thus far have contrasted active stimulation results in comparison with control tasks and active stimulation of control site locations on the scalp9; such measures would be beneficial in assessing the findings here and across the field. Additionally, it could be fruitful to replicate this experiment without the monetary incentive. Testing a larger and more representative sample including non-university students would also be informative. Furthermore, while performance improvements under stimulation are important, the clinical utilization of tDCS necessitates long lasting effects once stimulation has ceased. As many psychiatric and neurological illnesses are associated with deficits in executive function task performance, inclusion of a patient group may permit the assessment of the viability of tDCS as a neuroenhancement methodology for psychiatric illnesses. Recent research has indeed shown that tDCS can enhance cognitive control, a component of executive function, in MDD44; however, long-lasting cognitive ameliorative effects of stimulation in depression have yet to be demonstrated.\n\nIn conclusion, our results do not support the hypothesis that excitatory tDCS applied to the left DLPFC using a contralateral fronto-extracephalic electrode reference produces consistent improvements in executive function beyond the period of stimulation. Nonetheless, we found a beneficial effect of tDCS during task performance only when the task was relatively novel, which could be interpreted as indicating that this particular electrode montage, stimulation voltage and study design may be best suited to early stages of learning.",
"appendix": "Author contributions\n\n\n\nNL and CLN collected the data. NL and JPR analysed the data. All authors were involved in the writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by a Wellcome Trust PhD studentship awarded to Níall Lally (WT095465). Vincent Walsh is supported by a Royal Society Industry Fellowship.\n\n\nAcknowledgments\n\nThe authors thank Joseph Arizpe for assistance with the stimulation device and David Luckenbaugh for statistical advice.\n\n\nReferences\n\nHecht D, Walsh V, Lavidor M: Transcranial direct current stimulation facilitates decision making in a probabilistic guessing task. J Neurosci. 2010; 30(12): 4241–5. PubMed Abstract | Publisher Full Text\n\nDockery CA, Hueckel-Weng R, Birbaumer N, et al.: Enhancement of planning ability by transcranial direct current stimulation. J Neurosci. 2009; 29(22): 7271–7. PubMed Abstract | Publisher Full Text\n\nAndrews SC, Hoy KE, Enticott PG, et al.: Improving working memory: the effect of combining cognitive activity and anodal transcranial direct current stimulation to the left dorsolateral prefrontal cortex. Brain Stimul. 2011; 4(2): 84–9. PubMed Abstract | Publisher Full Text\n\nOhn SH, Park CI, Yoo WK, et al.: Time-dependent effect of transcranial direct current stimulation on the enhancement of working memory. Neuroreport. 2008; 19(1): 43–7. PubMed Abstract | Publisher Full Text\n\nKuo MF, Paulus W, Nitsche MA: Therapeutic effects of non-invasive brain stimulation with direct currents (tDCS) in neuropsychiatric diseases. Neuroimage. 2013. PubMed Abstract | Publisher Full Text\n\nO'Connell NE, Cossar J, Marston L, et al.: Rethinking clinical trials of transcranial direct current stimulation: participant and assessor blinding is inadequate at intensities of 2mA. PLoS One. 2012; 7(10): e47514. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoy KE, Emonson MR, Arnold SL, et al.: Testing the limits: Investigating the effect of tDCS dose on working memory enhancement in healthy controls. Neuropsychologia. 2013; 51(9): 1777–84. PubMed Abstract | Publisher Full Text\n\nMiranda PC, Lomarev M, Hallett M: Modeling the current distribution during transcranial direct current stimulation. Clin Neurophysiol. 2006; 117(7): 1623–9. PubMed Abstract | Publisher Full Text\n\nWalsh VQ: Ethics and social risks in brain stimulation. Brain Stimul. 2013; 6(5): 715–7. PubMed Abstract | Publisher Full Text\n\nSantiesteban I, Banissy MJ, Catmur C, et al.: Enhancing social ability by stimulating right temporoparietal junction. Curr Biol. 2012; 22(23): 2274–7. PubMed Abstract | Publisher Full Text\n\nCoffman BA, Trumbo MC, Clark VP: Enhancement of object detection with transcranial direct current stimulation is associated with increased attention. BMC Neurosci. 2012; 13: 108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClark VP, Coffman BA, Mayer AR, et al.: TDCS guided using fMRI significantly accelerates learning to identify concealed objects. Neuroimage. 2012; 59(1): 117–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSparing R, Dafotakis M, Meister IG, et al.: Enhancing language performance with non-invasive brain stimulation--a transcranial direct current stimulation study in healthy humans. Neuropsychologia. 2008; 46(1): 261–8. PubMed Abstract | Publisher Full Text\n\nFecteau S, Knoch D, Fregni F, et al.: Diminishing risk-taking behavior by modulating activity in the prefrontal cortex: a direct current stimulation study. J Neurosci. 2007; 27(46): 12500–5. PubMed Abstract | Publisher Full Text\n\nNitsche MA, Paulus W: Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation. J Physiol. 2000; 527(Pt 3): 633–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoliadze V, Antal A, Paulus W: Electrode-distance dependent after-effects of transcranial direct and random noise stimulation with extracephalic reference electrodes. Clin Neurophysiol. 2010; 121(12): 2165–71. PubMed Abstract | Publisher Full Text\n\nKirchner WK: Age differences in short-term retention of rapidly changing information. J Exp Psychol. 1958; 55(4): 352–8. PubMed Abstract | Publisher Full Text\n\nJaeggi SM, Buschkuehl M, Perrig WJ, et al.: The concurrent validity of the N-back task as a working memory measure. Memory. 2010; 18(4): 394–412. PubMed Abstract | Publisher Full Text\n\nOwen AM, McMillan KM, Laird AR, et al.: N-back working memory paradigm: a meta-analysis of normative functional neuroimaging studies. Hum Brain Mapp. 2005; 25(1): 46–59. PubMed Abstract | Publisher Full Text\n\nPascual-Leone A, Rubio B, Pallardo F, et al.: Rapid-rate transcranial magnetic stimulation of left dorsolateral prefrontal cortex in drug-resistant depression. Lancet. 1996; 348(9022): 233–7. PubMed Abstract | Publisher Full Text\n\nPizzagalli DA: Frontocingulate dysfunction in depression: toward biomarkers of treatment response. Neuropsychopharmacology. 2011; 36(1): 183–206. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeinberger DR, Berman KF, Zec RF: Physiologic dysfunction of dorsolateral prefrontal cortex in schizophrenia. I. Regional cerebral blood flow evidence. Arch Gen Psychiatry. 1986; 43(2): 114–24. PubMed Abstract | Publisher Full Text\n\nHarvey PO, Fossati P, Pochon JB, et al.: Cognitive control and brain resources in major depression: an fMRI study using the n-back task. Neuroimage. 2005; 26(3): 860–9. PubMed Abstract | Publisher Full Text\n\nMatsuo K, Glahn DC, Peluso MA, et al.: Prefrontal hyperactivation during working memory task in untreated individuals with major depressive disorder. Mol Psychiatry. 2007; 12(2): 158–66. PubMed Abstract | Publisher Full Text\n\nCannon TD, Glahn DC, Kim J, et al.: Dorsolateral prefrontal cortex activity during maintenance and manipulation of information in working memory in patients with schizophrenia. Arch Gen Psychiatry. 2005; 62(10): 1071–80. PubMed Abstract | Publisher Full Text\n\nElliott R: Executive functions and their disorders. Br Med Bull. 2003; 65(1): 49–59. PubMed Abstract | Publisher Full Text\n\nGoldberg MC, Mostofsky SH, Cutting LE, et al.: Subtle executive impairment in children with autism and children with ADHD. J Autism Dev Disord. 2005; 35(3): 279–93. PubMed Abstract | Publisher Full Text\n\nTate RL: Executive dysfunction and characterological changes after traumatic brain injury: two sides of the same coin? Cortex. 1999; 35(1): 39–55. PubMed Abstract | Publisher Full Text\n\nHasler G, Drevets WC, Manji HK, et al.: Discovering endophenotypes for major depression. Neuropsychopharmacology. 2004; 29(10): 1765–81. PubMed Abstract | Publisher Full Text\n\nHoran WP, Braff DL, Nuechterlein KH, et al.: Verbal working memory impairments in individuals with schizophrenia and their first-degree relatives: findings from the Consortium on the Genetics of Schizophrenia. Schizophr Res. 2008; 103(1–3): 218–28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaehle T, Sandmann P, Thorne JD, et al.: Transcranial direct current stimulation of the prefrontal cortex modulates working memory performance: combined behavioural and electrophysiological evidence. BMC Neurosci. 2011; 12: 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaatveit BC, Sundet K, Hugdahl K, et al.: The validity of d prime as a working memory index: results from the \"Bergen n-back task\". J Clin Exp Neuropsychol. 2010; 32(8): 871–80. PubMed Abstract | Publisher Full Text\n\nMull BR, Seyal M: Transcranial magnetic stimulation of left prefrontal cortex impairs working memory. Clin Neurophysiol. 2001; 112(9): 1672–5. PubMed Abstract | Publisher Full Text\n\nPalm U, Reisinger E, Keeser D, et al.: Evaluation of sham transcranial direct current stimulation for randomized, placebo-controlled clinical trials. Brain Stimul. 2013; 6(4): 690–5. PubMed Abstract | Publisher Full Text\n\nGlahn DC, Therman S, Manninen M, et al.: Spatial working memory as an endophenotype for schizophrenia. Biol Psychiatry. 2003; 53(7): 624–6. PubMed Abstract | Publisher Full Text\n\nMartin DM, Alonzo A, Mitchell PB, et al.: Fronto-extracephalic transcranial direct current stimulation as a treatment for major depression: an open-label pilot study. J Affect Disord. 2011; 134(1–3): 459–63. PubMed Abstract | Publisher Full Text\n\nPlazier M, Joos K, Vanneste S, et al.: Bifrontal and bioccipital transcranial direct current stimulation (tDCS) does not induce mood changes in healthy volunteers: a placebo controlled study. Brain Stimul. 2012; 5(4): 454–61. PubMed Abstract | Publisher Full Text\n\nKang EK, Kim YK, Sohn HM, et al.: Improved picture naming in aphasia patients treated with cathodal tDCS to inhibit the right Broca's homologue area. Restor Neurol Neurosci. 2011; 29(3): 141–52. PubMed Abstract | Publisher Full Text\n\nCohen Kadosh R, Soskic S, Iuculano T, et al.: Modulating neuronal activity produces specific and long-lasting changes in numerical competence. Curr Biol. 2010; 20(22): 2016–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeinzer M, Jähnigen S, Copland DA, et al.: Transcranial direct current stimulation over multiple days improves learning and maintenance of a novel vocabulary. Cortex. 2013. PubMed Abstract | Publisher Full Text\n\nFregni F, Boggio PS, Nitsche M, et al.: Anodal transcranial direct current stimulation of prefrontal cortex enhances working memory. Exp Brain Res. 2005; 166(1): 23–30. PubMed Abstract | Publisher Full Text\n\nTseng P, Hsu TY, Chang CF, et al.: Unleashing potential: transcranial direct current stimulation over the right posterior parietal cortex improves change detection in low-performing individuals. J Neurosci. 2012; 32(31): 10554–61. PubMed Abstract | Publisher Full Text\n\nFritsch B, Reis J, Martinowich K, et al.: Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning. Neuron. 2010; 66(2): 198–204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWolkenstein L, Plewnia C: Amelioration of cognitive control in depression by transcranial direct current stimulation. Biol Psychiatry. 2013; 73(7): 646–51. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2142",
"date": "15 Nov 2013",
"name": "Marian Berryhill",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors provide a between-subjects comparison of 3-back performances after active and anodal tDCS to the left DLPFC. The first question of interest was to identify the time course of potential tDCS performance modulations and the second, to determine whether extracephalic (contralateral cheek) influenced performance. A significant benefit of active tDCS was identified immediately after the first session, but was not seen not during the first or second sessions. The experiment appears to be very well done and the writing clearly explains their process. My primary concern is that the authors conducted this experiment using a very small sample size (which they acknowledge, and provide power estimates that are quite reasonable). However, tDCS effects in healthy young university students tend to be quite modest. Ideally, the authors would replicate the experiment with a larger sample and/or with a secondary WM task (apart from the baseline covariate) to identify any heterogeneities in the population that could be obscuring equal and opposite effects. More importantly, to provide greater generalizability in their data, it would be good for them to compare the cheek site with the commonly used orbital site. This would allow greater comparison between the current study and the existing research, and strengthen their point that different paradigms may elicit different effects.My second point is that the authors do not really return to the issue of the second electrode site as a potential explanation for their modest effects. One possible explanation would be that the previous work stimulating the contralateral supraorbital region provided stimulation elsewhere in the PFC which contributed to the WM benefit reported by others. Here, current modelling would be particularly helpful to compare the flow of current between the L. DLPFC-R. cheek vs. L-DLPFC-R.supraorbital montages. However, it is clear that the full parameter-space of tDCS effects requires increased clarity and a forum to avoid the 'file-drawer problem' associated with neurostimulation and small effect sizes. Overall therefore, these data will be of interest to the tDCS research community.",
"responses": [
{
"c_id": "640",
"date": "10 Dec 2013",
"name": "Niall Lally",
"role": "Author Response",
"response": "We thank Dr. Berryhill for the helpful comments. We agree that the issue of the return electrode is critical in the tDCS literature and remains to be fully evaluated. Therefore, we have revised the manuscript to include a discussion of the issue of the reference electrode in this experiment and identified issues which need to be addressed in future experiments."
}
]
},
{
"id": "2527",
"date": "21 Nov 2013",
"name": "Andrea Antal",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction: The authors cite previous studies (e.g., Zaehle et al., 2011) as an example that the applied electrode montage makes the interpretation of the effect of previous tDCS results difficult, due to the confounding factor coming from the cephalic or extracephalic position of the return electrode. However, the present study addresses this question relatively shortly, limiting the question to the extracephalic reference electrode position. Please rewrite this sentence.Method: Why did you include 21 subjects? Did the task last for exactly the same time for each participant (irrespectively of the individual differences in the reaction time)? Why did the participants receive performance-dependent extra monetary reward on Day 2 only? It makes the comparison between Day 2 and the previous session rather difficult.Results: First paragraph: line 1-5 and line 6 is seemingly contradictory.Discussion: page 5: ”no effect of tDCS “ delete stimulation, and: “was identified” on what?",
"responses": [
{
"c_id": "641",
"date": "10 Dec 2013",
"name": "Niall Lally",
"role": "Author Response",
"response": "We thank Dr. Antal for the comments and suggestions. We have amended the manuscript to address the majority of comments."
}
]
},
{
"id": "2398",
"date": "22 Nov 2013",
"name": "Angelo Alonzo",
"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 study investigates a topic of rapidly growing interest in the field of non-invasive brain stimulation techniques – namely using these techniques to improve cognitive abilities.This article is well referenced although I refer the authors to a recent article by Martin et al.1 which found enhancing effects of tDCS on a dual n-back task over 10 successive sessions. Although the present study found no clear difference between active and sham conditions except during tDCS on Day 1, a clearer advantage of tDCS may nonetheless have been observed with additional sessions.The statistical analyses conducted have been well detailed and I believe are appropriate for the questions of interest. The limitations of the study have also been adequately discussed. The authors rightly point out that the way in which the monetary incentive for participation was managed could have differentially affected performance between the two testing days, thus possibly obscuring any enhancing effects of tDCS. As also noted, the fact that the sample was primarily comprised of university students may have lessened the likelihood of observing tDCS effects, although Martin et al.1 similarly recruited a university student sample. Perhaps utilising a more difficult task, for example a dual n-back task or adjusting the n value based on participant performance on the previous trial block, may have produced greater differences in performance and allowed any potential effects of tDCS to be borne out.Regarding other methodological aspects, the authors applied quite an unusual placement for the reference electrode on the contralateral cheek; one that I have never encountered before. This would not be considered an extracephalic montage as conventionally, an extracephalic electrode is one that has been placed off the head (e.g., deltoid). I am curious as to why the authors chose this montage and if there is any modelling data to support this. Also, were participants asked to guess their stimulation condition after each session? I note that a double-blind design was used but the success of blinding should still be tested.In short, I commend the authors in investigating the potential uses of tDCS as a clinical tool but given the above limitations, changes in methodology would need to be implemented before drawing firmer conclusions.",
"responses": [
{
"c_id": "642",
"date": "10 Dec 2013",
"name": "Niall Lally",
"role": "Author Response",
"response": "We thank Dr. Alonzo for the detailed review, kind comments and suggestions. We have altered the title of the article to better reflect the electrode locations used here. Furthermore, we have attempted to address concerns surrounding the electrode montage by adding references to articles that have used the contralateral cheek as a reference, and added a paragraph to the discussion section detailing the issue of the reference electrode in tDCS experiments. As astutely indicated by Dr. Alonzo, the montage used is somewhat novel and current modelling studies comparing this configuration to other more established montages have yet to be performed. Finally, we thank Dr. Alonzo for the suggested article (Martin et al., 2013)."
}
]
}
] | 1
|
https://f1000research.com/articles/2-219
|
https://f1000research.com/articles/2-271/v1
|
09 Dec 13
|
{
"type": "Case Report",
"title": "Case Report: Resolution of submacular haemorrhage secondary to exudative age-related macular degeneration after a single intravitreal dobesilate injection",
"authors": [
"Pedro Cuevas",
"Luis Antonio Outeiriño",
"Carlos Azanza",
"Javier Angulo",
"Guillermo Giménez-Gallego",
"Luis Antonio Outeiriño",
"Carlos Azanza",
"Javier Angulo",
"Guillermo Giménez-Gallego"
],
"abstract": "Introduction: Submacular haemorrhage is not an unusual cause of acute central vision loss, particularly in older people. It may be caused by a number of conditions, most common of which is exudative age-related madular degeneration. In patients affected by this type of macular degeneration, choroidal neovascularization extends into the subretinal space, producing substantial bleeding in approximately 17% of cases, resulting in large haemorrhages in the subretinal space that detach the neurosensory retina from the supporting retinal pigment epithelial (RPE) layer. This leads to substantial vision loss because of a relatively fast process of extensive photoreceptor atrophy in the overlying neuroretina and formation of macular scarsCase presentation: We describe a patient with submacular haemorrhage secondary to exudative age-related macular degeneration, treated with intravitreal injection of dobesilate. Two months later, visual acuity in the treated eye reached 0.50 with a significant improvement of the distortion and an anatomical resolution of the haemorrhage, as confirmed by optical coherence tomography.Conclusions: Submacular haemorrhage secondary to exudative age-related macular degeneration can be successfully treated with intravitreal dobesilate. To our knowledge, this is the first case reporting a resolution of submacular haemorrhage after a single dobesilate injection.",
"keywords": [
"Fibroblast growth factor. Dobesilate. Submacular haemorrhage"
],
"content": "Case description\n\nA 75 year-old Caucasian man with a history of exudative age-related macular degeneration (AMD) in both eyes developed a sudden vision decrease in the right eye. The best corrected visual acuity (BCVA) on presentation was 0.05. Fundus examination showed a recent medium-sized submacular haemorrhage (SMH) (Figure 1A), and spectral-domain optical coherence tomography (SD-OCT) depicted a remarkable anomalous architecture of the retina and serious disturbances under the retinal pigment epithelium (RPE) without significant accumulation of intraretinal fluid (Figure 1D). The haemorrhage also seemed to have resulted in RPE detachment at some points (Figure 1D, asterisk). Treatment with dobesilate was recommended. After approval of our Institution Ethical Committee, the patient signed an informed consent form, which included a comprehensive description of dobesilate and the proposed procedure. Using a standard protocol, the patient received an intravitreal solution of dobesilate (150 μl) in his right eye under sterile conditions, following the International Guidelines for intravitreal injections1. Dobesilate was administered as a 12.5% solution of diethylammonium 2.5-dihydroxybenzenesulfonate (etamsylate; Dicynone® Sanofi-Aventis, Paris, France). No ocular side effects were observed upon the administration of dobesilate or during the following days. Over two months follow-up the haemorrhage signs gradually disappeared (Figure 1B,C), and the SD-OCT scans showed a progressive normalization of the retinal architecture with the disappearance of the RPE detachment (Figure 1E,F). At the end of this period of two months, the patient’s BCVA had reached a value of 0.50.\n\nThe asterisk in D indicates retinal pigment epithelium (RPE) detachment.\n\n\nDiscussion\n\nPatients with exudative AMD and SMH on initial presentation have more than a 50% chance of developing recurrent submacular bleeding within the first subsequent three years2. Retinal diseases characterized by an excess of vessel proliferation are sometimes treated with antibodies that inhibit vascular endothelial growth factor (VEGF). Recently, anti-VEGF antibodies have been also proposed for the management of SMH3,4. However, this therapy needs repeated injections in some patients and takes several months to clear SMH5. In addition, SMH may occur following the injection of intravitreal anti-VEGF agents6,7. As subretinal haemorrhages are, so far, inadequately addressed with the use of the current anti-angiogenic therapies, many different treatments have been used to physically remove or displace them, with inconclusive results8. Thus, safe and efficient therapy for SMH is needed. Here we present a patient with SMH secondary to exudative AMD who was successfully treated with a single intravitreal dobesilate injection, a synthetic inhibitor of fibroblast growth factor.\n\nUsing a murine model of subretinal haemorrhage it has been shown that, like other central nervous system locations such as the cerebral cortex, inactive microglia, which reside in the inner retina under quiescent conditions, acquire the reactive phenotype and rapidly migrate to the site of the haemorrhage8. Once there, microglia participate in the phagocytosis of blood products and promote inflammation at the injury site, together with leukocytes and monocytes of the extravasated blood, by secreting cytokines, chemokines and other immunomolecules. In these studies it was also shown that inhibition of microglial activation reduced microglial infiltration and photoreceptor cell loss caused by SMH8.\n\nMicroglial cells are activated by fibroblast growth factor (FGF) synthesized by astrocytes in the case of the central nervous system9. In the case of hemorrhages, extravasated blood cells like monocyte-derived macrophages also synthesize FGF10, which also acts as chemoattractant of microglial cells. Furthermore, microglia also synthesize FGF and other cytokines, once they have been activated. Since activated microglia also expresses FGF receptors (FGFRs), an autocrine loop could readily develop in the case of important lesions, which could sustain the appearance of chronic inflammation. In addition, recently published studies, point to the fundamental importance of activated microglia in re-shaping the vasculature during pathological insults9,11–16. This additional source of FGF may greatly contribute to the development and worsening of AMD.\n\nFGF was the first inducer of vasculogenesis and proliferation of endothelial cells to be isolated17. Later on, it was shown that it is a broad-spectrum mitogen18 and, recently, that FGF should be envisioned rather as an inflammation-triggering and -sustaining protein than as mere mitogen for mesoderm-derived cells19,20. These inflammatory activities are probably an important contributor to the sequelae of the brain and retinal haemorrhages, including development and worsening of AMD. FGF may further contribute to aggravate this scenario by directly promoting vascular permeability when it participates in neovessel formation17,21,22. We have shown that neovascular growth induced by FGF and the accompanying bleeding can be suppressed with inhibitors of FGF23. Consequently, it seems that inhibition of FGF could be an appropriate treatment to prevent retinal injuries in the aftermath of haemorrhages.\n\nWe have spent important efforts in the development of synthetic inhibitors of FGF. These studies led to the identification of a family of small-size chemical inhibitors, structurally similar to gentisic acid, the first member of the group that was identified. The most active of the family was a compound known in pharmacology as dobesilate, the active principle of Doxium, a drug that has been orally administered for more than 35 years for the treatment of diabetic retinopathy with a good safety profile, but inconclusive outcomes25,26. Contrarily, we have obtained clear positive results in different retinal pathologies27–31 by administering, off-label, dobesilate through intraocular injection.\n\nOur results may seem in contradiction with those of Haritoglou et al.26, who carried out an accurate statistical study to assess the real clinical benefits of calcium dobesilate in the treatment of diabetic retinopathy. They concluded that the oral administration of dobesilate did not show statistically significant clinical benefits for treating this disease. Perhaps, as we discussed in detail in previous articles, the different administration procedures employed in the Haritoglou et al. study and in our treatments, respectively, may explain the differences in outcome, since the intestinal flora and relative instability of dobesilate above pH 5 may significantly hamper the ability of the inhibitor to reach adequate concentrations in the retina27. FGF is involved in the homeostasis of the tissues of mesodermal and neuroectodermal origin. A detailed discussion about the apparent paradox that constitutes that the administration of a FGF inhibitor does not cause considerable distortion in those tissues has been carried out in previous articles27.\n\nVEGF is a protein that enhances the permeability of blood vessels (actually, it was also isolated under the name of vascular permeability factor). It is also, apparently, a paradox that the treatments with antibodies against VEGF had not shown significant therapeutic effects in the treatment of SMH. To shed some light on this apparent contradiction one has probably to take into account the serious drawbacks of depleting VEGF for a long time, since it is a key component in the homeostasis of the retina. The clearance of those antibodies from the vitreous is about 10 days, although some believe it to be as long as 2.5 months32–34. In contrast, inhibition of FGF is not accompanied by a prolonged presence of dobesilate at the eye, as it readily decomposes at physiological pH24. We have also shown that dobesilate can also be used to suppress VEGF-induced angiogenesis, as expected from the necessary synergism between VEGF and FGF for promoting angiogenesis35.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details and images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nPC and GGG wrote the paper. LO and CA were the physicians responsible for the patient in this case report. All authors have participated in the concept and design/analysis and interpretation of data, drafting and revising the manuscript, and they have given final approval for the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAiello LP, Brucker AJ, Chang S, et al.: Evolving guidelines for intravitreous injections. Retina. 2004; 24(5 Suppl): S3–S19. PubMed Abstract\n\nHwang JU, Yang SJ, Yoon YH, et al.: Recurrent submacular hemorrhage in patients with neovascular age-related macular degeneration. Retina. 2012; 32(4): 652–7. PubMed Abstract | Publisher Full Text\n\nStifter E, Michels S, Prager F, et al.: Intravitreal bevacizumab therapy for neovascular age-related macular degeneration with large submacular hemorrhage. Am J Ophthalmol. 2007; 144(6): 886–92. PubMed Abstract | Publisher Full Text\n\nMcKibbin M, Papastefanou V, Matthews B, et al.: Ranibizumab monotherapy for sub-foveal haemorrhage secondary to choroidal neovascularisation in age-related macular degeneration. Eye (Lond). 2010; 24(6): 994–8. PubMed Abstract | Publisher Full Text\n\nShienbaum G, García Filho CA, Flynn H Jr, et al.: Management of submacular haemorrhage secondary to neovascular age-related macular degeneration with anti-vascular endothelial growth factor monotherapy. Am J Ophthalmol. 2013; 155(6): 1009–13. PubMed Abstract | Publisher Full Text\n\nGoverdhan SV, Lochhead J: Submacular haemorrhages after intravitreal bevacizumab for large occult choroidal neovascularisation in age-related macular degeneration. Br J Ophthalmol. 2008; 92(2): 210–2. PubMed Abstract | Publisher Full Text\n\nKrishnan R, Goverdhan S, Lochhead J: Submacular haemorrhage after intravitreal bevacizumab compared with intravitreal ranibizumab in large occult choroidal neovascularization. Clin Exp Ophthalmol. 2009; 37(4): 384–8. PubMed Abstract | Publisher Full Text\n\nZhao L, Ma W, Fariss RN, et al.: Minocycline attenuates photoreceptor degeneration in a mouse model of subretinal hemorrhage microglial: inhibition as a potential therapeutic strategy. Am J Pathol. 2011; 179(3): 1265–77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee M, Kang Y, Suk K, et al.: Acidic fibroblast growth factor (FGF) potentiates glial-mediated neurotoxicity by activating FGFR2 IIIb protein. J Biol Chem. 2011; 286(48): 41230–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYum HY, Cho JY, Miller M, et al.: Allergen-induced coexpression of bFGF and TGF-β1 by macrophages in a mouse model of airway remodeling: bFGF induces macrophage TGF-β1 expression in vitro. Int Arch Allergy Immunol. 2011; 155(1): 12–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAraujo DM, Cotman CW: Basic FGF in astroglial, microglial, and neuronal cultures: characterization of binding sites and modulation of release by lymphokines and trophic factors. J Neurosci. 1992; 12(5): 1668–78. PubMed Abstract\n\nLiu X, Mashour GA, Webster HF, et al.: Basic FGF and FGF receptor 1 are expressed in microglia during experimental autoimmune encephalomyelitis: temporally distinct expression of midkine and pleiotrophin. Glia. 1998; 24(4): 390–7. PubMed Abstract | Publisher Full Text\n\nMantovani A, Schioppa T, Porta C, et al.: Role of tumor-associated macrophages in tumor progression and invasion. Cancer Metastasis Rev. 2006; 25(3): 315–22. PubMed Abstract | Publisher Full Text\n\nBaird A, Mormède P, Böhlen P: Immunoreactive fibroblast growth factor in cells of peritoneal exudate suggests its identity with macrophage-derived growth factor. Biochem Biophys Res Commun. 1985; 126(1): 358–64. PubMed Abstract | Publisher Full Text\n\nSchulze-Osthoff K, Risau W, Vollmer E, et al.: In situ detection of basic fibroblast growth factor by highly specific antibodies. Am J Pathol. 1990; 137(1): 85–92. PubMed Abstract | Free Full Text\n\nArnold T, Betsholtz C: The importance of microglia in the development of the vasculature in the central nervous system. Vasc Cell. 2013; 5(1): 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThomas KA, Rios-Candelore M, Giménez-Gallego G, et al.: Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1. Proc Natl Acad Sci U S A. 1985; 82(19): 6409–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThomas KA, Giménez-Gallego G: Fibroblast Growth Factors: broad spectrum mitogens with potent angiogenic activity. TIBS. 1986; 11(2): 81–84. Publisher Full Text\n\nPresta M, Andrés G, Leali D, et al.: Inflammatory cells and chemokines sustain FGF2–induced angiogenesis. Eur Cytokine Netw. 2009; 20(2): 39–50. PubMed Abstract | Publisher Full Text\n\nAndrés G, Leali D, Mitola S, et al.: A pro-inflammatory signature mediates FGF2–induced angiogenesis. J Cell Mol Med. 2009; 13(8B): 2083–108. PubMed Abstract | Publisher Full Text\n\nMcDonnell K, Bowden ET, Cabal-Manzano R, et al.: Vascular leakage in chick embryos after expression of a secreted binding protein for fibroblast growth factors. Lab Invest. 2005; 85(6): 747–55. PubMed Abstract | Publisher Full Text\n\nRibatti D, Gualandris A, Belleri M, et al.: Alterations of blood vessel development by endothelial cells overexpressing fibroblast growth factor-2. J Pathol. 1999; 189(4): 590–9. PubMed Abstract | Publisher Full Text\n\nCuevas P, Carceller F, Reimers D, et al.: Inhibition of intra-tumoral angiogenesis and glioma growth by the fibroblast growth factor inhibitor 1,3,6–naphthalenetrisulfonate. Neurol Res. 1999; 21(5): 481–7. PubMed Abstract\n\nFernández IS, Cuevas P, Angulo J, et al.: Gentisic acid, a compound associated with plant defence and a metabolite of aspirin, heads a new class of in vivo FGF inhibitor. J Biol Chem. 2010; 285(15): 11714–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAllain H, Ramelet AA, Polard E, et al.: Safety of calcium dobesilate in chronic venous disease, diabetic retinopathy and haemorrhoids. Drug Saf. 2004; 27(9): 649–60. PubMed Abstract | Publisher Full Text\n\nHaritoglou C, Gerss J, Sauerland C, et al.: CALDIRET study group. Effect of calcium dobesilate on occurrence of diabetic macular oedema (CALDIRET study): randomised, double-blind, placebo-controlled, multicentre trial. Lancet. 2009; 373(9672): 1364–71. PubMed Abstract | Publisher Full Text\n\nCuevas P, Outeiriño LA, Azanza C, et al.: Intravitreal dobesilate in the treatment of choroidal neovascularization associated with age-related macular degeneration. Report of two cases. BMJ Case Rep. 2012; 2012. pii: bcr2012006619. PubMed Abstract | Publisher Full Text\n\nCuevas P, Outeiriño LA, Angulo J, et al.: Treatment of Stargardt disease with dobesilate. BMJ Case Rep. 2012; 2012 pii: bcr2012007128. PubMed Abstract | Publisher Full Text\n\nCuevas P, Outeiriño LA, Angulo J, et al.: Treatment of dry age-related macular degeneration with dobesilate. BMJ Case Rep. 2012; 2012. pii: bcr0220125942. PubMed Abstract | Publisher Full Text\n\nCuevas P, Outeiriño LA, Angulo J, et al.: Chronic cystoid macular edema treated with intravitreal dobesilate. BMJ Case Rep. 2012; 2012. pii: bcr2012006376. PubMed Abstract | Publisher Full Text\n\nCuevas P, Outeiriño LA, Azanza C, et al.: Short-term efficacy of intravitreal dobesilate in central serous chorioretinopathy. Eur J Med Res. 2012; 17: 22–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArevalo JF, Sanchez JG, Saldarriaga L, et al.: Pan American Collaborative Retina Study Group. Retinal detachment after bevacizumab. Ophthalmology. 2011; 118(11): 2304.e3–7. PubMed Abstract | Publisher Full Text\n\nStewart MW: Clinical and differential utility of VEGF inhibitors in wet age-related macular degeneration: focus on aflibercept. Clin Ophthalmol. 2012; 6: 1175–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrohne TU, Eter N, Holz FG, et al.: Intraocular pharmacokinetics of bevacizumab after a single intravitreal injection in humans. Am J Ophthalmol. 2008; 146(4): 508–12. PubMed Abstract | Publisher Full Text\n\nAngulo J, Peiró C, Romacho T, et al.: Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial proliferation, arterial relaxation, vascular permeability and angiogenesis by dobesilate. Eur J Pharmacol. 2011; 667(1–3): 153–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "6200",
"date": "14 Oct 2014",
"name": "João Rafael de Oliveira Dias",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 my opinion the manuscript is well written and describes the effects of intravitreal dobesilate in the resolution of submacular haemmorrhage due to exudative AMD.I think the authors could describe the findings of the angiogram and ICG and how they ruled out other causes of SMH as polipoidal or RAP. As we know, polipoidal is always a diagnostic hypothesis that must be thought when a patient has SMH.Another point to be discussed is if the patient developed a new subretinal fluid and the possibility of a new dobesilate administration in refractory cases. Also something not clear is that the patient was or wasn't already submitted to intravitreal anti-VEGF injections before the dobesilate administration.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-271
|
https://f1000research.com/articles/2-270/v1
|
09 Dec 13
|
{
"type": "Short Research Article",
"title": "A novel role for syndecan-3 in angiogenesis",
"authors": [
"Giulia De Rossi",
"James R. Whiteford",
"James R. Whiteford"
],
"abstract": "Syndecan-3 is one of the four members of the syndecan family of heparan sulphate proteoglycans and has been shown to interact with numerous growth factors via its heparan sulphate chains. The extracellular core proteins of syndecan-1,-2 and -4 all possess adhesion regulatory motifs and we hypothesized that syndecan-3 may also possess such characteristics. Here we show that a bacterially expressed GST fusion protein consisting of the entire mature syndecan-3 ectodomain has anti-angiogenic properties and acts via modulating endothelial cell migration. This work identifies syndecan-3 as a possible therapeutic target for anti-angiogenic therapy.",
"keywords": [
"Angiogenesis",
"Endothelial Cell",
"Syndecan",
"Heparan Sulphate"
],
"content": "Introduction\n\nAngiogenesis is the process of new blood vessel formation from pre-existing vessels. This process is essential for embryonic development but it is also a feature of pathologies such as cancer and chronic inflammation (reviewed in1). For angiogenesis to occur, the release of pro-angiogenic factors, which promote the transition of endothelial cells (ECs) from a quiescent to a proliferative and migratory phenotype, is required2–4. The best characterized pro-angiogenic molecules are: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) (reviewed in5). Angiogenesis represents an attractive target for the treatment of cancer, as during tumour development new blood vessels permeate the tumour mass and provide oxygen and nutrients to further enhance tumour growth. Therapies aimed at blocking angiogenesis have focused on targeting VEGF and its related receptors, however the prohibitive cost of such treatments and their side effects mean that there is still a need to discover new therapeutic anti-angiogenic targets4.\n\nSyndecans are a four member family of transmembrane adhesion receptors with diverse expression and functionality6,7. Syndecan-3 is the least well understood of the four family members. Like other syndecans, it possesses a short, highly conserved cytoplasmic domain, a single pass transmembrane domain and a large extracellular domain containing 6 glycosaminoglycan attachment sites which can be substituted by both heparan sulphate (HS) and chondroitin sulphate (CS). Syndecan-3 has the largest extracellular core protein of the syndecans and, in addition to the HS and CS modifications, the molecule possesses a number of potential sites for O-linked glycosylation resembling a mucin-rich domain which may affect both the structure and molecular interactions of syndecan-38,9.\n\nSyndecan-3 Knock-Out mice, in common with other syndecan-KO animals, develop normally and only under conditions of challenge or insult are different phenotypes observed6. Syndecan-3 is mainly found in the nervous system and has been shown to be a co-receptor for a number of important growth factors, including Agouti related protein (AgRP), heparin binding growth associated molecule (HB-GAM), glial cell line derived growth factor (GDNF), neurturin (NRTN), artemin (PSPN) and NOTCH, via interactions with its HS chains10–14. Interactions with AgRP, an antagonist of melanocortin, lead to altered feeding behaviours in the syndecan-3 null mouse14,15. In addition, syndecan-3 deficiency leads to the mice exhibiting more addictive behaviours to opiates such as cocaine, and this occurs as a result of its interaction with GDNF16. Syndecan-3 is also expressed on satellite cells, adult skeletal muscle progenitors and has roles in the development of normal adult muscle13,17–19.\n\nAn important emerging feature of syndecans is that sequences contained within their extracellular core domains have biological activity which can influence cell adhesion and migratory responses20. Such domains have been identified in syndecan-1, 2 and -421. The syndecans can be divided into two subfamilies based on sequence homology: syndecan-1 and -3 and syndecans -2 and -422. Since regions of the syndecan-1 core protein are anti-angiogenic, we hypothesized that the core protein of syndecan-3 may exhibit similar properties23–25. In this study we demonstrate that the syndecan-3 extracellular core protein is able to inhibit angiogenesis by reducing the migratory potential of endothelial cells and as such may be a candidate for use in anti-angiogenic therapy.\n\n\nMaterials and methods\n\nCell culture: Brain endothelial cells (bEND3.1) and skin endothelial cells (sEND) were obtained from Health Protection Agency UK and were grown in DMEM (PAA, GE Healthcare) supplemented with 10% FBS, 2 mM L-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate and 5 μM β-mercaptoethanol (all Invitrogen), at 37°C, 10% CO2.\n\nSyndecan-3 GST fusion protein: The full length syndecan-3 cDNA was obtained from Source BioScience. The entire length of the mature syndecan-3 ectodomain (A45-L380) was amplified by PCR using the primers S3forEcoRI (ttaattgaattcgctcaacgctggcgcaatg) and S3revHindIII (ttaattaagcttctacagtatgctcttctgaggga) (Integrated DNA Technologies) and the resultant product was digested with EcoRI and HIndIII and ligated into the equivalent sites of pET41 (Novagen) according to Manufacturer’s instructions. Plasmids were verified by sequencing and transformed into the BL21 strain of Escherichia coli (Novagen). S3ED protein was purified from bacterial cultures which had reached an OD600 of 0.4 prior to the addition of 0.1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG) and subsequent outgrowth for 4 hours. Affinity purification of both GST and S3ED was performed using glutathione-sepharose 4B (GE Healthcare) as described by the manufacturers.\n\nAortic ring assay: Angiogenic sprouts were induced from rat thoracic aortas according to the method of Nicosia and Ottinetti26. Briefly, aortas were dissected from cervically dislocated 180–200g 12 male Wistar rats (Harlan Laboratories) and sliced into 0.5 mm sections and incubated overnight in serum free OptiMEM (Invitrogen) at 37°C. Aortic rings were embedded in type I collagen (1 mg/ml) in E4 media (Invitrogen) containing either GST or S3ED in 48 well plates (Corning). Wells were supplemented with OptiMEM with 1% FBS and 10 ng/ml VEGF (R and D systems) and incubated at 37°C, 10% CO2. Angiogenic sprouts from rat aortas were counted after 4 days and 8 days respectively. Animals were housed and treated in Accordance with UK Home Office Regulations.\n\nEC tubule formation assay: Endothelial cell microtubule formation was measured as follows: ECs (5 × 104) were seeded into 24 well plates coated with 150 µl of Matrigel (BD sciences) in the presence of either GST or S3ED. Using the Cell-IQ controlled environmental chamber (CM technologies), the plates were incubated at 37°C, 10% CO2 and images were captured every 15 minutes for 16 hours.\n\nScratch wound migration assay: Confluent monolayers of bEND cells were scratched with a pipette tip, cells were then washed twice with PBS prior to the addition of growth medium supplemented with 0.5 µM of either GST or S3ED. Wounds were monitored by time lapse microscopy using an Olympus IX81 Microscope Hamamatsu Orca ER digital camera. Images were acquired every 30 minutes and subsequently analysed using Adobe Photoshop. Cell speed was quantified by manually measuring the track of individual cells migrated for 9 hours (20 cells per conditions) using Adobe Photoshop, track lengths were then divided by the time to obtain cell speed.\n\nCell proliferation assay: EC proliferation was measured using the CellTiter 96 AQueous Cell proliferation assay kit as described by the manufacturer (Promega). bEND (5 × 103/spot) cells were seeded in a 96-well plate (Corning) and incubated in the presence of 0.5 µM either GST or S3ED.\n\nInvasion assay: EC invasion assays through collagen matrices were performed in 24-well plates with trans-well inserts (Millipore; 8 μm pore size, polyester (PET) membrane). Membranes were coated with 10 μl of Collagen Type I mixture (Millipore; 1 mg/ml in E4 medium) containing 0.5 μM GST or S3ED. sEND cells were seeded on the gel in a homogenous single cell suspension of 5 × 103 cells/ insert in 200 μl of DMEM + 10% FBS; 1 ml of the same medium was added to the bottom well. Invasion was measured after 6 hours after which time gels were removed with a cotton swab, the filter washed in PBS and stained with calcein (Invitrogen) and the number of cells attached to the filter was counted.\n\n\nResults\n\nIn keeping with previous studies from our group and others in the field25,27–29, we set out to determine whether the extracellular core protein of syndecan-3 (S3ED) had any effect on cellular responses such as cell adhesion or migration and in particular on angiogenesis. To do this we generated a fusion protein consisting of the entire length of the syndecan-3 extracellular core protein (A45-L380) fused at the N-terminus to GST (S3ED). S3ED was expressed and purified from bacteria and therefore was not substituted with GAGs, or O-linked sugars, since bacteria lack the necessary transferases to perform these post-translational modifications (Figure 1).\n\nDiagrammatic representation of the syndecan-3 fusion protein (S3ED) used in this study. The entire length of the syndecan-3 extracellular core protein (A45-L380) was fused at the N-terminus to GST.\n\nIn the aortic ring assay, angiogenic sprouts are formed from sections of rat aortas when embedded into a collagen I matrix (Figure 2a and b). Sprouting occurs in the absence of VEGF although a more robust response is observed when this growth factor is included in the medium (Figure 2c). We incorporated 0.5 µM of either GST or S3ED into the collagen I matrices to see if they affected sprout formation. Significantly fewer sprouts were observed in the presence of S3ED compared to either the untreated control or GST treated rings. This was true for both rings grown in the presence or in absence of VEGF (Figure 2c). We then demonstrated that this inhibition of angiogenesis is dose dependent; as little as 0.5 µM of S3ED had an inhibitory effect (Figure 3). Although 0.1 µM of S3ED also showed a statistically significant anti-angiogenic effect in this assay, we decided to use the higher dose (0.5 µM) for further experiments.\n\n(a) Low magnification (bar=500 µm) of a rat aortic ring and (b) high magnification (bar=100 µm) image of an angiogenic sprout grown under control conditions. (c) S3ED inhibits angiogenic sprout formation from rat aortic rings. Rat aortic rings were seeded in Collagen I gels with 0.5 μM of either S3ED or GST in the presence or absence of VEGF. Data is the mean taken from rings from 3 different animals and error bars represent the SEM. One-way ANOVA with Bonferroni multiple comparisons was used to compare S3ED to relative UT control.\n\nRat aortic rings were embedded in Collagen I with the indicated concentrations of either GST or S3ED in the presence of VEGF. Angiogenic sprouts were counted 4 days after seeding. Data is from 5 rings per condition. One-way ANOVA with Bonferroni multiple comparisons was used to compare S3ED 0.5 μM to S3ED 0 μM considered as a control.\n\nTubular network formation in response to matrigel is another measure of angiogenesis30. Brain endothelial cells form highly branched networks when seeded on matrigel and this was unaffected when GST was added to the culture medium (Figure 4a). In the presence of S3ED (0.5 µM) far fewer branch points were evident and the length of the microtubules was greatly reduced (Figure 4b and c; and Movie 1 and Movie 2). Together these data suggest that S3ED has anti-angiogenic properties.\n\nbEND3.1 cells were seeded on Matrigel in media containing GST or S3ED (0.5 µM) and micrographs were obtained after 16 hours (a). Tube length (b) and branch points (c) were calculated and both were reduced in the presence of S3ED. Error bar represents SEM. T-test was used to compare S3ED to GST control.\n\n\n\nThe ectodomains of syndecan-1,-2 and -4 have all been shown to affect cell behaviour through interactions with integrins (for review see21). For example, the anti-angiogenic properties of syndecan-1 have been demonstrated to occur through interactions with the αV sub-family25. Integrins are the major drivers of cell migration31,32 so we set out to determine whether the inhibitory properties of S3ED were due to effects on EC migration. Scratch wound cell migration assays revealed that S3ED significantly inhibited brain endothelial cell migration as compared to GST and untreated controls (Figure 5a). This was reflected in both percentage of wound closure and single cell speed measurements (Figure 5b and c). We further confirmed that S3ED inhibits EC migratory potential by using a 3D culture system where we observed that S3ED, when incorporated into a collagen I matrix, inhibited endothelial cell invasion through the collagen (Figure 6a and b). Although we demonstrate that S3ED inhibits EC migration, it could be argued that these effects may also be associated with anti-proliferative effects of this protein. To test this we performed proliferation assays on brain ECs in the presence of S3ED or GST and observed no differences in the proliferation of these cells compared with controls (Figure 7).\n\nScratch wounds were made to confluent monolayers of bEND3.1 cells and micrographs were captured after 9 hours clearly showing that wound closure is reduced in the presence of S3ED 0.5 μM (a). Scratch wound closure (b) and single cell speeds (c) were calculated. Single cell speed was calculated from 15–25 cells per condition. Error bars represent SD for (a) and SED for (b). One-way ANOVA with Bonferroni multiple comparisons was used to compare S3ED to relative UT control.\n\n5 × 103 sEND cells were seeded on 10 μl of Collagen I and were allowed to migrate for 6 hours. Numbers of transmigrated cells are shown in (a) and micrographs of transmigrated Calcein-labelled cells are in (b). Error bars represent SD. One-way ANOVA with Bonferroni multiple comparisons was used to compare S3ED to relative UT control.\n\n5 x 103 bEND 3.1 cells were incubated in the presence of 0.5 µM of either GST or S3ED. Error bars represent SD. One-way ANOVA with Bonferroni multiple comparisons was used to compare S3ED to relative UT control showing no statistically significant difference between the two at after 9 and 24 hours.\n\n\n\n\nDiscussion\n\nHere we show for the first time that the ectodomain of syndecan-3, like the other syndecan family members, has cell adhesion regulatory properties and acts as an inhibitor of angiogenesis. This strongly suggests that adhesion regulatory domains in syndecan extracellular core domains exist in all four family members and also provides further insight as to how these molecules function. Interestingly, there is very little sequence homology between syndecan ectodomains32, and the adhesion regulatory motifs identified in syndecan-1,-2 and -4 are not present in S3ED. In addition, syndecan-3 is unique amongst the family since its core protein possesses a mucin-rich domain and it is likely that these O-linked sugars will also have a role in its molecular interactions. Although syndecan-3 expression is mostly associated with cells of the nervous system, it has also been found to be associated with cells related to maternal and foetal circulation33. Vascular defects in the syndecan-3 null mouse have not been reported but the identification of S3ED as a regulator of angiogenesis suggests that this may be a fertile area for future research. This work identifies syndecan-3 as a potential therapeutic target in pathologies where angiogenesis is a feature.",
"appendix": "Author contributions\n\n\n\nGDR and JRW conceived of the study, designed the experiments and carried out the research.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by Arthritis Research-UK (Grant No. 19207) and funds from the William Harvey Research Foundation both to JRW.\n\n\nReferences\n\nPotente M, Gerhardt H, Carmeliet P: Basic and therapeutic aspects of angiogenesis. Cell. 2011; 146(6): 873–887. PubMed Abstract | Publisher Full Text\n\nHolderfield MT, Hughes CC: Crosstalk between vascular endothelial growth factor, notch, and transforming growth factor-beta in vascular morphogenesis. Circ Res. 2008; 102(6): 637–652. PubMed Abstract | Publisher Full Text\n\nChung AS, Ferrara N: Developmental and pathological angiogenesis. Annu Rev Cell Dev Biol. 2011; 27: 563–584. PubMed Abstract | Publisher Full Text\n\nCarmeliet P, Jain RK: Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011; 473(7347): 298–307. PubMed Abstract | Publisher Full Text\n\nWeis SM, Cheresh DA: Tumor angiogenesis: Molecular pathways and therapeutic targets. Nature Medicine. 2011; 17(11): 1359–1370. PubMed Abstract | Publisher Full Text\n\nAlexopoulou AN, Multhaupt HA, Couchman JR: Syndecans in wound healing, inflammation and vascular biology. Int J Biochem Cell Biol. 2007; 39(3): 505–528. PubMed Abstract | Publisher Full Text\n\nCouchman JR: Transmembrane signaling proteoglycans. Annu Rev Cell Dev Biol. 2010; 26: 89–114. PubMed Abstract | Publisher Full Text\n\nChernousov MA, Carey DJ: N-syndecan (syndecan 3) from neonatal rat brain binds basic fibroblast growth factor. J Biol Chem. 1993; 268(22): 16810–16814. PubMed Abstract\n\nAsundi VK, Carey DJ: Self-association of n-syndecan (syndecan-3) core protein is mediated by a novel structural motif in the transmembrane domain and ectodomain flanking region. J Biol Chem. 1995; 270(44): 26404–26410. PubMed Abstract | Publisher Full Text\n\nBespalov MM, Sidorova YA, Tumova S, et al.: Heparan sulfate proteoglycan syndecan-3 is a novel receptor for gdnf, neurturin, and artemin. J Cell Biol. 2011; 192(1): 153–169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCreemers JW, Pritchard LE, Gyte A, et al.: Agouti-related protein is posttranslationally cleaved by proprotein convertase 1 to generate agouti-related protein (agrp)83–132: Interaction between agrp83–132 and melanocortin receptors cannot be influenced by syndecan-3. Endocrinology. 2006; 147(4): 1621–1631. PubMed Abstract | Publisher Full Text\n\nNolo R, Kaksonen M, Raulo E, et al.: Co-expression of heparin-binding growth-associated molecule (hb-gam) and n-syndecan (syndecan-3) in developing rat brain. Neurosci Lett. 1995; 191(1–2): 39–42. PubMed Abstract | Publisher Full Text\n\nPisconti A, Cornelison DD, Olguin HC, et al.: Syndecan-3 and notch cooperate in regulating adult myogenesis. J Cell Biol. 2010; 190(3): 427–441. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReizes O, Benoit SC, Strader AD, et al.: Syndecan-3 modulates food intake by interacting with the melanocortin/agrp pathway. Ann N Y Acad Sci. 2003; 994: 66–73. PubMed Abstract | Publisher Full Text\n\nZheng Q, Zhu J, Shanabrough M, et al.: Enhanced anorexigenic signaling in lean obesity resistant syndecan-3 null mice. Neuroscience. 2010; 171(4): 1032–1040. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen J, Repunte-Canonigo V, Kawamura T, et al.: Hypothalamic proteoglycan syndecan-3 is a novel cocaine addiction resilience factor. Nat Commun. 2013; 4: 1955. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCornelison DD, Filla MS, Stanley HM, et al.: Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev Biol. 2001; 239(1): 79–94. PubMed Abstract | Publisher Full Text\n\nKosher RA: Syndecan-3 in limb skeletal development. Microsc Res Tech. 1998; 43(2): 123–130. PubMed Abstract | Publisher Full Text\n\nDealy CN, Seghatoleslami MR, Ferrari D, et al.: Fgf-stimulated outgrowth and proliferation of limb mesoderm is dependent on syndecan-3. Dev Biol. 1997; 184(2): 343–350. PubMed Abstract | Publisher Full Text\n\nDe Rossi G, Whiteford JR: Novel insight into the biological functions of syndecan ectodomain core proteins. BioFactors. 2013; 39(4): 374–382. PubMed Abstract | Publisher Full Text\n\nDe Rossi G, Whiteford JR: Novel insight into the biological functions of syndecan ectodomain core proteins. BioFactors. 2013; 39(4): 374–382. PubMed Abstract | Publisher Full Text\n\nCouchman JR, Woods A: Syndecans, signaling, and cell adhesion. J Cell Biochem. 1996; 61(4): 578–584. PubMed Abstract | Publisher Full Text\n\nRapraeger AC, Ell BJ, Roy M, et al.: Vascular endothelial-cadherin stimulates syndecan-1-coupled insulin-like growth factor-1 receptor and cross-talk between alphavbeta3 integrin and vascular endothelial growth factor receptor 2 at the onset of endothelial cell dissemination during angiogenesis. FEBS J. 2013; 280(10): 2194–2206. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRapraeger AC: Synstatin: A selective inhibitor of the syndecan-1–coupled igf1r-alphavbeta3 integrin complex in tumorigenesis and angiogenesis. FEBS J. 2013; 280(10): 2207–2215. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeauvais DM, Ell BJ, McWhorter AR, et al.: Syndecan-1 regulates alphavbeta3 and alphavbeta5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor. J Exp Med. 2009; 206(3): 691–705. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNicosia RF, Ottinetti A: Growth of microvessels in serum-free matrix culture of rat aorta - a quantitative assay of angiogenesis in vitro. Lab Invest. 1990; 63(1): 115–122. PubMed Abstract\n\nWhiteford JR, Couchman JR: A conserved nxip motif is required for cell adhesion properties of the syndecan-4 ectodomain. J Biol Chem. 2006; 281(43): 32156–32163. PubMed Abstract | Publisher Full Text\n\nWhiteford JR, Behrends V, Kirby H, et al.: Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways. Exp Cell Res. 2007; 313(18): 3902–3913. PubMed Abstract | Publisher Full Text\n\nWhiteford JR, Xian X, Chaussade C, et al.: Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor cd148. Mol Biol Cell. 2011; 22(19): 3609–3624. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPassaniti A, Taylor RM, Pili R, et al.: Methods in laboratory investigation A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement-membrane, heparin, and fibroblast growth-factor. Lab Invest. 1992; 67(4): 519–528. PubMed Abstract\n\nStreuli CH, Akhtar N: Signal co-operation between integrins and other receptor systems. Biochem J. 2009; 418(3): 491–506. PubMed Abstract | Publisher Full Text\n\nCouchman JR, Chen L, Woods A: Syndecans and cell adhesion. Int Rev Cytol. 2001; 207: 113–150. PubMed Abstract\n\nChui A, Zainuddin N, Rajaraman G, et al.: Placental syndecan expression is altered in human idiopathic fetal growth restriction. Am J Pathol. 2012; 180(2): 693–702. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2701",
"date": "10 Dec 2013",
"name": "Alberto Grossi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 conducted study which highlights an unforeseen role for the heparan sulphate proteoglycan syndecan-3. Additionally, beyond the realms of this study, these data suggest more detailed knowledge of the syndecan-3 expression profile is required.",
"responses": []
},
{
"id": "2703",
"date": "20 Dec 2013",
"name": "Mark Morgan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study highlighting the potential role of syndecan-3 in the regulation of angiogenesis. The study was well executed and the manuscript well written. The authors show that recombinant syndecan-3 ectodomain (S3ED) inhibits angiogenic sprouting in a dose-dependent manner, using ex vivo aortic ring assays. They further show that S3ED inhibits endothelial cell migration and suggest that this is responsible for its anti-angiogenic effect. This work warrants publication as it raises many new questions (beyond the realms of this project), about the non-neuronal roles of a relatively understudied syndecan. For example:What are the effects of S3ED in vivo and can syndecan-3 be targeted therapeutically?Does endogenous syndecan-3 (either shed or membrane-bound) serve a role for fine-tuning angiogenic processes in vivo?What is the specific mechanism by which S3ED inhibits endothelial cell migration and angiogenesis? Does it involve modulation of integrin and/or growth factor receptor function?No doubt these questions will form the basis of many future studies. Minor Points:In Fig 2 it would be useful to include a representative image of an S3ED-treated aortic ring. Are angiogenic sprouts completely absent or are they just stumps? The Transwell \"invasion\" assays described use a very thin coating (10ul) of non-crosslinked collagen type 1 and a relatively short time period (6Hrs). Consequently, they are not a good read-out of \"invasion\" per se, but rather of a mode of migration. Therefore the process should be described as \"migration through collagen\", or perhaps \"invasive migration\".",
"responses": []
},
{
"id": "2705",
"date": "07 Jan 2014",
"name": "Jonas Jacobsen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work described by researchers Whiteford and De Rossi is placed in the scientific field angiogenesis. The research group has identified the mature syndecan-3 ectodomain in fusion with a GST protein as a possible therapeutic target for anti-angiogenic therapy. A therapy that could prove relevant in the treatment of cancer.The biological activities of extracellular core domain of Syndecan-3 is both novel and interesting and deserve publication.The only minor point of criticism to the authors is the following:In the discussion it is mentioned that O-linked glycosylation in the mucin-rich region of Syndecan-3 may also engage in molecular interactions. While this may be true it is also a fact that the expressed syndecan-3 fusion protein is not glycosylated because in was produced in the BL21 strain of Escherichia coli.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-270
|
https://f1000research.com/articles/2-195/v1
|
23 Sep 13
|
{
"type": "Research Article",
"title": "Kairomonal communication in mice is concentration-dependent with a proportional discrimination threshold",
"authors": [
"Anand Vasudevan",
"Ajai Vyas",
"Anand Vasudevan"
],
"abstract": "Odors of predators are often co-opted by prey species to serve as warning signals. Perceptual properties of such kairomonal communication are under studied despite their common use in many mammals. We demonstrate that the kairomonal response in mice to rat odors varies monotonically with the volume of rat odor. Moreover, the ability of mice to differentiate between two strengths of rat odors is dependent on the ratio of the two concentrations. These results show that mice can compare kairomonal strength over a large range of values, and that kairomonal communication follows Weber’s law.",
"keywords": [
"Major urinary proteins",
"Olfaction",
"Pheromone",
"Predator",
"Rat",
"Weber’s law"
],
"content": "Introduction\n\nForaging animals continually face a conflict between 1) the need to seek opportunities such as food and mating partners; and, 2) the need to avoid exposure to predators. In response to predation pressure, many prey species have co-opted predator odors as kairomones; chemicals emitted by one species, usually for inter-species communication, but intercepted by other species resulting in benefit for the receiver and detriment of the emitter. In this role, predator odors such as urine, fecal material or body odors initiate a rapid avoidance response in prey, thus reducing the probability of successful predation1,2. Such avoidance of predator cues needs to be ‘traded-off’ against foraging opportunities. In view of this, it can be speculated that a kairomonal responses may not be an absolute all-or-nothing phenomenon. Rather avoidance is expected to be relative to the intensity of the predator cue. Implicit in this speculation is the idea that animals can quantitatively perceive differences in kairomonal strength.\n\nA wide variety of animals can make quantitative estimates of percepts such as time, foraging opportunities, efforts and rewards3–7. These quantitative estimates are often derived using a comparative representational system that is dependent on the ratios between opposing quantities3–7. The ability to make quantitative estimates is important because it allows calibration of behavioral responses to incipient environmental opportunities and challenges. In accordance with the comparative nature of such perceptual systems, it can be predicted that greater quantities of kairomones evoke greater response i.e. that the response is dose-dependent. More importantly, sensitivity to changes in the magnitude of a stimulus decreases when stimulus magnitude increases. In other words, the discrimination threshold (i.e. the ‘just-noticeable difference’ between two stimuli of different intensities) is smaller when both stimuli are weak compared to when both stimuli are strong. This formulation is often termed Weber’s law8, and is a fundamental property of many percepts.\n\nKairomonal communication has been widely studied in insects9–12. Additionally, the neurobiology and physiology of rodent kairomones has attracted significant scientific interest in the recent past1,13. Yet, the perceptual properties of kairomonal communication in mammals have so far been under studied, including, the dose-responsivity of kairomonal communication14 and the relationship of discrimination threshold to stimulus magnitude.\n\nHouse mice (Mus musculus) are predated by rats (Rattus norvegicus)15–17 and accordingly, the mice express innate avoidance to rat odors18,19. In this report, we investigate the dose-responsivity and discrimination threshold of kairomonal communication in mice.\n\n\nMaterials and methods\n\nThe Nanyang Technological University (IACUC number: ARF SBS/NIE-A-0106AZ) institutional animal care and use committee reviewed and approved all procedures. Fifteen male Balb/c mice (7–8 weeks old, housed five per cage (369 x 156 x 132 mm; 1145T, Tecniplast, UK)) were obtained from the vivarium of the National University of Singapore. Eight male Wistar rats (48 days old, housed two per cage (425 x 266 x 185 mm; 1291H, Tecniplast, UK)) were obtained from the same vivarium and used as a kairomonal source. Standard corn cob cage bedding was changed twice a week. Animals were maintained on a 12 hours light-dark cycle, with temperature and relative humidity ranging between 20–25 degree celsius and 70–80%, respectively. Experiments were conducted during the light phase. Food and water was available ad libitum. The diet consisted of standard laboratory chow (PicoLab Rodent Diet 20, 5053) with 20% protein content.\n\nRat urine was collected using metabolic cages (Harvard Apparatus). Rat urine contains volatile compounds and major urinary proteins (MUPs). The urine was treated with menadione (M5625 Sigma-Aldrich, Singapore) to competitively displace volatile compounds bound to the MUPs, followed by centrifugation (Millipore, 3000 g for 5 minutes) through a size-exclusion column (>3 kDa). Only the high molecular-weight fraction containing MUPs and devoid of volatiles was used, in accordance with the prior demonstration that rat MUPs serve as kairomones to mice13.\n\nThe response of mice (n = 10) to increasing doses of MUP fraction of rat urine ((henceforth referred to as rat urine) was studied (trial duration = 600s). Avoidance was quantified by comparing time spent by mice in two opposing bisects of an arena (76 × 9 cm; 15 cm high). Data on time spent in each bisect was collected by automated behavioral tracking software (ANY-maze, version 4.3, Stoelting). Opposing arms contained either rat urine or phosphate-buffered saline. The amount of rat urine was systematically varied from 1X to 16X (3.125, 6.25, 12.5, 25 and 50 µl). X was arbitrarily defined as 3.125 µl of rat urine. The same set of mice was used in successive testing for all doses (starting from lower to higher doses) with 24 hours elapsing between two successive trials).\n\nBoth arms of the arena contained rat urine in this condition. The amount of rat urine in one arm was varied in five discrete doses (6.25–25 μl), equidistant on a Log2 scale. The opposing arm contained volume that was greater by ratio of either 1.2 or 1.3. The percentage of time that mice (n = 15, the same mice that were used in the previous experiment) spent in the arm with the greater volume of urine, was quantified. The same set of mice were used in successive testing for all doses (starting from lower to higher doses), with two successive trials (24 hours apart).\n\nAll statistical tests were conducted using IBM SPSS software (version 20) One-way analysis of variance followed by ‘Fisher’s least significant difference’ (LSD) post-hoc test was used to analyze increasing doses of kairomones on mouse behaviour. A two-way analysis of variance was carried out to determine main effect and/or interaction of kairomone dose and its corresponding ratios.\n\n\nResults\n\nOne-way analysis of variance (ANOVA) revealed that the amount of time spent near rat urine decreased with an incremental increase in the amount of rat urine (Figure 1; n = 10; F(4,45) = 6.9; p = 0.0002). Animals spent significantly more time near the weakest odor, compared to the strongest (LSD, Fisher's least significant difference, p = 0.0002). The stimulus-response curve exhibited a robust fit to sigmoidal curve (Figure 1; R2 > 0.99; p < 0.01), showing a monotonic linear response between concentrations 2X to 8X.\n\nIn order to rule out carry-over effects during repeated trials, we tested a separate set of five mice repeatedly at a singular dose (4X) over five days. This set of mice exhibited a comparable aversion to rat urine across all trials, showing a lack of habituation, sensitization or conditioning during repeated testing (one-way ANOVA; F(4,20) = 0.314, p > 0.8).\n\nAversion of mice to increasing doses of rat urine was quantified by comparing the time spent in two opposing arms of an arena, with one arm containing incremental doses of rat urine and the other containing buffered saline (trial duration = 600 s, n = 10 mice). The graph depicts average time spent in rat urine arm. The gray line depicts sigmoidal fit. Abscissa depicts dose of rat urine employed (log2 scale; x arbitrarily set as 3.125 µl of rat urine).\n\nThe discrimination threshold (i.e the ‘just-noticeable difference’) was studied for five equidistant doses (Log2 scale) encompassing the linear part of the dose-response curve (Figure 2). One arm of the arena in this case contained the dose depicted in the abscissa. The discrimination around this dose was studied in two successive trials by providing a greater amount of kairomone in the opposing arm, differing by a ratio of either 1.2 or 1.3. A positive discrimination was noted as less time was spent in the arm containing the greater volume of urine.\n\nThe detection threshold at varying doses of rat urine was further examined by setting up an avoidance-avoidance conflict, where mice chose to spend time in arms containing either lower or higher amounts of rat urine. The higher dose was of either a 20% (un-shaded bars) or 30% (shaded bars) greater magnitude (e.g. 120% or 130% of 2X). Abscissa depicts the lower dose used in each of the comparisons (e.g. 2X). Ordinate depicts time spent in arm with the greater amount of rat urine divided by the sum of the time spent in both arms (gray line = 50% chance). N = 15 mice for all comparisons.Log2 scale; arbitrarily set as 3.125 µl of rat urine.\n\nA two-way ANOVA for dose and ratio revealed a significant main effect of the ratio (Figure 2A; F(1,138) = 32.1, p = 0.00000008). The main effect of doses themselves did not reach statistical significance (F(4,138) = 1.347, p = 0.256). Similarly, interaction between doses and ratios was not significant (F(4,138) = 1.214, p = 0.308). Thus, regardless of the dose studied, detection threshold was constantly proportional to the kairomone strength (Figure 2B) by a ratio ≤ 1.3 but above >1.2. In other words, the discrimination threshold was smaller for weaker stimuli and bigger for stronger stimuli\n\n\nDiscussion\n\nKairomones are compounds emitted by one species and co-opted by another receiving species, resulting in benefit for the receiver and detriment for the emitter. Kairomones can be used by both predators to locate prey and by prey to secure advanced warning of predator presence. Odors used as pheromones in intra-species communication are often the most vulnerable for co-option as kairomones. This is because use in conspecific communication requires robust expression of odors, making them more liable for eavesdropping by other species (reviewed in Kolluru and Zuk20). In agreement with this formulation, rats use urine marks to communicate status and sexual attractiveness21–23; and, proteins secreted with rat urine are sufficient to initiate innate avoidance in mice13, a prey species of rats15–17. The salient kairomone in this case has been identified to be a major urinary protein, MUP1313. These involatile rodent kairomones provide a unique opportunity to study kairomonal perception by virtue of their stability in the ambient environment and due to their ease of reliably controlling their dosing. This is in contrast with the volatile nature of many kairomones that require onerous delivery methods using olfactometers.\n\nSeveral pheromonal responses in insects and mammals show dose responsivity whereby a stronger pheromonal stimulus evokes a greater response (e.g. Coureaud et al., He et al. and Perna et al.24–26). In contrast, the dose responsivity of kairomonal communication has not been well-studied. In this report, we demonstrate that mice perceive rat kairomones in a dose-dependent manner. We speculate that the relative nature of kairomonal communication permits prey to calibrate foraging responses according to perceived predatory threats. For example, a weak kairomonal stimulus might signal the passage of a long period of time since the urine mark was laid, and thereby evoke lesser avoidance. In contrast, a stronger odor is likely to be fresh and a better indicator of predator presence. Similar dose-responsivity has been previously described during the perception of cat odors by rats14.\n\nThe ability to calibrate avoidance also suggests that mice are able to differentiate between various amounts of kairomones. Animals can indeed discriminate between different amounts of many percepts, including olfactory sensations3–7. In the absence of an absolute numerical system, these discriminations are often dependant on a relative estimation based on comparative perceptions. Weber and Fletcher8 formalized one of the hallmarks of such relative estimation by showing that the discrimination threshold is a constant fraction of the stimulus intensity in many perceptual systems (k = ∆I/I; where k is a constant, I is intensity and ∆I is just-noticeable difference). In this report, we demonstrate that kairomonal communication in mice follows Weber’s law with Weber’s fraction valued at greater than 0.2 but smaller than 0.3. Weber’s law has been previously studied in human olfaction for volatile odors, yielding a comparable sensitivity of 0.287. Similarly, pheromonal communication in Argentine Ants (Linepithema humile) has been recently shown to follow Weber’s law26. To the best of our knowledge, this is the first demonstration of Weber’s law applied to kairomones.\n\n",
"appendix": "Author contributions\n\n\n\nAnand Vasudevan (AV) and Ajai Vyas (AY) designed the experiments. AV collected the urine samples and carried out the experiments. AV and AY analyzed the data. AY prepared the manuscript. Both were involved in revising the script and approve the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded by Nanyang Technological University and the Ministry of Education, Singapore (MOE2011-T2-2-111).\n\n\nReferences\n\nApfelbach R, Blanchard CD, Blanchard RJ, et al.: The effects of predator odors in mammalian prey species: a review of field and laboratory studies. Neurosci Biobehav Rev. 2005; 29(8): 1123–1144. PubMed Abstract | Publisher Full Text\n\nWisenden BD: Olfactory assessment of predation risk in the aquatic environment. Philos Trans R Soc Lond B Biol Sci. 2000; 355(1401): 1205–1208. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaker JM, Shivik J, Jordan KE: Tracking of food quantity by coyotes (Canis latrans). Behav Processes. 2011; 88(2): 72–75. PubMed Abstract | Publisher Full Text\n\nMeck WH, Church RM: A mode control model of counting and timing processes. J Exp Psychol Anim Behav Process. 1983; 9(3): 320–334. PubMed Abstract | Publisher Full Text\n\nShafiei N, Gray M, Viau V, et al.: Acute Stress Induces Selective Alterations in Cost/Benefit Decision-Making. Neuropsychopharmacology. 2012; 37(10): 2194–209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMohring W, Libertus ME, Bertin E: Speed discrimination in 6- and 10-month-old infants follows Weber's law. J Exp Child Psychol. 2012; 111(3): 405–418. PubMed Abstract | Publisher Full Text\n\nStone H, Bosley JJ: Olfactory Discrimination and Weber's Law. Percept Mot Skills. 1965; 20: 657–665. PubMed Abstract | Publisher Full Text\n\nGlimcher PW: Foundations of neuroeconomic analysis. (Oxford University Press, New York) pp xix, 2011; 467. Publisher Full Text\n\nSvensson GP, Larsson MC, Hedin J: Attraction of the larval predator Elater ferrugineus to the sex pheromone of its prey, Osmoderma eremita, and its implication for conservation biology. J Chem Ecol. 2004; 30(2): 353–363. PubMed Abstract | Publisher Full Text\n\nLarsson MC, Svensson GP: Pheromone monitoring of rare and threatened insects: exploiting a pheromone-kairomone system to estimate prey and predator abundance. Conserv Biol. 2009; 23(6): 1516–1525. PubMed Abstract | Publisher Full Text\n\nRutledge C: A survey of identified kairomones and synomones used by insect parasitoids to locate and accept their hosts. Chemoecology. 1996; 7(3): 121–131. Publisher Full Text\n\nMiller DR, Asaro C, Crowe CM, et al.: Bark beetle pheromones and pine volatiles: attractant kairomone lure blend for longhorn beetles (Cerambycidae) in pine stands of the southeastern United States. J Econ Entomol. 2011; 104(4): 1245–1257. PubMed Abstract | Publisher Full Text\n\nPapes F, Logan DW, Stowers L: The vomeronasal organ mediates interspecies defensive behaviors through detection of protein pheromone homologs. Cell. 2010; 141(4): 692–703. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTakahashi LK, Nakashima BR, Hong H, et al.: The smell of danger: a behavioral and neural analysis of predator odor-induced fear. Neurosci Biobehav Rev. 2005; 29(8): 1157–1167. PubMed Abstract | Publisher Full Text\n\nGalef BG Jr: Aggression and timidity: responses to novelty in feral Norway rats. J Comp Physiol Psychol. 1970; 70(3): 370–381. PubMed Abstract | Publisher Full Text\n\nHsuchou H, Ho YJ, Shui HA, et al.: Effects of incisor-cutting on muricidal behavior induced by olfactory bulbectomy in rats. Physiol Behav. 2002; 76(4–5): 669–675. PubMed Abstract | Publisher Full Text\n\nKarli P: The Norway rat's killing response to the white mouse: an experimental analysis. Behaviour. 1956; 10: 81–103. Publisher Full Text\n\nAmaral VC, Santos Gomes K, Nunes-de-Souza RL: Increased corticosterone levels in mice subjected to the rat exposure test. Horm Behav. 2010; 57(2): 128–133. PubMed Abstract | Publisher Full Text\n\nYang M, Augustsson H, Markham CM, et al.: The rat exposure test: a model of mouse defensive behaviors. Physiol Behav. 2004; 81(3): 465–473. PubMed Abstract | Publisher Full Text\n\nKolluru GR, Zuk M: Exploitation of sexual signals by predators and parasitoids. Q Rev Biol. 1998; 73(4): 415–438. Publisher Full Text\n\nTaylor GT, Haller J, Bartko G, et al.: Conspecific urine marking in male-female pairs of laboratory rats. Physiol Behav. 1984; 32(4): 541–546. PubMed Abstract | Publisher Full Text\n\nTaylor GT, Regan D, Haller J: Sexual experience, androgens and female choice of a mate in laboratory rats. J Endocrinol. 1983; 96(1): 43–52. PubMed Abstract | Publisher Full Text\n\nTaylor GT, Haller J, Regan D: Female rats prefer an area vacated by a high testosterone male. Physiol Behav. 1982; 28(6): 953–958. PubMed Abstract | Publisher Full Text\n\nCoureaud G, Langlois D, Sicard G, et al.: Newborn rabbit responsiveness to the mammary pheromone is concentration-dependent. Chem Senses. 2004; 29(4): 341–350. PubMed Abstract | Publisher Full Text\n\nHe J, Ma L, Kim S, et al.: Distinct signals conveyed by pheromone concentrations to the mouse vomeronasal organ. J Neurosci. 2010; 30(22): 7473–7483. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerna A, Granovskiy B, Garnier S, et al.: Individual Rules for Trail Pattern Formation in Argentine Ants (Linepithema humile). PLoS Comput Biol. 2012; 8(7): e1002592. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1883",
"date": "14 Oct 2013",
"name": "Joanne Yew",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 addresses and characterizes rat urine kairomonal effects on mouse avoidance behavior. The study convincingly shows that 1) the effect is dose-dependent and 2) mice display proportional discrimination across several different concentrations.The title of the study and abstract provide an adequate summary. With respect to the methods, I would like some clarification on whether the different urine doses were made from the same initial sample. If several different urine collections were used, it should be stated how it was verified that the concentration of MUPs in the size exclusion fraction is similar from sample to sample.Otherwise, the conclusions are justified on the basis of the results and the paper is well-written.",
"responses": [
{
"c_id": "634",
"date": "30 Nov 2013",
"name": "Ajai Vyas",
"role": "Author Response",
"response": "The same urine sample was indeed used to create all doses during this experiment. The second version of the manuscript now clearly states this in material and methods section."
}
]
},
{
"id": "2187",
"date": "25 Oct 2013",
"name": "Kazumi Osada",
"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 paper showed the quantitative relationship between the amount of crude rat urinary kairomone fraction, and aversive behavior in mice. The figure 1 data is reasonable, but not interesting.The figure 2 data fails to demonstrate the relationship between the amount of rat urine and the \"detection threshold\" of the mice. On top of that, this evaluation system is not suitable for detecting the detection threshold of kairomones. This is because if mice spent time evenly in this system, you cannot say that the mice cannot distinguish between these two sources, you can only say that the mice did not distinguish between them.",
"responses": [
{
"c_id": "635",
"date": "30 Nov 2013",
"name": "Ajai Vyas",
"role": "Author Response",
"response": "We believe revisions to the script address the main concerns raised by referee #2.The referee posits that creating an avoidance-avoidance conflict in figure two does not differentiate between possibilities that mice cannot detect rat odor, versus that mice avoid both sources of odor with equal magnitude. Our goal in this figure is not the detection but the discrimination between sets of two odor source. The detection itself is being tested in Figure 1, which does not use avoidance-avoidance conflict. In other words, we are asking if mice can discriminate an odor (i.e. baseline) from another odor that is incrementally stronger and is presented simultaneously (baseline X ∆). Weber’s law predicts that increment in odor strength required for discrimination will be proportional to baseline (i.e. ∆ remains constant). Because Weber’s law pertains to discrimination and not detection, we tested our hypothesis in Figure 2 using avoidance-avoidance conflict.We have revised the manuscript to incorporate “discrimination” instead of “detection” in the appropriate results and figure sections."
}
]
},
{
"id": "2397",
"date": "12 Nov 2013",
"name": "Fernando Martínez-García",
"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 by Vasudevan and Vyas explores the interesting hypothesis that the response of mice to urine-borne kairomones of rats is dose dependent according to the Weber’s law. To do so the authors basically analyse the avoidance of mice to different amounts of urine. In fact, the authors purify the MUPs fraction (>3 kD mw), where the kairomone is contained according to Papes et al. (2010), and they treat this fraction with high concentrations of menadione to displace odorants contained in the lipophilic pockets of the MUPs. This strategy ensures that the reaction of mice to the stimulus is due to the major urinary proteins and not to urine-borne volatiles.The results apparently lend support to the authors’ hypothesis. There is a highly significant (negative) correlation between the amount of stimulus and the time spent near it by the mice, following a sigmoid dose-response curve.The authors further test their hypothesis by checking whether mice are able to discriminate different amounts of rat kairomone. To do so they design a simple two-choice test in which the mice can explore two arms of an arena in which two different amounts of MUPs are present, the ratio between the amount in both sides being 1.2 or 1.3. There is a clear, highly significant avoidance of the arm containing the higher amount, as compared to the other arm. This demonstrates that, throughout the linear region of the dose-response curve, mice discriminate two “doses” differing by x1.2 or x1.3.The main problem with this experimental design is that the authors are checking the response to different amounts (from 3.125 to 50 μL) of the putative kairomone, rather than to different concentrations of it. If the kairomone were volatile, there would be a direct relationship between amount of sample (volume) and the concentration of the substance in the air: the larger air-liquid interface, the more molecules would be in the air phase. However, a MUP is, due to its high molecular weight, virtually non-volatile. Being a vomeronasal stimulus (according to Papes et al., 2010, trp2-/- do not avoid rat urine), for the animal to detect the kairomone it must contact the “drop” of MUP solution and perform vomeronasal pumping.The important question here is how the MUP solution is presented, and no details are found in Material and Methods. Very likely, a drop of the MUP solution would be deposited in a piece of filter paper or a similar absorbent material. Depending on the volume of solution, a given area of the paper would become soaked by capillarity.In a regular piece of absorbent material, a drop of liquid would diffuse giving an approximately circular wet area. Accordingly, one can calculate the theoretical relationship between the volume (V) of MUP solution and the radius (r) of the resulting spot (circular wet area) which would be related to the function: V = π·r2. The radius of the spot of urine increases with the volume of the drop. Depending on the size of the area considered in each experiment as “near urine”, this fact, rather than the effect of the “dose”, may explain the results.In Material and methods, under the heading “Dose-responsivity to kairomones”, the authors explain that “avoidance was quantified by comparing the time spent by mice in two opposing bisects of the area (76x9 cm; 15 cm)”. The term bisect is confusing (it may mean a line sectioning something in two halves, or just sectioning it in two pieces of different size). It is difficult, therefore, to have a clear idea of what the authors are really measuring, for two reasons. First, in spite of what they say (“…comparing the time spent by mice in two opposing bisects”) in their Fig. 1 the authors only represent the time spent “near rat urine”. It seems that this is the measurement analysed statistically (no comparison is made with the side containing PBS).Second, the arena employed for the tests seems to be a long corridor 76cm long per 9 cm wide, but there is no mention of the actual size of the area that the authors consider “near rat urine”. I assume that they are using an identical area throughout the experiment, maybe a given region at the end of the arm where urine (or MUP solution) is presented. But, if so, as the volume of urine (or MUP solution) increases, the urine spot occupies a larger proportion of the area “near rat urine”. If we assume that contacting the urine allows detecting the kairomone (by vomeronasal pumping) and results in avoidance, the larger the urine spot (which would be the region actually avoided), the shorter the time in the area “near rat urine”. When the spot diameter approaches the size of the area “near rat urine” (e.g. the width of the arena), the time visiting this area reaches an asymptotic line at a minimum. This would explain Figure 1 of the manuscript.In the same vein, if two spots of different size are located in the two sides of the arena (experiment 2; discrimination threshold) and animals are avoiding contact with the MUP spot, the time in area “near rat urine” on the side having a larger spot (let’s say a 70% of the area “near rat urine”) will be shorter than the time in the side having a smaller spot (which may represent just a 20% of the area “near rat urine”).As a conclusion, the main problem of the work reported in the manuscript, otherwise well designed and performed, is that the authors are using the term “dose” in a misleading way. The volume of MUP solution is probably not a proper measurement of the “dose” of the stimulus, or the response of the sensory organ (VNO). Vomeronasal neurons respond to the stimulus (MUP) in a concentration-dependent manner (e.g. Leinders-Zufall et al., 2000; Leinders-Zufall et al., 2004), rather than in a way proportional to the volume of the MUP solution (and the area of the resulting “urine” spot). Therefore, varying the concentration of MUPs in the solution while using a fixed volume would really test the hypothesis that the dose-response to a kairomone follows Weber’s law.Anyway, the results are difficult to interpret due to the lack of details on procedure. The way in which the stimulus is presented is not properly described, and the size of the area “near rat urine” must be known in order to check if the explanation of the results by the authors is correct or not.",
"responses": [
{
"c_id": "636",
"date": "30 Nov 2013",
"name": "Ajai Vyas",
"role": "Author Response",
"response": "We have attempted to address the concerns of referee #3 in second version of the manuscript. The referee’s comments are based on two strands of criticisms, both of which arise because of our inadequate and unclear description of methods:The assumption that the authors varied volume in order to vary amount of the stimulus, rather than concentration.The bisect area was undefined in the original methods.The volume of urine stimuli used was constant; concentrations were varied instead to reach varying amounts. This distinction has now been made clearly in the revised version. Since the volume of the spot was constant, the radius/area of the spot of urine remains constant across the different doses, which addresses the question of variance in the area of the spot of urine affecting our results.In addition, we have defined the time spent near rat urine by providing dimensions of bisects. In this case, bisects were obtained by equal division of the arena. This point has been clearly mentioned now in the revision. A rather big difference in size of bisect and size of the stimulus spot, along with equal volume of stimuli across doses, avoids substantial interaction between doses and bisect definition.We believe additional clarifications and elaborations in the material and methods sections adequately respond to all of referee’s comments.In figure 1, we did not show data for PBS because time spent near PBS is directly dependent on time spent neat rat urine stimuli. We use two equal bisects to measure time spent near PBS and rat odor (i.e. the arena is virtually divided in two equal compartments). In this design, time spent near PBS is merely subtraction of time near rat odor from total trial duration. Dependence of these two measures then means that statistics for both of them will be redundant."
}
]
}
] | 1
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https://f1000research.com/articles/2-195
|
https://f1000research.com/articles/2-208/v1
|
08 Oct 13
|
{
"type": "Research Article",
"title": "A computational analysis of the long-term regulation of arterial pressure",
"authors": [
"Daniel A. Beard",
"Klas H. Pettersen",
"Brian E. Carlson",
"Stig W. Omholt",
"Scott M. Bugenhagen",
"Klas H. Pettersen",
"Brian E. Carlson",
"Stig W. Omholt",
"Scott M. Bugenhagen"
],
"abstract": "The asserted dominant role of the kidneys in the chronic regulation of blood pressure and in the etiology of hypertension has been debated since the 1970s. At the center of the theory is the observation that the acute relationships between arterial pressure and urine production—the acute pressure-diuresis and pressure-natriuresis curves—physiologically adapt to perturbations in pressure and/or changes in the rate of salt and volume intake. These adaptations, modulated by various interacting neurohumoral mechanisms, result in chronic relationships between water and salt excretion and pressure that are much steeper than the acute relationships. While the view that renal function is the dominant controller of arterial pressure has been supported by computer models of the cardiovascular system known as the “Guyton-Coleman model”, no unambiguous description of a computer model capturing chronic adaptation of acute renal function in blood pressure control has been presented. Here, such a model is developed with the goals of: 1. capturing the relevant mechanisms in an identifiable mathematical model; 2. identifying model parameters using appropriate data; 3. validating model predictions in comparison to data; and 4. probing hypotheses regarding the long-term control of arterial pressure and the etiology of primary hypertension. The developed model reveals: long-term control of arterial blood pressure is primarily through the baroreflex arc and the renin-angiotensin system; and arterial stiffening provides a sufficient explanation for the etiology of primary hypertension associated with ageing. Furthermore, the model provides the first consistent explanation of the physiological response to chronic stimulation of the baroreflex.",
"keywords": [
"Theoretical analysis and observations of the control of blood volume and salt content by the kidneys has led to the hypothesis that arterial pressure is determined in the long-term (over time scales of days or more) by the balance between the level of salt intake and the acute relationship between pressure and salt excretion by the kidneys. In fact",
"it is stated that the renal pressure/volume control system adjusts arterial pressure with “infinite gain” and that the renal function curve and rate of salt and water intake are the “two sole determinants of the long-term arterial pressure”1. Yet while it is certain that at any steady level of arterial pressure the rates of salt and water intake and excretion are balanced",
"there is considerable debate over how this balance is achieved",
"and thus what are the long-term determinants of arterial pressure2–5."
],
"content": "Introduction\n\nTheoretical analysis and observations of the control of blood volume and salt content by the kidneys has led to the hypothesis that arterial pressure is determined in the long-term (over time scales of days or more) by the balance between the level of salt intake and the acute relationship between pressure and salt excretion by the kidneys. In fact, it is stated that the renal pressure/volume control system adjusts arterial pressure with “infinite gain” and that the renal function curve and rate of salt and water intake are the “two sole determinants of the long-term arterial pressure”1. Yet while it is certain that at any steady level of arterial pressure the rates of salt and water intake and excretion are balanced, there is considerable debate over how this balance is achieved, and thus what are the long-term determinants of arterial pressure2–5.\n\nOne viewpoint is that the acute relationship between pressure and salt excretion—the acute pressure-natriuresis mechanism—represents a physiological input-output relationship and that alterations to this mechanism underlie most (if not all) chronic changes in pressure1,6. While competing ideas identify the nervous system and its influence on the heart and vasculature as the primary long-term controllers of arterial pressure3,4, the hypothesis that long-term control of blood pressure is achieved through the renal pressure-diuresis represents the dominant the thinking in the field7.\n\nThe most widely recognized model of long-term blood pressure control is the model developed over several decades by Guyton and colleagues. The 1972 realization of the model6 invokes approximately 160 variables, and several hundred adjustable parameters8. More recent versions of the model involve many thousands of variables and tens of thousands of parameters9. While several realizations of the Guyton model are or have been disseminated as computer programs, human readable expositions of the various versions of the model are lacking. Moreover, there is no published report of any of these models in which the governing equations are defined, the parameter values are provided, the data that were used for model identification are reported, and the model identification process and results are reported. Indeed, it is unlikely if not impossible that even the relatively simple 1972 version of the model was formally identified. These facts unfortunately make this seminal work largely impenetrable and possibly irreproducible. Most critically, the Guyton-Coleman model is based on the assertion that arterial blood pressure is, under all circumstances, controlled primarily by the kidney10. Therefore the Guyton-Coleman model cannot be used to explore alternative hypotheses.\n\nIn the Systems Approach for PHysiological Integration of Renal, cardiac and respiratory functions (SAPHIR) project, Thomas et al.8 aimed to develop and disseminate a core “model of human physiology targeting the short- and long-term regulation of blood pressure, body fluids and homeostasis of the major solutes” including “the main regulatory sensors (baro- and chemoreceptors) and nervous (autonomic control) and hormonal regulators (antidiuretic hormone, aldosterone and angiotensin)”. The project goal is to, for the first time, provide an open-source transparent model integrating these systems. In 2008, the SAPHIR group published a core model of circulation and volume exchange across fluid compartments that is derived from the Guyton models, consisting of 20 equations and perhaps less than 100 parameters8. This model does not account for adaptation of the acute pressure-diuresis/natriuresis function to chronic changes in arterial pressure or volume/salt loading. Thus, it does not capture the concept of the chronic renal function curve, which is thought to be of fundamental importance to the long-term regulation of arterial pressure. Recently Averina et al.4 reported a mathematical model of long-term control of arterial pressure that captures the concept of chronic adaptation of pressure-natriuresis and, to our knowledge, represents the only published model incorporating the concept of adaptation of the acute pressure-natriuresis relationship in response to changes in salt/volume loading. Thus, the model of Averina et al. represents the model of record of regulation of blood pressure accounting for the chronic renal function curve. Yet, this model was developed to illustrate that the chronic renal function curve need not represent a physiological input-output mechanism, and to introduce an alternative description of the long-term regulation of arterial pressure and the etiology of salt- and angiotensin-dependent hypertension.\n\nSimilar to the goals of the SAPHIR project, we developed a mathematical model of the Guyton concept of long-term control of arterial pressure, analysed its behavior and compared its predictions to experimental observations. Similarly, an open-source transparent model integrating these systems is provided. The approach pursued here differs from that of Thomas and colleagues in that while the SAPHIR project explicitly centers on translating the components of the Guyton model into an explicitly defined core model, our goal is to develop a model from scratch in which all model components and associated data are formally identified based on experimental data. Furthermore, given the demands of model identification and validation, our goals are necessarily more narrowly constrained than those of the SAPHIR project and of the original Guyton-Coleman models. In our study, a model is constructed in the spirit of the Guyton-Coleman models, adopting a practical approach to data-driven phenomenological representations of physiological systems, rather than aiming for physical and mechanistic realism.\n\nSpecifically, we have developed a model of the long-term control of arterial pressure that captures the Guyton concept of pressure-diuresis/natriuresis as physiological input-output relationships. The model is designed to meet the following criteria: 1. The model accounts for the effects of the baroreflex on the circulation, the heart, and the kidney, and the effects of the renin-angiotensin system on the circulation, the heart, and the kidney; 2. The model is explicitly documented for definition and reproducibility: all model equations are reported herein and justified based on experimental data; 3. Parameter values are reported and justified: All parameter values are estimated by comparing model simulations to measured data. The identified model is used to probe physiological mechanisms underlying the chronic renal function curve, to provide insight into how chronic stimulation of the baroreflex leads to chronic reductions in arterial pressure, and to generate hypotheses regarding the etiology of primary hypertension.\n\n\nMethods\n\nBased on the simple approximation of a thin-walled cylinder, the strain ε in the aorta is computed as a function of volume VAo\n\n\n\nwhere V0 is a parameter representing the unstressed volume, and dA/d0 is the ratio of diameter to unstressed diameter. (Here ε is defined to be equal to 1 in the unstressed state when dA = d0). The aortic pressure-volume relationship is assumed to be governed by\n\n\n\nwhere CAo represents and acute compliance and VsAo(t) accounts for creep mechanics of aortic wall, simulated according to\n\n\n\nwhere τcAo is the time constant of stress relaxation and CAo/C∞ is the ratio of acute to effective chronic compliance of the vessel. (Equation (2) and Equation (3) represent an alternative equivalent formulation of the standard linear model of vessel mechanics).\n\nThe parameters of the large-artery mechanics model component are identified based on measurement of aortic diameter and pressure in dogs reported by Coleridge et al.11. Figure 1 plots the measured aortic pressure wave in the upper panel and vessel diameter as a function of pressure in the lower. The experiment of Coleridge et al. is simulated using the governing equations\n\nA. The aortic pressure time course obtained from Coleridge et al.11 is used as the input to the aortic mechanics model module, Equation (4). B. The model-predicted relationship between aortic pressure and diameter is compared to the data reported by Coleridge et al.11. Model simulations are plotted as a solid black cure; data are plotted as shaded circles.\n\n\n\nwhere the measured aortic pressure waveform (Figure 1A) is used to numerically approximate dPA0(t)/dt in integrating Equation (4).\n\nThe parameters γAo, CAo, τcA, and d0 were adjusted to match the data in Figure 1B; the value of V0 was set arbitrarily by assuming a cylindrical vessel of length 30 mm. All parameter values are listed in Table 1.\n\nThe baroreceptor afferent firing rate is assumed to be governed by the rate of change of strain in the vessel wall. The model invokes a moving average strain value –ε(t), which is computed\n\n\n\nThe time constant τs is an adjustable parameter. The baroreceptor firing rate is assumed proportional to δε = max(ε––ε,0) via the saturable relationship\n\n\n\nwhere s(t) represents the fraction of baroreceptor afferents that are in an active/permissible state, and δε and f0 are additional adjustable parameters. Equation (6) is a static nonlinearity12 that enforces a saturating response. It is assumed that the baroreceptors within the population transition from an active to inactive state at a rate proportional to the firing rate, and transition to the active state at a constant rate:\n\n\n\nThe adjustable parameters in the baroreflex afferent model (τs, δε, f0, a, and b) are identified based on measurements following step changes in non-pulsatile carotid sinus pressure13 and ramps in in vivo pulsatile aortic pressure11.\n\nThe data in Figure 2A, obtained from Chapleau et al.13, correspond to the multi-fiber response of the carotid sinus baroreceptors following a step change in pressure from 40 mmHg to 93 mmHg. To simulate this experiment, the pressure is assumed to follow a time course described by\n\nA. The response of the baroreflex model to a step increase in pressure is compared to data from Chapleau et al.13. The model simulations are based on Equation (4)–Equation (7), as described in the text. B. Simulated baroreflex output based on input aortic pressure wave obtained from Coleridge et al.11.\n\n\n\nwhere pressure is expressed in units of mmHg and time in seconds. Thus it is assumed that pressure is increased by +53 mmHg over an interval from -1 to 0 seconds. Since the overall model does not differentiate between aortic and carotid baroreflex signals, data from the carotid pressure step are matched to the model of Equation (4)–Equation (7) in Figure 2A. Following the step pressure increase the afferent firing rate fBR rapidly increases to a maximum value before decaying over a time scale of several hundred seconds. This decay is determined in the model by the combined action of mechanical relaxation governed by Equation (5) and inactivation governed by Equation (7).\n\nFigure 2B plots model-predicted baroreflex firing rate elicited by the normal aortic pressure waveform of Coleridge et al.11. The model produces the characteristic bursting pattern where peaks in baroreceptor afferent firing occur in systole, with the firing rate dropping to zero in diastole.\n\nResponses to ramps of pressure in vivo are compared to data of Coleridge et al. in Figure 3. Coleridge et al. adjusted in vivo pressure in the aortic sinus by placing hydraulic occlusion at various positions along the aorta. Model simulations are based on applying a constant increase/decrease to measured PAo(t), resulting in the pressure time series plotted in Figure 3A and 3C. It is assumed that mean pressure is adjusted at a rate of ±10 mmHg sec-1, corresponding to the experimental measurements. The top panel in the figure corresponds to simulations and data associated with the baseline state, where initial mean pressure is 100 mmHg. Data on afferent firing rate for this experiment are plotted as open circles in Figure 3B. The solid line in Figure 3B represent simulated fBR(t) versus PAo(t). The red line represents fBR(t) and PAo(t) averaged over each beat. The lower panel in Figure 3 corresponds to a hypertensive state where the aortic pressure was held at an elevated level with mean of 125 mmHg for 20 minutes prior to the pressure ramp experiment. Thus the system has had 20 minutes to adapt or “reset” to a new mean pressure, resulting in a right shift of the baroreflex response fBR versus PAo.\n\nA. Applied pressure transients for increasing and decreasing pressure starting at a baseline mean pressure of 100 mmHg. Pressure is increased/decreased so that the mean pressure changes at the rate of ±10 mmHg sec-1. B. Simulated baroreceptor firing response to pressure ramps from A is compared to data from Coleridge et al.11. C. Applied pressure transients for increasing and decreasing pressure starting at a baseline mean pressure of 125 mmHg. D. Simulated baroreceptor firing response to pressure ramps from C is compared to data from Coleridge et al.11. See text for details on simulation protocol. In B and D, experimental data are plotted as circles; black line represents the simulation prediction; red line represents simulation predictions averaged over each heart beat.\n\nThe values of the parameters τs, δ0, f0, a, and b were adjusted to match the data in Figure 2A and Figure 3.\n\nThe circulation is modeled as a closed-loop lumped-parameter circuit illustrated in Figure 4, which ignores the pulmonary circulation and treats the heart as time-varying elastance representing the left ventricle14. The left-ventricular pressure is described by\n\n\n\nFlows entering and exciting the heart are denoted Fin and Fout; pressure in the aorta and arterial and venous capacitors are denoted PAo, PA and PV; left-ventricular pressure is denoted PLV.\n\nwhere ELV(t) is the left-ventricular elastance and VLV(t) is the volume of blood in the ventricle. The elastance is simulated using a smooth function that increases in systole and decreases as the heart relaxes:\n\n\n\nwhere θ Є (0,1) is the fraction of a heart beat that has elapsed at a given time. The factor (0.75 + ϕSN) multiplying Emax accounts for the effect of sympathetic tone on contractility, where ϕSN (t) Є (0,1) is a model variable (see below) representing the sympathetic drive. Under baseline conditions ϕSN ≈ 0.25 and therefore, under maximal sympathetic stimulation, cardiac contractility is approximately 175% of baseline.\n\nThe variable θ is simulated via\n\n\n\nwhere H = H0 + H1 (ϕSN -0.25) is the heart rate, and θ(t) is reset to 0 each time the value reaches 1. The parameters H0 and H1 are set to give a baseline heart rate of 75 beats min-1 and a heart rate of 150 beats min-1 under maximal sympathetic tone.\n\nThe circuit model is simulated based on equations for the state variables θ(t), VLV(t), VAo(t), VA(t), VV(t), VsA(t), and VsV(t). The governing equations for the six volume variables are\n\n\n\nwhere PLV(t) is determined by Equation (9).\n\nThe flows Qin and Qurine represent the rates of volume uptake/infusion and urine production, described below.\n\nThe max(0,∙) terms in Equation (12) account for the valves, which permit flow only in the direction indicated in Figure 4.\n\nThe RAo and RV resistances are set to constant values, while other resistances and capacitances vary with sympathetic tone and angiotensin II level. Specifically, CA(t), CV(t), and RA(t) are determined by\n\n\n\nwhere CA0, CV0, and RA0 are constants, and α1, α2, α3, and α4 are constants that determine the magnitude of the effects of vasoconstriction via sympathetic tone and angiotensin II. The variable ϕA2 represents the plasma angiotensin II activity. The governing equations for ϕA2 are described below.\n\nThe RA resistance is further governed by a whole-body autoregulation phenomenon, as incorporated into the Guyton-Coleman models6,8,9. The function rAR(t) Є (0,1) accounts for autoregulatory effects on systemic arterial conductivity based on\n\n\n\nwhere –F(t) is a variable that averages the mean cardiac output with a moving average defined by a first-order process:\n\n\n\nThe pressures are computed from the relationships\n\n\n\nin which venous compliance is simulated using a linear formulation of stress relaxation similar to that used for the aorta. Specifically, venous stress relaxation kinetics are governed by τcV(VsV/dt) = VsV∞ – VsV, where\n\n\n\nThe constant VV01 in Equation (17) represents an unstressed volume for the overall cardiovascular system.\n\nThere are a total of 23 parameters associated with this component of the model. Assignment of the values listed in Table 1 was guided by a variety of data sets and previous computational models. The values of Emax and Emax were obtained by scaling the model of14 to provide reasonable pressure under baseline conditions at ventricular volumes appropriate for dog. The cardiac cycle timing parameters TM and TR were set to the values used by Beard14. Heart rate parameters H0 and H1 were determined as described above. The baseline resistance and compliance parameters Rout, RAo, RA0, RV, CA0, CV0, and VV01 were chosen so that under baseline conditions (Figure 5), the mean pressure is 100 mmHg, the diastolic and systolic pressures are 85 and 115 mmHg, and the ejection fraction is 0.58. (The values for these seven parameters do not represent a unique set that gives these outputs under baseline conditions). The parameters γV and τcV were set to match measurement of stress relaxation in the canine jugular15.\n\nA. Model-predicted aortic pressure and baroreflex firing rate, obtained with Qin = 0.5835 ml min-1. This simulation represents a period steady-state of the model, in which Qin = Qout and average pressure is 100 mmHg. B. Model-predicted aortic and left-ventricular pressures are plotted for the baseline period steady state.\n\nThe remaining seven parameters in the cardiac and circulation model component (α1, α2, α3, α4, F0, F1, and τAR) were identified by simulating the responses of the system to volume infusion and hemorrhage, as detailed below in Results.\n\nThe whole-body sympathetic tone is represented in the model by the variable ϕSN(t) Є (0,1) and is determined by the baroreflex arc:\n\n\n\nThus, in the absence of baroreflex firing, the sympathetic tone will approach the maximum value of 1. The constant parameter fSN is set so that under baseline conditions ϕSN(t) = 0.25. Thus, following a rapid severe drop in pressure ϕSN(t) will approach a value that is four times the baseline value.\n\nThe state of the renin-angiotensin system is captured by two variables: ϕR(t) Є (0,1) and ϕA2(t) Є (0,1), which represents plasma renin and angiotensin II activity governed by the combined action of sympathetic tone and pressure on renin release.\n\nThe plasma renin variable ϕR(t) is governed by\n\n\n\nwhere ϕR∞ decreases with increasing time-averaged arterial pressure –P. This formulation assumes that the relationship between steady-state ϕR and pressure –P is shifted by the sympathetic tone. Based on observations showing that plasma renin activity and angiotensin II levels are nearly perfectly linearly related in vivo16, we assumed that ϕA2(t) follows ϕR(t) according to\n\n\n\nThe five parameters invoked in this model component (τR, τA2, g, P1, and P2) were identified based on comparing simulations to measurements of pressure, heart rate, and plasma renin activity in rabbit during graded hemorrhage, as detailed below in Results. (The simulation parameter τP was arbitrarily set at 15 seconds).\n\nRegulation of body-fluid volume is assumed governed by a linear pressure-diuresis relationship:\n\n\n\nwhere Qurine is rate of volume output via the kidneys and k1 is the slope of the relationship between Qurine and pressure. The model variable Ps(t) is the variable offset of the pressure-diuresis relationship, which is controlled by sympathetic tone and angiotensin II level:\n\n\n\nThe constant parameters Ps,min, Ps,max, ϕ0, and ϕ1 determine how the acute pressure-diuresis relationship shifts in response to changes in the tone variables ϕSN and ϕA2. Thus it is assumed that ϕSN and ϕA2 have equal and additive effects on renal function. Equation (22) assumes that changes in Ps governed by sympathetic tone and the renin-angiotensin system occur with a time constant τk. Furthermore, the maximal rate of urine production is set to 10 ml min-1.\n\nThe parameters k1, Ps,max, Ps,min, ϕ0, and ϕ1, were set by matching model predictions to data on the pressure diuresis relationship under physiological conditions, with angiotensin II infusion, and with administration of an angiotensin converting enzyme (ACE) inhibitor. To compare predictions of Equation (21) and Equation (22) to renal output measurements under ACE inhibition, we assumed that for the chronic measurements, sympathetic tone was maintained at its baseline value and ϕA2 = 0. Fitting data from Hall et al.17, it was estimated that Ps∞ (ϕSN = 0.25, ϕA2 = 0) = 75 mmHg. Similarly, with angiotensin II infusion, we assumed that sympathetic tone was maintained at its baseline value and ϕA2 = 1. The data of Hall et al. yield an estimate of Ps∞ (ϕSN = 0.25, ϕA2 = 1.0) = 125 mmHg for these conditions. Finally, under baseline conditions, with –P = 100 mmHg, urine output was estimated to be Qurine = 0.5835 ml min-1, based on volume infusion experiments described in the Results. With k1 = 0.125 ml sec-1 mmHg-1 (see Results), and using the baseline ϕA2 value of 0.1864 (which is determined from blood withdrawal experiments; see Results), it follows that Ps∞ (ϕSN = 0.25, ϕA2 = 0.1864) = 95.3 mmHg. These estimates of Ps∞ at three different values of ϕSN + ϕA2 were used to estimate the values of Ps,max, Ps,min, ϕ0, and ϕ1.\n\nThe time constant τk was determined from data on the rate of urine production following infusion of blood leading to an acute increase in pressure and drop in ϕSN and ϕA2. Analysis of data from these experiments is detailed below in the Results together with a direct comparison between the data of Hall et al.17 and model simulations.\n\n\nResults\n\nGuyton and colleagues conducted experiments in which a large amount of blood was infused into an anesthetized dog, resulting in a rapid increase in total blood volume of 45% compared to initial baseline value. The model of Equation (1)–Equation (22) was simulated and compared to data from this experiment to identify adjustable model parameters and to probe the predicted response of unmeasured model variables to this protocol.\n\nFigure 6 shows the predicted effects of infusing 45% of initial baseline blood volume in a normal animal on arterial pressure, cardiac output, rate of urine formation, blood volume, sympathetic tone, and renin-angiotensin level together with experimental data obtained from Dobbs et al.18, Guyton et al.1, and Prather et al.19 on the first four variables. (For these experiments Guyton et al. reported a baseline mean arterial pressure of approximately 115 mmHg. Since all other data sets analyzed here have a baseline pressure of 100 mmHg, 15 mmHg was subtracted from the data from these experiments to achieve consistency). The initial steady state of the model was obtained based on a constant infusion of Qin = 0.5835 ml min-1, which matches the mean reported rate of volume output for the baseline data. For times 0 < t < 5 min, Qin was increased to 0.5835 + 126.82 ml min-1, to result in a total excess volume of 634 ml. After the five-minute infusion, Qin was returned to the baseline value of 0.5835 ml min-1. The model is able to effectively reproduce the trends in the experimentally measured variables, and predicts that ϕSN and ϕRA drop to less than one half of their resting values following the volume infusion in response to the transient increase in pressure. These reductions in ϕSN and ϕR confer increases in vascular compliances helping to accommodate the substantial volume increase. As volume is removed from the system ϕSN and ϕR return toward the baseline values, with ϕSN responding substantially faster than ϕR.\n\nData on arterial pressure, cardiac output, rate of urine formation, and blood volume are obtained from Dobbs et al.18, Guyton et al.1, and Prather et al.19. The initial steady state of the model was obtained based on a constant infusion of Qin = 0.5835 ml min-1. For times 0 < t < 5 min, Qin was set to 0.5835 + 126.82 ml min-1, to result in a total excess volume of 634 ml. The initial condition for the simulation was obtained by setting Qin = 0.5835 ml min-1 and running the model to obtain the steady state.\n\nThe data plotted in Figure 6, along with data observations on graded hemorrhage, were used in identifying several adjustable parameters in the model. (See following section for details).\n\nData from Quail et al.20 on heart rate, pressure, and plasma renin activity following graded blood withdrawal in rabbits were used to identify parameters associated with the cardiovascular mechanics and the renin-angiotensin system. Figure 7 shows data measured in normal rabbits, where the heart rate measurements have been scaled to a baseline value of 75 beats min-1, renin activity is scaled to maximum value of 1, and mean pressure is scaled to an initial baseline value of 100 mmHg. In the experiments, blood was withdrawn from the animals at a rate of 2% blood volume per minute. In model simulations, blood is withdrawn at the experimental rate starting at time 0 and with withdrawal stopped at time 17.5 minutes. For this experiment, the initial condition was identical to that used for the volume infusion experiments of Figure 6, and volume infusion and urine output were set to zero for the time course.\n\nThe experimental and simulation protocol is to withdraw blood at a rate of 2% of initial total volume per minute, starting at time 0. The end of the withdrawal period, 17.5 minutes, is indicated in by dashed line in all plots. Model simulations are compared to data for mean pressure (A), heart rate (B), and plasma renin activity (D). Panel C plots the model-predicted sympathetic tone during the protocol. The initial condition is the same baseline condition used for the simulations of Figure 6. Data from heart rate, pressure, and plasma renin activity following graded blood withdrawal are from Quail et al.18 with pressure and heart rate scaled as described in the text.\n\nModel predictions of H, –P, and ϕR compare favorably to experimental data, with mean arterial pressure dropping from the initial value of 100 mmHg to approximately 93 mmHg at the end of the blood withdrawal. The observed increase in heart rate is associated with an increase in ϕSN from baseline level of 0.25 to 0.81, and an increase of ϕR from baseline level of 0.186 to 0.668, at the termination of the withdrawal period. During this period, arterial blood pressure is protected from a more severe reduction in large part by an increase in vascular tone mediated by increases in sympathetic tone and angiotensin II level. Simulated cardiac output (not shown) also drops during blood withdrawal, reaching a minimum value of 690 ml min-1 (approximately 46% of baseline flow) at 17.5 minutes.\n\nThe model is not able to capture the sharp, almost step-like, observed increase in renin that occurs following the 10-minute time point (corresponding to 20% of blood withdrawn). Specifically, the model predicts a more graded response, without the delay observed in the data. A better match to the data might be achieved by incorporating a delay into the governing equations, or by simulating the renin-angiotensin system using a higher-order system of differential equations than Equation (19) and Equation (20). The model predicts that ϕSN and H peak at the end of the withdrawal period and partially recover toward baseline levels within a few minutes after the end of blood withdrawal. The renin-angiotensin tone is predicted to remain at an elevated level after the withdrawal. The partial drops in ϕSN and H are associated with a graded decrease in pressure continuing over the entire simulated time course, with pressure reaching approximately 85 mmHg at 30 minutes. Pressure continues to drop after the end of the withdrawal period due in part to the action of the whole-body autoregulatory mechanism governed by Equation (16) and Equation (17). Via this mechanism, the drop in flow associated with hemorrhage causes a reduction in arterial resistance, resulting in a recovery in flow and a continuing drop in pressure.\n\nThe model predictions in Figure 6 and Figure 7 are most sensitive to the values of 13 parameters from the model components of circulatory mechanics, the renin-angiotensin system, and the kinetics of pressure-diuresis: α1, α2, α3, α4, F0, F1, τAR, τR, τA2, g, P1, P2, and tK. The values of these parameters were adjusted to match the data plotted in Figure 6 and Figure 7.\n\nThe steady-state relationship between arterial pressure and rate of urine output is obtained from model simulations by varying Qin, the rate of volume infusion, and attaining steady-state model predictions of steady state pressure where Qin = Qurine. Figure 8A plots the predicted relationship between rate of urine output and mean arterial pressure under three different conditions: (1) the normal physiological state; (2) ϕR clamped at 0, representing complete block of angiotensin converting enzyme (ACE); and (3) ϕR clamped at 1, representing infusion of saturating levels of angiotensin II. Note that the pressure-diuresis relationship is plotted in the traditional manner with arterial pressure on the abscissa and rate of volume excretion on the ordinate, even though in these simulations Qin is varied and steady-state –P is computed as a function of Qin.\n\nA. Model predictions are compared to data on the steady-state relationship between mean arterial pressure and rate of volume infusion (equal to rate of urine output) for normal conditions, for angiotensin converting enzyme inhibition (ϕR = 0), and for angiotensin II infusion (ϕR = 1). Data are obtained from Hall et al.17, in which net salt output is reported under these three conditions. Rate of urine volume production is assumed proportional to rate of sodium excretion, and normalized to the rate of urine production at baseline conditions (–P = 100 mmHg) for the normal case. B. Model prediction for steady-state renin and angiotensin II activities (ϕR and ϕA2) and sympathetic tone (ϕSN) are plotted as functions of Qin for the normal case (without ϕR clamped).\n\nModel predictions are compared to data from Hall et al.17 on steady-state pressure at different levels of salt/volume loading in normal dogs, dogs infused with ACE inhibitor, and dogs infused with angiotensin II. Model simulations effectively match experimental data, validating the assignment of parameter values described above under “Neurohumoral Control of Pressure-Diuresis/Natriuresis”.\n\nFigure 8B illustrates model-predicted steady-state renin and angiotensin II activities (ϕR and ϕA2) and sympathetic tone (ϕSN) as functions of Qin for the normal case. (Recall that from Equation (20) angiotensin II activity ϕA2 is equal to ϕR in the steady state). As Qin is increased ϕR and ϕA2 decrease to maintain pressure at nearly a constant level of the simulated range of volume/salt loading. The sympathetic tone on the other hand remains nearly constant around the baseline level of 0.25. Sympathetic tone remains constant because in the model the only determinant of ϕSN is the baroreceptor afferent firing rate, which effectively adapts to the small changes in pressure that are associated with the simulated range of Qin. (Thus, the model does not capture suppression of sympathetic tone typically observed with chronic salt/volume loading).\n\nNumerous studies have demonstrated that chronic stimulation of the carotid baroreceptor afferent nerve with implantable devices results in a prolonged decrease in mean arterial pressure21,22. Figure 9 illustrates a representative data set from Lohmeier et al.23,24 for which baroreflex stimulation was applied in dogs for a seven day period, followed by several days of recovery. During the stimulation period, mean arterial pressure drops by approximately 20%, renin activity by approximately 35%, and sympathetic tone (experimentally assayed indirectly by plasma norepinephrine) drops by approximately 55%.\n\nTo simulate this experiment, a constant was added to the model-predicted baroreceptor firing rate in the equation for sympathetic tone. Thus Equation (18) was replaced by\n\n\n\nwhere fstim is a parameter adjusted to match the experimental data. Using fstim = 6.5 sec-1 yields predictions that effectively match the observations of Lohmeier et al.23,24 (Figure 9). Indeed, given that only one parameter was adjusted, the comparison of model predictions to the five variables plotted in the figure represents a strong validation test for the model. In particular, the observed reduction in renin activity with baroreflex stimulation represents a phenomenon that has not been captured by previous modeling efforts22,24. Here, the phenomenon emerges as a property of the integrated computational model: stimulation of the baroreflex and associated drop in sympathetic tone is predicted to result in a drop in renin production even with the long-term drop in mean pressure.\n\nElectrical stimulation of the carotid baroreflex afferent nerve is simulated by modifying the normal model by replacing Equation (18) in the normal model with Equation (23) during the baroreflex stimulation period (for a 1-week period starting on day 0). Data on mean pressure, heart rate, urine output, plasma norepinephrine (plotted as ϕSN), and plasma renin activity (plotted as ϕRA) are obtained from Lohmeier et al.23,24.\n\nPettersen et al.25 demonstrate that impairment of the baroreflex caused by stiffening of arterial vessels represents a viable hypothesis for the etiology of primary hypertension. Specifically, using a closed-loop cardiovascular mechanics model coupled to a detailed model of the baroreflex arc26, Pettersen et al.25 show that when the stiffness of the aorta is increased to represent changes in mechanical properties associated with ageing, the model based on their mechanogenic hypothesis predicts a substantial increase in mean arterial pressure with age. By assuming a constant blood volume for all age groups, the model explicitly did not account for the regulation of plasma volume and salt through the kidney and the renin-angiotensin system following from the hypothesized shift in the renal pressure-diuresis/natriuresis function curve. The rationale for not including adaptive mechanisms likely to partially ameliorate the effects of arterial stiffening on blood pressure was to test the explanatory sufficiency of the mechanogenic hypothesis by showing that it would predict a stronger relation between blood pressure increase and age than empirically observed. As a first step towards a complete merge of physiological renal function with the model developed by Pettersen et al. to expand the prediction space of the mechanogenic hypothesis, we studied how the current model responded to changes in the aortic compliance.\n\nUsing the current model, which does account for blood volume regulation by the kidneys, the hypothesis of Pettersen et al. may be further analyzed by determining how simulations of the current model respond to changes in the aortic compliance. Figure 10A (solid line) plots predicted steady-state mean arterial pressure as a function of relative aortic stiffness, CAo0/CAo, where CAo0 is the baseline normal value of aortic compliance, and CAo is the value used to obtain the pressures reported in the Figure. The maximum simulated relative stiffness, CAo0/CAo = 4, is approximately the average relative stiffness for the 75-year-old population simulated in Pettersen et al.25. This 4-fold increase in aortic stiffness increases the predicted mean pressure from 100 to 128 mmHg, with systolic/diastolic ratio increasing from 115/90 to 139/117. Furthermore, over the simulated range of stiffness model-predicted ϕSN increases from 0.25 to 0.45 and ϕR increases from 0.186 to 0.59 (Figure 10B) with a heart rate increase of approximately 25% as mean pressure increases from 100 to 128 mmHg.\n\nA. The model predicted for the steady-state mean arterial pressure is plotted as a function of relative aortic stiffness, CAo0/CAo, where CAo0 is the baseline normal value of aortic compliance, and CAo is the value used to obtain the simulated pressure. As stiffness is increased (as compliance is decreased), predicted mean pressure increases. Calculations assume normal salt/volume loading, Qin = 0.5835 ml min-1, resulting in a mean pressure of 100 mmHg at CAo0/CAo = 1. B. Model-predicted steady-state sympathetic tone ϕSN, plasma renin activity ϕR, and angiotensin-II activity ϕA2 are plotted as functions of CAo0/CAo. C. Predicted blood volume is plotted as a function of CAo0/CAo.\n\nAs expected, this predicted pressure increase is less than that predicted for a model that does not account for blood volume regulation. (The model of Pettersen et al. predicts a mean arterial pressure 150 mmHg for 75-year-old group). The difference between the predictions of the current model and that of Pettersen et al. is explained primarily by blood volume regulation: with a 4-fold increase in aortic stiffness the current model predicts the active blood volume to decrease from 1364 to 1050 ml (Figure 10C). This result supports the view that normal functioning kidneys are able to partially compensate for age-related increases in arterial stiffness25.\n\nTherefore this model demonstrates that it is possible to capture the age-related phenotype of primary hypertension as emerging solely from changes to the mechanical properties of the aorta and carotid arteries. This may seem surprising since the simulation results plotted in Figure 10 are obtained with mechanical properties of all components of the vasculature other than large vessels associated with the baroreflex remaining normal. Indeed, it is expected that vascular stiffening would directly impact other physiological processes. For example, Beard and Mescam demonstrated that stiffening of renal arteries can explain the shift in the acute pressure-diuresis curve associated with changes in mean pressure in Dahl S rats27. For the model results illustrated in Figure 10 (representing normal kidney function), the predicted increase in mean pressure associated with increased vascular stiffness is partly due to an increase in the pressure that is required to elicit a given baroreceptor firing rate. As a result, the relationship between baroreceptor firing and mean arterial pressure is shifted to higher pressure in hypertension, as has been observed clinically and in animal models7. This phenomenon has been called “adaptation” or “resetting”. Yet, even though model predictions are in agreement with the observed adaptation of the baroreflex, model predictions are not in agreement with the interpretation that, since baroreceptors adapt, “they cannot participate in the long-term control of arterial pressure”7. By contrast, the observed adaptation is a crucial component of the mechanogenic mechanism simulated here. Because stiffening causes an adaptation of the relationship between pressure and baroreceptor firing rate, vascular stiffening can cause a chronic increase in stable mean pressure. In other words, since baroreceptors adapt, they can contribute to the etiology of hypertension.\n\n\nSummary and conclusions\n\nA computational model (Source code is permanently available on: http://zenodo.org/record/7126 (10.5281/zenodo.7126)) was developed and identified to serve as a representation of the major physiological processes controlling arterial blood pressure. The design of the model was based on balancing the compromise between complexity/physiological fidelity and identifiability, yielding a model of 16 state variables and 46 adjustable parameters. The model captures physiological phenomena occurring on times scales ranging from milliseconds (e.g. response of baroreceptor firing to arterial pressure) to days (e.g. response of mean pressure to chronic stimulation of baroreflex). It also effectively captures the response of the cardiovascular system to drastic perturbations, including infusion of 45% of blood volume, withdrawal of 35% of blood volume, electrical stimulation of the baroreflex, and the partial compensatory response of the kidneys to blood pressure increase accompanying arterial stiffening.\n\nThe major conclusions from this work are:\n\n1. Over time frames ranging from seconds to weeks, arterial blood pressure is regulated primarily through the baroreflex arc, the renin-angiotensin system, and the interoperation of the baroreflex arc and the renin-angiotensin system.\n\n2. It is demonstrated how renal function is not the central or primary determinant of long-term arterial pressure in a model system that captures the observed behavior of the renin-angiotensin system, and chronic adaptation of the acute pressure-diuresis/natriuresis phenomena. This result contradicts the classical ‘renocentric’ view of the long-term control of blood pressure even though the model is built on the core assumption of the renocentric view that the pressure-diuresis relationship acts as a physiological input-output relationship in which pressure determines renal output. As a result, it is shown that hypertension does not necessarily indicate any degree of renal dysfunction.\n\n3. The physiological response to chronic stimulation of the baroreflex can be explained by a mechanism in which arterial blood pressure is regulated primarily through the baroreflex arc and the interoperation of the baroreflex arc and the renin-angiotensin system.\n\n4. Model simulations are consistent with the mechanogenic hypothesis25 that arterial stiffening represents a contributing factor causing changes in pressure, sympathetic tone, and renal function associated with ageing. Through pressure-diuresis, normal kidney function is predicted to be able to partially ameliorate the effects of arterial stiffening on blood pressure.\n\nRegarding the forth conclusion, model simulations predict that stiffening of the large arterial vessels that are associated with baroreceptors can contribute substantially to long-term changes in pressure. This result contradicts the ‘renocentric’ theory of blood pressure control even though the core assumption of the Guyton ‘renocentric’ theory of blood pressure control (that the pressure-diuresis relationship acts as a physiological input-output relationship in which pressure determines renal output) is a core assumption of this model. Although it has been argued that because its effective gain resets to a given mean arterial pressure the baroreflex cannot play an important role in long-term pressure regulation, this analysis shows how a dynamic system representing the baroreflex and capturing the observed resetting phenomenon can play a key role in the long-term control of pressure and the etiology of hypertension. Model simulations reveal that vascular stiffening increases sympathetic tone and shifts the effective baroreflex response to an increased pressure baseline. Because these changes cause an increase in angiotensin II activity and associated shift in the acute pressure-diuresis relationship, the renocentric7 and mechanogenic25 hypotheses for the etiology of primary hypertension could potentially be interpreted as compatible. However, as changes to renal function arise as downstream consequences of the mechanical remodeling, the mechanogenic explanation subsumes more of the biology involved in the etiology of hypertension compared with a renocentric explanation. This is a major conceptual advance that also provides a new interpretational framework for available experimental and clinical data.\n\nThe model developed here represents a simplification and several components are represented largely in phenomenological terms. A more detailed model may be constructed through integration of more biophysically based and detailed component models26–28 into the framework developed here. Regardless, the simplifications invoked in the model do not impact the major conclusions.",
"appendix": "Author contributions\n\n\n\nAll authors contributed to model conception, development of the hypotheses to be tested, and design of computational experiments to analyze data and test hypotheses. DAB and SMB constructed the mathematical formulation of the model and wrote the computer code. DAB drafted the manuscript with all authors contributing to the content and/or revisions.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests to disclose.\n\n\nGrant information\n\nThis work was funded by the VPR Project, supported by NIH grant P50GM094503. KHP is supported by the Research Council of Norway under the eVITA program, project number 178901/V30.\n\n\nAcknowledgements\n\nThe authors are grateful to Allen Cowley and David Mattson for valuable discussions.\n\n\nReferences\n\nGuyton AC: Circulatory physiology iii:Arterial pressure and hypertension. Philadelphia, London, Toronto: W. B. Saunders Company; 1980. Reference Source\n\nMontani JP, Van Vliet BN: Understanding the contribution of guyton's large circulatory model to long-term control of arterial pressure. Exp Physiol. 2009; 94(4): 382–388. PubMed Abstract | Publisher Full Text\n\nOsborn JW, Averina VA, Fink GD: Current computational models do not reveal the importance of the nervous system in long-term control of arterial pressure. Exp Physiol. 2009; 94(4): 389–396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAverina VA, Othmer HG, Fink GD, et al.: A new conceptual paradigm for the haemodynamics of salt-sensitive hypertension: A mathematical modelling approach. J Physiol. 2012; 590(Pt 23): 5975–5992. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHall JE, Mizelle HL, Hildebrandt DA, et al.: Abnormal pressure natriuresis. A cause or a consequence of hypertension? Hypertension. 1990; 15(6 Pt 1): 547–559. PubMed Abstract | Publisher Full Text\n\nGuyton AC, Coleman TG, Granger HJ: Circulation: Overall regulation. Annu Rev Physiol. 1972; 34: 13–46. PubMed Abstract | Publisher Full Text\n\nCowley AW Jr: Long-term control of arterial blood pressure. Physiol Rev. 1992; 72(1): 231–300. PubMed Abstract\n\nThomas SR, Baconnier P, Fontecave J, et al.: Saphir: A physiome core model of body fluid homeostasis and blood pressure regulation. Philos Trans A Math Phys Eng Sci. 2008; 366(1878): 3175–3197. PubMed Abstract | Publisher Full Text\n\nAbram SR, Hodnett BL, Summers RL, et al.: Quantitative circulatory physiology: An integrative mathematical model of human physiology for medical education. Adv Physiol Educ. 2007; 31(2): 202–210. PubMed Abstract | Publisher Full Text\n\nBeard DA: Tautology vs. Physiology in the etiology of hypertension. Physiology (Bethesda). 2013; 28(5): 270–271. PubMed Abstract | Publisher Full Text\n\nColeridge HM, Coleridge JC, Poore ER, et al.: Aortic wall properties and baroreceptor behaviour at normal arterial pressure and in acute hypertensive resetting in dogs. J Physiol. 1984; 350: 309–326. PubMed Abstract | Free Full Text\n\nSharpee TO, Miller KD, Stryker MP: On the importance of static nonlinearity in estimating spatiotemporal neural filters with natural stimuli. J Neurophysiol. 2008; 99(5): 2496–2509. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChapleau MW, Lu J, Hajduczok G, et al.: Mechanism of baroreceptor adaptation in dogs: Attenuation of adaptation by the k+ channel blocker 4-aminopyridine. J Physiol. 1993; 462: 291–306. PubMed Abstract | Free Full Text\n\nBeard DA: Biosimulation: Simulation of living systems. Cambridge; New York: Cambridge University Press; 2012. Reference Source\n\nPorciuncula CI, Armstrong GG Jr, Guyton AC, et al.: Delayed compliance in external jugular vein of the dog. Am J Physiol. 1964; 207: 728–732. PubMed Abstract\n\nCowley AW Jr, Guyton AC: Quantification of intermediate steps in the renin-angiotensin-vasoconstrictor feedback loop in the dog. Circ Res. 1972; 30(5): 557–566. PubMed Abstract | Publisher Full Text\n\nHall JE, Guyton AC, Smith MJ Jr, et al.: Blood pressure and renal function during chronic changes in sodium intake: Role of angiotensin. Am J Physiol. 1980; 239(3): F271–280. PubMed Abstract\n\nDobbs WA Jr, Prather JW, Guyton AC: Relative importance of nervous control of cardiac output and arterial pressure. Am J Cardiol. 1971; 27(5): 507–512. PubMed Abstract | Publisher Full Text\n\nPrather JW, Taylor AE, Guyton AC: Effect of blood volume, mean circulatory pressure, and stress relaxation on cardiac output. Am J Physiol. 1969; 216(3): 467–472. PubMed Abstract\n\nQuail AW, Woods RL, Korner PI: Cardiac and arterial baroreceptor influences in release of vasopressin and renin during hemorrhage. Am J Physiol. 1987; 252(6 Pt 2): H1120–1126. PubMed Abstract\n\nIliescu R, Irwin ED, Georgakopoulos D, et al.: Renal responses to chronic suppression of central sympathetic outflow. Hypertension. 2012; 60(3): 749–756. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLohmeier TE, Iliescu R: Lowering of blood pressure by chronic suppression of central sympathetic outflow: Insight from prolonged baroreflex activation. J Appl Physiol (1985). 2012; 113(10): 1652–1658. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLohmeier TE, Irwin ED, Rossing MA, et al.: Prolonged activation of the baroreflex produces sustained hypotension. Hypertension. 2004; 43(2): 306–311. PubMed Abstract | Publisher Full Text\n\nIliescu R, Lohmeier TE: Lowering of blood pressure during chronic suppression of central sympathetic outflow: Insight from computer simulations. Clin Exp Pharmacol Physiol. 2010; 37(2): e24–33. PubMed Abstract | Publisher Full Text\n\nPettersen KH, Bugenhagen SM, Nauman J, et al.: Arterial stiffening provides sufficient explanation for primary hypertension. 2013. Reference Source\n\nBugenhagen SM, Cowley AW Jr, Beard DA: Identifying physiological origins of baroreflex dysfunction in salt-sensitive hypertension in the dahl ss rat. Physiol Genomics. 2010; 42(1): 23–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeard DA, Mescam M: Mechanisms of pressure-diuresis and pressure-natriuresis in dahl salt-resistant and dahl salt-sensitive rats. BMC physiology. 2012; 12: 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeard DA, Neal ML, Tabesh-Saleki N, et al.: Multiscale modeling and data integration in the virtual physiological rat project. Ann Biomed Eng. 2012; 40(11): 2365–2378. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2028",
"date": "04 Nov 2013",
"name": "John Osborn",
"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 by Beard et al makes three major claims: 1) to provide a mathematical description for Guyton’s existing model; 2) to show how stimulation of the baroreflex arc can explain long-term changes in blood pressure via the renal function curve employed in Guyton’s model; and 3) to show how arterial stiffening can lead to hypertension without renal injury. I commend the authors for making a much needed mathematical contribution to the field of computational physiology, where one of the most widely recognized and used models of long-term control of arterial pressure has only very rudimentary mathematical description and analysis published. Moreover, the authors are bridging the gap between the renocentric view of hypertension etiology and the emerging experimental data that points to an independent long-term role of neural regulation. For these reasons the paper is a valuable addition to the existing body of knowledge.There are several issues however, that need to be addressed in a revised version of the paper. The overarching recommendation is to correct the paper’s focus and claims to make them more consistent with the model design, as well as to make the model’s description easier to understand. In particular, I believe the model should not be presented as a generic model with the “representation of the major physiological processes controlling arterial blood pressure” but instead be positioned as a conceptual framework which aims to illustrate how the baroreflex arc may be involved in long-term blood pressure control. Phenomenological mathematical representation, a small amount of detail, limited analysis, and quality of shown simulations are appropriate for a conceptual model but are insufficient for a model from which one could be extracting conclusions on the particular physiological mechanisms to be implicated in hypertension development.Suggested revisions:There are several versions of Guyton’s model ranging from very simplistic to very complex 1. Some models are extremely detailed but have a closed architecture (e.g. hummod.org). The model created by the authors is not sophisticated enough to correspond to most of these versions. Please state clearly which version of Guyton’s model is being considered here. In my opinion, the model proposed here seems of approximately the same level of complexity to the one presented by Guyton and Coleman 'Long-term regulation of the circulation: interrelationships with body fluid volumes' in the text Physical bases of circulatory transport: Regulation and exchange. (1967) 2Focus the goals according to what was accomplished in the paper:“capturing the relevant mechanisms in an identifiable mathematical model” is too over-reaching since the three key equations (18, 19, and 21) do not capture mechanisms but are of phenomenological nature.“probing hypotheses regarding the long-term control of arterial pressure” – the model is both too limited and phenomenological to allow for any testing of various hypotheses. It is, however, fit for providing a possible explanation for two hypotheses: roles of the baroreflex involvement and arterial stiffening. Otherwise, for testing, one would require a more mechanistic description, inclusion of other key organs and regulatory mechanisms, and a more rigorous mathematical analyses. The authors provided an explanation of how the baroreflex may be involved in long-term control of blood pressure. This is a novel contribution to the field and should be the focus of the discussion:Add the discussion and references of experimental knowledge of baroreflex stimulation, inhibition, and especially long-term resetting.Describe how Guyton’s models incorporate neural control and explain the above finding, especially the effects of chronic stimulation and how it is different from the proposed model (e.g. 3)Discuss existing knowledge or a possible physiological explanation for the finding that renin activity will be diminished with baroreflex stimulation.Discuss what other important physiological mechanisms are not reflected in the model and how they may play a role. In particular, explain why macula densa sodium delivery is thought not to be important enough to include in the model, despite the fact that it is known to be one of the key regulators of renin activity.Do not confuse the model’s predictions with its assumptions when discussing simulation results. The model assumes and is explicitly designed to tie the baroreflex, renin, and SNA with long-term pressure control. An explicitly designed assumption is not the same as prediction. Thus, statements like Conclusion 1 in “Summary and Conclusions” are inappropriate. The fact that the model’s assumptions allow for physiologically plausible simulations merely supports the notion that baroreflex may be important in the way the authors have described it.\n\nImprove the clarity of the mathematical description: Provide motivation for the chosen elements of the model and outline the model in its entirety first. For example, a flowchart or a diagram of the model may help readers to grasp the overall idea.Add at least a basic analysis of parameter sensitivity, with clear description in the Methods section.In Table 1 please provide physiologically-based description of parameters rather than referring to the equations where they are used (e.g. Emax is the maximal left-ventricular elastance, rather than “Varying elastance heart model, Equation (10)”). Otherwise, the definitions are circular: parameters are used in the equations without an explanation of their meaning in either the text or the table.",
"responses": [
{
"c_id": "632",
"date": "26 Nov 2013",
"name": ". .",
"role": "Author Response",
"response": "We are grateful to the reviewers for paying such serious attention to our paper and offering many constructive criticisms. While we have revised the paper to address the concerns and clarify the presentation, we disagree with the general interpretation of our model as “a conceptual framework which aims to illustrate how the baroreflex arc may be involved in long-term blood pressure control.” As stated in the summary, the model “was developed and identified to serve as a representation of the major physiological processes controlling arterial blood pressure.” For example, there is no equation or parameter value artificially injected into the model to require pressure-diuresis to not be able to return the pressure to a baseline value during baroreflex stimulation. In fact, we could parameterize the model so that the chronic pressure-diuresis phenomenon acts as an “infinite-gain” control system, always returning pressure to its set point. But in doing so, the model would not be able to match the data used to identify it, or to predict the data used to validate it. Regarding the level of detail captured by the model, the reviewers are correct that at some level, all components of the model are phenomenological in nature. (This is true of all mathematical models.) Yet regarding the question around the level of detail and complexity of the model, we stand by the assertion that this model represents the most detailed model of long-term control of arterial pressure that is identified in the literature. (Here we are invoking the word “identified” to indicate that the model equations and associated parameter values are reported along with comparisons of model simulations of data used to identify the parameters.) In fact, one of the unstated goals of this paper is to inject some clarity and rigor into this field with precise and concrete language. Therefore we do not know how to respond to the statement that our model “is not sophisticated enough to correspond to most of” the versions of the Guyton model because it is not clear, precisely, what is meant by “sophisticated” or “correspond”. In discussing the Guyton models, we clearly state that: “The 1972 realization of the model invokes approximately 160 variables, and several hundred adjustable parameters. More recent versions of the model involve many thousands of variables and tens of thousands of parameters.” Although it is true that our model has less variables and parameters than any version of the Guyton model that we are aware of, is the sophistication of a model measured purely by the number of variables or adjustable parameters? Since no version of the Guyton model captures pulsatile mechanics, no version of the model can capture the baroreceptor resetting phenomena illustrated in Figures 2 and 3 of our paper. Nor does the HumMod model capture the system response to baroreflex stimulation illustrated in Figure 9. Therefore one could argue that our 16-variable model is more sophisticated than the 5,000-variable Guyton model. Regarding the specific suggestions: “capturing the relevant mechanisms in an identifiable mathematical model” is too over-reaching since the three key equations (18, 19, and 21) do not capture mechanisms but are of phenomenological nature. More specifically, we stated that one of the “goal” of the study is “capturing the relevant mechanisms in an identifiable mathematical model.” We believe we have done that. However, in the revised paper we have changed the goal to “representing the relevant mechanisms in an identifiable mathematical model.” “probing hypotheses regarding the long-term control of arterial pressure” – the model is both too limited and phenomenological to allow for any testing of various hypotheses. Again, we counter that we have met the stated goal. We are not claiming to have proved any hypothesis. Yet we have introduced some important disproofs, as outlined in some of our major conclusions. Specifically, it is stated in the literature as ‘scientific facts’ that; the renal function curve represents the dominant long-term controller of blood pressure and, that since the baroreflex “resets” it cannot play a role in chronic changes in mean pressure. Our model analysis has cast significant doubt upon the validity of both of these hypotheses. Admittedly, these findings represent somewhat of a double negative i.e., a model capturing baroreflex resetting is used to demonstrate how a baroreflex that resets could play a role in long-term changes in pressure. Therefore, we have shown that the fact that the baroflex resets, does not mean that the baroreflex cannot play a role in long-term pressure control. Therefore we are confident that it is appropriate to state that we have constructed a model with the goal of “probing hypotheses regarding the long-term control of arterial pressure.” Add the discussion and references of experimental knowledge of baroreflex stimulation, inhibition, and especially long-term resetting. Data in Figures 2 and 3 represent direct experimental interrogation of baroreflex stimulation, inhibition, and long-term resetting. The resetting phenomenon is also indirectly probed in Figure 6 where, over the time course of the experiment, the baroreflex response to the acute pressure increase gradually diminishes. The phenomenon and experimental observations are discussed in association with our simulations of these experiments. These are also discussed in the section on arterial stiffening and the etiology of hypertension. Finally, we have added a couple of sentences (end of Results Section 5) comparing our model predictions with the long-term observations of Thrasher on baroreceptor unloading. Describe how Guyton’s models incorporate neural control and explain the above finding, especially the effects of chronic stimulation and how it is different from the proposed model... A discussion on this point has been added to the section “Results 4”. Discuss existing knowledge or a possible physiological explanation for the finding that renin activity will be diminished with baroreflex stimulation. It is not our finding that renin activity will diminish with baroreflex stimulation. That is the prior experimental observation. The mechanism in the model is through the observed dependence of renin release on sympathetic tone. At a deeper level, the mechanism may simply be explained as follows: decreased sympathetic tone leads to decreased renal arterial tone, leading to increased glomerular filtration and/or medullary blood flow, leading to increased flux of sodium to the macula densa, leading to decreased renin production. While we would rather leave such speculation out of the manuscript, it is published here as part of the open review process. Discuss what other important physiological mechanisms are not reflected in the model and how they may play a role... explain why macula densa sodium delivery is thought not to be important enough to include in the model, despite the fact that it is known to be one of the key regulators of renin activity. It is not that we believe that “macula densa sodium delivery is…not...important enough to include in the model.” Rather the phenomenon is captured in a simplified way. To clarify this point, we have added the following text to the section on “Neurohumoral control of pressure-diuresis/natriuresis”: “These expressions simplify the known (and unknown) mechanisms governing renin production into a simple phenomenological relationship between pressure, sympathetic tone, and the kinetics of the renin-angiotensin system.” A more biophysically detailed model would be required to represent the direct link between sodium transport and renin production. At the bare minimum, such a model would represent the biophysical processes linking pressure and solute transport in the kidney, including effects of sympathetic innervation on arterial smooth muscle stress development, mechanics of blood and filtrate flow, solute advection, diffusion, permeation, exchange, and pumping. Do not confuse the model’s predictions with its assumptions when discussing simulation results... Conclusion 1 in “Summary and Conclusions” is inappropriate. The fact that the model’s assumptions allow for physiologically plausible simulations merely supports the notion that baroreflex may be important in the way the authors have described it. We agree that this first stated conclusion does not represent a conclusion and what we are trying to say is now better captured in the revised text for Conclusion 1. This same sentiment applies to the other conclusions, which are theoretical. For example, we conclude that we have demonstrated that “that hypertension does not necessarily indicate any degree of renal dysfunction.” It may seem like an equivocation to insert the word “necessarily”. However, the literature is full of claims that due to the phenomenon of pressure-diuresis, hypertension necessarily does indicate some degree of renal dysfunction. Our theoretical analysis however would disprove that widespread claim. Provide motivation for the chosen elements of the model and outline the model in its entirety first. For example, a flowchart or a diagram of the model may help readers to grasp the overall idea. We have added a diagram illustrating the control processes captured by the model. (Figure 4B.) Add at least a basic analysis of parameter sensitivity, with clear description in the Methods section. This is an excellent idea. A detailed sensitivity analysis is not feasible because the model is expensive to simulate (particularly for the long time frames of Figure 9, for example), the number of parameters is large and would require calculation of 46x46 Fischer Information matrix (or cross-correlation matrix), and we do not have standard error estimates for all of the data used to identify the model. However, as suggested we have done a basic analysis, and reported a sensitivity index in Table 1. Results are discussed in a new section on parameter sensitivity. (Also, the sensitivity analysis facilitated as a slightly better parameterization of the model. The results have change imperceptibly.) In Table 1 please provide physiologically-based description of parameters rather than referring to the equations where they are used... Otherwise, the definitions are circular: parameters are used in the equations without an explanation of their meaning in either the text or the table. We believe that it useful to the reader to indicate in the table where the parameter is used in the model. Therefore, the indication of which equations given parameters appear in has been retained in the table. In addition, descriptions of the meaning of all parameters have been added wherever they were missing in the text."
}
]
},
{
"id": "2446",
"date": "14 Nov 2013",
"name": "Johnny Ottesen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComplex physiologically systems are often subdivided into parts and studied separately by experimental methods. But in experimental physiology, subsystems are not easily put back together and conclusions made concerning subsystems have limited validity for the original undivided system as their function and dynamics may have changed when isolated from one another. Parts that cannot be isolated experimentally may be studied (separately) using modeling. Mathematical modeling is able to unfold the influence that each of the processes has on the overall dynamic and thus modeling is highly adept at separating systems into components simple enough for their structures and functions to be studied in isolation. Mathematical modeling is the only controlled way to put the pieces back together by using equations that represent the system's components and processes, as well as its structures and interactions. In “A computational analysis of the long-term regulation of arterial pressure” the authors follow this strategy nicely in a successful attempt to investigate what they denote the ‘renocentric’ theory by Guyton. Here the authors combine cardiovascular and baroreflex models with models of the renin-angiotensin system whereby some important conclusions are reached. Among these conclusions the authors emphasize that the baroreflex may play a key role in the long-term regulation of the arterial pressure and especially in hypertension in contrast to the Guytonian view. The authors follow the physiologically structure well in their models as they carefully validate the different sub-models/elements by comparing them with existing various animal experiments. These sub-models are then integrated into a well-behaved and solid overall model. However, there are some methodic drawbacks I would like to emphasize: Most model equations are in some sense ad-hoc choices (or phenomenological, as the authors mention at the very end). These equations are not based on first principles and hence they do not represent the underlying mechanisms. Instead the approach is to assume a first order transient toward some static sigmoidal relationship. In addition, the authors make a lot of self-references, which shadows the fact that many of the used sub-models are adjustment taken from elsewhere and ideas from other sources. Strengthening the paper with such information would definitely support the approach taken. The paper is very well structured and written but a few misprints have sneaked in: Formula (3): Should the fraction be / CAo instead of CAo/?Formula (4): An index o is missing on VsAOn page 4, coulomb 2, line 2: should beOn page 4, coulomb 2, line 5: ‘… the population transition from an active state at a rate …’ Please state reference (or state that this is a hypothetical assumption)!",
"responses": [
{
"c_id": "631",
"date": "26 Nov 2013",
"name": ". .",
"role": "Author Response",
"response": "Thank you for the attention to our paper and the constructive criticisms. Most model equations are in some sense ad-hoc choices (or phenomenological, as the authors mention at the very end). These equations are not based on first principles and hence they do not represent the underlying mechanisms... In addition, the authors make a lot of self-references... We agree with the reviewer’s assessment. To reiterate a statement from the paper summary, the model represents a balance complexity/physiological fidelity and identifiability. The sensitivity analysis added to the revised paper indirectly addresses the issue of the phenomenological nature of model components. Finally, we have revised the text to clearly state the nature of the model earlier than the last paragraph of the paper. See third paragraph on page two, section on “Model Components 5”, and first paragraph of the summary. Also, we point out that while we have cited some previous modelling works, the majority of components used here have been constructed de novo, with the overall goal in mind. The varying elastance heart model is adapted from the cited reference. And of course there is nothing new about lumped-parameter models of the circulation. But most of the other (phenomenological) formulae are constructed to match the observed dynamics of responses of these systems to the experiments used to identify the model.Formula (3): Should the fraction be / CAo instead of CAo/?No, it is correct in the paper. One way to see this is to think about the limit where CAo goes to infinity. In this case the vessel becomes infinitely compliant if we wait long enough and thus does not support any finite pressure.Formula (4): An index o is missing on VsA Fixed.On page 4, column 2, line 2: should be d0 FixedOn page 4, column 2, line 5: ‘… the population transition from an active state at a rate …’ Please state reference (or state that this is a hypothetical assumption)! The assumption of Equation (7) is stated: “It is assumed that the baroreceptors within the population transition from an active to inactive state at a rate proportional to the firing rate”. We do not know what factors are responsible for the slow “resetting” of the baroreflex response. But, it is compelling that this simple equation is able to capture the nonlinear phenomena of Figures 2 and 3."
}
]
},
{
"id": "2540",
"date": "26 Nov 2013",
"name": "Patrick Hannaert",
"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\nSUMMARYThis work by Beard et al., is an interesting, consequential and mind-tickling work, definitely worth considering and being published, provided that several issues are addressed.Strong points:Sound “horizontal” (multi-organ) physiological modelling & simulation (following a systems/engineering analysis and conception).Modular, integrative, multi-dynamic approach.Targeted, specific hypothesis - proposes one possible/sufficient etiogenic mechanism in the context of age–related hypertension.Weak points:Phenomenological equations.Some conclusions drawn are too assertive (especially with respect to such a complex, multi-factorial, and evolving, pathology: hypertension); the likeliness of the conclusion appears strongly dependent on the limited physiology (and pathology) that is implemented.1. GENERALIn bio-medical research, it has become understood that mathematical modelling and simulation (MS) can provide a complementary approach to experiments and are a necessary response to extreme biological complexity. Indeed, biochemical and genetic circuits modelling are becoming mature and productive. On the other hand, there remains a very serious “modelling gap” to be filled between the (“low-level”) biological/physiological processes and (“higher-level”) pathophysiology.Simply put, the “CV-circulatory-pressive” construct by Guyton and colleagues (started in the late 60’s, with 1972 and 1992 milestones; Guyton et al., 1972; see Montani & Van Vliet, 2009, Moss et al., 2012) remains a landmark, to the point that most, if not all modelling endeavours refer to it in one way or another. This is because it was an exhaustive attempt to describe the CV circulatory system in its physiological entirety, including high-level interactions such as an integrated CV and renal system.Many versions, declinations (and improvements) do exist, including the late HumMod (Hester et al, 2011 should perhaps be cited, instead of Abran et al., 2007, since it is the current evolution of QCP/QHP). However, as mentioned by the Authors, for various reasons it is difficult or impossible to rationally understand and exploit those models (e.g. “too large” in the case of HumMod, or “not identified” as in the case of Guyton’s models of 1972 or 1992).Beard et al. propose here a “top-down”, modular, “engineering” high-quality MS work, as a rationally exploitable (numerically identifiable) alternative to Guyton’s model, in order to explore hypertension etiogenic hypothesis related to arterial stiffness, baroreflex and the renin-angiotensin system (RAS). The main incentive relates to the controversed Guytonian view that long-term regulation of arterial pressure is driven by the kidney. In that context the Authors try to demonstrate that arterial stiffening (as in ageing) and/or the central nervous system (here simplified as the “baroreflex”) could provide a sufficient explanation(s) of hypertension, through interactions with the kidney pressure-natriuresis function and the RAS.The significance and relevance of this work for CV regulations and hypertension is obvious, although(i) it is limited by the model itself (i.e. effectively implemented features, simplifications, phenomenological approach for equations, etc.) and(ii) some of the conclusions/assertions should be tempered (among other reasons: given the multifactorial/multi-dynamic nature of HT, that it has been resistant to investigation for more than a century now, and given the simplicity of the proposed construct). 2. MAJOR COMMENTSIn the targeted domain of the pathophysiology of blood pressure regulation and primary hypertension, this work proposes a rigorously constructed and parameterized mathematical model of the “long-term control of arterial pressure” (but see below). The authors should emphasize however that the model numerical instantiation centers on dog (or rabbit). Also of important note (this should be addressed in the revised version), no mention is made of the parameterization procedure and tools or sensitivity analysis.Moreover, the parameterization is done under the (quite frequent) implicit hypothesis that arterial pressure regulation and other CV-related processes only differ quantitatively (e.g. in parameter values) and are otherwise qualitatively similar e.g. in dog, rabbit and man. This very point is rarely, if ever, addressed. We do feel that in the present turmoil of “physiological” models targeting *human* pathology, this should be, at minimum, mentioned and preferably justified. Given that the Authors target human hypertension, and especially given the phenomenological approach chosen, why did they not directly parameterize their model as a “human” CVR model?As a matter of fact, the anatomy and mechanical/physiological/biochemical properties and functions of the human CV system, arteries, heart, kidney and RAS are, to the best of our knowledge, now understood and numerically characterized enough to allow for such a human quantitative approach to be adopted (especially with this articles chosen phenomenological “input-output” approach). This would have given some more resonance and relevance to the work. In particular, even if all CV-pressive, renal and hormonal processes proved to be the same between all these species, the possibility would remain that their relative contributions (and dynamics) to arterial pressure (dys-) regulations are different.As their objectives, the Authors aimed to:develop a \"computer model capturing chronic adaptation of acute renal function in blood pressure control\" as a more rigorous alternative to Guyton’s circulatory model (Guyton et al., 1972) and to Guyton’s-derived approaches, particularly SAPHIR (Thomas et al., 2008, - Hernandez et al., 2011, could be cited too here, as well as Moss et al., 2012)thus \"capturing the relevant mechanisms\".These are very strong and ambitious objectives, and possibly ab initio flawed by the phenomenological (but otherwise legitimate) approach chosen.Further, that this is “a model capturing chronic adaptation of acute renal function in blood pressure” cannot be claimed with such a simple kidney function, for example: the pressure-natriuresis ignoring actions of the ANS (autonomic nervous system) upon renal hemodynamics and tubular function and the angiotensin action on glomerular and tubular function (for instance Glomerular Tubulo Balance and Na/water reabsorption). Kidney nerve afferents are also ignored.What is really addressed in this work are the interactions (possibly etiogenic) between the physiological (input/output) modules that are implemented.Finally, after more than a century of physiological and clinical research, the etiology of primary hypertension remains unresolved; although it is established that (numerous), genetic, environmental, age-related and adaptive factors do intervene. Moreover, when in the realm of age-related hypertension the Authors do select putative etiogenic processes (arterial stiffening, reduced BR receptors sensitivity) and explore potential interactions with RAS and kidney function (phenomenological pressure-natriuresis) in a simplified CV construct. Driven by the objective (age related HT/arterial stiffening), the proposed model features six central physiological functionalities, as modules (model components) implementing:MC1. “Aorta-large artery mechanics”MC2. “Kinetics of baroreflex afferent firing”MC3. “Mechanics of the heart and circulation”MC4. “Autonomic system”MC5. “Neuro humoral control of pressure diuresis/natriuresis”MC6. “Renin-angiotensin system” For each one of these model components (physiological organ/process), the Authors should give, where possible, at least one published “first-principle” (physiologically-based) model as a reference, e.g. amongst others (i) Moss and Thomas, 2013 for the kidney/pressure-natriuresis under hormonal influence, (ii) Guillaud and Hannaert, 2010 and/or Claassen et al., 2013 for the RAS. The model is first validated (short-term) vs haemorrhage and then volume expansion. Of note, no real validation criterion is given (except for visual adequation through the figures).Secondly, the model is validated (long-term = days to weeks) using published steady-state pressure-natriuresis kidney function data. Although the Authors target long-term processes, 2 weeks (maximal simulated time here) is far too insufficient to refer to the CV, renal and central physiological mechanisms truly involved (months-to-years in dogs, years and decades in man). OTHER COMMENTSIs one of the input arrows missing in the model figure (fig 4)?A model diagram (rather than the electrical analog presented), as well as for the main hypothesis (vs alternative(s)) would be welcome.To what extent does this work differ from the previous work by the same group (cited, see Pettersen et al., 2013)?The Matlab(C) model code and scripts proposed (http://zenodo.org/record/7126#.UpOLNOJ21pg) have been tested and verified by Dr F. Guillaud (ModTeam/Inserm U1082). The scripts could be run, the model could be simulated and results that were visually identical to those in the paper were obtained.",
"responses": [
{
"c_id": "630",
"date": "26 Nov 2013",
"name": ". .",
"role": "Author Response",
"response": "The reviewers have provided an extensive and insightful commentary on our paper. We agree with most of the points raised. We have extracted specific criticisms and responded to them below. Weak points: Phenomenological equations; This point is addressed in some detail in the responses to the first two reviewers, as well in response to specific points below. Many versions, declinations (and improvements) do exist, including the late HumMod (Hester et al, 2011 should perhaps be cited, instead of Abran et al., 2007, since it is the current evolution of QCP/QHP). The more recent citation has been inserted into the revised text. ...some of the conclusions/assertions should be tempered... The validity of our conclusions is addressed extensively in our response to the first set of reviewers. In brief, we agree that some statements needed to be clarified and rephrased. See responses above for details. The authors should emphasize however that the model numerical instantiation centers on dog (or rabbit); the parameterization is done under the (quite frequent) implicit hypothesis that arterial pressure regulation and other CV-related processes only differ quantitatively (e.g. in parameter values) and are otherwise qualitatively similar e.g. in dog, rabbit and man. This is an important point that cuts across all areas of computational physiology. Large-scale models are built based on data from multiple sources, multiple species, different conditions (in vitro, in vivo, etc.) In the end, all of our models are chimeras. We have taken the liberty of adapting the reviewers’ language at the end of the discussion section of the revised paper. (We do point out that our implicit assumption is the central assumption of all biomedical research using animal models. It might not be a perfect assumption, but it does give us a starting point.) Also of important note... no mention is made of the parameterization procedure and tools or sensitivity analysis. Please see our detailed response to this point in the response to the first review. For each one of these model components (physiological organ/process), the Authors should give, where possible, at least one published “first-principle” (physiologically-based) model as a reference This is an excellent idea in principle. However, it is not clear if it is possible. For example, we are unclear as to how the Moss and Thomas paper could be characterized as representing the hormonal influence on pressure-natriuresis from first principles. Specifically, the Moss and Thomas paper represents the action of angiotensin-II “via the parameter FrANG, which adjusts the response of the pressure-natriuresis mechanism.” The parameter FrANG appears in the model equations as a factor multiplying a phenomenological expression for the fractional reabsorption, and the effect of the hormone is simulated by varying FrANG between “a maximal value of 1 (minimal angiotensin II) and a minimal value of 1/6 (maximal angiotensin II)”. Thus, the model is every bit as “phenomenological” as the one presented here. (This point is not to be taken as a criticism of the excellent study of Moss and Thomas.) The papers by Guillaud and Hannaert, and Claassen et al. do account for the RAS system with more physiological fidelity/detail than our model. We do disagree with the assertion that any of the models discussed here are built from “first” principles. All of these papers invoke the same sort of phenomenological expressions for certain components. Although the Authors target long-term processes, 2 weeks (maximal simulated time here) is far too insufficient to refer to the CV, renal and central physiological mechanisms truly involved... The reviewers may be correct that our model is far too insufficient to provide insight into a pathophysiological mechanism that emerges over decades. Yet, we refer the reviewers (and the readers) to the stated conclusions in the paper. Is one of the input arrows missing in the model figure (fig 4)? The net volume output rate is Q_urine – Q_in. The figure has been revised to clarify this. A model diagram (rather than the electrical analog presented), as well as for the main hypothesis (vs alternative(s)) would be welcome. This has been added. (See Figure 4B.) The main hypothesis here is that pressure-diuresis acts as a physiological input-out relationship, and that renal function is controlled through the RAS and the autonomic system. Thus, fundamentally, the model is constructed to capture the Guyton concept of the long-term control of arterial pressure. (Therefore, it is not clear what alternatives we might indicate in a model diagram.) To what extent does this work differ from the previous work by the same group..?Please see the Section “Results 5: Exploration of the etiology of primary hypertension” in the revised text."
}
]
}
] | 1
|
https://f1000research.com/articles/2-208
|
https://f1000research.com/articles/2-268/v1
|
06 Dec 13
|
{
"type": "Opinion Article",
"title": "Cancer quasispecies and stem-like adaptive aneuploidy",
"authors": [
"Domenico Napoletani",
"Michele Signore",
"Daniele C Struppa",
"Michele Signore",
"Daniele C Struppa"
],
"abstract": "In this paper we develop a theoretical frame to understand self-regulation of aneuploidy rate in cancer and stem cells. This is accomplished building upon quasispecies theory, by leaving its formal mathematical structure intact, but by drastically changing the meaning of its objects. In particular, we propose a novel definition of chromosomal master sequence, as a sequence of physically distinct whole or fragmented chromosomes, whose length is taken to be the sum of the copy numbers of each whole or fragmented chromosome. This fundamental change in the functional objects of quasispecies theory allows us to show that previously measured aneuploidy rates in cancer populations are already close to a formally derived aneuploid error threshold, and that any value of aneuploidy rate larger than the aneuploid error threshold would lead to a loss of fitness of a tumor population. Finally, we make a phenomenological analysis of existing experimental evidence to argue that single clone cancer cells, derived from an aneuploid cancer subpopulation, are capable of self-regulating their aneuploidy rate and of adapting it to distinct environments, namely primary and metastatic microenvironments. We also discuss the potential origin of this self-regulatory ability in the wider context of developmental and comparative biology and we hypothesize the existence of a diversification factor, i.e. a cellular mechanism that regulates adaptation of aneuploidy rates, active in all embryo, adult and cancer stem cells.",
"keywords": [
"Chromosomal instability",
"Aneuploidy",
"Cancer",
"Stem Cells",
"Quasispecies",
"Error Threshold"
],
"content": "Introduction\n\nIn normal cells the number of chromosomes and the total DNA content depends on the phase of the cell cycle1. Non-diploid chromosome content, also known as aneuploidy, is instead the most common feature of human tumor cells2–4. This feature of tumor cells is commonly associated with acquired resistance to various kinds of treatments such as radio- or chemotherapy2–5. Nonetheless, it is not completely clear whether aneuploidy per se contributes to and drives tumor development, or if instead it is deleterious. In fact, individuals carrying an extra copy of chromosome 21 have a 50% lower probability of developing solid tumors than do individuals with the correct chromosome number6,7 and although aneuploidy is compatible with organism and cell viability, the presence of additional copies of chromosomes decreases the overall cellular fitness8. What is clear is that chromosomal instability, i.e. the tendency to gain or lose parts of the genome during cell replication, seems to give an advantage to tumor cells9, and clonal heterogeneity within tumors is one of the main causes of tumor dormancy and resistance to anti-cancer therapies5,10. Indeed, it has been demonstrated that even a small population of cells derived from a single cancer cell clone (diversified population from a single cell), could be responsible for drug resistance and, upon removal of the drug, the population spontaneously reverted to a sensitive state11,12.\n\nThe development of single-cell sequencing techniques13 has recently allowed a wide range of studies analyzing chromosomal variability in primary and metastatic tumor cells, as well as healthy tissues14,15. In particular in Navin et al.16 it is shown that metastatic tumors are likely to be the product of single clones proliferating from the primary tumor by observing the microvariation of the integer copy number of consensus sequences in individual tumor cells through single-cell sequencing.\n\nIn this paper we are concerned with measurable changes in the aneuploid heterogeneity of populations, due to different levels of chromosomal instability, and to emphasize even more the close link between aneuploidy and chromosomal instability. We will refer to the latter as aneuploidy rate, formally defining aneuploidy rate as the average probability that there is at least one new aneuploid modification per chromosome during cell replication. We will demonstrate in the Discussion that simple and verifiable statistical consequences of the findings of Navin et al.16 are logically bound to imply the following proposition.\n\nAdaptive Aneuploidy in Cancer Cells: Single clone cancer cells derived from an aneuploid cancer subpopulation are capable of adapting their aneuploidy rate and display distinct aneuploid rates in distinct environments, namely primary and metastatic microenvironments.\n\nThe full impact of this proposition hinges on the potential ability to harness adaptive aneuploidy and potentially alter the aneuploidy rate of cancer cells. The theoretical frame for such a program can be built on quasispecies theory, a general evolutionary model for error-prone self replicative systems17, first introduced by Eigen18. The potential relevance of quasispecies theory to cancer biology has already been suggested19,20 but unlike previous attempts20–26, we do not recommend in this paper alternative, structurally diffnt quasispecies models of cancer cell subpopulations dynamics. Instead, after a quick review of the relevant elements of standard quasispecies theory, we note the limits of this theory for the study of eucaryotic cells, and show that only by defining a novel notion of chromosomal master sequence it is possible to radically shift the whole standard quasispecies theory to a new categorical context (i.e. the scenario of cell populations with variable aneuploidy rates) and properly justify a valid aneuploid quasispecies theory. This new context allows us to predict (similarly to what the standard quasispecies theory does for single base error rates) a maximal aneuploidy rate, an error threshold, after which each cancer subpopulation loses its identity, and therefore its ability to pass on its selectively advantageous genetic traits to future cell generations, which is referred to as error catastrophe27.\n\nFinally, in the Discussion we perform a phenomenological analysis of some especially illuminating single cell analysis experiments, from Navin et al.16, that seem to support our proposition on the existence of adaptive aneuploidy in cancer cells. We then speculate on the possible biological basis of self-regulation of aneuploidy rates in cancer cells. Such refined ability is unlikely to be the product of a single, specific tumor evolutionary history, rather, we will argue that embryo and adult stem cells already display very finely regulated rates of aneuploidy, suggesting that a common cellular mechanism of adaptation of aneuploidy is at play, a diversification factor, which is possibly reactivated in cancer stem cells.\n\n\nAneuploid quasispecies\n\nAs noted in the Introduction, we need to redefine the main quantities at play in quasispecies theory to make it an appropriate ground for the biology of aneuploidy, and we begin by briefly sketching the argument that leads to the error threshold inequality.\n\nThe concept of quasispecies was first introduced by Eigen18,28, and it is a powerful way to relate the structure of population dynamics to the error rate of single base replication in viruses or unicellular organisms27,29. The most important consequence of the theory is that it is possible to determine theoretically a threshold on the error rate such that, if the error rate of replication of genomic sequences is pushed above the threshold, the subpopulation will not be able to retain its identity, for a wide range of models for fitness distribution in the population (cf. Schuster30, page 81).\n\nAssuming there are N subpopulation types within a population, we start by writing down the differential equation that describes the rate of change dxm of type m in terms of the instantaneous size xk(t), k = 1, ..., N of all types:\n\n\n\nWmm is the rate of effective excess production of sub-population type m, and if we consider the genetic sequence associated to type m, we can write Wmm = QmmAm − Dm, with Qmm the probability of precise reproduction of sequence m, Am the growth rate of type m, and Dm its mortality rate. Ē(t) = Σk Ek xk is the average, over all types, of the excess reproduction rate, with Ek = Ak − Dk and, finally, Wmk is the rate of production of type m by erroneous reproduction of type k.\n\nAssuming a steady state in which dxm/dt = 0 and neglecting in first approximation the contributions Σk≠m Wmk xk(t), it is possible to derive a condition that constrains the probability of precise reproduction of sequence m:\n\n\n\nwhere σ¯m = Am/(Dm + Ēk≠m), with Ēk≠m = Σk≠m Ek xk, is the average superiority of a master sequence associated to a dominant subpopulation versus competitor sequences, essentially, σ¯m is an index of relative fitness27. If a master sequence has length vm, and we denote by q¯ the average fidelity of single nucleotide reproduction, then Qmm = q¯vm and the error threshold can be written as\n\n\n\nRemarkably, Equation 3 establishes a phase transition on the information content; if the error rate of single nucleotide reproduction goes above lnσ¯mvm the information contained in the master sequence will disintegrate, in the sense that the loss of information in the sequence due to reproduction errors will not be compensated by a sufficiently high fitness relative to other subpopulations and the subpopulation associated to the master sequence will implode27,30.\n\nIn complex organisms, the quasispecies model is potentially applicable only in specific scenarios, such as competition among embryo stem cells during development, adult stem cells and progenitor cells proliferation, and, crucially, cancer cells, where subpopulations compete with each other under limited resources and changing environment. However, the applicability of the basic quasispecies model, originally devised in the setting of virus RNA replication, has been put into question as appropriate for eucaryotes and specifically for cancer cells. Eucaryotic cells reproduce semi-conservatively meaning that the parental double strand degenerates in the process of generating two daughter double strands, which led to Brumer et al.21 to raise the possibility that, for high enough replication error rates, the master sequence, seen here as the double strand of DNA, would eventually disappear, and they suggested more refined quasispecies models that take into consideration this phenomenon.\n\nEven more seriously, the applicability of quasispecies theory to human cells is put into question by the exceedingly high size of the human genome as compared to RNA viruses. In fact, in order for the quasispecies not to undergo genetic drift, the neutral space around a fitness peak should be sufficiently small to be completely explored by the population. The complexity and the inherent mutational and phenotypical robustness of the human genome amplifies its neutral space, preventing quasispecies evolution even at higher than normal mutation rates, as it is the case in cancer cells31. This fact, together with very low single nucleotide errors for humans, implies that the fitness of mutants of an hypothetical master sequence does not change significantly, and the fitness distribution of mutants around the master sequence is likely to decay linearly, or sub-linearly, a scenario under which no error threshold is possible30.\n\nIndeed, the existence of Lynch syndrome or hereditary non-polyposis colorectal carcinomas (HNPCC), which are characterized by a higher risk of colon cancer, show the inability of the basic quasispecies theory to predict the maximum single nucleotide error that is viable for a tumor. HNPCC tumors, as well as all microsatellite instable (MSI) colon cancers, arise because of a break down of the mismatch repair mechanism32,33. Therefore, MSI cancer cells display increased error rates of single nucleotide replication by 1 to 3 orders of magnitude, with respect to the baseline single nucleotide error rate 1 − q¯ in healthy cells, estimated to range between 10−9 and 10−10 for the human genome (see Alberts et al.34, page 271, and Gundry & Vijg35, Lange et al.36 and Jiricny37).\n\nNow recall that the human genome has roughly 3.2 × 109 nucleotides (see Alberts et al.34, page 206), and note that for organisms with very large genomes, the relative superiority σ¯m of a master sequence associated to a given subpopulation cannot be very large (vis a vis other subpopulations), as any given mutation will only affect its fitness marginally27, and therefore σ¯ m ≈ 1. Given these numerical estimates, according to the error threshold inequality in Equation 3, MSI tumors would fail to satisfy the error threshold inequality to such an extent that they should not even exist. This is true even if we restrict our attention, in defining the master sequence, to conserved DNA, i.e. the 5% of the human genome that is known to be coding and essential to cell function (see again Alberts et al.34, page 206).\n\nWe believe that the inconsistences of the basic quasispecies model, when applied to human cells, completely disappear if we replace single nucleotide errors with aneuploidy errors. Notably, in all scenarios where quasispecies theory could potentially apply, i.e. stem and progenitor cells proliferation and cancer cells, aneuploidy rates far exceed single nucleotide error rates in frequency and impact on the cell. This means the leading cause in the evolution of a population will be the aneuploidy error, rather than the single nucleotide error, which can be neglected, especially when the mismatch repair genes are intact as happens in the overwhelming majority of cancers and all healthy stem cells.\n\nWe need now to reinterpret the notion of reproduction fidelity of a sequence adequately to properly define error thresholds in the presence of aneuploidy. Since we can neglect nucleotide errors, we assume a faithful reproduction of the genetic material when the copy number of each chromosome in a sequence (two, for example, in a diploid cell) is kept constant during replication, both numerically (number of physically distinct whole chromosomes or fragments) and structurally (translocations, deletions and amplifications of DNA). Although complex aneuploidy landscapes may arise, characterized by concurrent numerical and structural chromosomal changes, most of the somatic copy-number alterations (SCNAs) frequently found in tumor cells involve whole chromosomes or whole-arms (25% of the genome), with only 10% of the cancer cell genome being affected by focal SCNAs9,38,39.\n\nTherefore, we now make a series of definitions consistent with these arguments, whose objective is to change the domain of applicability of quasispecies theory, without altering its formal mathematical structure:\n\nChromosomal Master Sequence. A chromosomal master sequence is the collection of physically distinct whole and fragmented chromosomes in the cell.\n\nChromosomal Master Sequence Length. The chromosomal master sequence length cm of a cell is the sum of the copy numbers of each whole or fragmented chromosome in its nucleus.\n\nAneuploid Fidelity. The aneuploid fidelity Ām is the average probability that each whole or fragmented chromosome is reproduced exactly once in cell division, with no gain or loss of sub-chromosomal regions.\n\nIn this aneuploid scenario, the chromosomal master sequence length cm can fluctuate depending on the number of aneuploid copies of whole chromosomes or fragments, and the underlying nucleotide sequence will clearly differ according to which chromosomes or individual genes are affected by copy-number alterations in each cell. Although tumors vary widely in the number and type of copy number changes, most of these comprise low-level alterations and only a few genes reach more than 20 copy numbers, mainly due to their oncogenic or drugresistance functions16,40–42.\n\nAneuploid events can cause large phenotypical variations43–46, even a single error leading to chromosomal loss or addition can have large effects, therefore the fitness distribution around a master sequence is expected to display a sharp decay from the master sequence peak, in line with the types of fitness distribution known to express the error threshold30. At least for cancer cells, sub-populations are sharply defined in terms of their aneuploid profile, as evinced from single cell analysis works16 commented in the Discussion. This is further evidence of the strong concentration of fitness distributions around a few chromosomal sequence types.\n\nMutants of the master sequence, generated by even a single aneuploid error, and individual cells belonging to other sub-populations, are exceedingly unlikely to be able to mutate into cells expressing the master sequence, since any additional (erroneous) chromosome copy is subject to a wide variety of further partial deletions/additions, and only very few of them would correspond to a return to the master sequence configuration. Essentially, we can assume that the contribution of cells belonging to other subpopulation types to the dynamical evolution of the master sequence subpopulation is very small. This is exactly the condition that led to the error threshold in the first place, since Equation 3 is derived as a limiting stationary behavior of an interacting family of subpopulations described by Equation 1, where the rate of growth of each of them is weakly affected by the cross-mutations derived from the other subpopulations27,30.\n\nGiven these caveats regarding distribution of fitness for chromosomal master sequences in the presence of aneuploidy and regarding sub-population interactions, we reach the conclusion that quasispecies theory is indeed applicable to cancer and stem cells, but only in the context of aneuploid chromosomal master sequences, neglecting the underlying nucleotide errors.\n\nWe can now replace variables in the error threshold inequality in Equation 3 to take into account not only the varivariable length cm of the chromosomal master sequence associated to all whole and fragmented chromosomes, but also the correction to the probability Qmm of precise reproduction of a sequence that aneuploidy entails. The probability of precise reproduction of a specific sequence of chromosomes m can be expressed as Qmm = Ācmm, and the aneuploid error threshold inequality can be written as σ¯mĀcmm > 1, which eventually gives us a standard form (formally identical to Equation 3) for the aneuploid error threshold inequality:\n\n\n\nHowever each term in this equation has drastically different orders of magnitudes than the threshold inequality for DNA or RNA master sequences. To start with, as already stressed above, the relative superiority σ¯m will have considerable fluctuations, since the chromosomal master sequence is much shorter than a nucleotide sequence, and even small variations in copy numbers can effect large phenotypical variations.\n\nAt the same time, the diversity of subpopulations in primary tumors16,47–51 implies that, in a fully developed tumor, different subpopulations do not have extremely different relative superiority σ¯m, a scenario that would lead to a single, highly dominant subpopulation. It is therefore reasonable to assume at the very most σ¯m ∈ [102, 103], for the subpopulations of highest relative superiority. We know moreover that highly aneuploid tumors have higher fitness52, so larger values of σ¯m are likely to be associated with large values of cm, up to the order of 10213,39,53,54.\n\nLet Em = 1 – Ām, with Em denoting the aneuploidy error rate, i.e. the average probability that there is at least one new aneuploid defect for each chromosome or fragment of chromosome during cell replication. If we call T(Em) the threshold aneuploidy error rate above which a chromosomal master sequence is not viable, and if we take σ¯m ∈ [102, 103], cm ≈ 102, then Equation 4 gives T(Em) ≈ 10–2, which is consistent with the estimates of Em for cancer cells, in the range [10–3, 10–1]3,55–58. Our argument implies that the more a cell is aneuploid, the tighter the error threshold bound is, and that highly aneuploid cancer cells, known to be most adaptable5,52,59,60, are already working with aneuploid error rates close to the limit of a viable quasispecies.\n\nThere is some evidence that indeed aneuploidy rates in the tumor can affect the prognosis of cancer patients9,61. It is suggested that a moderate tumor aneuploidy rate worsens the prognosis, while a very high aneuploidy rate is associated with improved patient outcomes9, consistent with the quasispecies and error threshold catastrophe approach.\n\n\nDiscussion\n\nWe would like to revisit and comment upon some specific experimental evidence for our proposal of a self-regulated aneuploidy rate in cancer cells, focusing on several measures of aneuploidy rate, and showing how the variability observed in tumor subpopulations subject to distinct micro-environments can be given a far-reaching interpretation. Our analysis is phenomenological, meaning that we explain and reinterpret existing experimental work, showing how the inner logic of our argument can severely constraint the causes and interpretation of the data we review. We chose to first perform an in-depth analysis of a single recent study16, so that the flow of our discourse is unified and made coherent by constant reference to the same context. At the same time, we support our arguments with related experimental works, when appropriate.\n\nThe in-depth analysis and commentary of the literature that we perform here is meant to show that there is rigorous, logically compelling, and experimentally testable biological evidence for the usefulness of an aneuploid quasispecies theory. Indeed, we argue that it is possible to envision a precise, and functionally in-built mechanism of self-regulation for aneuploidy rates in normal and cancer stem cells. The evolutionary role and the structure of this self-regulation mechanism could be studied and conceptualized within the framework we developed for aneuploid quasispecies. At the same time, any experimental validation of aneuploid quasispecies predictions on threshold aneuploidy error rates, would be essentially related to the presence of this self-regulatory mechanism.\n\nThe main focus and objective of Navin et al.16 was to show that metastatic tumors are likely to be the product of single clones proliferating from the primary tumor by observing the microvariation of the integer copy number of consensus sequences in individual tumor cells through single-cell sequencing. A coarse ploidy distribution of a large number of cells from a breast tumor and one of its metastasis was plotted in Navin et al.16 (Figure 3a-b in that paper) as an histogram with respect to the total DNA content. These ploidy distributions showed, for both primary and metastatic tumors, two peaks, one around twice and another at four times the total amount of DNA. This double peaked distribution is accounted by the presence of roughly 50% of normal diploid cells in each tumor tissue sample. Importantly, whereas in the primary tumor a significant fraction of the gated normal cells was pseudodiploid, in the paired metastasis the normal population was likely to derive from the stromal content of the tissue (see ref.16, Figure 4 in their paper).\n\nNavin et al.16 performed very refined measurements of copy number profiles, across all chromosomes, from a small subset of cells (hundreds) in sections of primary and metastatic tumors and generated a neighbor-joining tree of these profiles. This analysis showed that metastatic and primary aneuploid cells were closely related in the neighbor-joining tree derived from the clustering, and yet they produced clearly distinct subclusters. The conclusion of this single cell study was that the metastasis proliferated from a single cell derived from the aneuploid subpopulation of the primary tumor, since no pseudodiploid cancer cells were observed in the metastatic tumor.\n\nOther recent studies on myeloproliferative disorders62 and melanoma54, kidney47,48 and pancreas49 tumors or, again, in breast cancer50, arrived at similar conclusions, pointing to a late, metastasis-specific diversification of primary tumor-derived cells (see Wu et al.63, Figure S14, and Clifford64). The presence of aneuploid cells in most primary tumors examined in Gerlinger et al.49, is well documented by ploidy analysis (see supplementary Figure 10 in their paper) and metastases show a marked increase in allelic imbalance as compared to primary tumor regions. The authors conclude that tumor heterogeneity is probably driven by aneuploidy and that chromosomal aberrations contribute substantially to genetic intratumor heterogeneity. Notably, even in an evolutionary context where the primary tumor and the metastasis share most of the sequenced regions, there is a striking variation in copy number specifically in the metastatic counterpart (see ref.54, Figures S5 to S11 in Lengauer et al.54). This concept is best exemplified in ref.16, but there, both the coarse ploidy distribution analysis and the refined, single cell copy number count for primary and metastatic tumors, drive us to an additional conclusion: the genetic variability of the aneuploid clone in the metastasis is greater than its corresponding variability in the primary tumor from which it came. Even if we took into account a parallel progression model as opposed to a punctuated or linear evolution model51,65, the final result would not change. The metastatic population described in Navin et al.16 has diversified more than its parental population in the primary tumor, regardless of whether the metastasis developed in a later, much shorter time than the primary tumor, or whether its origins date back to the first stages of primary tumor dissemination. This higher diversification of the metastatic population holds even though the aneuploid cells’ compartment in the primary tumor does not represent a minority of the population, and has expanded considerably at some point during the tumor evolution history.\n\nTo justify our claim of adaptive aneuploidy, we note that in Figure 4 of Navin et al.16 the Euclidean distances in the neighbor-joining tree for the aneuploid cells from the metastatic tumor showed much greater variability than the Euclidean distances for the corresponding aneuploid cells in the primary tumor. We note that these distances were calculated with respect to a common root profile, and that mutual distances among individual profiles are likely to be ever greater. Granted that this study dealt with very small sample populations, a closer inspection and analysis of the tightness of the variance of Euclidean distances in the primary tumor subpopulation, as opposed to the variance of Euclidean distances of the metastatic tumor subpopulation, would almost certainly reveal a statistically significant discrimination of the two. Indeed, distances inferred from their Figure 4 of ref.16 using a Levene test for equality of variance66 suggest a low probability that the underlying distributions of Euclidean distances for metastatic aneuploid cells and for primary aneuploid cells have the same variance (p-value ≈ 0.01).\n\nNote, crucially, that even if we assume high experimental noise in the data, such noise would affect equally both Euclidean distributions and the identification of distinct variances for distances of metastatic aneuploidy cells and primary aneuploidy cells would be even more unlikely to be observed by chance.\n\nThe hypothesis of a larger variability of the copy number profiles of the metastatic subpopulation is also supported by a closer inspection of the tails of the ploidy distributions of primary and metastatic tumor populations analyzed in Navin et al.16. The right-hand sides of the tetraploid peaks for the metastatic tumor have distinctly thicker and longer tails than the corresponding tetraploid peaks in the primary tumor, suggesting greater variability of aneuploidy in the former. Other works point to a similar conclusion. For example, it has been shown recently that, although sharing most of the examined somatic single-nucleotide variants, in vitro cultured low passage melanoma cells have higher copy number variation when compared to the parental tumor67.\n\nPloidy distributions, in their simplicity, offer even more scope for interpretation and testing of the hypothesis that cancer cells have the ability to self-regulate their aneuploidy rate. Indeed, a single cell clone could be capable of generating a diverse metastasis either because of inherent chromosomal instability, or because its rate of aneuploidy is somehow increased under the stress conditions of a new tissue embedding.\n\nLet’s assume first that the single clone from the primary tumor has chromosomal instability. The dispersion of metastatic cells should not be in any way preferential to such cells (even if their successful embedding in a tissue may be), so there will be a, possibly small, subpopulation of cells in the primary tumor with similar or higher aneuploidy rate than the cell generating the metastasis. This subpopulation of the primary tumor, by its greater aneuploidy rate, will be more adaptable and likely self-sustaining, and it should be observable as a long tail in the ploidy distribution of the primary tumor population. The tail will be much thinner for the primary tumor than the metastatic tumor, since high aneuploidy rate cells are only a sub-population of the primary tumor. However, no such long and thin tail is observed experimentally for the primary tumor in Navin et al.16. It is still possible, if highly unlikely, that the metastatic cell is an extreme outlier, with no comparable cells left in the primary tumor, but then we would see a much more pronounced evolutionary difference between primary and metastatic aneuploid populations than what is observed. This logically implies that the single metastatic cell clone was not essentially different from its primary population before starting to proliferate in the new environment.\n\nThis argument leaves only one other option: some cancer cells are capable of altering and self-regulating their aneuploidy rate under stress, or under changes in the environment. In conclusion, both ploidy distribution analysis and single cell analysis of Navin et al.16 give strong evidence for the proposition on adaptive aneuploidy in cancer cells presented in the Introduction, i.e. that single clone cancer cells derived from an aneuploid cancer subpopulation, adapt their aneuploid rates in distinct environments, namely primary and metastatic microenvironments.\n\nThe Discussion, up to now, centered on the inference, from some experimental results of ref.16 and other published supportive information48,54,63, that metastatic cancer cells have higher aneuploidy rates than the corresponding original subpopulation of the primary tumor. We concluded that this differential could only be explained by assuming an adaptive, self-regulatory cellular response sensitive to changes in the environment. As much as tumor populations undergo extensive evolution during their development, it seems highly unlikely that such a refined property could arise by chance only in the specific population studied in Navin et al.16, without a preexisting dormant ability to self-regulation.\n\nIndeed, high levels of aneuploidy are associated with increased adaptability in plants and yeast68,69, and a certain rate of aneuploidy, leading to precise percentages of mosaic aneuploidy, is common in several mammals’ embryos (see van Soom & Boerjan70 chapter 10), including humans71,72. Similarly, it is speculated that the significant mosaic aneuploidy in adult human organs such as the liver and brain is instrumental to an increased plasticity and adaptability of such organs73,74. Lang et al.75 raise the possibility that the extensive aneuploidy in the embryo may transfer into similarly widespread copy number variations in all human tissues.\n\nObservable levels of aneuploidy have been found in adult cells76, and while this widespread aneuploidy could already originate during embryo development75, adult, non-transformed stem cells continue to have distinct levels of aneuploidy rates according to their type. For example, mesenchymal stem cells are likely to have very low aneuploidy rates77, while hepatocytes together with small intestine and pancreas cells display within-tissue extensive copy number variation (CNV)78,79.\n\nThese strikingly different aneuploidy rates among embryo stem cells and adult stem cells, raise the possibility that the finely tuned, and distinct, high aneuploidy rates observed in embryos and adult tissues are regulated by some mechanism specific to stem cells, rather than being a simple byproduct of aberrant or sustained cell division. This is a simple, fundamental observation, and yet one that is rarely emphasized in the literature. We formalize its essence in a proposition:\n\nDifferentially Expressed Aneuploidy in Stem Cells. Embryo and adult stem cells display finely tuned, and distinct, aneuploidy rates, unrelated to the replication rates of the stem cells themselves.\n\nAnd we refer to Section 2 of our preprint ‘Stem-like Adaptive Aneuploidy and Cancer Quasispecies’ (available at arxiv.org/pdf/1303.6374.pdf) for a much more thorough review of literature supporting the idea of a differentially expressed aneuploidy in stem cells.\n\nSince many types of cancers partially inherit the hierarchical structure of the tissues they have derived from and are assumed to be propagated by stemlike tumor cells80, it is possible that increased aneuploidy rates are used actively, to the population advantage, to increase the adaptability of stem or fast-dividing progenitor cells. It is worth noting that, to date and to our knowledge, only two studies assessed the relationship between aneuploidy and cancer stem cells (CSCs)81,82. In the first paper, Kusumbe and Bapat evaluated the expression of stem-cell markers and the DNA content distribution of fluorescently labeled ovarian cancer cells after subcutaneous injection into immunodeficient mice. The authors found that, unlike label-free tumor cells, the label-retaining (quiescent) cells displayed stem-cell markers and were embedded with a small fraction of aneuploid cells. Treatment with chemotherapy increased the percentage of quiescent cells in the overall population and selectively stimulated the proliferation of the aneuploid fraction, which retained stem-like properties upon removal of the drug81. A second study by Fujimori et al., reveals again that stressful conditions favor the emergence of CSC-like clones from differentiating embryo stem cells in in vitro culture82.\n\nAnd this brings us to the proposition we stated in the introduction on adaptive aneuploidy in cancer cells: in light of the refined use of aneuploidy in stem cells, self-regulation of aneuploidy as argued in this Discussion could be a reactivation, in cancer stem cells, of a preexisting cellular mechanism common to embryo and adult stem cells - a diversification factor. Note that the proposition on differentially expressed levels of aneuploidy in normal stem cells is not compatible with a random, self-catalytic increment of aneuploidy first suggested in Rasnick & Duesby45, since otherwise we would not be able to observe consistently similar levels of aneuploidy within each stem cell type, and among different individuals. Moreover, we would see a correlation between fast replicating stem cells and levels of aneuploidy in the corresponding tissue. This is not necessarily the case. For example the percentage of cells undergoing DNA replication in solid tumors, which are mostly aneuploid, varies between 2% to 8%, whereas a normal renewing epithelium such as the intestine exhibits a DNA replication index of approximately 16%83.\n\nA random progression of aneuploidy would display much more pronounced variability within each stem cell class, while the spread of aneuploidy would be similar in all stem cell classes, and not differentially expressed in each of them. Note that the theory of a self-catalytic origin of aneuploidy in principle could be made consistent with the proposition on adaptive aneuploidy in cancer. It is only by taking together the proposition on adaptive aneuploidy in cancer (stem) cells, and the proposition on differentially expressed aneuploidy in (all) stem cells that we argue the following hypothesis is strongly implied:\n\nA Diversification Factor: There exists a cellular mechanism that regulates adaptation of aneuploidy rates, active, to different degrees and in different modalities, in all embryo, adult and cancer stem cells.\n\n\nConclusions\n\nWhile it was not the main objective of this theoretical paper to explore the experimental consequences of our hypothesis on adaptive aneuploidy and aneuploid quasispecies, we note that all our statements are open to, indeed they invite, simple forms of validation, either through single cell analysis or ploidy distribution analysis. Elucidating the mechanisms underlying self-regulation of aneuploidy rates in stem cells under specific microenvironmental stresses, would provide crucial insight into the developmental and evolutionary processes of complex organisms.\n\nFinally, we emphasize that validation of the hypothesis that stem cells can adapt their aneuploidy rate through a diversification factor would have significant therapeutical implications, when specialized to cancer stem cells. Indeed, it would provide a biological way, mostly inactive or less sensitive in healthy adult cells, to induce an aneuploid quasispecies error catastrophe to weaken cancer populations, a long held hope that may yet prove itself true.",
"appendix": "Author contributions\n\n\n\nDN, MS and DCS conceived the study and carried out the research. DN and MS prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThis paper is dedicated to Rosanna Gonzales. She never ceased to hope.\n\n\nReferences\n\nCooper GM: The cell: a molecular approach. ASM Press, Washington, D.C., 1997; 673. Reference Source\n\nTorres EM, Williams BR, Amon A: Aneuploidy: cells losing their balance. Genetics. 2008; 179(2): 737–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeaver BA, Silk AD, Montagna C, et al.: Aneuploidy acts both oncogenically and as a tumor suppressor. Cancer Cell. 2007; 11(1): 25–36. PubMed Abstract | Publisher Full Text\n\nPavelka N, Rancati G, Li R: Dr jekyll and Mr Hyde: role of aneuploidy in cellular adaptation and cancer. Curr Opin Cell Biol. 2010; 22(6): 809–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKreso A, O’Brien CA, van Galen P, et al.: Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer. Science. 2013; 339(6119): 543–8. PubMed Abstract | Publisher Full Text\n\nHasle H, Clemmensen IH, Mikkelsen M: Risks of leukaemia and solid tumours in individuals with Down’s syndrome. Lancet. 2000; 355(9199): 165–9. PubMed Abstract | Publisher Full Text\n\nSatgé D, Sasco AJ, Lacour B: Are solid tumours different in children with Down’s syndrome? Int J Cancer. 2003; 106(2): 297–8. PubMed Abstract | Publisher Full Text\n\nWilliams BR, Prabhu VR, Hunter KE, et al.: Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science. 2008; 322(5902): 703–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcGranahan N, Burrell RA, Endesfelder D, et al.: Cancer chromosomal instability: therapeutic and diagnostic challenges. EMBO Rep. 2012; 13(6): 528–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandau DA, Carter SL, Stojanov P, et al.: Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell. 2013; 152(4): 714–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharma SV, Lee DY, Li B, et al.: A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell. 2010; 141(1): 69–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorkman P, Travers J: Cancer: drug-tolerant insurgents. Nature. 2010; 464(7290): 844–5. PubMed Abstract | Publisher Full Text\n\nZong C, Lu S, Chapman AR, et al.: Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. 2012; 338(6114): 1622–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOwens B: Genomics: The single life. Nature. 2012; 491(7422): 27–9. PubMed Abstract | Publisher Full Text\n\nWang J, Fan HC, Behr B, et al.: Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell. 2012; 150(2): 402–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNavin N, Kendall J, Troge J, et al.: Tumour evolution inferred by single-cell sequencing. Nature. 2011; 472(7341): 90–4. PubMed Abstract | Publisher Full Text\n\nOjosnegros S, Perales C, Mas A, et al.: Quasispecies as a matter of fact: viruses and beyond. Virus Res. 2011; 162(1–2): 203–15. PubMed Abstract | Publisher Full Text\n\nEigen M: Selforganization of matter and the evolution of biological macromolecules. Naturwissenschaften. 1971; 58(10): 465–523. PubMed Abstract | Publisher Full Text\n\nCahill DP, Kinzler KW, Vogelstein B, et al.: Genetic instability and darwinian selection in tumours. Trends Cell Biol. 1999; 9(12): M57–60. PubMed Abstract | Publisher Full Text\n\nSolé RV, Deisboeck TS: An error catastrophe in cancer? J Theor Biol. 2004; 228(1): 47–54. PubMed Abstract | Publisher Full Text\n\nBrumer Y, Michor F, Shakhnovich EI: Genetic instability and the quasispecies model. J Theor Biol. 2006; 241(2): 216–22. PubMed Abstract | Publisher Full Text\n\nDiego D, Calvo GF, Pérez-García VM: Modeling the connection between primary and metastatic tumors. J Math Biol. 2013; 67(3): 657–92. PubMed Abstract | Publisher Full Text\n\nItan E, Tannenbaum E: Effect of chromosomal instability on the mutation-selection balance in unicellular populations. PLoS One. 2012; 7(5): e26513. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIwami S, Haeno H, Michor F: A race between tumor immunoescape and genome maintenance selects for optimum levels of (epi)genetic instability. PLoS Comput Biol. 2012; 8(2): e1002370. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMichor F, Iwasa Y, Nowak MA: Dynamics of cancer progression. Nat Rev Cancer. 2004; 4(3): 197–205. PubMed Abstract | Publisher Full Text\n\nNowak MA, Michor F, Iwasa Y: Genetic instability and clonal expansion. J Theor Biol. 2006; 241(1): 26–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiebricher CK, Eigen M: The error threshold. Virus Res. 2005; 107(2): 117–27. PubMed Abstract | Publisher Full Text\n\nEigen M, Schuster P: The hypercycle, a principle of natural self-organization. Springer-Verlag, Berlin, 1979; 92. Reference Source\n\nBiebricher CK, Eigen M: What is a quasispecies? Curr Top Microbiol Immunol. 2006; 299: 1–31. PubMed Abstract\n\nSchuster P: Mathematical modeling of evolution. Solved and open problems. Theory Biosci. 2011; 130(1): 71–89. PubMed Abstract | Publisher Full Text\n\nJenkins GM, Worobey M, Woelk CH, et al.: Evidence for the non-quasispecies evolution of RNA viruses [corrected]. Mol Biol Evol. 2001; 18(6): 987–94. PubMed Abstract\n\nVilar E, Gruber SB: Microsatellite instability in colorectal cancer-the stable evidence. Nat Rev Clin Oncol. 2010; 7(3): 153–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde la Chapelle A, Hampel H: Clinical relevance of microsatellite instability in colorectal cancer. J Clin Oncol. 2010; 28(20): 3380–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlberts B, Wilson JH, Hunt T: Molecular biology of the cell. Garland Science, New York, 5th ed., reference ed edition, 2008; 1392. Reference Source\n\nGundry M, Vijg J: Direct mutation analysis by high-throughput sequencing: from germline to low-abundant, somatic variants. Mutat Res. 2012; 729(1–2): 1–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLange SS, Takata K, Wood RD: DNA polymerases and cancer. Nat Rev Cancer. 2011; 11(2): 96–110. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiricny J: The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol. 2006; 7(5): 335–46. PubMed Abstract | Publisher Full Text\n\nGordon DJ, Resio B, Pellman D: Causes and consequences of aneuploidy in cancer. Nat Rev Genet. 2012; 13(3): 189–203. PubMed Abstract | Publisher Full Text\n\nBeroukhim R, Mermel CH, Porter D, et al.: The landscape of somatic copy-number alteration across human cancers. Nature. 2010; 463(7283): 899–905. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlbertson DG: Gene amplification in cancer. Trends Genet. 2006; 22(8): 447–55. PubMed Abstract | Publisher Full Text\n\nGajduskova P, Snijders AM, Kwek S, et al.: Genome position and gene amplification. Genome Biol. 2007; 8(6): R120. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantarius T, Shipley J, Brewer D, et al.: A census of amplified and overexpressed human cancer genes. Nat Rev Cancer. 2010; 10(1): 59–64. PubMed Abstract | Publisher Full Text\n\nChen G, Rubinstein B, Li R: Whole chromosome aneuploidy: big mutations drive adaptation by phenotypic leap. Bioessays. 2012; 34(10): 893–900. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSheltzer JM, Amon A: The aneuploidy paradox: costs and benefits of an incorrect karyotype. Trends Genet. 2011; 27(11): 446–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRasnick D, Duesberg PH: How aneuploidy affects metabolic control and causes cancer. Biochem J. 1999; 340((Pt 3): 621–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPavelka N, Rancati G, Zhu J, et al.: Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast. Nature. 2010; 468(7321): 321–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu X, Hou Y, Yin X, et al.: Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor. Cell. 2012; 148(5): 886–95. PubMed Abstract | Publisher Full Text\n\nGerlinger M, Rowan AJ, Horswell S, et al.: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012; 366(10): 883–92. PubMed Abstract | Publisher Full Text\n\nCampbell PJ, Yachida S, Mudie LJ, et al.: The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature. 2010; 467(7319): 1109–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShah SP, Roth A, Goya R, et al.: The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature. 2012; 486(7403): 395–9. PubMed Abstract | Publisher Full Text\n\nCaldas C: Cancer sequencing unravels clonal evolution. Nat Biotechnol. 2012; 30(5): 408–10. PubMed Abstract | Publisher Full Text\n\nBakhoum SF, Compton DA: Chromosomal instability and cancer: a complex relationship with therapeutic potential. J Clin Invest. 2012; 122(4): 1138–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVogt N, Lefèvre SH, Apiou F, et al.: Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas. Proc Natl Acad Sci U S A. 2004; 101(31): 11368–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTurajlic S, Furney SJ, Lambros MB, et al.: Whole genome sequencing of matched primary and metastatic acral melanomas. Genome Res. 2012; 22(2): 196–207. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLengauer C, Kinzler KW, Vogelstein B: Genetic instability in colorectal cancers. Nature. 1997; 386(6625): 623–7. PubMed Abstract | Publisher Full Text\n\nRajagopalan H, Nowak MA, Vogelstein B, et al.: The significance of unstable chromosomes in colorectal cancer. Nat Rev Cancer. 2003; 3(9): 695–701. PubMed Abstract | Publisher Full Text\n\nBurns EM, Christopoulou L, Corish P, et al.: Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein. J Cell Sci. 1999; 112((Pt 16): 2705–14. PubMed Abstract\n\nCamps J, Ponsa I, Ribas M, et al.: Comprehensive measurement of chromosomal instability in cancer cells: combination of fluorescence in situ hybridization and cytokinesis-block micronucleus assay. FASEB J. 2005; 19(7): 828–30. PubMed Abstract | Publisher Full Text\n\nLee AJ, Endesfelder D, Rowan AJ, et al.: Chromosomal instability confers intrinsic multidrug resistance. Cancer Res. 2011; 71(5): 1858–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuesberg P, Stindl R, Hehlmann R: Origin of multidrug resistance in cells with and without multidrug resistance genes: chromosome reassortments catalyzed by aneuploidy. Proc Natl Acad Sci U S A. 2001; 98(20): 11283–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBakhoum SF, Danilova OV, Kaur P, et al.: Chromosomal instability substantiates poor prognosis in patients with diffuse large B-cell lymphoma. Clin Cancer Res. 2011; 17(24): 7704–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHou Y, Song L, Zhu P, et al.: Single-cell exome sequencing and monoclonal evolution of a JAK2–negative myeloproliferative neoplasm. Cell. 2012; 148(5): 873–85. PubMed Abstract | Publisher Full Text\n\nWu X, Northcott PA, Dubuc A, et al.: Clonal selection drives genetic divergence of metastatic medulloblastoma. Nature. 2012; 482(7386): 529–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClifford SC: Cancer genetics: Evolution after tumour spread. Nature. 2012; 482(7386): 481–2. PubMed Abstract | Publisher Full Text\n\nKlein CA: Parallel progression of primary tumours and metastases. Nat Rev Cancer. 2009; 9(4): 302–12. PubMed Abstract | Publisher Full Text\n\nSchultz BB: Levene’s test for relative variation. Syst Biol. 1985; 34(4): 449–456. Publisher Full Text\n\nParker SC, Gartner J, Cardenas-Navia I, et al.: Mutational signatures of de-differentiation in functional non-coding regions of melanoma genomes. PLoS Genet. 2012; 8(8): e1002871. PubMed Abstract | Publisher Full Text | Free Full Text\n\nte Beest M, Le Roux JJ, Richardson DM, et al.: The more the better? the role of polyploidy in facilitating plant invasions. Ann Bot. 2012; 109(1): 19–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen G, Bradford WD, Seidel CW, et al.: Hsp90 stress potentiates rapid cellular adaptation through induction of aneuploidy. Nature. 2012; 482(7384): 246–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Soom A, Boerjan M: Assessment of mammalian embryo quality: invasive and non-invasive techniques. Kluwer Academic Publishers, Dordrecht, 2002; 406. Reference Source\n\nLedbetter DH: Chaos in the embryo. Nat Med. 2009; 15(5): 490–1. PubMed Abstract | Publisher Full Text\n\nPeterson SE, Westra JW, Rehen SK, et al.: Normal human pluripotent stem cell lines exhibit pervasive mosaic aneuploidy. PLoS One. 2011; 6(8): e23018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuncan AW, Hanlon Newell AE, Bi W, et al.: Aneuploidy as a mechanism for stress-induced liver adaptation. J Clin Invest. 2012; 122(9): 3307–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRehen SK, Yung YC, McCreight MP, et al.: Constitutional aneuploidy in the normal human brain. J Neurosci. 2005; 25(9): 2176–80. PubMed Abstract | Publisher Full Text\n\nLiang Q, Conte N, Skarnes WC, et al.: Extensive genomic copy number variation in embryonic stem cells. Proc Natl Acad Sci U S A. 2008; 105(45): 17453–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbyzov A, Mariani J, Palejev D, et al.: Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature. 2012; 492(7429): 438–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrimes BR, Steiner CM, Merfeld-Clauss S, et al.: Interphase fish demonstrates that human adipose stromal cells maintain a high level of genomic stability in long-term culture. Stem Cells Dev. 2009; 18(5): 717–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuncan AW, Taylor MH, Hickey RD, et al.: The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature. 2010; 467(7316): 707–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Huallachain M, Karczewski KJ, Weissman SM, et al.: Extensive genetic variation in somatic human tissues. Proc Natl Acad Sci U S A. 2012; 109(44): 18018–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNguyen LV, Vanner R, Dirks P, et al.: Cancer stem cells: an evolving concept. Nat Rev Cancer. 2012; 12(2): 133–43. PubMed Abstract | Publisher Full Text\n\nKusumbe AP, Bapat SA: Cancer stem cells and aneuploid populations within developing tumors are the major determinants of tumor dormancy. Cancer Res. 2009; 69(24): 9245–53. PubMed Abstract | Publisher Full Text\n\nFujimori H, Shikanai M, Teraoka H: Induction of cancerous stem cells during embryonic stem cell differentiation. J Biol Chem. 2012; 287(44): 36777–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTannock I, Hill RP: The Basic science of oncology. Pergamon Press, New York, 1987; 398. Reference Source"
}
|
[
{
"id": "2916",
"date": "30 Dec 2013",
"name": "Jan Oxholm Gordeladze",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 emphasize in their introduction to the present paper that: “The theoretical frame to understand such self-regulation resides with quasispecies theory”, however, they introduce a change in its definition or the meaning of its objects. In particular, they propose a novel definition of the chromosomal master sequence. Finally, they discuss the potential origin of this self-regulatory ability in the wider context of developmental and comparative biology with emphasis on a putative “diversification factor”, defined as a cellular mechanism regulating the adaptation of aneuploidy rates, which is active in all embryonic and adult cells, as well as cancer stem cells. Furthermore, they refer to literature asserting that single clone cancer cells derived from an aneuploid cancer subpopulation, are capable of adapting their aneuploidy rate, and they display distinct aneuploid rates in distinct environments, namely primary and metastatic microenvironments. This literature survey indicates that embryonic and adult stem cells display finely tuned, and distinct, aneuploidy rates, which are unrelated to the replication rates of the stem cells in question. Since many types of cancer cells partially inherit the hierarchical structure of the tissues from which they emanate, and are hypothesized to develop from stem like tumor cells, it seems highly probable that increased aneuploidy rates are used as an advantage to a given cell population, e.g. to increase the adaptability of stem or fast-dividing progenitor cells. In this article the authors introduce the concept of a “diversification factor”, which is probably the key issue of the article. This factor may as well be extended to a cluster of “impacting signals”, which may fit the modern definition of epigenetics, encompassing signals defined as “epigenators”, “initiators” and “maintainers”, of which transcription factors (TFs), microRNAs and histone modifying enzymes (like histone deacetylases = HDACS) are known to play important parts, since they constitute a strongly interwoven network with feed-forward- and feed-back regulatory loops. This network is under the influence of environmental/positional “factors” (epigenators, e.g. surface molecules, mechanical stimulation, environmental chemicals and others) recognized by the “initiators/maintainers” (e.g. TFs and microRNAs), and most importantly, this regulatory web is common to embryonic cells, stem cells, cancer stem cells, and adult (i.e. differentiated) cells. It is therefore highly probable that the degree of aneuploidy of cancer cells is highly dependent on environmental phenomena, and not solely on intrinsic or “inherited” traits localized within the cancerous cells themselves.",
"responses": []
},
{
"id": "4034",
"date": "10 Mar 2014",
"name": "Samuel F Bakhoum",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this interesting opinion article, Napoletani et al. attempt to apply the quasispecies theory to the evolution of anueploidy in cancer. This article proposes some interesting points that attempt to address the origin of genetic heterogeneity in tumors. An important claim made therein is that aneuploidy is likely to be the major source of genetic heterogeneity in later stages of tumor development particularly when compared to the rate of individual DNA mutations. Further, the authors also posit that cancer cells must maintain a given rate of chromosome missegregation beyond which a meltdown occurs.I think this article is acceptable, but feel the authors should address the following points:Please change the \"aneuploidy rate\" into \"chromosome mis-segregation rate\" or something of that nature. Aneuploidy by definition is a state and not a rate. What the authors imply is \"the rate of change in aneuploid karyotypes which is synonymous to chromosome mis-segregation (which includes whole chromosome missegregation as well as partial i.e. translocation etc...) It is important throughout the article to make the distinction between tolerance for aneuploidy in normal vs. cancer tissues. While evidence suggests that aneuploidy may lead to cellular stress in normal tissues, it is unclear whether this is the case in cancer cells. Either cancer cells preferentially maintain aneuploidy at the expense of increased cellular stress and slower proliferation rate or they might instead have also evolved to overcome cellular stress in response to aneuploidy. Furthermore, constitutional aneuploidy (for instance in individuals with trisomy) may have gone through a stem-cell adaptation phase that is different when compared to cancer cells who acquire aneuploidy de novo. The concept of error catastrophe is very interesting and rather important in the field of aneuploidy and chromosome segregation. I would encourage the authors to further elaborate on the optimal T(Em) value being near 10-2. Can the authors use the quasispecies model to make predictions for the fitness of the tumor cell populations if the T(Em) values were much higher? This would be extremely valuable. In addition to chromosome segregation errors (which include whole and partial chromosome copy number changes) it would be useful to incorporate the newly uncovered massive rearrangements known as chromothripsis and chromoplexy. In some cases, these massive rearrangements can lead to double minute chromosomes carrying oncogenes that are present in hundreds of copies (a lot higher than the maximum rate of 20 copies for any given gene that the authors presume). The authors do not account for these possibilities in their model. A concept that the authors bring up is the presence of a feedback loop from the environment that can \"fine tune\" chromosome segregation. While there is no data to necessarily refute this assumption, this reviewer believes that it is not currently supported by experimental data. What is much more likely is that mis-segregation rates occur somewhat independently and aneuploid cells are under constant selection in vivo which may ultimately favor cells that have a given missegregation rate to become more viable under certain conditions.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-268
|
https://f1000research.com/articles/2-267/v1
|
05 Dec 13
|
{
"type": "Case Report",
"title": "A close call: does the location of incision at cesarean delivery matter in patients with vasa previa? A case report.",
"authors": [
"Werner M Neuhausser",
"Laxmi V Baxi",
"Werner M Neuhausser"
],
"abstract": "We present here a case of vasa previa in a multipara, diagnosed at the time of her late second trimester ultrasonogram. The patient subsequently underwent an elective cesarean section after 37 weeks gestation, giving birth to a healthy child with an uneventful post-partum, neonatal and infant course. At the time of cesarean section, the incision was gradually deepened in layers through the myometrium by utmost care allowing the amniotic sac to protrude through the uterine incision hereby avoiding laceration of the vasa previa and its branches. Fetal exsanguination and a need for blood transfusion as well as a possible adverse neonatal course were therefore avoided.",
"keywords": [
"The CARE checklist submitted with this case report is accessible here."
],
"content": "Case\n\nThe CARE checklist submitted with this case report is accessible here.\n\nA 27 year old, Caucasian gravida 4 para 3, was diagnosed with vasa previa at a 22 week anatomy scan. On ultrasound her right lateral placenta had a significant anterior component with a marginal cord insertion at the inferior margin of its anterior aspect. A vessel coursing over the internal os between the anterior placental cord insertion and a posterior succenturiate lobe was identified on ultrasound (type 2 vasa previa, see Figure 1). The patient’s antenatal course was otherwise uneventful. She was counseled for Cesarean delivery at 35–36 weeks to avoid the risk of inadvertent rupture of the vasa previa and fetal exsanguination1,2. However, she believed that considering given her past obstetrical history of post –term pregnancies and her closed long cervix, she is was less likely to go into preterm labor or sustain preterm premature rupture of membranes with a disastrous outcome secondary to vasa previa. She therefore declined hospitalization and requested delivery after 37 weeks gestation.\n\n\nTreatment\n\nPrimary low transverse Cesarean section was performed at 37 weeks and 1 day gestation as per the patient’s request. At the time of surgery, the uterine incision was gradually and carefully deepened to allow the membranes to remain intact and bulge out from the incision. This was to avoid making an incision into the amniotic sac prior to localization of the course of the vasa previa vessels in the exposed membranes underneath the uterine incision. Indeed, extensive vasa previa vessels were identified in the intact membranes directly underneath the uterine incision in the lower uterine segment (Figure 2). After identification of the vasa previa vasculature the amnion was incised about one cm away, remaining parallel to the vessel leaving these vessels intact. Additionally, hemostats were kept available to clamp the vessel on either side if possible extension of the incision into one of the vessels occurred; and followed by expeditious delivery of the baby. A healthy 2980 g male fetus was delivered with Apgar scores 9, 9 and umbilical cord artery (Ua) pH 7.3.\n\nThe site of amnion incision (black arrows) in the vicinity of but distinct from the vasa previa vessels (yellow arrows).\n\n\nDiscussion\n\nVasa previa is a rare (1:2500) but important and potentially fatal cause of bleeding in the second and third trimester as well as in labor. The condition carries a risk of fetal exsanguination and death when rupture of the membranes involves tearing of vasa previa vessels running within the membranes and carrying fetal blood. Fortunately, the condition can often be diagnosed prenatally by ultrasound examination. Type I vasa previa refers to velamentous insertion of the cord with resultant vasa previa and Type II indicates interconnecting vessels between two lobes of placenta in a bipartite placenta or connecting vessel with a succenturiate lobe of the placenta. Nomiyama et al. identified placental cord insertion site with great degree of certainty at 18–20 weeks gestation and Sepluveda confirmed that gray scale with color Doppler has significant and better accuracy in diagnosing potential abnormal cord insertion and exclude vasa previa than 3D3,4. Situations, where vasa previa should be specifically looked for, include a larger placental mass as it is seen in multiple gestations, particularly those with a high number of fetuses, where there is a greater likelihood of velamentous insertion of the cord, succenturiate lobe or bipartite placenta. Patients with a low lying placenta, particularly if the placental margin appears at the internal os, and pregnancies conceived following in-vitro-fertilization constitute ‘at risk’ groups as well. Favorable outcomes depend on prenatal diagnosis and Cesarean delivery before the rupture of membranes. However, transection of vasa previa vessels during Cesarean delivery itself may cause significant fetal blood loss or exsanguination given the small fetal blood volume. Although the chance of fetal exsanguination during Cesarean section is less common than during vaginal delivery this complication, requiring blood transfusion to the newborn, has been reported4. This can be avoided by careful selection of the site of amniotic incision prior to delivery of the fetus. Canterino et al. recommend use of 3D sonography with power Doppler imaging with surface rendering and 3D multiplannar reconstruction to confirm vasa previa and also to map the path of this fetal vessel to prevent laceration of the vessel at cesarean section and fetal exsanguination5. According to Oylese et al., although 2D and color Doppler sonography is mostly adequate, 3D “allowed precise depiction of complex spatial relationship and confirmed vasa previa” and has an important role in placental abnormalities with uncertain diagnosis6. Vaginal ultrasonography (USG) with color Doppler is a recommended way to identify or confirm vasa previa7. In a recent case report, the authors published fetoscopic laser coagulation of a type II vasa previa at 32 5/7 weeks gestation and thereby facilitated an uneventful vaginal delivery, though the authors rightly caution benefits vs. risk of fetoscopy8. Earlier, Quintero et al. and others have published early third trimester in-utero laser treatment of type II vasa previa, however these patients had preterm delivery and needed cesarean section9,10. Although pre-op mapping of vasa previa by USG is an excellent measure to identify the course of the vasa previa vasculature, considering the recommendations made in this case report could be of additional help in the management of these patients.\n\n\nPatient perspective\n\nI would like to thank Dr. Baxi for the most professional, delicate care she provided during my pre-natal care and through the high risk cesarean section.\n\n\nConsent\n\nThe patient has given written consent for publication of these findings and believes sharing of this information would benefit other patients.",
"appendix": "Author contributions\n\n\n\nBoth authors have equally participated in preparation of this manuscript. LVB took care of the patient, determined the importance of the topic and prepared the case for publication and modified, proof read the manuscript after preparation by Dr. Neuhausser. Dr. Neuhausser, reviewed the literature, prepared the manuscript, including pictures and in the final format and determined the title of the case report.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGagnon R, Morin L, Bly S, et al.: Diagnosic Imaging Committee; Maternal Fetal Medicine Committee Guidelines for the management of vasa previa. J Obstet Gynaecol Can. 2009; 31(8): 748–760. PubMed Abstract | Publisher Full Text\n\nRobinson BK, Grobman WA: Effectiveness of timing strategies for delivery of individuals with vasa previa. Obstet Gynecol. 2011; 117(3): 542–549. PubMed Abstract | Publisher Full Text\n\nNomiyama M, Toyota Y, Kawano H: Antenatal diagnosis of velamentous umbilical cord insertion and vasa previa with color Doppler imaging. Ultrasound Obstet Gynecol. 1998; 12(6): 426–9. PubMed Abstract | Publisher Full Text\n\nSepluveda W, Rojas I, Robert JA, et al.: Prenatal detection of velamentous insertion of the umbilical cord: a prospective color Doppler Ultrasound study. Ultrasound Obstet Gynecol. 2003; 21(6): 564–569. PubMed Abstract | Publisher Full Text\n\nCanterino JC, Mondestin-Sorrentino M, Muench MV, et al.: Vasa previa: prenatal diagnosis and evaluation with 3-dimensional sonography and power angiography. J Ultrasound Med. 2005; 24(5): 721–724. PubMed Abstract\n\nOyelese Y, Chavez MR, Yeo L, et al.: Three-dimensional sonographic diagnosis of vasa previa. Ultrasound Obstet Gynecol. 2004; 24(2): 211–5. PubMed Abstract | Publisher Full Text\n\nOyelese KO, Schwärzler P, Coates S, et al.: A strategy for reducing the mortality rate from vasa previa using transvaginal sonography with color Doppler. Ultrasound Obstet Gynecol. 1998; 12(6): 434–438. PubMed Abstract | Publisher Full Text\n\nJohnston R, Shrivastava VK, Chmait RH: Term vaginal delivery following fetoscopic laser photocoagulation of type II vasa previa. Fetal Diagn Ther. Online publication Nov 15, 2013. PubMed Abstract | Publisher Full Text\n\nQuintero RA, Kontopoulos EV, Bornick PW, et al.: In utero laser treatment of type II vasa previa. J Matern Fetal Neonatal Med. 2007; 20(12): 847–851. PubMed Abstract | Publisher Full Text\n\nChmait RH, Chavira E, Kontopoulos EV, et al.: Third trimester fetoscopic laser ablation of type II vasa previa. J Matern Fetal Neonatal Med. 2010; 23(5): 459–462. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2724",
"date": "30 Jan 2014",
"name": "Conrad Chao",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 with useful suggestions for clinical practice.",
"responses": []
},
{
"id": "5127",
"date": "16 Jun 2014",
"name": "Everett F. Magann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is well written, insightful, and addresses the problems linked with the cesarean delivery for a vasa previa. It describes a technique to ensure the integrity of the fetal vessels located within the membranes as a cesarean delivery is undertaken.",
"responses": []
},
{
"id": "4572",
"date": "10 Jul 2014",
"name": "Ryu Matsuoka",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the case report of vasa previa. It was well described in detail and very helpful to the physician.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-267
|
https://f1000research.com/articles/2-265/v1
|
05 Dec 13
|
{
"type": "Web Tool",
"title": "PanFunPro: PAN-genome analysis based on FUNctional PROfiles",
"authors": [
"Oksana Lukjancenko",
"Martin Christen Thomsen",
"Mette Voldby Larsen",
"David Wayne Ussery",
"Martin Christen Thomsen",
"Mette Voldby Larsen",
"David Wayne Ussery"
],
"abstract": "PanFunPro is a tool for pan-genome analysis that integrates functional domains from three Hidden Markov Models (HMM) collections, and uses this information to group homologous proteins into families based on functional domain content. We use PanFunPro to compare a set of Lactobacillus and Streptococcus genomes. The example demonstrates that this method can provide analysis of differences and similarities in protein content within user-defined sets of genomes. PanFunPro can find various applications in a comparative genomic study, starting with the basic comparison of newly sequenced isolates to already existing strains, and an estimation of shared and specific genomic content. Furthermore, it can potentially be used in the determination of target sequences for in silico bacterial identification, as well as for epidemiological studies.",
"keywords": [
"Whole genome sequencing continues to become faster and less expensive with time",
"currently there are more than 2000 complete microbial genomes that are publically accessible",
"and the number of sequences is still growing exponentially. The availability of numerous strains from the same species has led to the development of new analyses",
"such as the bacterial species pan-genome1. Pan-genomic studies aim to determine differences in protein content between organisms and characterize the complete genomic repertoire of certain taxonomic groups. Therefore",
"comparative genomics is the first fundamental step in pan-genome analysis."
],
"content": "Introduction\n\nWhole genome sequencing continues to become faster and less expensive with time; currently there are more than 2000 complete microbial genomes that are publically accessible, and the number of sequences is still growing exponentially. The availability of numerous strains from the same species has led to the development of new analyses, such as the bacterial species pan-genome1. Pan-genomic studies aim to determine differences in protein content between organisms and characterize the complete genomic repertoire of certain taxonomic groups. Therefore, comparative genomics is the first fundamental step in pan-genome analysis.\n\nProteins can be naturally classified into families of homologous sequences that derive from a common ancestor through a speciation event, or a duplication event2,3. As a result, comparative genomics usually starts with a sequence similarity search using standard approaches, such as a local alignment search (BLAST4, FASTA5); orthology detection and clustering (CD-HIT6, OrthoMCL7, Inparanoid8); or search tools based on Hidden Markov Models (HMM)9. The comparison of homologous sequences and analysis of their phylogenetic relationships has important implications in understanding evolutionary processes and provides very useful information regarding the structure and function of proteins10.\n\nHere we present a tool for pan-genome analysis. It is a stand-alone tool providing several functionalities such as homology detection and genome annotation by three HMM-collections, pan-/core genome calculation within a set of proteomes, pairwise pan-/core-genome analysis, specific genome estimation for different sets of genomes as well as pairwise analysis of specific proteomes, basic statistics for the output proteins from the pan-/core-/specific-genome calculation, and finally analysis of available Gene Ontology (GO) information for the output proteins from the pan-/core-/specific-genome calculation.\n\n\nDesign and implementation\n\nThere are four basic steps in the PanFunPro approach, as shown in Figure 1: (1) genome selection; (2) functional domain collection; (3) construction of functional profiles and and protein grouping; (4) and finally, analysis of the pan, core and accessory genomes.\n\nThe method includes four basic steps: (1) genome selection; (2) functional domain collection; (3) construction of functional profiles and and protein grouping; (4) and finally, analysis of the pan, core and accessory genomes. Boxes in blue explain the profile construction steps, while green boxes indicate the possible types of analysis.\n\nThe PanFunPro programme first imports a list of genomes, selected for analysis. Each genome is represented by a FASTA file of amino acid sequences for all the encoded proteins. In the case of DNA sequences with no annotated genes, prediction of open-reading frames (ORFs) from the DNA sequence of the genome is carried out using Prodigal software11.\n\nTo form a set of functional profiles for each genome, all proteins are scanned against three collections of HMMs: PfamA12, TIGRFAM13, and Superfamily14 using InterProScan software15.\n\nBriefly, the functional profile or architecture is a combination of non-overlapping functional domains (HMMs) found in a particular protein. Only HMM hits with an E-value below 0.001 are considered significant and are used to create functional architectures. Furthermore, domains of only one database at a time are considered, meaning that if the protein has any matches in the PfamA database, the hits in the TIGRFAM and Superfamily databases are not considered. However, if the scan against the PfamA database does not result in any hit, analogously TIGRFAM and Superfamily databases are checked. HMM collections are searched in the following order: PfamA, TIGRFAM, and then Superfamily.\n\nFor each protein the functional profile name is created based on alphabetically sorted non-repeating accession numbers of all non-overlapping domains found in the protein sequence. Multiple proteins can belong to a single protein family if they share the same functional architecture, resulting in a lower number of families per genome than the reported number of proteins. Sequences with no significant matches to any searched HMM-database are collected from each of analysed genomes and clustered using the CD-HIT tool6. Clustering is implemented with a five amino acid window search, allowing two proteins to be in the same protein family if similarity between sequences is at least 60%. Resulting clusters are considered to be protein families, where the profile name is prefixed with ‘CL‘ (stands for clustering) and followed by the cluster identification number. Later, HMM-based and clustering-based protein families for each genome are joined together to form a whole genome profile collection.\n\nThe analysis is divided into two parts, as shown in Figure 1, and described in the two sections below. First, the pan-genome is estimated, along with the core-genome. A pan-/core-genome plot is created, along with a pan-/core-genome matrix. From this GO terms are determined for the core- and pan-genomes. The accessory genome is then estimated, and specific or enriched genes are determined, and as before, GO terms are calculated.\n\nThe pan-genome is defined as the complete collection of all proteins found in a set of genomes1; in our case, this is represented by the collection of all unique functional profiles found in those genomes. Starting with the first genome, as more genomes are added, an accumulative pan-genome is constructed and the resulting pan-genome number increases with the addition of more genomes. Similarly, the core-genome is the collection of conserved proteins (functional profiles) that are conserved across the analysed genomes, and the size of the core-genome decreases as more genomes are added. Conservation data are stored as table and can be visualised in an accumulative pan-/core-genome plot. Additionally, lists of profiles, comprising the pan- and core-genomes, can be visualised as a table.\n\nPairwise comparison between genome is visualised as a triangle-shaped ‘matrix‘, showing the number of protein families that are shared between two proteomes, both as percentage and absolute number; as well as the total number of protein families found in both genomes. When a strain is compared to itself, the fraction of protein families with more than one member is provided. The blue colour gradient indicates homology between different genomes, and the red triangles at the bottom of the figure represent homology within a genome (e.g., duplicate proteins).\n\nDifferences between proteomes can be assessed by identification of accessory profiles. The accessory genome includes proteins that are present in several, but not all analysed genomes; or are specific to a particular genome or to a group of genomes. A protein is considered to be ‘specific‘ if the functional profile is present in the query set of genomes and is absent in subject set of organisms. Estimation of accessory or specific genomes requires two sets of organisms and can fit one of the following descriptions: (1) proteins present in the core-genome of the first set of genomes, and absent in the core-genome of the second set of genomes; (2) proteins present in the pan-genome of the first set of genomes, and absent in the core-genome of the second set of genomes; (3) proteins present in the core-genome of the first set of genomes, and absent in the pan-genome of the second set of genomes; (4) and proteins present in the pan-genome of the first set of genomes, and absent in the pan-genome of the second set of genomes. Descriptions (1) and (2) introduce the specific-core-genome, while descriptions (3) and (4) describe the specific-pan-genome. Given that the first and the second sets of genomes are the same, application of options (3) and (4) will yield as accessory genome of input set of genomes.\n\nPairwise analysis of specific content can be visualised as a square-shaped matrix, where each row represents the specific genome of one organism compared to another, while the diagonal shows the comparison to the same genome. In the matrix cells, the amount of non-shared sequences is provided as a ratio of specific genome to a total number of proteins in the query strain. When compared to the same genome result is 0. The colour intensity indicates the level of similarity, where darker green shows more specific gene families, lighter green indicates less specific gene families, and white colour shows no specific gene families.\n\nFor a given collection of genomes, the set of core, pan, and accessory proteins is calculated, and the share of PfamA-, TIGRFAM-, Superfamily-, and CD-HIT-based profiles, as well as protein length distribution are visualised using the R ggplot2 package and can be visualised as a table.\n\nIn addition, available GO16 information can be extracted. The Interproscan tool provides possible GO identification numbers (GO ID) for each domain in the profile. Consequent GO IDs for each of the profiles are searched for GO term description and grouped by more common functional category using the map2slim tool, part of the GO::Parser module. Results are visualised using the R package ggplot2.\n\n\nResults\n\nThe PanFunPro approach was tested on genomes of members of the genera Lactobacillus and Streptococcus, previously used in comparative genomics study by Lukjancenko et al.17, further mentioned as a BLAST-based study. All of the Lactobacillus genomes used were from probiotic strains, whereas the genomes of the Streptococcus strains contained both pathogenic and probiotic species.\n\nHere, we focus on the types of results PanFunPro (further mentioned as PanFunPro-based analysis) can generate: a pan-/core-genome plot; a pairwise pan-/core-genome matrix; a pairwise specific-genome matrix; distribution of database source by which protein was annotated; and finally, the distribution of predicted GO terms among profiles.\n\nAccumulative pan- and core-genomes were calculated for both example genera and are shown in Figure 2. Analysis of the strains of the Lactobacillus genus resulted in a total of 467 core and 7009 pan gene families (Figure 2A). Most of the shared architectures consisted of PfamA domains and GO terms were available for 73% of them (Figure S1.A), whereas only 37% of the pan-genome gene families were HMM-based profiles and barely half of them had GO information available (Figure S1.B). Analysis of GO IDs distribution among the 3 general functional groups: biological process, molecular function, and cellular component, resulted in 239, 176 and 26 GOs, respectively, in the core-genome; and 470, 418 and 60 GOs, respectively, in the pan-genome.\n\nA. Analysis performed on Lactobacillus genomes. B. Analysis performed on Streptococcus genomes.\n\nA similar analysis, performed on the genomes of the strains from the genus Streptococcus, yielded 576 shared functional profiles and a total amount of 6263 architectures found within the genus (Figure 2B). Similarly to the Lactobacillus results, core-genome profiles consisted of PfamA domains and 72% of them contained GO information (Figure S2.A), whereas only 23% pan-genome profiles were based on HMM-domains and for more than half of them pathway information was accessible (Figure S2.B). Analysis of GO IDs distribution among the 3 general functional groups: biological process, molecular function, and cellular component, resulted in 269, 211 and 36 GOs, respectively, in the core-genome; and 492, 434 and 56 GOs, respectively, in the pan-genome.\n\nPairwise pan- and core comparison of strains within the Lactobacillus genus showed that pairs of genomes from different species share 30–60% of the protein families (profiles), while 70–90% are shared within the same species (Figure 3). Homology estimation within single proteomes revealed that approximately 20% of protein families in each genome had more than 1 member.\n\nComparison of core- and pan-genome analyses, performed by BLAST-based and PanFunPro-based approaches, found that typically HMM-based grouping of homologous sequences is more sensitive than BLAST-based grouping, and result in significantly reduced number of pan-genome families, 7,009 compared to 13,069 for the genus Lactobacillus, and 6,263 compared to 9,785 in the genus Streptococcus. Furthermore, the number of shared profiles increased fo the r Lactobacillus genus (363 to 467); however the core of Streptococcus genus did not follow the expansion tendency, and yielded 576 compared to 638 profiles.\n\nStreptococcus genomes were used as an example of accessory genome analysis. The genus contains twelve species for which complete sequenced genomes are available. S. thermophilus is used in making yoghurt, and considered to be probiotic, while the other strains are pathogenic. Single representatives of each pathogenic species and all probiotic genomes were selected for specific genome analysis. Proteomes were compared in pairs to estimate the fraction of specific profiles, which are present in one genome and absent in another. The resulting overview is visualised in Figure 4. On average each pathogenic proteome contained 30–40% specific profiles compared to other species and 6–20% within the non-pathogenic species.\n\nFurthermore, proteomes from pathogenic genomes were compared to non-pathogenic proteomes. Profiles, conserved in each pathogenic strain and absent in probiotic Streptococcus genomes, were considered to form specific core profiles. Specific-core-genome estimation resulted in 23 functional architectures formed from PfamA domains (Figure 5A), 14 of them contained GO information. Each protein could serve multiple functions, though more than one GO ID was available. The classification of proteins into three common gene ontology groups, as well as GO slims, are shown in Figure 5B. Specific core protein families were involved in metabolic processes, transport, signal transduction, and various binding and enzyme activity. Similar analysis of specific pan-genome for pathogenic Streptococcus strains yielded in 4,603 profiles, 31% of which were based on HMM-domains and 703 contained pathway information (Figure S3A). An overview of the GO functional groups (Figure S3B) reveals a broader collection of processes that proteins of pathogenic strains are involved in, however, they are not shared among all the pathogenic Streptococcus strains and are most likely to be species-specific. The BLAST-based analysis included pathogenic strains from other genera, and thus cannot be comparable.\n\nA. Specific core-genome profile distribution. B. Specific core-genome GO functional categories distribution.\n\n\nPerformance\n\nThe PanFunPro method was designed to integrate the information of functional domains from three HMM-based databases and group proteins into families according to the domain content within the protein. Further it can be used to analyse differences and similarities within defined groups of genomes based on functional architectures and visualise them. The approach includes a complex construction and assignment of functional profiles step. Therefore, we have measured the time required to collect functional domain information and perform profile formation for a set of 21 Lactobacillus genomes17. The test was performed both on MacBookPro, 2.4 GHz Intel Core i5, 8GB 1067 MHz DDR3; and on a Cluster with x86_64 architecture using 1 processor per genome and the default InterProScan settings. As illustrated in Table 1, single genome annotation by the PanFunPro approach takes about 25 and 14 min, on a laptop and cluster, respectively. To prepare profiles for the whole genus of 21 genomes, scanning one genome at a time, took more than 8h on MacBookPro and approximately 5h on the cluster. However if we allow scanning of genomes to run simultaneously on the cluster, the pan-genome calculation takes less than an hour.\n\n*NA - Analysis could not be performed\n\n\nAvailability and future directions\n\nThe source code for PanFunPro is developed in the Perl programming language for UNIX systems, and requires access to the following programs: BioPerl, GO Parser, HMMER packages, R program, Interproscan, Oracle/Sun Java 1.6, CD-HIT clustering tool. The software and instructions are available via http://www.cbs.dtu.dk/~oksana/PhD_Thesis/PanFunPro/ and permanently accessible through 10.5281/zenodo.7583.\n\nPanFunPro has been also implemented as a web server (http://cge.cbs.dtu.dk/services/PanFunPro/). The user can select a set of genomes from the provided database, including 1982 Bacterial and 128 Archaeal strains; or can upload a genome sequence and compare it to the genomes listed in the database (optional). The input file can be uploaded either in Genbank/FASTA format, or can already contain predicted proteins. The web server provides 6 analysis possibilities: core-, pan-, specific-genomes, pan-/core-plot, pan-/core-matrix, and specific-matrix. The results of analyses can be downloaded as a table and postscript file. For core-, pan-, and specific-gene families basic statistics and GO information can additionally be predicted as described above. More detailed instructions and output examples are provided on the server web page.\n\nIn the future we plan to update the approach with the analysis features and data visualization possibilities. Moreover, a web-interface will provide the possibility to compare known genomes to multiple user-submitted isolates.",
"appendix": "Author contributions\n\n\n\nOL planned the study, carried out all the bioinformatics analysis and drafted the manuscript. MCT carried out the web-server set-up. MVL and DWU participated in the design of the study and drafted the manuscript. All authors have read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAuthors received support from the Center for Genomic Epidemiology at the Technical University of Denmark; part of this work was funded by grant 09-067103/DSF from the Danish Council for Strategic Research.\n\n\nConflict-of-interest statement\n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\n\n\nAcknowledgments\n\nThe authors are grateful to all research groups that have submitted their genome sequences to public databases, without which this analysis would not have been possible. We are also grateful to John Damm Sørensen for the excellent technical assistance.\n\n\nSupplementary figures\n\nA. Core-genome GO functional categories and profile distribution. B. Pan-genome GO functional categories and profile distribution.\n\nA. Core-genome GO functional categories and profile distribution. B. Pan-genome GO functional categories and profile distribution.\n\nA. Specific pan-genome profile distribution. B. Specific pan-genome GO functional categories distribution.\n\n\nReferences\n\nTettelin H, Riley D, Cattuto C, et al.: Comparative genomics: the bacterial pan-genome. Curr Opin Microbiol. 2008; 11(5): 472–7. PubMed Abstract | Publisher Full Text\n\nKuzniar A, van Ham RC, Pongor S, et al.: The quest for orthologs: finding the corresponding gene across genomes. Trends Genet. 2008; 24(11): 539–51. PubMed Abstract | Publisher Full Text\n\nFouts DE, Brinkac L, Beck E, et al.: PanOCT: automated clustering of orthologs using conserved gene neighborhood for pan-genomic analysis of bacterial strains and closely related species. Nucleic Acids Res. 2012; 40(22): e172. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAltschul SF, Gish W, Miller W, et al.: Basic local alignment search tool. J Mol Biol. 1990; 215(3): 403–10. PubMed Abstract | Publisher Full Text\n\nPearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988; 85(8): 2444–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi W, Godzik A: Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006; 22(13): 1658–9. PubMed Abstract | Publisher Full Text\n\nLi L, Stoeckert CJ Jr, Roos DS: OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003; 13(9): 2178–89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Brien KP, Remm M, Sonnhammer EL: Inparanoid: a comprehensive database of eukaryotic orthologs. Nucleic Acids Res. 2005; 33(Database issue): D476–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEddy SR: Hidden Markov models. Curr Opin Struct Biol. 1996; 6(3): 361–5. PubMed Abstract | Publisher Full Text\n\nGabaldón T, Dessimoz C, Huxley-Jones J, et al.: Joining forces in the quest for orthologs. Genome Biol. 2009; 10(9): 403. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHyatt D, Chen GL, Locascio PF, et al.: Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11: 119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPunta M, Coggill PC, Eberhardt RY, et al.: The Pfam protein families database. Nucleic Acids Res. 2012; 40(Database issue): D290–301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaft DH, Selengut JD, White O: The TIGRFAMs database of protein families. Nucleic Acids Res. 2003; 31(1): 371–373. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilson D, Pethica R, Zhou Y, et al.: SUPERFAMILY-sophisticated comparative genomics, data mining, visualization and phylogeny. Nucleic Acids Res. 2009; 37(Database issue): D380–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZdobnov EM, Apweiler R: InterProScan-an integration platform for the signature-recognition methods in InterPro. Bioinformatics. 2001; 17(9): 847–8. PubMed Abstract | Publisher Full Text\n\nGene Ontology Consortium. The Gene Ontology project in 2008. Nucleic Acids Res. 2008; 36(Database issue): D440–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLukjancenko O, Ussery DW, Wassenaar TM: Comparative genomics of Bifidobacterium, Lactobacillus and related probiotic genera. Microb Ecol. 2012; 63(3): 651–73. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2660",
"date": "06 Dec 2013",
"name": "Granger Sutton",
"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\nPan-genome analysis always starts with some form of clustering of genes/features. It is fairly standard to limit this to protein coding genes as is done here. It is also fairly standard to cluster paralogs with orthologs as is done here, but I prefer ortholog clustering as is done with my tool PanOCT. The clustering method presented here is somewhat unique in its use of HMMs followed by CD-hit for proteins without HMM hits. This would make no attempt to separate in-paralogs from out-paralogs as some tools attempt. This approach also does not use a specific identity or similarity cutoff for homology clustering as some tools do - although for the CD-hit phase the parameters used tend to enforce a 60% similarity. There is no real justification given for the clustering used here. I tend to like the use of HMM domain architectures but have little to justify that - especially with what seems to be an arbitrary specificity cutoff. Many other tools also provide little justification for their clustering approach. It is not clear how much the clustering affects the post-clustering analysis. I think it would be wise for the authors to decouple the clustering from the post-clustering analysis so that competing clustering methods could make use of the post-clustering analysis presented here.The choice of .001 as the cutoff for significance of HMM domain hits seems overly generous and unjustified. This generosity is compounded when any PfamA hit without regard to significance is given precedence over TIGRFAM and Superfamily hits. Certainly it would be more difficult to try combining hits from the three HMM sets but some justification for this should be given.In figure 2 the bars for total and new gene families are not explained, and the new gene family bars are not consistent with what I expect them to be and the pan-genome blue line. I assume that the total family bar is the number of families/clusters in the given genome and that the new family bar is the number of families in the given genome which have not been seen in the genomes to the left. Given this, the new family bar should be equal to the amount of increase in the blue pan-genome line form the last point which it does not appear to be.A more major point is that the plots in figure 2 represent a single order of the genomes and how that order is determined is not specified. Many plots of this type choose to randomize the order over multiple trials and present the average possibly with standard deviation shown as well.In the \"Accessory genome analysis\" section, it is far from clear what is meant by: \"Descriptions (1) and (2) introduce the specific-core-genome, while descriptions (3) and (4) describe the specific-pan-genome. Given that the first and the second sets of genomes are the same, application of options (3) and (4) will yield as accessory genome of input set of genomes.\" This section talks about comparing two groups of genomes but in later sections the analysis seems to be only pairwise.The figure captions are not very descriptive and sometimes the text which describes the basic information about the figure does not reference the figure, such as for figures 2 and 4. The asymmetry in figure 4 is due to which genome is the denominator, whereas in figure 2 a symmetric measure is used. This difference is not well motivated or discussed. The asymmetry is the result of genome size which is a property of the individual genome - shouldn't we be seeking to normalize away this effect in pairwise comparisons if possible?Figure 5 was very disappointing. I had thought that the major benefit of this tool would be for analysis of different groups within the pan-genome such as pathogen versus non-pathogen. Either the GO terms are not appropriate for this or there wasn't much to be found. Not sure the set of non-pathogens was diverse enough for the question either.In summation, while I like the HMM based clustering approach there does not appear to be any justification for it over other approaches. The HMM approach does give you a level of annotation transfer from the HMMs to the cluster but how valuable this is in the post-analysis is not brought out very strongly in this article. The post-clustering analysis tools presented here appear to be useful but not overly unique. The most unique part is the transferance of the annotation to make judgments about core vs non-core and different groupings of the genome. It is not clear to me if GO is sufficient for this or if more specific pathway annotation would be more helpful. Finally, I would suggest a separation of the clustering modules and the post-clustering analysis with a defined input for the clusters with generalized or specific GO annotation.",
"responses": []
},
{
"id": "3191",
"date": "20 Jan 2014",
"name": "Bruno Contreras-Moreira",
"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 “PanFunPro: PAN‐genome analysis based on FUNctional PROfiles” by OksanaLukjancenko et al. presents a standalone application and a web server designed for the task of i)estimating the protein domain composition of proteomes and ii) the calculation of core andpangenomes inferred from the resulting functional profiles of those proteomes. The title and theabstract simply describe these features, which then they apply to test sets of Lactobacillus andStreptococcus genomes.I think the general idea behind the manuscript is appealing, in the sense that this approach could in theorycircumvent the need for BLAST reciprocal hits (BRHs), which are present in the majority of alternative software choices that can be applied to the same problem, and are known to have issues. However, in myview the authors should have done more to convince the readers of the performance of theirsolution, thoroughly comparing their results to those based on the de facto standard BRHs, whichare reported in their own reference 17 and summarized in a very short paragraph. I believe thatpart of the work required when introducing new software is to do a critical analysis of thepotential benefits and drawbacks of the new solution, and unfortunately this was not satisfactorilyaccomplished in this paper. I further elaborate on these and other points in the following list ofcomments:Why are functional profiles made by alphabetically sorting domains? I can see that this choice clearly reduces the numbers of architectures, but aren’t the authors assuming that domain order along the sequence is not important? Surely they can think of example proteins where the ordering of domains is indeed important. For proteins with no matches in Pfam, InterPro or Superfamily, CDHIT was used for clustering sequences, with a fixed %60 sequence identity cut‐off. Have the authors benchmarked this cut‐off to make sure it replicates the equivalent clustering based on HMM matches? Or perhaps was this value taken based on previous work? This should have been clearly stated in the text. I invite the authors to further expand the most important part of the work in my opinion, in particular the paragraph:“Comparison of core‐ and pan‐genome analyses, performed by BLAST‐based and PanFunPro‐based approaches, found that typically HMM‐based grouping of homologous sequences is more sensitive than BLAST‐based grouping, and result in significantly reduced number of pan‐genome families, 7,009 compared to 13,069 for the genus Lactobacillus, and 6,263 compared to 9,785 in the genus Streptococcus. Furthermore, the number of shared profiles increased fo the r Lactobacillus genus (363 to 467); however the core of Streptococcus genus did not follow the expansion tendency, and yielded 576 compared to 638 profiles.”These numbers indeed suggest that pangenomes are greatly reduced, but the conclusion is less obvious for coregenomes. The manuscript would benefit if the authors select and discuss a few protein clusters produced by both BRH and PanFunPro to show what kind of proteins are merged together, to check and discuss whether the merging always make sense. Until this analysis is carried out it would not be possible to fairly evaluate PanFunPro. Please make sure that the standalone version can effectively be downloaded from http://www.cbs.dtu.dk/~oksana/PhD_Thesis/PanFunPro/ (it does nevertheless work OK from the other provided URL). In addition, please make sure that the PanFunPro_v1.0.tar file contains binaries for all target architectures. The version I could examine only contained prodigal for Mac OS, so it currently does NOT work on Linux systems, as claimed in the text. Please make available the raw data used in this work so that users can reproduce these results using the standalone version of the software. Alternatively, demo buttons with these datasets could be added to the web tool.",
"responses": []
},
{
"id": "4218",
"date": "25 Mar 2014",
"name": "Cameron Thrash",
"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 and abstract are appropriate, but the abstract would be better if it could be more specific about the total set of analyses provided by the pipeline.In general, the article is well constructed but lacks significant information on the methods. I would prefer to see an actual methods section with more detail and the parameters used for each of the programs. I couldn’t find this at the web-server either, but that information should be in the manuscript. Also, much of the information needed to reproduce these analyses is missing. For example, what are the accession numbers of the genome sequences used? What were the settings and versions of the software? How were the HMM and BLAST searches compared? What settings were used in the BLAST part of that comparison? The authors describe “scanning” one genome, or 21 (“Performance” section)- what is meant by this and how is it done?Like the other reviewers (Granger Sutton and Bruno Contreras-Moreira), I share the concern about a blanket cutoff for similarity. This may not be appropriate for all protein families, so being able to adjust this as a user would be helpful. Regardless, the authors should provide better justification for the cutoff (literature-based or experimental: investigating how changing parameters affects the outcome of these analyses). If the results could be provided at a range of cutoffs, the user could inspect the effects of this change and make decisions accordingly. In the same vein, allowing some flexibility to add or change databases might improve the longevity of this work. One obvious additional database would be the Sifting Families database (Sharpton et al., 2012).There are now several pan-genome analysis pipelines available, for example see the following recent publications: Contreras-Moreira & Vinuesa (2013) Benedict et al. (2014)This situation is analogous to that of microbial ecology, where competing pipelines are common. It would be very helpful for the authors to point out specifically what distinguishes this pipeline from existing tools and why readers should use it. This not only will improve the usefulness of the manuscript, but encourage adoption of your software.I have some additional specific edits that I think will improve things further:“…speciation event, or a duplication event…” Add: (orthologs and paralogs respectively). …“the” subject set of organisms. …ggplot2 package (citation/website needed). Supplementary figures: legends should specify what the numbers are inside the bar plots. Also, some additional specificity describing what is being shown in the four internal plots for A and B would be helpful. The type in S1/S2 is almost too small to be legible on my screen even in the expanded view. Finally, keeping consistent color coding between A and B would speed comparisons, e.g., Pfam is green in three plots, red in one. Figure 3- what are the numbers inside the cells? Why are the pairwise comparisons of genomes with themselves included? “S. thermophilus is used in making yoghurt, and considered to be probiotic, while the other strains are pathogenic.” (Citations needed) Please define “GO slims” What is the purpose of showing the distribution of gene length according to different databases in figures 5, and S1, S2, S3? What is meant by “single genome annotation by the PanFunPro approach”? What are the methods for genome annotation? This sounds like a different analysis than that demonstrated in the current manuscript. Please provide more details on annotation and how this approach is done with the pipeline. The code is downloadable from the second link under “Availability…” but the first (http://www.cbs.dtu.dk/~oksana/PhD_Thesis/PanFunPro/) did not allow me permission to download.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-265
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https://f1000research.com/articles/2-224/v1
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23 Oct 13
|
{
"type": "Short Research Article",
"title": "Amplicon pyrosequencing of wild duck eubacterial microbiome from a fecal sample reveals numerous species linked to human and animal diseases",
"authors": [
"Thomas Strong",
"Scot Dowd",
"Alexander F. Gutierrez",
"Jonathan Coffman",
"Thomas Strong",
"Scot Dowd",
"Alexander F. Gutierrez"
],
"abstract": "Our investigation into the composition of the wild duck, Aythya americana, eubacterial microbiome from a fecal sample using amplicon pyrosequencing revealed that the representative bacterial species were quite distinct from a pond water sample, and we were able to classify the major operational taxonomic units with Fusobacterium mortiferum, Streptobacillus moniliformis, Lactobacillus intermedius, Actinomyces suimastitidis, Campylobacter Canadensis, Enterococcus cecorum, Lactobacillus aviarus, Actimomyces spp., Pseudobutyrivibrio spp. and Helicobacter brantae representing the majority of the eubacterial fecal microbiome. Bacterial species present in the analysis revealed numerous organisms linked to human and animal diseases including septicemia, rat bite fever, pig mastitis, endocarditis, malar masses, genital infections, skin lesions, peritonitis, wound infections, septic arthritis, urocystitis, gastroenteritis and drinking water diseases. In addition, to being known carriers of viral pathogens wild ducks should also be recognized as a potential source of a range of bacterial diseases.",
"keywords": [
"Throughout the history of medicine there has been an awareness of animal to human transmission of disease",
"and the etiological pathogens have been collectively described as zoonoses1. Water fowl and wild birds have been identified as reservoirs for the virus Influenza A2",
"3",
"a highly mutable and infectious pathogen that infects avian and mammalian species4. Ducks are observed in a multitude of fresh water sources including ponds",
"water fountains and pools where they can defecate",
"bacteria have been shown to be distributed through aerosols from ornamental fountains5",
"6 and reclaimed water dispensed through an irrigation system7. Humans may also have direct contact with ducks and their excrement through the recreational sport of duck hunting8. Ducks can also shed pathogens near chicken farms or other animals—such as pigs—that have access to outside areas. An avian influenza A virus (H7N7) epidemic in the Netherlands in 2003 thought to be initiated from a migratory water fowl resulted in the culling of 30 million poultry in an area of the country where free-range poultry farming was common9. Due to the migratory nature and unrestrained behavior of the wild duck",
"Aythya Americana",
"our study set out to investigate the bacterial microbiome of a wild duck and to identify its bacterial flora relative to the same bacterial species that have been reported to cause disease in farm animals and humans."
],
"content": "Introduction\n\nThroughout the history of medicine there has been an awareness of animal to human transmission of disease, and the etiological pathogens have been collectively described as zoonoses1. Water fowl and wild birds have been identified as reservoirs for the virus Influenza A2,3; a highly mutable and infectious pathogen that infects avian and mammalian species4. Ducks are observed in a multitude of fresh water sources including ponds, water fountains and pools where they can defecate; bacteria have been shown to be distributed through aerosols from ornamental fountains5,6 and reclaimed water dispensed through an irrigation system7. Humans may also have direct contact with ducks and their excrement through the recreational sport of duck hunting8. Ducks can also shed pathogens near chicken farms or other animals—such as pigs—that have access to outside areas. An avian influenza A virus (H7N7) epidemic in the Netherlands in 2003 thought to be initiated from a migratory water fowl resulted in the culling of 30 million poultry in an area of the country where free-range poultry farming was common9. Due to the migratory nature and unrestrained behavior of the wild duck, Aythya Americana, our study set out to investigate the bacterial microbiome of a wild duck and to identify its bacterial flora relative to the same bacterial species that have been reported to cause disease in farm animals and humans.\n\n\nMethods\n\nAmplicon pyrosequencing (bTEFAP) was originally described by Dowd et al.10 and has been used in describing a wide range of environmental and health related microbiomes including the intestinal populations of a variety of animals and their environments including cattle11–15. A fecal sample obtained from a wild duck, Aythya americana, that was killed during duck hunting season (December 2012) by a licensed hunter, was aseptically swabbed onto a Whatman FTA card (GE Healthcare Life Sciences) using a sterile swab and gloves being careful to avoid environmental contamination. The flap of the FTA card was placed over the FTA paper and placed into a sterile pouch, and the FTA card was stored at room temperature prior to DNA amplification. 2 mm punches were washed with FTA reagent and TE (10 mM Tris-HCL, 1 mM EDTA, pH 8.0) according to the manufacturer’s protocol, and the dried punches were used as template DNA for thermal cycling. DNA was also isolated from pond water as a negative comparison and sampled from a source of water visited by numerous avian species but not at the source of the fecal sampling but within the migratory range of Aythya americana. The pond water DNA was isolated using water RNA/DNA purification kit (0.45 µm) [Norgen Biotek Corp, Thorold, ON, Canada]. For thermal cycling and DNA amplification we used the 16S universal Eubacterial primers 27f 5´-AGAGTTTGATCCTGGCTCAG-3´ and 1492r primer 5´-ACGGCTACCTTGTTACGACTT-3´ (Integrated DNA Technologies). A single-step 30 cycle PCR using EconoTaq PLUS 2X Master Mix (Lucigen, Middleton, WI) were used under the following conditions: 94°C for 2 minutes, followed by 30 cycles of 95°C for 120 seconds; 42°C for 30 seconds and 72°C for 4 minutes; after which a final elongation step at 72°C for 20 minutes was performed. Following PCR, DNA products were resolved in a 1% agarose, 1X TAE gel stained with ethidium bromide and 1.5 Kb products were excised from the gel purified using a cyclo-prep spin column (Amresco, Solon, OH). All the DNA products were purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA). Samples were sequenced using Roche 454 FLX titanium instruments and reagents following manufacturer’s guidelines. The Q25 sequence data derived from the sequencing process was processed using a proprietary analysis pipeline (www.mrdnalab.com, MR DNA, Shallowater, TX). Sequences were depleted of barcodes and primers. Next, short sequences < 200bp, sequences with ambiguous base calls, and sequences with homopolymer runs exceeding 6bp were removed. Sequences were then denoised and chimeras removed. Operational taxonomic units (OTUs) were defined after removal of singleton sequences, clustering at 3% divergence--97% similarity10,15. OTUs were then taxonomically classified using BLASTn against a curated GreenGenes database16,17 and compiled into each taxonomic level into both “counts” and “percentage” files.\n\n\nResults\n\nDue to the aquatic nature of the animal, we initially expected that the biodiversity of bacterial species in the duck feces would reflect numerous bacterial species present in the pond water, and since we observed multiple species of aquatic birds in the pond we expected to find eubacteria in common. Figure 1 is a modified heat map showing differences and similarities among the classes of eubacteria sequenced and identified. The figure demonstrates clear differences at the taxonomical level of Class with few common classes of bacteria namely Actinobacteria, Clostridia and Gammaproteobactera.\n\nDarker colors represent a higher representation of the bacterial class.\n\nHowever, similarities at the level of Genus and species included only Agrobacterium tumefaciencs and a species of Porphyromonas and a species of Ruminococcaceae (Figure 2). This analysis indicated distinct differences between the eubacteria present in the duck fecal sample and the pond water sample, and it also indicated that our sampling of the duck feces was devoid of any obvious pond water eubacterial constituents.\n\nThe taxonomical classification of OTU at the level of genus and species was compiled in relation to percentages of the Eubacterial microbiome (Table 1). In Table 2, we referenced reported cases of diseases related to the bacteria sequenced from the duck’s fecal sample reflecting the eubacterial microbiome’s potential to cause disease in humans and other mammals. The largest representative bacterial species—relative to percentage—was Fusobacterium mortiferum at 31.6%. Fusobacterium mortiferum reports related to human disease are sparse, but Fusobacterium have been associated with rare but serious cases of bacteremia18,19, and a 6 year study of “other gram-negative anaerobic bacilli” (OGNAB) isolated from anaerobic infections at the Wadsworth Clinical Anaerobic Clinical Anaerobic Bacteriology Research Laboratory in Los Angeles, CA reported that most strains of Fusobacteria—outside of Fusobacterium nucleatum—were resistant to erythromycin20. The pathogen, Fusobacterium nucleatum, on the other hand, is well-known for its association with disease and its ability to adhere to Gram-positive and Gram-negative bacteria in dental biofilms such as plaque21.\n\nStreptobacillus moniliformis was also identified as a major constituent of the duck fecal eubacterial microbiome at 30.1%. Several well-studied and documented cases of disease are attributed to S. moniliformis including rat bite fever or Haverhill disease22, osteomyelitis23, epidural abscesses24, fever and polyarthralgia25, bacteremia26 and contaminated drinking water related disease27.\n\nOther organisms and their respective illnesses included Lactobacillus intermedius (11.02%) in a renal transplant infection28, Actinomyces suimastitidis (4.47%) in pig mastitis29 and Campylobacter canadensis (3.69%) in drinking water related disease27. Enterococcus cecorum was another identified pathogen at 3.59% of the sequenced Eubacterial microbiome, and E. cecorum has been reported to cause disease in chicks30,31 and humans including aortic valve endocardititis32, empyema thoracis33, septicemia in a malnourished adult34 and recurrent bacteremic peritonitis in a patient with liver cirrhosis35. Actinomyces odontolyticus (0.70%) has recently been reported to cause bacteremia in immunosuppressed patients36, and members of the genus Actinomyces have been known to cause actinomycosis for some time. A. odonolyticus was reported by Michell, Hintz and Haselby in 1997 to be the cause of a malar mass in soft tissue in a human37. A species of the genus Leptotrichia (0.36%) was also identified, a genus that has been associated with bacteremia in multiple myeloma patients receiving chemotherapy38. Another Actinomyces present in the wild duck eubacterial microbiome was Actinomyces turicensis at 0.3%, a bacterium associated with a spectrum of diseases including genital infections, urinary tract infections, skin infections, post-operative wound infection, abscesses, appendicitis, ear and nose and throat infection and bacteremia39. In addition, Actinomyces europaeus (0.14%) was reported in human abscesses40, Actinomyces neuii (0.03%) was reported to cause endophthalmitis41 and periprosthetic infection42, Actinomyces vaccimaxillae (0.01%) was isolated from a cow jaw lesion43 and Actinomyces hongkongensis (0.004%) was reported to cause high-mortality bacteremia in humans44.\n\n0.24% of the eubacterial population was composed of Plesiomonas shigelloides a well-documented pathogen associated with Travelers’ diarrhea, dysentery and gastroenteritis45–48. Arcanobacterium pyogenes was also present (0.18%), a pathogen reported to cause soft tissue infections in humans49. Atopobium vaginae (0.12%) was reported to cause bacterial vaginosis in a human50 and Varibaculum cambriense (0.01%) was reported to cause complications with intrauterine devices and vaginal infections in Hong Kong51, Parvimonas micra (0.08%) was associated with odontogenic infection52 and human bacteremia was reported with Atopobium rima53, Fusobacterium nucleatum54, Corynebacterium freneyi55 and Streptococcus suis56. Finally, Veillonella dispar (0.02%) was reported in a case of septic arthritis57 and Porphyromonas gingivalis (0.02%) is a well-studied pathogen reported decades earlier and associated with periodontitis58.\n\n\nDiscussion\n\nNumerous pathogenic eubacterial species have been identified in the fecal sample obtained from the wild duck, Aythya Americana, using amplicon pyrosequencing, a widely accepted method for analyzing the bacterial composition of microbial ecosystems. We were surprised to find that most of the species of eubacteria sequenced the duck feces were not present in a pond water sample from a water source that was known to be visited by numerous water fowl. Perhaps, the analyses of small samples from a pond or lake are not adequate when investigating the presence of avian contamination.\n\nThe summary in Table 2 indicates that many of the bacteria that are listed are clinically important causing severe diseases such as bacteremia and septicemia. The potential to cause disease can be appreciated when one considers that wild-duck feces can contaminate food, drinking water and open wounds. In addition, bird feces can easily contaminate ornamental fountains--where aerosols are produced—and the aerosols can carry the bacteria in a similar way to what has been reported for Legionella pneumophila47. It is possible that many of the bacterial entities when disseminated to humans and other animals could also cause subclinical respiratory illnesses that are not reported due to patient resolution.\n\nIt is only prudent to recommend that immunocompromised humans and animals should limit their exposure to environments where ducks may have polluted the water source—this includes outdoor pools and fountains. That realization also supports the practice of adequately chlorinating or sanitizing artificial pools and fountains to prevent opportunistic infections through aerosols or breaks in the skin. Duck hunters should also be aware of the risk of bacterial contamination in addition to the risk posed by the influenza virus. Additionally, reclaimed water poses a threat to the elderly and other immunocompromised humans who might be exposed to aerosols that are produced when the reclaimed water is used as a source of irrigation such as in golf courses and gardens, a common practice that might warrant further inquiry.\n\nWhen determining the cause of disease it is difficult—if not impossible—to identify the source of infection, and whether it has indeed originated from an animal that is migratory or aquatic in nature. Many of the bacterial species that were cited to cause infections among humans were also found in the excrement of a migratory and aquatic bird that defecates in water supplies and around other animals. However, since our analysis was limited to the careful analysis of a single, wild duck’s eubacterial microbiome, the disease potential was relative to that animal only and cannot be extrapolated to all ducks of the same species. Thus, the disease potential is relative to this studied microbiome and further statistical studies will be needed to determine the global risks associated with duck excrement among different species.",
"appendix": "Author contributions\n\n\n\nTS carried out the majority of the molecular biology techniques in the laboratory, AG was instrumental in obtaining the wild ducks specimens. SD provided expertise in pyrosequencing and bioinformatics. JC conceived the study and wrote the first draft of the manuscript. All authors were involved in the revision of the draft and have agreed to the final content. The study was an active learning exercise that helped bridge the understanding of Medical Microbiology with field research, molecular biology and bioinformatics for graduate students seeking their Masters (MS) degree in Biomedical Sciences under the guidance of Dr. Coffman.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work was funded by the Barry University Faculty Incentive Grant 10-110106 awarded to Dr. Jonathan Coffman.\n\n\nAcknowledgements\n\nWe would like to acknowledge Barry University for providing the infrastructure to conduct the experiments.\n\n\nReferences\n\nSpickler AR, Roth JA, Galyon J, et al.: Emerging and Exotic Diseases of Animals Textbook. 4th ed. Center for Food Security and Public Heatlh. 2010; 383. Reference Source\n\nAlexander DJ: A review of avian influenza in different bird species. Vet Microbiol. 2000; 74(1–2): 3–13. PubMed Abstract | Publisher Full Text\n\nBahl J, Krauss S, Kuhnert D, et al.: Influenza a virus migration and persistence in north american wild birds. PLoS Pathog. 2013; 9(8): e1003570. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKocer ZA, Krauss S, Stallknecht DE, et al.: The potential of avian H1N1 influenza A viruses to replicate and cause disease in mammalian models. PLoS One. 2012; 7(7): e41609. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Loughlin RE, Kightlinger L, Werpy MC, et al.: Restaurant outbreak of Legionnaires’ disease associated with a decorative fountain: an environmental and case-control study. BMC Infect Dis. 2007; 7(1): 93–99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEscartin EF, Lozano JS, Garcia OR, et al.: Potential Salmonella transmission from ornamental fountains. J Environ Health. 2002; 65(4): 9–12. PubMed Abstract\n\nHerzog A, Bhaduri P, Stedtfeld RD, et al.: Detection and occurrence of indicator organisms and pathogens. Water Environ Res. 2010; 82(10): 883–907. Publisher Full Text\n\nDishman H, Stallknecht D, Cole D: Duck hunters’ perceptions of risk for avian influenza, Georgia, USA. Emerg Infect Dis. 2010; 16(8): 1279–1281. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStegeman A, Bouma A, Elbers AR, et al.: Avian influenza A virus (H7N7) epidemic in The Netherlands in 2003: course of the epidemic and effectiveness of control measures. J Infect Dis. 2004; 190(12): 2088–2095. PubMed Abstract | Publisher Full Text\n\nDowd SE, Callaway TR, Wolcott RD, et al.: Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiol. 2008; 8: 125. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaporaso JG, Lauber CL, Walters WA, et al.: Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A. 2011; 108(Suppl 1): 4516–4522. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCapone KA, Dowd SE, Stamatas GN, et al.: Diversity of the human skin microbiome early in life. J Invest Dermatol. 2011; 131(10): 2026–2032. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDowd SE, Sun Y, Wolcott RD, et al.: Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: bacterial diversity in the ileum of newly weaned Salmonella-infected pigs. Foodborne Pathog Dis. 2008; 5(4): 459–472. PubMed Abstract | Publisher Full Text\n\nEren AM, Zozaya M, Taylor CM, et al.: Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation. PLoS One. 2011; 6(10): e26732. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDowd SE, Hanson JD, Rees E, et al.: Survey of fungi and yeast in polymicrobial infections in chronic wounds. J Wound Care. 2011; 20(1): 40–47. PubMed Abstract\n\nDeSantis TZ, Hugenholtz P, Larsen N, et al.: Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Enviro Micro. 2006; 72(7): 5069–5072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdgar RC: Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010; 26(19): 2460–2461. PubMed Abstract | Publisher Full Text\n\nAfra K, Laupland K, Leal J, et al.: Incidence, risk factors, and outcomes of Fusobacterium species bacteremia. BMC Inf Dis. 2013; 13(1): 264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nProut J, Glymph R: Anaerobic septicemia secondary to Fusobacterium mortiferum. J Natl Med Assoc. 1986; 78(4): 334–337. PubMed Abstract | Free Full Text\n\nGeorge WL, Kirby BD, Sutter VL, et al.: Gram-negative anaerobic bacilli: their role in infection and patterns of susceptibility to antimicrobial agents. II. Little-known Fusobacterium species and miscellaneous genera. Rev Infect Dis. 1981; 3(3): 599–626. PubMed Abstract\n\nVan der Velden U, Van Winkelhoff AJ, Abbas F, et al.: The habitat of periodontopathic micro-organisms. J Clin Periodontol. 1986; 13(3): 243–248. PubMed Abstract | Publisher Full Text\n\nHimsworth CG, Parsons KL, Jardine C, et al.: Rats, cities, people, and pathogens: a systematic review and narrative synthesis of literature regarding the ecology of rat-associated zoonoses in urban centers. Vector Borne Zoonotic Dis. 2013; 13(6): 349–359. PubMed Abstract | Publisher Full Text\n\nFlannery DD, Akinboyo I, Ty JM, et al.: Septic arthritis and concern for osteomyelitis in a child with rat bite fever. J Clin Microbiol. 2013; 51(6): 1987–1989. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAddidle M, Pynn J, Grimwade K, et al.: Epidural abscess caused by Streptobacillus moniliformis. J Clin Microbiol. 2012; 50(9): 3122–3124. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrlev A, Miskin I, Temper V, et al.: A 70-year-old man with fever and polyarthralgia. Streptobacillus moniliformis. Clin Infect Dis. 2011; 53(10): 1037–1038. PubMed Abstract | Publisher Full Text\n\nJoshi RM, Al Sweih N, Bin Nakhi HA, et al.: Streptobacillus moniliformis bacteremia in a child: case report. Med Princ Pract. 2010; 19(5): 409–411. PubMed Abstract | Publisher Full Text\n\nNichols G, Lane C, Asgari N, et al.: Rainfall and outbreaks of drinking water related disease and in England and Wales. J Water Health. 2009; 7(1): 1–8. PubMed Abstract | Publisher Full Text\n\nDomann E, Hong G, Imirzalioglu C, et al.: Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients. J Clin Microbiol. 2003; 41(12): 5500–5510. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoyles L, Falsen E, Holmstrom G, et al.: Actinomyces suimastitidis sp. nov., isolated from pig mastitis. Int J Syst Evol Microbiol. 2001; 51(4): 1323–1326. PubMed Abstract | Publisher Full Text\n\nStalker MJ, Brash ML, Weisz A, et al.: Arthritis and osteomyelitis associated with Enterococcus cecorum infection in broiler and broiler breeder chickens in Ontario, Canada. J VET Diagn Invest. 2010; 22(4): 643–645. PubMed Abstract | Publisher Full Text\n\nBorst LB, Suyemoto MM, Robbins KM, et al.: Molecular epidemiology of Enterococcus cecorum isolates recovered from enterococcal spondylitis outbreaks in the southeastern United States. Avian Pathol. 2012; 41(5): 479–485. PubMed Abstract | Publisher Full Text\n\nAhmed FZ, Baig MW, Gascoyne-Binzi D, et al.: Enterococcus cecorum aortic valve endocarditis. Diagn Microbiol Infect Dis. 2011; 70(4): 525–527. PubMed Abstract | Publisher Full Text\n\nWoo PC, Tam DM, Lau SK, et al.: Enterococcus cecorum empyema thoracis successfully treated with cefotaxime. J Clin Micrbiol. 2004; 422): 919–922. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreub G, Devriese LA, Pot B, et al.: Enterococcus cecorum septicemia in a malnourished adult patient. Eur J Clin Microbiol Infect Dis. 1997; 16(8): 594–598. PubMed Abstract | Publisher Full Text\n\nHsueh PR, Teng LJ, Chen YC, et al.: Recurrent bacteremic peritonitis caused by Enterococcus cecorum in a patient with liver cirrhosis. J Clin Microbiol. 2000; 38(6): 2450–2452. PubMed Abstract | Free Full Text\n\nCone LA, Leung MM, Hirschberg J: Actinomyces Odontolyticus bacteremia. Emerg Infect Dis.[serial online]. 2003; 9(12): 1629–32. PubMed Abstract | Publisher Full Text\n\nMitchell PD, Hintz CS, Haselby RC: Malar mass due to Actinomyces odontolyticus. J Clin Microbiol. 1977; 5(6): 658–60. PubMed Abstract | Free Full Text\n\nCouturier MR, Slechta ES, Goulston C, et al.: Leptotrichia bacteremia in patients receiving high-dose chemotherapy. J Clinical Micro. 2012; 50(4): 1228–1232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSabbe LJ, Van De Merwe D, Schouls L, et al.: Clinical spectrum of infections due to the newly described Actinomyces species A. turicensis, A. radingae, and A. europaeus. J Clin Micro. 1999; 37(1): 8–13. PubMed Abstract | Free Full Text\n\nFunke G, Alvarez N, Pascual C, et al.: Actinomyces europaeus sp. nov., isolated from human clinical specimens. Int J Syst Bacter. 1997; 47(3): 687–692. PubMed Abstract | Publisher Full Text\n\nGraffi S, Peretz A, Naftali M: Endogenous endophthalmitis with an unusual infective agent: Actinomyces neuii. Eur J Ophthalmol. 2012; 22(5): 834–835. PubMed Abstract | Publisher Full Text\n\nRieber H, Schwarz R, Kramer O, et al.: Actinomyces neuii subsp. neuii associated with periprosthetic infection in total hip arthroplasty as causative agent. J Clin Microbiol. 2009; 47(12): 4183–4184. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHall V, Collin MD, Hutson R, et al.: Actinomyces vaccimaxillae sp. nov., from the jaw of a cow. Int J Syst Evol Microbiol. 2003; 53(Pt 2): 603–606. PubMed Abstract | Publisher Full Text\n\nLau SK, Fan RY, Lo HW, et al.: High mortality associated with Catabacter hongkongensis bacteremia. J Clin Microbiol. 2012; 50(7): 2239–2243. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShah N, DuPont HL, Ramsey DJ: Global etiology of travelers’ diarrhea: systematic review from 1973 to the present. Am J Trop Med Hyg. 2009; 80(4): 609–614. PubMed Abstract\n\nPfeiffer ML, DuPont HL, Ochoa TJ: The patient presenting with acute dysentery—a systematic review. J Infect. 2012; 64(4): 374–386. PubMed Abstract | Publisher Full Text\n\nKhan AM, Faruque AS, Hossain MS, et al.: Plesiomonas shigelloides-associated diarrhoea in Bangladeshi children: a hospital-based surveillance study. J Trop Pediatr. 2004; 50(6): 354–356. PubMed Abstract | Publisher Full Text\n\nChan SS, Ng KC, Lyon DJ, et al.: Acute bacterial gastroenteritis: a study of adult patients with positive stool cultures treated in the emergency department. Emerg Med J. 2003; 20(4): 335–338. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKavitha K, Latha R, Udayashankar C, et al.: Three cases of Arcanobacterium pyogenes-associated soft tissue infection. J Med Microbiol. 2010; 59(Pt 6): 736–739. PubMed Abstract | Publisher Full Text\n\nFerris MJ, Masztal A, Aldridge KE, et al.: Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis. BMC Infect Dis. 2004; 4(5): 1–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu YW, Wong CH, Chu MY, et al.: Varibaculum cambriense infections in Hong Kong, China, 2006. Emerg Infect Dis. 2009; 15(7): 1137–1139. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlynn TR, Paster BJ, Stokes LN, et al.: Molecular methods for diagnosis of odontogenic infections. J Oral Maxillo fac Surg. 2012; 70(8): 1854–1859. PubMed Abstract | Publisher Full Text\n\nAngelakis E, Roux V, Raoult D, et al.: Human case of Atopobium rimae bacteremia. Emerg Infect Dis. 2009; 15(2): 354–355. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSu CP, Huang PY, Yang CC, et al.: Fusobacterium bacteremia: clinical significance and outcomes. J Microbiol Immunol Infect. 2009; 42(4): 336–342. PubMed Abstract\n\nAuzias A, Bollet C, Ayari R, et al.: Corynebacterium freneyi bacteremia. J Clin Microbiol. 2003; 41(6): 2777–2778. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLun ZR, Wang QP, Chen XG, et al.: Streptococcus suis: an emerging zoonotic pathogen. Lancet Infect Dis. 2007; 7(3): 201–209. PubMed Abstract | Publisher Full Text\n\nHillier DI, Ballal MS, Sanger R, et al.: Veillonella dispar and Streptococcus bovis septic arthritis in a native shoulder joint. Shoulder & Elbow. 2011; 3(4): 219–221. Publisher Full Text\n\nGriffen AL, Becker MR, Lyons SR, et al.: Prevalence of Porphyromonas gingivalis and periodontal health status. J Clin Microbiol. 1998; 36(11): 3239–3242. PubMed Abstract | Free Full Text\n\nGrattarola C, Bellino C, Tursi M, et al.: Helcococcus kunzii isolated from a sow with purulent urocystitis. J Clin Microbiol. 2010; 48(8): 3019–3020. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2348",
"date": "06 Nov 2013",
"name": "Jorge Santo Domingo",
"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 of Strong et al. focuses on the description of the bacterial community of a duck fecal sample. In the study the authors analyzed 16S rDNA pyrosequencing data to determine the identity and diversity of the bacteria present in the sample. They also collected water samples and performed similar studies in order to determine if the water was influenced by the duck staging over the area were the sample was collected. The presence of pathogenic bacteria in avian feces is well documented, although most of the work has been conducted with a relatively limited number of species. It is also known that birds can influence the microbial quality of surface waters. Thus any additional data on the occurrence and relative abundance of potential pathogens from birds and their relationship to human health risks is welcomed. The study, as presented, suffers from some relevant limitations and in my opinion the data should be considered preliminary in nature. Perhaps the most important one is the low number of samples analyzed (n=1) for the fecal and water samples. Thus in this regard it would be difficult to determine if this sample is representative of the duck fecal microbiota or not. It is also not clear how many sequences were analyzed in this study and which area of the 16S rRNA gene was used in the analysis. The authors mentioned that they amplified the entire 16S rRNA gene but normally such large amplicons are not used for next generation sequencing. To this reviewer it seems that there is some details missing on the method, e.g. perhaps the sequencing facility performed a second amplification with primers that generated a smaller PCR product which was compatible with the sequencing technology. There are many other details on the methods used that are not clear, such as how long was the fecal sample at room temperature before it was processed, holding time for water sample before processed, the total volume of water filtered and how it was filtered. They also collected and analyzed one water sample on one data, which again it would be difficult to be representative of the pond. Typically, more samples would be needed to arrive at any conclusions.There is also the issue of using relatively short fragments of the 16S rRNA to accurately identify a bacterium at the species levels. What was the length of the sequences used in each of the cases and what was the sequence identity for each of the sequences that were identified as closely related to the identified pathogens? The authors would benefit from performing additional assays to further confirm the presence of potentially pathogenic populations. 16S rDNA-based assays might be available for some of the “pathogens” identified but preferably functional genes should be used if considered for such an exercise. The authors should also considered to compare this dataset (albeit limited in scope) with other studies in which 16S rDNA sequencing information has been used to describe avian fecal microbiota.As a minor comment, there are two data points in Figure 1 (which actually is a table) for Fusobacteria.",
"responses": [
{
"c_id": "609",
"date": "06 Nov 2013",
"name": "Jonathan Coffman",
"role": "Author Response",
"response": "The method for pyrosequencing including the variable regions of the 16S rRNA gene was referenced and previously described by:Scot E. Dowd, Yan Sun, Randy D. Wolcott, Alexander Domingo, and Jeffery A. Carroll. Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: bacterial diversity in the ileum of newly weaned Salmonella-infected pigs. Foodborne Pathogens and Disease. August 2008, 5(4): 459-472.We have obtained additional samples from Aythya americana and sequenced the samples using ion torrent sequencing and made a rarefaction graph.We understand we had a limited sample size and are trying to increase it, despite being hindered by rather limited resources and time constraints.The Whatman FTA card information (manufacturer's website):Advantages and benefitsCapture nucleic acid in one easy step.Captured nucleic acid is ready for downstream applications in less than 30 minutes.DNA collected on FTA Cards is preserved for years at room temperature.FTA Cards are stored at room temperature before and after sample application, reducing the need for laboratory freezers.Suitable for virtually any cell type."
}
]
},
{
"id": "2257",
"date": "13 Nov 2013",
"name": "Jonas Waldenström",
"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 novel sequencing techniques now allow for assessing a much larger fraction of the microorganisms a host is carrying. In the past, an investigation of the gastrointestinal microbiota of an animal would rely either on the culturable fraction of microorganisms, or laborious cloning and sequencing. The paper by Strong et al. uses amplicon sequencing of the 16sRNA gene for detecting and characterizing the microbiome of a wild duck fecal sample. The resulting data is compared to a water sample from another pond, and referenced to the existing literature to identify possible human and animal pathogens. I have some concerns with this paper. First, naturally, is the very limited sample size. With only a single sample from each of the two entities, it is problematic to tackle within-host and between host variability, and any temporal variation present in the data. Furthermore, the duck and the pond water sample were collected by different methods, which also may affect template DNA purity and yield, and the samples are not from the same site. The authors focus the majority of the paper on what potential human pathogens the duck is carrying. This is interesting, as many human pathogens have animal reservoirs and identifying them may lead us to better understand their epidemiology. There is a range of human pathogens identified, and the authors make recommendations based on these findings. However, recommendations need to be thoroughly justified by data, and in at least one case I did not find the link to human infection proven. This is Campylobacter canadensis that in table 2 is said to cause drinking water related disease. However, the reference cited for this claim does not discuss C. canadensis, but waterborne outbreaks in general. The methods/result sections could be made more informative. Every method has its cons and pros, and it would help the reader to illustrate how it was made sure the primers and protocol used were suitable for bird fecal microbiota. Have they been validated on avian fecal samples before? It is not presented clearly how many sequences that were obtained, how many that were possible to bin to different species/genera etc. Surprisingly, the duck host is not presented in any detail at all. I am not familiar with Aythya americana, as it is restricted to North America, but there are a number of Aythya species in Europe, all of which are diving ducks, often feeding to a large extent on bivalves and other mollusks, and not really dabbling in the surface water. This will likely influence the potential exposure of A. americana feces to humans, and should be taken into account in the recommendations given in the paper. As a biologist, I am also curious to know why these bacteria are in the bird, and what they do there – are they commensals, or pathogens? What has been found in other bird species? This is not discussed in the present version. Taken together, the data should be considered with some caution as it is based on such a small sample set and conclusions from comparisons should therefore be toned down. Preferentially, the study should be extended to include more avian samples. Minor things: Title: Very little in this article actually refers to animal disease, which makes the title slightly misleading. Introduction: It would be good if the host could be put in a larger perspective here, and that previous work on ducks as carriers of human-associated bacterial pathogens are cited to a larger extent. The ornamental/decorative fountains are unlikely significant duck habitats. Figure 1: Are the numbers referring to percentages? And is it not more a table than a figure? Please check bird names and bacterial names and make sure species names are not starting with capital letters (e.g. Aythya americana, not Americana etc).",
"responses": [
{
"c_id": "649",
"date": "13 Dec 2013",
"name": "Jonathan Coffman",
"role": "Author Response",
"response": "Thank you for your comments and recommendations. We are continuing our study with additional samples to increase our N."
}
]
}
] | 1
|
https://f1000research.com/articles/2-224
|
https://f1000research.com/articles/2-264/v1
|
04 Dec 13
|
{
"type": "Software Tool Article",
"title": "illuminaio: An open source IDAT parsing tool for Illumina microarrays",
"authors": [
"Mike L Smith",
"Keith A. Baggerly",
"Henrik Bengtsson",
"Matthew E. Ritchie",
"Kasper D. Hansen",
"Keith A. Baggerly",
"Henrik Bengtsson",
"Matthew E. Ritchie"
],
"abstract": "The IDAT file format is used to store BeadArray data from the myriad of genomewide profiling platforms on offer from Illumina Inc. This proprietary format is output directly from the scanner and stores summary intensities for each probe-type on an array in a compact manner. A lack of open source tools to process IDAT files has hampered their uptake by the research community beyond the standard step of using the vendor’s software to extract the data they contain in a human readable text format. To fill this void, we have developed the illuminaio package that parses IDAT files from any BeadArray platform, including the decryption of files from Illumina’s gene expression arrays. illuminaio provides the first open-source package for this task, and will promote wider uptake of the IDAT format as a standard for sharing Illumina BeadArray data in public databases, in the same way that the CEL file serves as the standard for the Affymetrix platform.",
"keywords": [
"computer software"
],
"content": "Introduction\n\nThe DNA microarray field is dominated by the three manufacturers: Affymetrix, Illumina and Agilent. While the basic premise behind their competing products is the same (i.e. the measurement of hybridisation between sample and immobilised probes on arrays via fluorescence), the formats in which these data are presented to end users are quite different, with each manufacturer electing to use their own proprietary format. The most ubiquitous of these is the CEL file, which has been accepted as a standard format for publishing the raw data generated on the Affymetrix platform. A search of the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database finds over 90% of submissions of Affymetrix data include one or more CEL files as supplementary material. The format itself is well documented by the manufacturer, who also provides an open-source software development kit (SDK). As a result, in addition to Affymetrix’s own software suite, a large number of CEL parsing tools exist, including a parser implemented based on the file format documentation: affyio1 and a parser based on the SDK: affxparser2.\n\nThe same is not true of the primary IDAT format from Illumina, with only 1.5% (49 out of 3208) of the submissions in GEO that use Illumina BeadArrays including IDAT files as supplementary material. Given that IDATs are the standard file type generated during BeadArray processing, it seems reasonable to assume that the relative dearth of IDAT files in the public domain is due to the lack of widespread support for the format. The development of alternative parsing tools has proven more challenging for IDATs for a number of reasons. The foremost amongst these is a lack of public documentation, leaving tool developers to determine the file structure themselves. A further hurdle has been the encryption of IDAT files generated from expression chips. These barriers initially left researchers reliant on the output from Illumina’s GenomeStudio software to convert the data into a more convenient format. Existing open source tools, particularly those that focus on gene expression analysis such as beadarray3, lumi4 and limma5, all require that the IDAT files have been processed using GenomeStudio to generate a plain-text ASCII file before any analysis can take place (Figure 1). The GenePattern6 software suite includes support for reading expression IDAT files, although it is limited to extracting only a subset of the array information. GenomeStudio output also omits various information that is available from the IDAT, such as control probe intensities (for SNP and methylation platforms), so-called out-of-band probes (methylation 450k)7 and meta information including software versions and scan date (all platforms).\n\nScanning of BeadChips is performed using the iScan or BeadScan control software, producing IDAT files. Currently, these are read by GenomeStudio where the user has the choice of performing an analysis using that software, or outputting the intensities to a text file for processing by alternative means. illuminaio removes this reliance on GenomeStudio. The intention is for analysis packages to incorporate its routines, effectively merging the dashed region and simplifying the analysis process for end users.\n\nHere we introduce the Bioconductor8 package illuminaio that can handle IDAT files from any Illumina BeadArray platform, providing a simple unified interface to various low-level data extraction routines.\n\n\nData format\n\nThe IDAT file format varies depending upon the array platform (Table 1). IDATs generated during the scanning of genotyping and methylation BeadArrays are binary files (one for each of the red and green channels). The bulk of each file is comprised of four fields: the ID of each bead-type on the array, the mean and standard deviation of their intensities, and the number of beads of each type. Additionally, metadata including the date the array was scanned, specific software versions used and the type of BeadChip are also included. Once the structure of the file is understood these binary values can be read directly.\n\nOn the other hand, gene expression IDAT files are produced as encrypted XML files. Once decrypted the majority of the data are found as ten Base64 encoded strings. These ten fields include the ID, mean and standard deviation values as found in genotyping IDATs, as well as median and trimmed-mean intensity values, the mean and standard deviation of local background intensities, and the number of beads both before and after outliers have been excluded.\n\nEach array type is also associated with a manifest file (with file extension BPM or BGX) that provides details of probe sequences, intended genomic targets and whether it is a control probe or not, information that is necessary to correctly interpret the data.\n\n\nImplementation\n\nilluminaio is an R package9. The reading of IDAT files is achieved using the readIDAT function. This routine is able to determine the type of IDAT file that has been passed and calls the appropriate code to read the file and return the data as a R list object (Figure 1). This not only contains intensity data, but also the meta information such as scan date that are not routinely extracted and can be useful for detecting batch effects10.\n\nDecryption of expression IDATs is performed using the open-source DES decryption routine available in Gnulib11. There is no official documentation of this file format, but illuminaio includes a document describing our findings in detail. Source code for the appropriate routines has been adapted and included in illuminaio, removing any requirement for specific external libraries to be installed on a user’s computer. Thus the package can be built and run on all three major operating systems (Linux, Windows and Mac).\n\nThe illuminaio package also supports the parsing of non-encrypted IDAT files compressed by gzip and the reading of manifest files describing the array design (readBGX and readBPM).\n\nThe summarised intensity values obtained by illuminaio are nearly identical to those reported using GenomeStudio. Small discrepancies related to rounding performed by GenomeStudio are observed. The package vignette contains a detailed comparison. The time taken to read an IDAT depends on the platform, with encrypted expression arrays taking around 1 second per file (for 50,000 probes), and methylation and SNP platforms between 1 to 6 seconds depending on the chip density (which can range between a few hundred thousand and several million probes).\n\n\nDiscussion\n\nThe availability of an open-source IDAT reader through illuminaio that can read files from any of Illumina’s BeadArray technologies will promote greater use of the IDAT file as a primary data format in the analysis and sharing of results from BeadArray based profiling studies. The illuminaio package is intended for use by developers to efficiently extract the content of both IDAT and bead-manifest files, thereby expanding the possibilities for conducting reproducible research with these data.\n\nOne exception to the dearth of IDAT files noted in the introduction is the The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov/). IDAT files from Illumina methylation and genotyping arrays are available in large numbers as Tier 1 data from the TCGA website (https://tcga-data.nci.nih.gov/tcga/). Of particular interest is the Illumina 450k methylation array, for which Triche et al.7 has shown improvements in background correction by using out-of-band probes, information that is only available through IDAT files and not the GenomeStudio output. For this work Triche et al. used illuminaio to access the out-of-band probes, which shows the advantage of having access to low-level data.\n\nilluminaio is currently used in the minfi12, methylumi13 and crlmm14,15 packages for importing IDAT files from the Infinium methylation and genotyping platforms respectively, demonstrating its utility.\n\n\nSoftware availability\n\nilluminaio is an R package available from the Bioconductor project (http://www.bioconductor.org) and from 10.5281/zenodo.7588.",
"appendix": "Author contributions\n\n\n\nKAB developed the first version of the IDAT reader for unencrypted IDAT files. This work was later improved by HB, MER and KDH. MLS developed the IDAT reader for encrypted files. All authors wrote and approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the European Community’s Seventh Framework Programme under grant agreement No. 305626 (Project RADIANT) (MLS), NHMRC Project grant 1050661, Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS (MER) and Grant Number R01 GM103552 from the National Institute of General Medical Sciences, National Institutes of Health (KDH).\n\n\nReferences\n\nBolstad BM: affyio: Tools for parsing Affymetrix data files. R package version 1.30.0. Reference Source\n\nBengtsson H, Bullard J, Hansen KD: affxparser: Affymetrix File Parsing SDK, R package version 1.34.0. 2013. Reference Source\n\nDunning MJ, Smith ML, Ritchie ME, et al.: beadarray: R classes and methods for Illumina bead-based data. Bioinformatics. 2007; 23(16): 2183–2184. PubMed Abstract | Publisher 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\nSmyth GK: Limma: linear models for microarray data. In R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, and W. Huber, editors, Bioinformatics and Computational Biology Solutions Using R and Bioconductor, 2005; 397–420. Springer, New York. Reference Source\n\nReich M, Liefeld T, Gould J, et al.: GenePattern 2.0. Nat Genet. 2006; 38(5): 500–501. PubMed Abstract | Publisher Full Text\n\nTriche TJ Jr, Weisenberger DJ, Van Den Berg D, et al.: Low-level processing of Illumina Infinium DNA Methylation BeadArrays. Nucleic Acids Res. 2013; 41(7): e90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nR Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2013. Reference Source\n\nLeek JT, Scharpf RB, Bravo HC, et al.: Tackling the widespread and critical impact of batch effects in high-throughput data. Nat Rev Genet. 2010; 11(10): 733–739. PubMed Abstract | Publisher Full Text\n\nGNU Project. Gnulib - The GNU Portability Library, 2013. Reference Source\n\nHansen KD, Aryee M: minfi: Analyze Illumina’s 450k methylation arrays, 2013. R package version 1.8.3. Reference Source\n\nDavis S, Du P, Bilke S, et al.: methylumi: Handle Illumina methylation data, R package version 2.8.0. 2013. Reference Source\n\nRitchie ME, Carvalho BS, Hetrick KN, et al.: R/Bioconductor software for Illumina’s Infinium whole-genome genotyping BeadChips. Bioinformatics. 2009; 25(19): 2621–2623. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScharpf RB, Irizarry RA, Ritchie ME, et al.: Using the R Package crlmm for Genotyping and Copy Number Estimation. J Stat Softw. 2011; 40(12): 1–32. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "3094",
"date": "27 Jan 2014",
"name": "Jan Oosting",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describes illuminaio, an R package to process the raw data files produced by the Illumina scanning software. This tool is valuable, because it enables researchers to use a completely open analysis workflow, without having to use a closed source, blackbox, analysis step.I have tried the package on IDAT files from several chiptypes, and only files from the beginning of 2005 scanned with beadscan 2.2 were rejected. Files scanned with beadscan 2.3 (Nov 2005) and onwards were processed properly.",
"responses": [
{
"c_id": "691",
"date": "06 Feb 2014",
"name": "Kasper Daniel Hansen",
"role": "Author Response",
"response": "Dr. Oosting,Thank you for your review. I am especially happy that you have taken the effort to apply illuminaio on output from older versions of beadscan.We have contacted Dr. Oosting offline and have received a number of files from old scans. We have started implementing support for these older files, and initial results are promising. We expect to release a new version of illuminaio shortly, which will read these files.Best,Kasper D. Hansen"
}
]
},
{
"id": "3091",
"date": "29 Jan 2014",
"name": "Tiffany J. 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 manuscript written by Smith et al. presents an R package to process IDAT files, the file format of Illumina BeadArrays. The package illuminaio fills an important gap in data processing by enabling the analysis of Illumina data without Illumina software. The gap is illustrated when the authors cite the disparity between the availability of CEL files (Affymetrix file format) and IDAT files in public data repositories. In addition to eliminating the need to use Illumina software, illuminaio extracts additional information from the IDAT files allowing improved quality control analyses and additional data correction algorithms. Figure 1 of the manuscript illustrates the simplified analysis pipeline enabled by illuminaio. The authors briefly describe the IDAT file format that was not publically documented before illuminaio. Figure 2 of the manuscript summarises the details of the file format for each of the BeadArray platforms. As such this is not just the development of a new tool, as the authors have provided thorough documentation for a widely used platform. In addition, the vignette accompanying illuminaio clearly explains the packages methods and how to use them. This package, illuminaio, has already led to the development of many new analysis packages that utilise IDAT files and the additional information they provide. In turn these packages are leading to the increased exchange and availability of the raw IDAT files in collaborations and data repositories.",
"responses": [
{
"c_id": "690",
"date": "06 Feb 2014",
"name": "Kasper Daniel Hansen",
"role": "Author Response",
"response": "Dr. Morris,Thank you for your positive review. We are happy that you find our tool useful.Best,Kasper D. Hansen"
}
]
}
] | 1
|
https://f1000research.com/articles/2-264
|
https://f1000research.com/articles/2-180/v1
|
09 Sep 13
|
{
"type": "Short Research Article",
"title": "Long term delivery of pulsed magnetic fields does not improve learning or alter dendritic spine density in the mouse hippocampus",
"authors": [
"Matthew Sykes",
"Kalina Makowiecki",
"Jennifer Rodger",
"Matthew Sykes",
"Kalina Makowiecki"
],
"abstract": "Repetitive transcranial magnetic stimulation (rTMS) is thought to facilitate brain plasticity. However, few studies address anatomical changes following rTMS in relation to behaviour. We delivered 5 weeks of daily pulsed rTMS stimulation to ephrin-A2-/- and wildtype mice (n=10 per genotype) undergoing a visual learning task and analysed learning performance, as well as spine density, in the dentate gyrus molecular and CA1 pyramidal cell layers in Golgi-stained brain sections. We found that neither learning behaviour, nor hippocampal spine density was affected by long term rTMS. Our negative results highlight the lack of deleterious side effects in normal subjects and are consistent with previous studies suggesting that rTMS has a bigger effect on abnormal or injured brain substrates than on normal/control structures.",
"keywords": [
"Repetitive transcranial stimulation (rTMS) generates electrical currents in the brain by electromagnetic induction and has been shown to induce synaptic plasticity in human and animal models1. Importantly",
"rTMS induces long term potentiation (LTP) in rodent hippocampus in vitro2 and several sessions of high-frequency rTMS increases the capacity to induce LTP (metaplasticity) compared to untreated controls3",
"4. Because rTMS acts on the same plasticity mechanisms as learning and memory",
"it has been hypothesised that rTMS may serve as a \"priming\" mechanism to facilitate long-term synaptic and structural modifications5",
"6. The implication is that repeated rTMS stimulation sets up a \"plastic\" brain state that is conducive to long term functional and structural changes5. For this reason",
"rTMS is being explored in combination with behavioural training tasks to see whether it can be used to prime or improve learning and cognitive performance in humans7",
"8. However",
"the potential mechanisms whereby rTMS might accelerate learning remain unknown."
],
"content": "Introduction\n\nRepetitive transcranial stimulation (rTMS) generates electrical currents in the brain by electromagnetic induction and has been shown to induce synaptic plasticity in human and animal models1. Importantly, rTMS induces long term potentiation (LTP) in rodent hippocampus in vitro2 and several sessions of high-frequency rTMS increases the capacity to induce LTP (metaplasticity) compared to untreated controls3,4. Because rTMS acts on the same plasticity mechanisms as learning and memory, it has been hypothesised that rTMS may serve as a \"priming\" mechanism to facilitate long-term synaptic and structural modifications5,6. The implication is that repeated rTMS stimulation sets up a \"plastic\" brain state that is conducive to long term functional and structural changes5. For this reason, rTMS is being explored in combination with behavioural training tasks to see whether it can be used to prime or improve learning and cognitive performance in humans7,8. However, the potential mechanisms whereby rTMS might accelerate learning remain unknown.\n\nDeclarative and spatial learning tasks are strongly associated with the hippocampus. More specifically, hippocampal dendritic spines have been identified as the likely loci of activity-dependent synaptic plasticity and possible structural correlates of memory and learning9,10. High dendritic spine density in hippocampal neurons is associated with learning ability and higher performance on cognitive tasks11–14. Furthermore, LTP and LTD induce structural changes in dendritic spines, with LTP induced electrically or via learning increasing dendritic length and spine density15,16. Because higher spine density is associated with higher spine mobility and turnover rates15,16, this measure is thought to reflect a greater capacity for synaptic reorganisation.\n\nTo date, the only study to examine changes in dendritic spines after rTMS did so following a single stimulation and showed no change in spine density, although the size of the smallest spines was increased2. Therefore, very little has been done to investigate the impact of long term rTMS on spine density in the hippocampus, or how it might interact with the learning process. Given the significant structural changes induced in the mouse visual system following repeated stimulation sessions, we hypothesised that a similar long-term rTMS regime in combination with a hippocampal learning task, would rescue impaired learning strategies previously found in ephrin-A2-/- mice17 and alter spine density in the hippocampus.\n\nWe delivered 5 weeks of daily pulsed rTMS stimulation to ephrin-A2-/- and wildtype mice undergoing a visual learning task and analysed learning performance, as well as spine density in the dentate gyrus molecular and CA1 pyramidal cell layers in Golgi-stained material. Although the mice learned the task, their performance remained suboptimal due to lack of motivation to obtain food rewards through insufficient food restriction18 and neither learning behaviour, nor hippocampal spine density were affected by long term rTMS. Our negative results are consistent with previous data showing that rTMS has a selective effect on abnormal or injured brain circuitry19, and the lack of deleterious side effects observed in normal human subjects8,20.\n\n\nMethods\n\nThis experiment used 10 wildtype (C57Bl/6J) and 10 ephrin-A2-/- knockout mice, with equal number of males and females. Wildtype mice were purchased from Animal Research Centre (Canning Vale, WA, Australia). Ephrin-A2-/- mice were a generous gift from David Feldheim21 and carry a homozygous null mutation of the ephrin-A2 gene. Ephrin-A2-/- mice were bred from heterozygous parents at the Biomedical Research Facility (The University of Western Australia) and backcrossed for >10 generations on a C57Bl/6J background. Mice were genotyped at weaning, as described previously21. Mice were age matched, aged 8–10 weeks old when commencing the experiment. For the duration of the study, mice were kept in standard caging in a controlled environment (12/12 light/dark cycle; temperature 22°C±2°C, separated into cages with clear plastic walls (17 cm x 19 cm base, 16 cm high) based on sex and genotype (2–4 per cage)). Food restriction began two days prior to commencing training. This aimed to reduce mice to 90% of their free-feeding body weight. Mice were weighed daily and food intake adjusted using a daily-based controlled diet to reach and maintain target body weights and ensure animals remained healthy. Water was available ad libitum throughout the experiment. All procedures in this study were conducted in accordance with US NIH guidelines and approved by The University of Western Australia Animal Ethics Committee.\n\nMice completed a visual discrimination task in two phases. Mice were initially rewarded for one stimulus (‘learning phase’). After mice learned the task (defined as 75% correct responses for three consecutive days), the rewarded stimulus was switched to the opposite, previously incorrect stimulus (‘reverse phase’). rTMS was applied (as described below) for 10 minutes daily immediately following the task during the reverse phase. Because most mice in the study failed to learn the reverse task, we decided to focus on the relationship between rTMS and dendritic spine density, therefore, mice were terminally anesthetised with pentobarbitone (Lethabarb, Virbac Australia, 160 mg/kg, i.p.) 24 hours after 35 days of rTMS so that all mice received the same amount of stimulation.\n\nThe visual discrimination task was carried out using a Y-maze fitted into a 50 cm2 box, with visual stimuli at each end of the Y maze arms (25 cm long)18. Stimuli consisted of two 5 cm2 laminated black and white striped cards at 0.37 cycles per degree oriented horizontally or vertically. Both genotypes are capable of distinguishing this spatial frequency22. The position of the horizontal and vertical stimuli (left vs. right maze arm) was randomly altered across 30 trials with the constraint of equal number of trials in right and left arms. The 30 trial schedule changed each day, repeating every seven days. Random allocation determined which stimulus was rewarded in the learning phase, with the constraint that half the mice in each genotype received rewards for the horizontal and half for vertical. The rewarded stimulus was also counter-balanced across cage groups (i.e. mice housed together were rewarded for opposite stimuli) and sex. Inferential statistics confirmed no significant performance differences between sexes or stripe orientation first rewarded (Data File). Mice were placed at the start of the Y-maze, and received a peanut butter reward immediately after approaching the correct stimulus. If mice did not approach a stimulus after 30 seconds, the trial was deemed a non-response (included in analyses as an incorrect response). Each mouse completed 30 trials per day, in a single session. The reverse phase commenced the day after mice reached criterion performance (75% correct for three consecutive days). In the reverse phase the opposite, previously incorrect stimulus was rewarded. All other aspects of the reverse task were identical to the initial learning phase.\n\nTo deliver rTMS, we built a small coil created for mice (0.25 mm copper wire (Jaycar, Australia) 300 windings, 16 Ω, outer diameter 8 mm)19. The coil was designed to ensure a similar coil-to brain ratio as is used for induction of focal electric fields in humans23 and was driven by an electromagnetic pulse generator (Global Energy Medicine, Australia) that delivered a magnetic field of 10 mT. This relatively low intensity was imposed by the constraints of the coil’s size but had the benefit of allowing us to evaluate the effects of stimulation without the confounding factors of stimulation-induced movement or the use of anaesthetic or restraint, with their associated changes to neuronal excitability and circulating stress hormones24. Furthermore, low intensity magnetic fields are clinically relevant for two reasons. Firstly, in humans, fields in the millitesla range delivered to the brain induce analgesia25–27, and alleviate depression28. Secondly, even though traditional rTMS is considered to be focal, magnetic fields of lower intensity are delivered outside of the focal area, raising the possibility that low intensity stimulation may be contributing to therapeutic effects by acting on interconnected brain regions.\n\nWe chose a complex pattern of stimulation because it has been shown to induce structural changes in mice19 (59.9-ms trains of 20 pulses at 3 different frequencies as follows: 1 min warm-up at 6.71 Hz, 8 min treatment at 10.1 Hz, and 1 min cool down at 6.26 Hz. The pulse duration was 200 µs, which is within the range used in human rTMS. A Hall device probe (Jaycar, Australia inserted into different brain regions of a euthanized mouse estimated that the dorsal hippocampus received roughly 6 mT. The surface temperature of the coil was measured after 10 min of stimulation and did not exceed 35°C.\n\nAs mice had completed the initial learning phase of the visual discrimination task before commencing rTMS they were accustomed to handling and remained relatively still without restraint. This allowed the stimulation coil to be held by the experimenter above, but not in contact with, the top of the animal’s head for reproducible rTMS delivery in the alert animal (as for cat studies29). Consistent with the low intensity of the magnetic field, mice did not display any head-eye or gross motor movements, nor altered behaviour in response to the stimulation. Sham stimulation involved the same procedure but with the stimulator switched off. This control was chosen as our coil did not produce any audible sound19.\n\nTerminally anesthetised mice were transcardially perfused with 4% paraformaldehyde (Sigma Aldrich; Montana USA); Right hemispheres of brains underwent a silver impregnation staining protocol, and a Golgi stain (according to manufacturer’s instructions: FD NeuroTechnologies, Maryland, USA), which allows the visualisation of morphology on a subset of neurons30. Briefly, hemispheres were incubated in the dark in solutions A+B for 8 days with a change into fresh solution after the first 24 hours. Hemispheres were then incubated in solution C for 4 days with a change into fresh solution after 2 days. The impregnated hemispheres were then cryosectioned at 100 µm on a Leica Cryostat CM1900 at -19°C, mounted onto glass slides (Thermo Fisher Scientific, Australia) subbed with 0.5% gelatin (Sigma Aldrich, Montana USA). Sections were dried in the dark for 2–7 days, washed in distilled water and developed in solution D+E for 10 minutes. Sections were dehydrated in increasing concentrations of ethanol and mounted in Entilin (Merck, Darmastadt Germany).\n\nSlides were analysed by a researcher blinded to stimulation condition and genotype. Sections were photographed by an Olympus DP70 digital camera at a 4× objective zoom, which encompassed the entire section. We analysed dendrites that could definitively be attributed to cells in the CA1 pyramidal and the molecular dentate layer of the hippocampus because dendritic spines on these cells have previously shown changes in dendritic spine density in response to various interventions31. Dendrites were deemed suitable for analysis if they had a relatively flat orientation and were uniformly and strongly stained. Photographs of the dendritic arbour were taken throughout multiple planes ensuring the entire arbour was photographed in focus for later image analysis. Between 2 and 12 cells were analysed per animal and values averaged within animals for statistical analysis. Due to variability in Golgi staining, the number of dendrites counted varied between regions (CA1 pyramidal layer number of cells, Mean = 5; dentate molecular layer, Mean = 4.4).\n\nThe images for each dendritic arbour were combined into a single image, using the Image J plug-in \"Stack Focuser\" and dendrite length and number of dendritic spines counted using the Image J (\"Cell counter\" plug-in). The number of spines and the dendrite length data were then used to calculate a dendritic spine density value, defined as the number of spines per unit length (10 µm).\n\nWe examined the effect of long-term rTMS on reverse learning performance in ephrin-A2-/- and wildtype mice. Inferential statistics confirmed no pre-existing differences between groups in the learning phase, before commencing rTMS (data not shown). The first day of reverse phase training was excluded from analyses as rTMS commenced after this training session. Two mice reached the learning criterion in the reverse phase and were terminally anesthetised before 35 days (one wildtype and one ephrin-A2-/-, both received sham stimulation (negative/handling controls)) thus there was non-random reduction in sample size over time, precluding use of daily performance measures in statistical analyses. To overcome this problem, data were divided into three blocks for each subject, with one third of total days training included in each block, reflecting early, middle, and late stages of the reverse learning phase. Mean percentage correct was analysed by a two-way mixed ANOVA to assess differences between stimulation conditions (rTMS vs. sham) and between genotypes (ephrin-A2-/- vs. wildtype) and changes over time (changes between early, middle and late blocks). As circuitry abnormalities and connections between measured hippocampal regions have not been characterised in ephrin-A2-/- mice, it is unknown whether these measures should be considered independent for statistical analyses. Accordingly, a MANOVA was conducted to assess effects of stimulation condition (rTMS vs. sham) and genotype (wildtype vs. ephrin-A2-/-) on dendritic spine densities in both regions. Pillai’s Trace (V) was used as the multivariate test statistic. Follow-up ANOVAs were conducted separately for each region, testing the same factors as used in the MANOVA. The F-test statistic (F) and probability (p) values are reported for each ANOVA. When the assumption of sphericity was violated degrees of freedom were adjusted with Greenhouse-Geisser correction. Data were analysed using SPSS statistics software (IBM Corporation, New York, USA, v.20).\n\n\n\n\nResults\n\nAs shown in Figure 1, groups had similar means in each block, with no significant difference between rTMS and sham, F (1, 16) = 0.28, p = 0.60, nor between genotypes, F (1, 16) = 0.86, p = 0.37, and no significant interactions across blocks (all p values >0.05). Within groups, the percentage correct increased significantly across blocks, indicating that mice adjusted to the rule reversal and successfully learned the task although this was not to the desired criterion (F (1.43, 22.87) = 71.80, p < 0.001 (Greenhouse-Geisser corrected)).\n\nEarly, middle (mid) and late blocks were delineated by the first, second and final third of total number of days training in the reverse-learning task. Within groups, scores increased significantly between blocks (p <0.001), but there were no significant differences between rTMS and sham or between genotypes (p values >0.05; ANOVA). Error bars = SEM.\n\nSpine density (spine number/10 µm) measures were obtained for each dendrite and averaged within mice. Figure 2 presents mean dendritic spine density in CA1 pyramidal layer and dentate molecular layer for ephrin-A2-/- and wildtype mice, contrasting rTMS to sham. There was a slight (non-significant) trend towards rTMS increasing spine density in ephrin-A2-/- mice in both regions. In wildtypes rTMS appeared to have no effect on spine densities in CA1 pyramidal cells, as means were almost identical. However, wildtypes showed a similar (non-significant) trend to ephrin-A2-/- mice in the dentate molecular layer.\n\n(A) Mean dendritic spine density (number of spines per 10 μm) for hippocampal regions: CA1 pyramidal layer and dentate gyrus molecular layer (DG Molecular). There were no significant effects of rTMS nor genotype on spine densities in either region (p values >0.05; ANOVA). Error bars = SEM. (B) Right hemisphere Golgi stained section of dorsal hippocampus representative of those used in analyses. Scale bar represents 500 μm. (C) Dendrite representative of those selected for analysis, with spines visible. Scale bar represents 10 μm.\n\nMANOVA, using Pillai’s trace showed there was no significant effect of stimulation condition, V = 0.13, F (2, 9) = 0.67, p = 0.54, nor genotype, V = 0.08, F (2, 9) = 0.39, p = 0.69, on spine densities. Follow up two-way ANOVAs were also performed separately for each region, with no significant differences between stimulation conditions or between genotypes in either region, all p values were >0.05 (CA1 pyramidal layer: stimulation condition, F (1, 10) = 0.03, p = 0.86; genotype, F (1, 10) = 0.86, p = 0.38. Dentate molecular layer: stimulation condition, F (1, 10) = 1.03, p = 0.34; genotype, F (1, 10) = 0.42, p = 0.53).\n\n\nDiscussion\n\nWe investigated the effects of long-term daily rTMS on learning and hippocampal dendritic spine density using ephrin-A2-/- mice and wildtype controls. We show that rTMS had no significant effect on learning and no significant effect on hippocampal dendritic spine densities. Although ephrin-A2-/- mice have abnormal brain circuitry and associated abnormal behaviours, in the present study, the previously reported learning deficit17 was not observed due to low levels of food deprivation18. The absence of observed behavioural and structural change is consistent with previously reported rTMS specificity for abnormal systems8,19. Furthermore, the lack of adverse effects in our long term study contributes evidence that rTMS is safe to use in healthy control participants20.\n\nWe originally hypothesized that rTMS would rescue the learning strategy deficit in ephrin-A2-/- mice17 with minimal or no effect on wildtype mice. However, both genotypes failed to demonstrate the strategy deficit, due to insufficient food restriction18. Our results nonetheless indicate that rTMS does not adversely affect performance, but nor does it improve motivation or accelerate learning when deficits are absent. This is consistent with previous reports that long-term rTMS effects are specific to abnormal brain circuitry: two weeks of rTMS improved visual tracking, visual electrophysiological function and topographical accuracy in mice with abnormal circuitry but produced no lasting effects in wildtype mice19. Human studies also support specificity of rTMS for abnormal brain circuits, as a meta-analysis of rTMS effects on cognitive performance found patients tend to improve more than healthy participants8. Although some human studies using healthy participants show a single-session of rTMS enhances cognitive task performance, such as analogous reasoning32 and reaction time7, results are mixed, with other studies showing no effect of rTMS on knowledge acquisition33 or accuracy in a go/no-go task34. To our knowledge, there have been no studies assessing cognitive effects of long-term rTMS in patients and healthy controls, which presents a large gap in knowledge. Although more research is needed, our results support that rTMS is safe to use in healthy control participants, invaluable in assessment of rTMS effects clinically.\n\nEach mouse received a controlled amount of daily rTMS, allowing us to investigate how long-term rTMS combined with daily training influences spine density. Importantly, we found similar spine densities in sham wildtype and ephrin-A2-/- mice, suggesting that if present, deficits in spines are subtle in ephrin-A2-/- mice. Although ephrin-A2 is expressed in the mouse hippocampus throughout life and has been implicated in its topographic organisation35,36, to our knowledge, there is no evidence that ephrin-A2 is involved in synaptic plasticity or spine dynamics. Rather, these processes involve ephrin-A3, ephrin-A5 and members of the ephrin-B family37. As such, the null effect of rTMS on dendritic spine density is in line with our behavioural results and may be attributed to the absence of a specific spine deficit for rTMS to correct in both wildtype and ephrin-A2-/- mice. It will be important to examine other brain regions to determine whether the selectivity of rTMS for normal and abnormal brain circuits is also observed.\n\nOur result that spine density was not significantly altered is in agreement with a previous study, showing no change in spine density in CA1 pyramidal neurons following a single rTMS stimulation2. However, it is surprising that dendritic spine density remains unaffected after long-term stimulation, given our previous results using the same stimulation parameters, demonstrating structural reorganisation in abnormal axon terminals following multiple, but not single rTMS stimulation sessions19. As neither long-term nor short-term rTMS results in dendritic spine density changes, these negative results may suggest different susceptibility of axons and dendrites to rTMS. The functional characteristics of these neuronal compartments require different expression of ion channels and growth factor receptors38, which could provide a molecular basis for differential rTMS effects on excitability and spatially localised structural and functional change.\n\nAn alternative interpretation of our null finding is that our rTMS treatment changes spine dynamics without affecting their final density, a result that would not be possible to detect in our fixed post-mortem tissue. Hence, these results highlight the limitations in Golgi staining of fixed tissue, a technique still commonly used in examining dendritic spine density. Sensory manipulation (either enrichment or withdrawal) strongly alters spine dynamics in vivo in various areas of the cortex of adult mice39. As a rule, established spines are pruned during the initial experience of the new stimulus, while new ones are established, which may result in some studies of fixed tissue showing no net change in spine density40,41. Consistent with the change in spine dynamics initiated by enrichment, a recent imaging study in the hippocampus identified two phases in spine dynamics following repeated induction of LTP. Initially both generation and retraction of spines increased, followed by a cessation of spine retraction42. This is consistent with post-mortem studies showing an initial period of apparent spine stability, followed by a detectable increase in density. The possibility that rTMS changes spine dynamics, as opposed to density, is further supported by an increase in the size of small spines following a single stimulation, which the authors suggested may indicate the activation of silent synapses by membrane recruitment of AMPA receptors, precluding the need for de novo synapse generation2. Future live imaging studies of spine dynamics in animals that have received single or multiple rTMS stimulation, potentially in combination with learning tasks will provide much needed insight into the mechanisms underpinning the plastic changes elicited by rTMS in humans.",
"appendix": "Author contributions\n\n\n\nJR conceived the study, JR and KM designed the experiments, MS and KM carried out the research and analysed results. JR and KM wrote the manuscript and all authors were involved in the revision of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nExperiments were funded by project funds to KM from the University of Western Australia (School of Animal Biology). Aspects of the project were funded by grants to JR from the NHMRC Australia (no 634386) and the Neurotrauma Program of Western Australia. JR is a NHMRC Senior Research Fellow (APP1002258).\n\n\nAcknowledgements\n\nWe are grateful to Marissa Penrose for technical assistance and Leah Attwood for expert animal care. We thank Global Energy Medicine (Australia) for providing pulse generators and the complex frequency pattern.\n\n\nReferences\n\nPell GS, Roth Y, Zangen A: Modulation of cortical excitability induced by repetitive transcranial magnetic stimulation: Influence of timing and geometrical parameters and underlying mechanisms. Prog Neurobiol. 2011; 93(1): 59–98. PubMed Abstract | Publisher Full Text\n\nVlachos A, Muller-Dahlhaus F, Rosskopp J, et al.: Repetitive magnetic stimulation induces functional and structural plasticity of excitatory postsynapses in mouse organotypic hippocampal slice cultures. J Neurosci. 2012; 32(48): 17514–23. PubMed Abstract | Publisher Full Text\n\nOgiue-Ikeda M, Kawato S, Ueno S: The effect of repetitive transcranial magnetic stimulation on long-term potentiation in rat hippocampus depends on stimulus intensity. Brain Res. 2003; 993(1–2): 222–6. PubMed Abstract | Publisher Full Text\n\nTokay T, Holl N, Kirschstein T, et al.: High-frequency magnetic stimulation induces long-term potentiation in rat hippocampal slices. Neurosci Lett. 2009; 461(2): 150–4. PubMed Abstract | Publisher Full Text\n\nMozzachiodi R, Byrne JH: More than synaptic plasticity: role of nonsynaptic plasticity in learning and memory. Trends Neurosci. 2010; 33(1): 17–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLisman J: A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory. Proc Natl Acad Sci U S A. 1989; 86(23): 9574–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEvers S, Bockermann I, Nyhuis PW: The impact of transcranial magnetic stimulation on cognitive processing: an event-related potential study. Neuroreport. 2001; 12(13): 2915–8. PubMed Abstract | Publisher Full Text\n\nGuse B, Falkai P, Wobrock T: Cognitive effects of high-frequency repetitive transcranial magnetic stimulation: a systematic review. J Neural Transm. 2010; 117(1): 105–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarms KJ, Dunaevsky A: Dendritic spine plasticity: looking beyond development. Brain Res. 2007; 1184: 65–71. PubMed Abstract | Publisher Full Text\n\nCalverley RK, Jones DG: Contributions of dendritic spines and perforated synapses to synaptic plasticity. Brain Res Brain Res Rev. 1990; 15(3): 215–49. PubMed Abstract | Publisher Full Text\n\nMoser MB, Trommald M, Egeland T, et al.: Spatial training in a complex environment and isolation alter the spine distribution differently in rat CA1 pyramidal cells. J Comp Neurol. 1997; 380(3): 373–81. PubMed Abstract | Publisher Full Text\n\nKolb B, Cioe J, Comeau W: Contrasting effects of motor and visual spatial learning tasks on dendritic arborization and spine density in rats. Neurobiol Learn Mem. 2008; 90(2): 295–300. PubMed Abstract | Publisher Full Text\n\nAvila-Costa MR, Colin-Barenque L, Fortoul TI, et al.: Memory deterioration in an oxidative stress model and its correlation with cytological changes on rat hippocampus CA1. Neurosci Lett. 1999; 270(2): 107–9. PubMed Abstract | Publisher Full Text\n\nWallace M, Luine V, Arellanos A, et al.: Ovariectomized rats show decreased recognition memory and spine density in the hippocampus and prefrontal cortex. Brain Res. 2006; 1126(1): 176–82. PubMed Abstract | Publisher Full Text\n\nMcKinney R, Thompson S: Glutamate Regulation of Dendritic Spine Form and Function. In: Larry R editor. Encyclopedia of Neuroscience. Oxford: Academic Press; 2009; 905–11.\n\nDur-e-Ahmad M, Imran M, Gul A: Calcium dynamics in dendritic spines: a link to structural plasticity. Math Biosci. 2011; 230(2): 55–66. PubMed Abstract | Publisher Full Text\n\nArnall S, Cheam LY, Smart C, et al.: Abnormal Strategies During Visual Discrimination Reversal Learning in ephrin-A2-/- Mice. Behav Brain Res. 2010; 209(1): 109–13. PubMed Abstract | Publisher Full Text\n\nMakowiecki K, Hammond G, Rodger J: Different levels of food restriction reveal genotype-specific differences in learning a visual discrimination task. PLoS ONE. 2012; 7(11): e48703. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodger J, Mo C, Wilks T, et al.: Transcranial pulsed magnetic field stimulation facilitates reorganization of abnormal neural circuits and corrects behavioral deficits without disrupting normal connectivity. Faseb J. 2012; 26(4): 1593–606. PubMed Abstract | Publisher Full Text\n\nRossi S, Hallett M, Rossini PM, et al.: Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research. Clin Neurophysiol. 2009; 120(12): 2008–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeldheim DA, Kim Y-I, Bergemann AD, et al.: Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping. Neuron. 2000; 25(3): 563–74. PubMed Abstract | Publisher Full Text\n\nHaustead DJ, Lukehurst SS, Clutton GT, et al.: Functional topography and integration of the contralateral and ipsilateral retinocollicular projections of ephrin-A-/- mice. J Neurosci. 2008; 28(29): 7376–86. PubMed Abstract | Publisher Full Text\n\nWeissman JD, Epstein CM, Davey KR: Magnetic brain stimulation and brain size: relevance to animal studies. Electroencephalogr Clin Neurophysiol. 1992; 85(3): 215–9. PubMed Abstract | Publisher Full Text\n\nGersner R, Kravetz E, Feil J, et al.: Long-term effects of repetitive transcranial magnetic stimulation on markers for neuroplasticity: differential outcomes in anesthetized and awake animals. J Neurosci. 2011; 31(20): 7521–6. PubMed Abstract | Publisher Full Text\n\nShupak NM, Prato FS, Thomas AW: Human exposure to a specific pulsed magnetic field: effects on thermal sensory and pain thresholds. Neurosci Lett. 2004; 363(2): 157–62. PubMed Abstract | Publisher Full Text\n\nRobertson JA, Theberge J, Weller J, et al.: Low-frequency pulsed electromagnetic field exposure can alter neuroprocessing in humans. J R Soc Interface. 2010; 7(44): 467–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCook CM, Thomas AW, Keenliside L, et al.: Resting EEG effects during exposure to a pulsed ELF magnetic field. Bioelectromagnetics. 2005; 26(5): 367–76. PubMed Abstract | Publisher Full Text\n\nMartiny K, Lunde M, Bech P: Transcranial low voltage pulsed electromagnetic fields in patients with treatment-resistant depression. Biol Psychiatry. 2010; 68(2): 163–9. PubMed Abstract | Publisher Full Text\n\nValero-Cabre A, Pascual-Leone A, Rushmore RJ: Cumulative sessions of repetitive transcranial magnetic stimulation (rTMS) build up facilitation to subsequent TMS-mediated behavioural disruptions. Eur J Neurosci. 2008; 27(3): 765–74. PubMed Abstract | Publisher Full Text\n\nSeress L, Pokorny J: Structure of the granular layer of the rat dentate gyrus. A light microscopic and Golgi study. J Anat. 1981; 133(Pt 2): 181–95. PubMed Abstract | Free Full Text\n\nvon Bohlen Und Halbach O: Structure and function of dendritic spines within the hippocampus. Ann Anat. 2009; 191(6): 518–31. PubMed Abstract | Publisher Full Text\n\nBoroojerdi B, Phipps M, Kopylev L, et al.: Enhancing analogic reasoning with rTMS over the left prefrontal cortex. Neurology. 2001; 56(4): 526–8. PubMed Abstract | Publisher Full Text\n\nRoth HL, Nadeau SE, Triggs WJ: Effect of repetitive transcranial magnetic stimulation on rate of memory acquisition. Neurology. 2004; 63(8): 1530–1. PubMed Abstract | Publisher Full Text\n\nHuang CC, Su TP, Shan IK, et al.: Effect of 5 Hz repetitive transcranial magnetic stimulation on cognition during a Go/NoGo task. J Psychiatr Res. 2004; 38(5): 513–20. PubMed Abstract | Publisher Full Text\n\nZhou R: Regulation of topographic projection by the Eph family receptor Bsk (EphA5) and its ligands. Cell Tissue Res. 1997; 290(2): 251–9. PubMed Abstract | Publisher Full Text\n\nGerlai R, Shinsky N, Shih A, et al.: Regulation of learning by EphA receptors: a protein targeting study. J Neurosci. 1999; 19(21): 9538–49. PubMed Abstract\n\nHruska M, Dalva MB: Ephrin regulation of synapse formation, function and plasticity. Mol Cell Neurosci. 2012; 50(1): 35–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArnold DB: Polarized targeting of ion channels in neurons. Pflugers Arch. 2007; 453(6): 763–9. PubMed Abstract | Publisher Full Text\n\nFu M, Zuo Y: Experience-dependent structural plasticity in the cortex. Trends Neurosci. 2011; 34(4): 177–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJung CK, Herms J: Structural Dynamics of Dendritic Spines are Influenced by an Environmental Enrichment: An In Vivo Imaging Study. Cereb Cortex. 2012. PubMed Abstract | Publisher Full Text\n\nXu T, Yu X, Perlik AJ, et al.: Rapid formation and selective stabilization of synapses for enduring motor memories. Nature. 2009; 462(7275): 915–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOe Y, Tominaga-Yoshino K, Hasegawa S, et al.: Dendritic spine dynamics in synaptogenesis after repeated LTP inductions: Dependence on pre-existing spine density. Sci Rep. 2013; 3: 1957. PubMed Abstract | Publisher Full Text | Free Full Text"
}
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[
{
"id": "1747",
"date": "26 Sep 2013",
"name": "Antoni Valero-Cabre",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 outstandingly planned and well-executed study that shows that the rodent brain, after a daily 10 minute exposure to low field pulsed magnetic stimulation for 5 weeks (i.e. 35 sessions) exhibits null effects on learning behavior and hippocampus dendritic spine density in both Wild Type and Ephrine A2-/- mice. The effort of the authors deserves a lot of credit, as studies like this one are extremely hard, long and laborious to perform and the possibility of significant results always uncertain. In spite of the current null results, this kind of work is greatly needed in the field of non-invasive brain stimulation in order to understand the cellular underpinnings of these techniques and assess the safeness and real therapeutic potential of stimulation technologies in clinical neurorehabilitation. Overall, the neurostimulation community should strongly thank the authors for investing their know-how, time and effort to carry over this type of animal research and we all should encourage them to pursue their efforts in the future.GENERAL POINTS The only limitation of the current manuscript is related to the difficulties in interpreting null outcomes; in this case a triple negative result. Not only did low field pulsed magnetic stimulation prove unable to modify visual learning behavior or hippocampus spine density in these two population of mice but neither of the two showed (as should have been at least expected) significant differences in any of these two measures. The authors do an excellent job going over the different possibilities that could explain this null pattern of outcomes, even if they do not have much basis to rule out any of the hypotheses in particular. Among other important issues, the study emphasizes once more the importance of task titration. Task titration is strongly related to animal motivation (and thus intake restriction) and is a key variable and therefore needs to be manipulated carefully to make sure that the potentially effective impact of magnetic pulses can be demonstrated behaviorally. With regards to the question however of whether or not increasing food restriction could have rendered, as the authors suggest, behavioral learning tests more sensitive, the answer remains unknown. It could also be that highly motivated animals would rapidly show ceiling learning effects and render the task less sensitive to modulation. To this reviewer, the performance of both groups of animals in the inversion phase of the task seems to show enough room for behavioral improvement, even after 5 weeks of stimulation; thus it is tempting to also speculate that such a complex task for a rodent (which relies on several cognitive processes such as visuo-spatial attention, visual perception, visual, spatial and declarative memory, associative rewarded learning, rule understanding and switching and decision making) may not have been well suited to the functions of the brain regions aimed at in this study and/or that their impact could have rapidly compensated for by other systems or regions.Adding to the authors’ arguments with regards to the lack of spine density effects, one could also argue that a direct impact of the stimulation (and not an indirect effect of the stimulation on learning behavior driving subsequent effects on memory related regions) on such measures could and should have occurred primarily in the cortical regions overlying the hippocampus (which have not been analysed in this study) rather than in the more deeply located hippocampus CA1 and DG neurons. Of course, if the targeted region theoretically related to the behavior that is being measured is not at least directly or indirectly impacted and modified in activity by the pulses, then no behavioral differences between sham and real rTMS patterns should be expected in either population of mice. All these explanations are directly or indirectly mentioned in the discussion and when not explicitly mentioned in the text, they emerge logically from the results. However any effort to nuance and expand such justifications even further would be welcome. To this reviewer, the only possibility (that would also be compatible with the negative outcomes of this study) that is not explicitly mentioned in the discussion is that the delivered low field pulsed magnetic patterns (even if estimated by means of a Hall device in postmortem brains as reaching intensities of ~6 mT in the hippocampus) were not effective at all. Several reasons that could account for that eventuality are; that the field was too weak to penetrate deep enough with a minimal intensity, imprecise widely distributed targeting or a field strongly attenuated by the space left, as indicated in the manuscript, between the stimulating coil and the top of the head. This reviewer cannot demonstrate that this possibility is more likely than any of the others already mentioned in the manuscript. Nonetheless, in the absence of any positive sign of a stimulatory impact, this possibility cannot be ruled out and should probably be briefly discussed. Additionally, it should be clear throughout the manuscript, that the ability of the current experiment to highlight the innocuity of 5 weeks of low field pulsed magnetic brain stimulation needs to be interpreted carefully and associated with the field strengths delivered by the modified e-cell device by Global Energy Medicine, which is far from the normal field strength for TMS/rTMS devices operated in humans for either research or therapeutic purposes. MINOR COMMENTS AND SUGGESTIONSIntroduction, Page 1, Col 1, Par 1, Line 6. Could the authors re-evaluate the adequacy of the term “metaplasticity” with regards to TMS-induced LTP effects, as to this reviewer, the main effect of high frequency stimulation are LTP or LTP-like effects. Metaplasticity is a consequence of forcing homeostatic plasticity beyond a particular boundary, which may result in paradoxical modulatory effects.Introduction, Page 1, Col 1, Par 2, Lines 7-9. With regards to this issue, in this particular study were the authors hypothesizing a direct impact of low field pulsed magnetic stimulation on specific cortical locations translated through connectivity into a declarative and spatial memory region such as the hippocampus, or a cortical effect of stimulation leading to subsequent changes in behavior that could leave a distinctive memory trace in the hippocampus?Introduction, Page 1, Col 1, Par 3. The paper by May et al. 2007, Cereb. Cortex, in which the effect of 5 straight days of rTMS on humans assessed with MRI methods and regional size increases (hypothetically attributed to increases in spine density) could be relevant for this introduction. Methods, Page 1, Col 2, Par 1, Lines 7-8. Could the authors provide an idea of how far in their development (if possible in age compared to humans) are 8-10 week old mice? It seems that in a prior study by the same authors that showed significant results after 14 days of low field rTMS on the visual cortex by Rodger et al. 2012, FASEB J, the mice were slightly younger (6-8 weeks). Is this period crucial in terms of postnatal development for mice? Could a less plastically sensitive brain of 8-10 week old mice vs. 6-8 week old mice explain the current null results? Please comment briefly in the manuscript.Methods, Page 2, Col 2, Par 2, Lines 6-7. Why were mice tested before the low field pulsed magnetic stimulation session and not also immediately thereafter, when the impact of the stimulation should have been stronger? Although I understand the authors sought a long-term effect, such a measure could have proven useful to reveal at least an immediate day-to-day impact of stimulation and become a proof of their efficacy? Given the order of events, one could be tempted to speculate the possibility of non-synergistic interaction between the lasting effects of task practice and immediately subsequent low field rTMS stimulation, cancelling the modulatory effects.Methods, Page 3, Col 1, Par 2, Lines 7-8. As this is not a standard rTMS stimulation device, some additional information on the stimulation source should be given to be able to compare the efficiency of low field pulsed magnetic stimulation with current human rTMS equipment. More specifically, what is the shape of the stimulating pulse (monophasic, biphasic?) and what is the rise time of the field? The pattern of stimulation used seems essentially an “excitatory” 10 Hz rTMS pattern, preceded and followed by short instances of ~6 Hz stimulation. Was this high frequency employed as a way to induce LTP phenomena in the hippocampus or as a tool to enhance cortical excitability and facilitate learning behavior leading then to functional and anatomical modulations of the hippocampus? Please comment briefly.Methods, Page 3, Col 1, Par 2, Lines 7-8. Navigation of the TMS coil of course could have been improved given the high-focality of the device used by the authors and the fact that any small head movements in a small rodent brain could easily lead to a completely different area of stimulation. It is true that prior studies in awake felines by our own group (see Valero-Cabré et al. 2006, Exp Brain, Valero-Cabre A et al. 2008, Eur J Neurosci.) have used a similar manual procedure, but it is also the case that the coils and brains in those studies were larger and precise location on a specific region of the posterior parietal cortex was guided day to day by stereotaxic based measures and references. In many instances, we additionally benefited from parietal bone transparency during surgery, and identified in each animal the sulci/gyral pattern and labelled the exact area of interest (see Valero-Cabré et al. 2005 Exp Brain Res, Valero-Cabré et al.2007 Exp Brain Res). Furthermore, in groups that were to be followed longitudinally, we placed a dental acrylic plug, that could be palpated through the skin of the scalp (see Afifi et al. 2012 Eur J Neurosci) which was then used as consistent localization marker. The correct positioning of those plugs with regards to the stimulated areas was often verified pre-treatment by anatomical MRI methods and also in post-mortem brain dissections at the end of the follow up. In the current study, given the elongated shape of the hippocampus in rodents, which part of the rodent cortex or which stereotaxic level was aimed at during the stimulation? Also, in contrast with the quoted feline study, the authors indicate here that the coil was not in direct contact with the scalp. As air is a strong isolator, could that have attenuated the strength of such a low intensity field even further? Were Hall probe field measures in the hippocampus performed with the stimulating cell also separated from the scalp?Discussion, Page 5, Col 1, Par 1, Lines 4-6. The statement “The absence of observed behavioral and structural change is consistent with previously reported rTMS specificity for abnormal systems” needs to be expanded upon as the Ephrin-A2-/- mice have indeed abnormal systems. Maybe a short comment on how to reconcile the current findings with those reported in Rodger et al. 2012, FASEB J, would be important as in that study by the same authors similar stimulation patterns were able to correct cortico-collicular connectivity only in Ephrin-A2-/- and not in Wild type mice, supporting the above-mentioned statement.Discussion, Page 5, Col 1, Par 1, Lines 6-8. I would strongly advise the authors to alter the contents of the sentence “Furthermore, the lack of adverse effects in our long term study contributes evidence that rTMS is safe to use in healthy control participants”. Could they rephrase to something like “Furthermore, the lack of adverse effects in our long term study suggests that up to 5 weeks of daily sessions (35 session) of low field pulsed magnetic stimulation at the parameters used in this study appears safe to use in healthy participants”. This is important as the intensity levels employed are very different of those employed in human rTMS, which are much higher, and this information could have some public health implications for future use of rTMS in humans.Discussion, Page 5, Col 2, Par 1, Lines 19-21. Please check the accuracy of the sentence “To our knowledge, there have been no studies assessing cognitive effects of long-term rTMS in patients and healthy controls, which presents a large gap in knowledge”Discussion, Page 5, Col 2, Par 1, Lines 21-22. As indicated above (comment 10), would the authors agree to rewrite the sentence “Although more research is needed, our results support that rTMS is safe to use in healthy control participants…” and revise the second part of it “… invaluable in assessment of rTMS effects clinically” which to this reviewer is confusing? Discussion, Page 5, Col 2, Par 2, Lines 5-8. This sentence can be a bit surprising to readers, why did the authors chose a knock out for Ephrine A2 in a study that aimed to analyze spine density in the hippocampus if as they affirm in this paragraph, this molecule is not involved in spine dynamics? Maybe the rational for that choice needs to be explained more carefully.",
"responses": []
},
{
"id": "2135",
"date": "21 Oct 2013",
"name": "Anthony Hannan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written paper describing the effects (or lack thereof) of rTMS over 5 weeks in wild-type and ephrin-a2 knockout (KO) mice. The data are clearly presented in a usable format and are discussed appropriately. The initial referee Antoni Valero-Cabre, who is an expert in TMS and neuro-stimulation, has already extensively covered the key points, both positive and negative. I am therefore only going to make a few additional comments.The title and abstract should ideally reflect the findings of the study more precisely, for example:‘Long-term delivery of pulsed magnetic fields does not alter visual discrimination learning or dendritic spine density in mouse CA1 pyramidal or dentate gyrus neurons’.The nature of such negative findings means that the authors cannot rule out potential significant effects of this rTMS protocol on other learning tasks or other classes of neurons/dendrites in the mouse hippocampus. Furthermore, the abstract should mention that they were adult mice and the background strain used was C57Bl/6J, as it is possible that different aged mice and/or a different genetic strain of mice might respond differently to the same rTMS protocol.One concern regarding the methods and design is that the authors appear to have used a separate wild-type mouse colony and compared them to ephrin-a2 KO mice inbred via a backcrossed colony. I strongly feel that all such experiments should always involve wild-type littermates randomised from age-matched litters to control for both genetic (e.g. sub-strain and genetic drift) and epigenetic differences between different colonies. This is particularly important for behavioural experiments where even subtle genetic and epigenetic differences can often have significant impacts. The authors report no significant gene effects, and it is therefore not a major confound in this case, however if randomised littermates were not used (as the methods imply) this should be noted.The discussion and conclusions are balanced. However, it might be worth noting that an extensive battery of behavioural tests (for example including sensory, cognitive, affective and motor protocols) on wild-type mice would be worth pursuing to assess whether this rTMS protocol (or others with different spatial and temporal specificities) has any effects on brain function analogous to human studies. If any positive results were found, then cellular, physiological and molecular follow-up studies could be targeted towards understand specific cognitive/behavioural effects of rTMS (thus nicely complementing human studies).If such comprehensive TMS animal studies are done but still show inconsistencies with matching human TMS studies then one possibility is that the animal TMS needs to better match the exact spatial, temporal and biophysical aspects of human TMS. For example, the human brain is much larger, the skull much thicker and the spatial extent and physical effects of a given TMS coil are no doubt difficult to match. The need to further improve such ‘construct validity’ for animal TMS may also be worthy of further discussion.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-180
|
https://f1000research.com/articles/2-86/v1
|
13 Mar 13
|
{
"type": "Research Article",
"title": "Nigerian secondary school adolescents’ perspective on abstinence-only sexual education as an effective tool for promotion of sexual health",
"authors": [
"Mfrekemfon P Inyang",
"Obonganyie P Inyang",
"Obonganyie P Inyang"
],
"abstract": "The success of any type of sexual education programme depends on the knowledge and preparedness for practice by adolescents. A recent study has found that an ‘abstinence-only’ sexual education programme is effective in reducing sexual activity among adolescents. Knowledge of abstinence-only sexual education and preparedness for practice as an effective tool for promotion of sexual health among Nigerian secondary school adolescents was studied. An analytic descriptive survey design was used for the study. The research population comprised of all public secondary schools in three southern geopolitical zones of the Niger Delta Region of Nigeria. A multistage sampling technique was used to select 2020 senior secondary school (SS1-SS3) students as sample for the study. A partially self-designed and partially adapted questionnaire from an 'abstinence-only versus comprehensive sex education' debate, from debatepedia (http://wiki.idebate.org/), entitled 'Questionnaire on Nigerian Secondary School Adolescents’ Perspective on Abstinence-Only Sexual Education (QNSSAPAOSE)' was used in eliciting information from respondents. Hypotheses were formulated and tested. Frequency counts, percentage and Pearson Product Moment Correlation were used in analysing data. A greater proportion of secondary school adolescents in this study lacked knowledge of sexual education. About 80% of the respondents could not define sexual education. The general perspective on abstinence-only sexual education was negative, as revealed by the larger number of respondents who demonstrated unwillingness to practice abstinence-only sexual education. Specifically, of those who responded in favour of abstinence-only sexual education, the youngest group of adolescents (11-13 years) and the male respondents were more likely to accept this type of education than the other groups. Poor knowledge of sexual education could be responsible for unwillingness to practice abstinence-only sexual education. Sexual education should, therefore, be introduced into the secondary school curriculum and taught by well-prepared teachers to enable an informed decision on practice.",
"keywords": [
"Sexual education is a lifelong process of acquiring information on sex and forming attitudes",
"values and beliefs. It involves sexual development",
"sexual and reproductive health",
"interpersonal relationships",
"affection and intimacy1. Abstinence-only sexual education teaches the adolescents to abstain from premarital sexual intercourse because of the advantages it offers. Such advantages include prevention of unintended pregnancies and prevention of contracting HIV/AIDS and other sexually transmitted infections. According to the Federal Law of the United States of America (USA)",
"every school-aged child should not engage in sexual activity2",
"3. The USA Federal law further advocates sexual activity within the confines of monogamous marital relationship to guard against adverse psychological and physical effects associated with premarital sexual activity. Bearing children outside marriage has serious effects on the child",
"the mother and the society as a whole3–6. The Federal Law of United States of America also posited that",
"abstaining from sexual activity outside marriage allows for maturity and understanding of self3",
"7."
],
"content": "Introduction\n\nSexual education is a lifelong process of acquiring information on sex and forming attitudes, values and beliefs. It involves sexual development, sexual and reproductive health, interpersonal relationships, affection and intimacy1. Abstinence-only sexual education teaches the adolescents to abstain from premarital sexual intercourse because of the advantages it offers. Such advantages include prevention of unintended pregnancies and prevention of contracting HIV/AIDS and other sexually transmitted infections. According to the Federal Law of the United States of America (USA), every school-aged child should not engage in sexual activity2,3. The USA Federal law further advocates sexual activity within the confines of monogamous marital relationship to guard against adverse psychological and physical effects associated with premarital sexual activity. Bearing children outside marriage has serious effects on the child, the mother and the society as a whole3–6. The Federal Law of United States of America also posited that, abstaining from sexual activity outside marriage allows for maturity and understanding of self3,7.\n\nHowever, numerous studies oppose abstinence-only in favour of comprehensive sexual education as being capable of preventing sexually transmitted diseases, including HIV/AIDS and unintended pregnancies. Studies further disclose that it is comprehensive sexual education and not abstinence-only that will delay first sexual activity2,3,8–14. Abstinence-only sexual education lacks strong evidence of effectiveness because of faulty designs3,15,16a. Abstinence-only sexual education does not positively affect the sexual behaviour of adolescents, lacks the message of sexually transmitted infections to its recipients and the positive effect in a few cases does not last for a long time16b,17.\n\nAccording to Bruckner and Bearman18 and a study by the Alan Guttmacher Institute19 those that embrace abstinence-only sexual education still have sex before they get married. The authors further posited that adolescents also resort to anal and oral sex as alternatives to vaginal sexual intercourse. There is no difference between adolescents that accept abstinence-only sexual education and those who do not in terms of number of sexual partners and ages of first sexual intercourse17. Abstinence-only sexual education does not reduce the scourge of HIV/AIDS16b. Bennett and Assefi saw the failure to provide adolescents with information about contraception as a serious weakness of abstinence only sexual-education20.\n\nThe proponents of abstinence-only sexual education frown at the role of comprehensive sexual education in emphasising so much on the reliability of contraceptives while de-emphasising their failure rates and the possibility of contracting new sexually transmitted diseases including HIV/AIDS21. They further frowned at the double message of comprehensive sexual education such as encouraging the delay of first sexual intercourse and promoting the use of contraceptives22. On the other hand, the proponents of abstinence-only sexual education are accused of overemphasising the failure rate of condoms. They are also blamed for stressing the possibility of contracting sexually transmitted infections to the extent of falsifying information to establish the negative aspect of comprehensive sexual education1,23,24. Studies further reveal that sexual educators do not stress enough on sexual intercourse or bring in sensitive issues such as homosexuality and abortions. The adolescents posited that the basic message is that they should not have sex19,25–27. The proponents of comprehensive sexual education attributed the ineffectiveness of condoms and contraceptives to poor-quality research28.\n\nAbstinence-only sexual education studies are criticised for exaggerations of findings. Such exaggerations include over-blowing the prevalent rate of HIV/AIDS and other sexually transmitted infections and failure rates of condom. They are also blamed for withholding information on the positive aspects of sexual relationships, while magnifying the emotional risks and pitfalls associated with premarital sexual activity1,24. Many adolescents may not buy into abstinence-only sexual education because they want to align with the modern life style in industrial societies where people marry later in life, while some do not marry at all. The modern life-style is also characterised by a high rate of broken marriages, predisposing an individual to having many sexual partners1. According to some studies the age of first marriage has risen to 30 years, with a fifth of such marriages ending in divorce within a period of five years29. Conversely the age at first sexual intercourse has dropped to 16 years with very few people having their husbands as their first sexual partners30,31. The data on adolescents’ sexual behaviour in the developing world with a high prevalence of HIV/AIDS suggests unacceptability of abstinence-only sexual education. Some countries do not accept abstinence-only sexual education even in the face of the high prevalence of HIV/AIDS. Some countries expect sexual educators to encourage adolescents to delay their first sexual experience while also providing education on contraception and sexual health services32. In some countries, the requirements for teaching any type of sexual education are clearly outlined for the sexual educators33.\n\nMost studies reveal comprehensive sexual education as the preference of parents and adolescents34–36. Studies advocate comprehensive sexual education for unmarried sexually active adolescents3. Studies also recommend abstinence-only sexual education with information on contraception and risk-reduction behaviour for the few sexually inactive adolescents. These align with the position of Collins and Priya that parents and adolescents prefer comprehensive sexual education to abstinence-only sexual education37. It can be deduced from the position of the proponents of comprehensive sexual education that abstinence-only sexual education denies adolescents the necessary information necessary to empower them for premarital sexual activity. Most studies revealed that the opinion of adults differs on the type of sexual education to teach adolescents. Most of the adults feel 7th to 9th graders should be taught ‘abstinence only’ while some advocate the teaching of contraception use38. Most studies also show that a greater percentage of adolescents prefer sexual orientation that will teach them about the use of contraception and sexually transmitted infections than abstinence-only sexual education39. Most studies reported the positive outcome of abstinence-only sexual education ranging from reduced sexual activity, pregnancies, abortions to more successful deliveries40. In line with this, John and Jemmott disclosed the success of abstinence-only sexual education in reducing sexual activity among youths41. In support of the findings of this new study, Rector submitted that out of 15 scientific evaluations of abstinence-only sexual education, 11 of them demonstrated its effectiveness in reducing adolescent sexual activity42.\n\nA survey of the National Campaign to Prevent Teen Pregnancy in 2001 showed that 93% of abstinence sexual education came from the society43. Studies showed that proponents of abstinence-only sexual education frown seriously at sex before marriage. They considered it not right and immoral. They concluded that this is the only 100% effective method to prevent teenage pregnancy and sexually transmitted diseases4. They further reminded that condoms cannot provide 100% protection against unplanned pregnancy and sexually transmitted infections and also that premarital sex can lead to life threatening health problems such as abortion and its associated complications5. They feel burdened that sexual, contraceptive and HIV information can provoke early sexual initiation among the adolescents5. Abstinence-only sexual education has positively produced a corresponding decrease in teenage pregnancy4. Studies have demonstrated that religion acts as a deterrent to early sexual activity5. In line with this, many adolescents submitted that morals, values and religious beliefs significantly influence the decision to have sex or not.\n\nIn deciding whether or not to have sex, the Organization of Concerned Women for America also decried the outcome of sex without love or responsibility supported by public policies. This results in the breakdown of nuclear families, increases crime, poverty, teen births and AIDS which in turn negatively affects the health of the general public. This only shows lack of values5.\n\n\nStatement of the problem\n\nAdolescents are the future and so they require proper guidance that will propel them into responsible productive adults useful to themselves and their nations. Adolescence is a remarkable period characterised by the quest for experimentations with drugs, alcohol and sexual activity saddled with numerous life threatening adverse effects. The Nigerian Association for the Promotion of Adolescent Health and Development, (NAPAHD) found that, a hospital based research study revealed that 80% of patients with abortion complications in hospitals are adolescents. Studies in Nigeria have also shown that most female adolescents by the age of 15 have already had their first sexual intercourse6,45. The same applies to male adolescents. In Nigeria, complicated abortion, sexually transmitted infections and HIV/AIDS, sexual coercion, unplanned and unwanted sexual activity and unwanted pregnancies and babies, drop outs from schools and homelessness abound and are very common features with the adolescents6. This ugly situation can be attributed to ignorance of sexual information and the implications of premarital sexual activities. Most Nigerian adolescents do not receive correct sexual information while some are ignorant. They also might not be aware that engaging in premarital sexual activities can terminate their educational career, hold back their potentials and also affect the nation negatively6. The intense outcome associated with adolescent sexual activity necessitates the search for a positive way out and thus, the main objective of this study.\n\n\nPurpose of the study\n\nAccording to Focus on the Family group, sex should be avoided the same way as the use of guns, tobacco, alcohol and drink-driving46. They sternly condemn the advocacy for the use of condoms against unwanted pregnancies and sexually transmitted diseases in favour of abstinence-only sexual education47. Previous studies document the advantages of abstinence-only sexual education in reducing adolescent sexual activities and the associated health problems. This study investigated the perspective of secondary school adolescents in Nigeria on abstinence-only sexual education as an effective tool for promoting adolescent sexual health. The findings will help in planning informed corresponding intervention programmes which will hopefully yield positive results in Nigeria as other countries documented in previous studies. Adolescents all over the world face the common problem of media pressure and hormonal surges, which is natural.\n\n\nMaterial and methods\n\nAn analytic descriptive survey design was used for the study. The research population comprised of all public secondary schools in three southern geopolitical zones of the Niger Delta Region of Nigeria. The States were Rivers, Akwa Ibom and Cross River. A multistage sampling technique was used for selecting 2020 senior secondary school (SS1-SS3) male and female students from the three states. Proportionate sampling technique was used in selecting the number of participating schools from the metropolis of each state. Five schools were selected from each of Cross River and Akwa Ibom States. Ten schools were selected from Rivers State. Respondents from each school were also selected proportionately. A total of 702 respondents were drawn from Akwa Ibom State, 510 from Cross River State and 808 from Rivers State. Participants were within the age range of 10–19 years. A questionnaire entitled Questionnaire on Nigerian Secondary School Adolescents Perspective on Abstinence-Only Sexual Education (QNSSAPAOSE) was used in eliciting information from respondents. The test/re-test reliability method was used to establish the internal consistency of the instrument. The instrument had a reliability coefficient of 0.75 established with Pearson Product Moment Correlation Coefficient (r). The instrument was divided into sections A and B. Section ‘A’ sought information on respondents’ demographic characteristics. Section ‘B’ sought information on respondents’ perspectives on Abstinence-Only sexual education. Questions were closed and open ended. The YES/NO questions attracted two points for positive responses and one point for negative responses. Questionnaires were administered with the aid of ten trained research assistants. Questionnaires were administered and collected on the spot to enhance a high return rate. Completion of the questionnaire was voluntary. Out of a total of 2020 questionnaires sent out, 2013 were returned and 13 questionnaires were not usable yielding a usable number of 2000 questionnaires. The return rate, therefore, was 99.1% (2000/2020). Hypotheses were formulated and tested. Frequency counts, percentage, Standard Deviation and Pearson Product Moment Correlation were used in analysing data. Approval of individual school management was obtained prior to execution of this study in their schools. Participants’ consent was also obtained. Participation was voluntary and anonymity was also assured and maintained.\n\n\nResults\n\nAll of the participants in this study were adolescents between the ages of 10 and 19 years (Table 1). Age distributions of the participants were the following: 11–13 years were 8.8% (N=176), 14–16 years were 61.15% (N=1223) while 17–19 years were 30.05% (N=601). Gender distribution showed males 35.1% (N=702) while females were 64.9% (N=1298). Class distribution showed Senior Secondary1 (SS1) were 70.7% (N=1413), Senior Secondary2 (SS2) were 24.3% (N=486) while Senior Secondary3 (SS3) were 5.1% (N=101) respectively. In religious distribution, 93.9% (N=1877), were found to be Christians, 4.3% (N=86) were Muslims, 1.6% (N=31) were Pagans and 0.3% (N=6) were traditional worshippers. The ethnic distribution of the participants was 40% (N=800) Ikwere’s, 35% (N=700) Ibibios and 25% (N=500) Efiks. This study also revealed that about 80% of the study participants could not define sexual education.\n\nThree research questions and six hypotheses were formulated in this study. Each one of them is addressed as follows:\n\nSeven positive statements representing the advantages of abstinence-only sexual education were made (Table 2). The general perspective of Nigerian secondary adolescents on the advantages of abstinence-only sexual education was negative. A greater number of the respondents did not agree with the statements highlighting the advantages of abstinence-only sexual education. However, taking into consideration the number of respondents from each age group that agreed with the statements highlighting the advantages of abstinence-only sexual education, it was found that the youngest age group (11–13 years) of adolescents ranked first. This implies that this group had the highest number of positive respondents in line with their total number when compared with the young (17–19 years) and younger (14–16 years) groups. Age group 11–13 years therefore demonstrated the likelihood of accepting abstinence-only sexual education.\n\nThe same number of statements was also made on the disadvantages of abstinence-only sexual education (Table 3). Generally, more respondents agreed with five while disagreeing with the last two of the seven statements highlighting disadvantages of abstinence-only sexual education. Of the total number of respondents that disagreed with the statements of disadvantages, the young adolescents (17–19 years) ranked first followed by the younger ones (14–16 years). More of the young adolescents out of their total number disagreed with the statements of disadvantages when compared with the responses of the other two groups. This might be a factor of a better understanding than the younger age groups. Moreover, the problem might not be with the type of sexual education but mostly the willingness to practice.\n\nThe general perspective of the respondents to five statements representing the acceptance of abstinence-only sexual education was negative. More respondents were against abstinence-only sexual education demonstrating a negative perspective on the acceptance of abstinence-only sexual education (Table 4). For instance 1686 out of the total respondents of 2000 objected to wanting a strong abstinence-only message. The youngest adolescent group (11–13 years) had the highest number of respondents out of their total number of those in favour of abstinence-only sexual education. For instance, more of them when compared with other age groups wanted sex to be saved until marriage. More of them (11–13 years) also wanted a strong abstinence message and education. This indicates that the youngest group of adolescents were more likely to accept abstinence-only sexual education than other age groups.\n\nA significant relationship existed between age and the students’ perspective on abstinence-only sexual education. (r = 0.123**, N=2000, P < 0.01) (Table 7). Young age specifically had an influence on the respondents’ perspective on abstinence-only sexual education in this study. Null hypothesis is rejected.\n\nRespondents belonged to different religious organisations but only 31 out of the total respondents of 2000 were pagans (Table 6). A greater proportion were Christians which numbered up to 1877 out of 2000 total number of respondents. Muslims were 86 while traditional worshippers were only 6. According to the total number of each age group, the highest number of Christians came from age group 14–16 years followed by age group 17–19. Age group 11–13 years which has demonstrated the likelihood of accepting abstinence-only sexual education had the least number of Christians relatively.\n\nNull hypothesis 1: There is no significant relationship between age and their perspective on abstinence-only sexual education.\n\n*No of participants.\n\n**Sig. at 0.01 level.\n\n***Pearson’s.\n\n****‘p’ value.\n\nNull hypothesis 2: There is No significant relationship between gender and their perspective on abstinence-only sexuality education.\n\nA significant negative relationship existed between religion and perspective on abstinence-only sexual education. (r=-0.122**, N=2000, P < 0.01) (Table 12). Religion had negatively influenced perspective on abstinence-only sexual education in the study. Null hypothesis is rejected.\n\nA greater number of male and female adolescents demonstrated a negative perspective to the advantages of abstinence-only sexual education. This is deduced from their responses to the statements reflecting the advantages of abstinence-only sexual education. Out of the total number according to gender that responded in favour of the advantages of abstinence-only sexual education, more females were in favour of three statements while more males were in favour of four statements.\n\nMore male and female respondents disagreed with five of the statements reflecting the disadvantages of abstinence-only sexual education and agreed with two of the statements. Out of the total number of those that did not agree with the disadvantages of abstinence-only sexual education, more males responded to four of the statements while more females responded to three of the statements.\n\nAn outright negative perspective on the acceptance of abstinence-only sexual education was demonstrated by both male and female adolescents. A greater number of male and female respondents reacted negatively to statements which were in favour of abstinence-only sexual education.\n\nMore females responded in favour of acceptance of abstinence-only sexual education in two statements out of five. More males responded to three of the statements. Specifically, more males than females advocated for sex to be saved until marriage and also wanted a strong abstinence message and education. This study found that boys are more likely to accept abstinence-only sexual education than females. This finding might be connected with the larger number of males among age 11–13 year group of adolescents with a likelihood to accepting abstinence-only sexual education than other older adolescents (Table 16).\n\nThere was no significant relationship between gender and perspective of the adolescents on abstinence-only sexual education. (r = 0.051, N=2000, P <0.05) (Table 11). Gender had no influence on perspective of abstinence-only sexual education in the study. Null hypothesis is therefore retained.\n\n*Not. Significant. at 0.05 level.\n\n**No of participants.\n\n***Pearson’s.\n\n****‘p’ value.\n\nNull hypothesis 3: There is No significant relationship between religion and their perspective on abstinence-only sexual education.\n\n*No of participants.\n\n**Sig. at 0.01 level.\n\n***Pearson’s.\n\n****‘p’ value.\n\nA significant joint effect existed between the independent variables (age, gender, religion, ethnicity and parent’s occupation) and perspective on abstinence-only sexual education (F (5, 1994) = 13.085; R = 0.178, R2 = 0.032, Adj. R2 = 0.029; P < 0.05) (Table 13). About 3% of the variation was jointly accounted for by the independent variables. The null hypothesis is therefore rejected.\n\nR = 0.178.\n\nR2 = 0.032.\n\nAdj R2 = 0.029.\n\n*Degree of freedom.\n\n**F-ratio.\n\nThere is a relative contribution of each of the independent variables on the dependent: age (β = 0.115, P <0.05), gender (β = 0.042, P >0.05), religion (β = -0.117, P <0.05), ethnicity (β = -0.016, P >0.05) and Parent’s occupation (β = 0.021, P >0.05) (Table 14). Hence, while age and religion made a significant contribution, gender, ethnicity and parent’s occupation were not significant.\n\nA positive significant relationship existed between perspective on abstinence-only sexual education and age, a negative significant relationship between perspective of abstinence-only sexual education and religion but no relationship between perspective of abstinence-only sexual education and gender, ethnicity and parents’ occupation (Table 15).\n\n**Sig. at 0.01 level.\n\n*Sig. at 0.05 level.\n\n\nDiscussion\n\nYoung age is an important factor in the success of abstinence-only sexual education. According to Massey, three significant periods exist where values are learnt. They are the imprint period, modelling period and socialisation period48.\n\nDuring the imprint and modelling periods, children learn through instructions and modelling. During these periods, the behaviour of children is formed from instructions given to them and examples before them. Therefore, if abstinence-only sexual education is taught between the imprinting to modelling period, it might produce positive results in adolescent lives. During the period of socialisation which starts from 13–21 years, a child is already exposed to different types of views and influences. Introducing abstinence-only sexual education might not be successful at this stage because for several reasons. Probably, most of the adolescents would have become sexually active by this age. Secondly, they might have a conviction already for sexual intercourse outside the confines of marriage as an ideal way of life. It is possible to accept instructions at a younger age. Those within the age range of 11–13 years were 8.8% (N=176) of the total population and they were the youngest group in this study. Most of them would have constituted the population in senior secondary one. The largest number of respondents was within the age bracket of 14–19 years. They might have established their beliefs about premarital sex and are probably sexually active already. This could be an explanation for the larger number of respondents that opposed abstinence-only sexual education.\n\nThe major finding of this study shows that Nigerian secondary school adolescents generally have a negative perspective towards abstinence-only sexual education. Out of a total population of 2000 respondents, abstinence-only sexual education was accepted by only 314 respondents and rejected by 1686. Those that had the tendency to accept abstinence-only sexual education were within the age bracket of 11–13 years and are the youngest group of adolescents The highest number of those that advocated for sex to be saved until marriage and also wanted abstinence-only sexual message and education belonged to the youngest group of adolescents. A greater number of respondents that were not in favour of abstinence-only sexual education belong to the 14–19 years age bracket and are the older adolescents. These are the ones within the age of socialisation already. With their exposure to different views about premarital sex, it is likely that most of them are already sexually active and have already taken a position for premarital sex. The message of abstinence-only sexual education at this stage might not be accepted with ease.\n\nReligion had a negative significant influence on the adolescents’ perspective on abstinence-only sexual education. Previous studies revealed that almost no religion supported premarital sexual activities. The studies of Concerned Women for America, demonstrated that religion acts as a deterrent to early sexual activity5. Many adolescents in previous studies posited that morals, values and religious beliefs significantly influence the decision of whether to have sex3–5. Probably, those that accepted abstinence-only sexual education would have been influenced by their religious beliefs. This also implies that teaching of religious values could be a useful tool for inculcating the values of sexual intercourse within the confines of marriage. Fortunately 93.9% (N=1877) of the total respondents were Christians 4.3% (N=86) were Muslims and 1.6% (N=31) belonged to traditional religion. Only 0.3% (N=6) were pagans. Christianity should therefore teach abstinence-only sexual education effectively with positive results.\n\nMale and female Nigerian secondary school adolescents generally have a negative perspective towards abstinence-only sexual education. Out of the number that favoured abstinence-only sexual education, more females than males were in favour of some statements and in other statements more males than females were in agreement. Surprisingly, more males than females advocated for sex to be saved until marriage and also wanted abstinence-only sexual message and education. One would have expected a more positive response from the females than the males because a greater proportion of the respondents in this study were females 64.9% (N=1298) of the total respondents. c. This finding is also sad because it is the females that suffer most from the adverse effects of premarital sexual activities. They are the ones that drop out from schools in the advent of pregnancy. They suffer the adverse effects of early pregnancy and child birth or abortion so one would have expected more females than males to advocate for sex to be saved until marriage. At the other hand more males than females advocating for sex to be saved until marriage and also wanting strong abstinence message and education might not be unconnected with the high number of males among the 11–13 years adolescents (Table 16). This study has revealed age 11–13 years as the group with a more likelihood of accepting abstinence-only sexual education than the other two groups of adolescents.\n\nEthnicity did not make any significant contribution to perspective of adolescents on abstinence-only sexual education. This shows that adolescents are all the same anywhere and everywhere. This study also revealed that about 80% of the total respondents could not define sexual education. Probably most of the responses would have been informed by ignorance.\n\n\nConclusion\n\nAbstinence-only sexual education can work with a timely and early introduction in the lives of adolescents. The findings of this study reveal the need to teach abstinence-only sexual education at an early age as what is right and obtainable by parents who are the first contacts, in religious organisations by the Sunday school teachers and then teachers in school for possible positive results. This will also form part of the values and belief system of the child which might not be easily compromised. It is very important to teach abstinence-only sexual education as an ideal standard of life before a child gets to the age of socialisation of 13–21 years. Teaching abstinence-only sexual education after a child might have been exposed to different views about sex might not yield a good result as revealed by the findings of this study. This can be attributed to the difficulties associated with changing of values and beliefs or habits when once they are formed.\n\n\nTranslations to health education practice\n\nKnowing the perspective of adolescents on abstinence-only sexual education is the key to knowing the right intervention programme to design and the approach to adopt for the implementation of such programme. This study reveals that abstinence-only sexual education will not work for majority of the respondents because it is not acceptable to them. For instance, only 314 out of 2000 respondents agreed with statements reflecting acceptance of abstinence-only sexual education while 1686 respondents disagreed. A very important finding of this study is that abstinence-only sexual education might only impact positively on those within the imprint and modelling periods of development. This include those within the age range of 11–13 years. The earlier abstinence-only sexual education is introduced in life, the more promising the result. Those within the age bracket of 14–19 years seem to have formed their opinion already from different socialisation processes. This finding suggests the need for a second study only for those within the imprint and modelling period. The intervention programme based on the findings of this study recommends a two- dimensional approach. Comprehensive sexual education for the older age group (14–19 years) and abstinence only for the younger ones (11–13 years).\n\nThe implications of this study are directed specifically to parents who are the primary caretakers of the children. They have the first contact with the children during the early years. The religious organisations and the school health educators are also very important role players. Findings from this study suggest the introduction of different programming strategies aimed at teaching abstinence-only sexual education as a way of life within the early ages. This promises positive results capable of safeguarding adolescents especially the females from the numerous life threatening adverse effects associated with premarital sexual activities.\n\nParents must have a sound knowledge of sexual education so that they can serve as effective teachers to the children. This is the reason it is very good also to empower today’s adolescents with the knowledge of sexual education and the benefits as future parents. Preparing the adolescents of today to become knowledgeable parents of tomorrow can assist in creating a subsequent future of reduced cases of teenage pregnancies, deliveries, abortions and sexually transmitted infections. Whatever is wrong today can be corrected through adequately prepared adolescents who are the future of any nation.\n\nReligious organisations should never relent in teaching the morals and values of abstinence-only sexual education early enough to the children right from their Sunday school classes. School health professionals should design appropriate programmes to internalise the need for abstinence-only sexual education in adolescents. The health educators should devise ways of identifying the children with the right foundations already and continue to build on them. They should also identify those with negative beliefs and design some remedial programmes for them to reduce the negative influence on other adolescents through the process of socialisation. It can be seen from this study that many problems faced by adolescents as a result of pre-marital sexual activity are avoidable.",
"appendix": "Author contributions\n\n\n\nObonganyie P Inyang trained research assistants who helped in administering the questionnaire. She coordinated the administration, retrieval and sorting to separate useful copies of questionnaires from those not useful. The title of the work was conceived by Mfrekemfon P Inyang. She planned the research, reviewed the work, did the analysis and wrote the article.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nSexuality Information and Education Council of United States (SIECUS). Sexuality Education and Abstinence-Only-Until-Marriage Programs in the States: An Overview Fiscal Year 2008 Edition. 2008. Reference Source\n\nKirby D: Impact of Sex and HIV Education Programs on Sexual Behaviors of Youth in Developing and Developed Countries. [Youth Research Working Paper, No. 2] Research Triangle Park, NC: Family Health International, 2005. Reference Source\n\nCollins C, Alagiri P, Morin SF, et al.: Abstinence only vs. comprehensive sex education: What are the arguments? What is the evidence? University of California, San Francisco: AIDS Research Institute 2002. Reference Source\n\nAbstinence Clearinghouse. Choosing Abstinence-only Way to Protect America’s Youth [Internet]. Undated. Reference Source\n\nConcerned Women for America. Abstinence: Why Sex is Worth the Wait [Internet]. 1998, Accessed October 16, 2001. Reference Source\n\nInyang MP: Socio-cultural factors as predictors of sexual behaviour of female secondary school adolescents on Port-Harcourt Metropolis, Nigeria. PhD thesis: University of Ibadan. 2009.\n\n42 U.S.C. section 710 (b)(2): (A)–(H).\n\nKirby D, Short L, Collins J, et al.: School-based programs to reduce sexual risk behaviors: a review of effectiveness. Public Health Rep. 1994; 109(3): 339–360. PubMed Abstract | Free Full Text\n\nDickson R, Fullerton D, Eastwood A, et al.: Effective Health Care: Preventing and reducing the adverse effects of unintended teenage pregnancies. National Health Service Centre for Reviews and Dissemination University of York 1997; 3(1). Reference Source\n\nDennison C: Teenage Pregnancy: An overview of the research evidence. London: Health Development Agency 2004. Reference Source\n\nDicenso A, Guyatt G, Willan A, et al.: Interventions to reduce unintended pregnancies among adolescents: systematic review of randomised controlled trials. BMJ. 2002; 324(7351): 1426–1435. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWann C, McCormick G, Kosmin M: Teenage Pregnancy and Parenthood: A Review of Reviews. London: Health Development Agency 2003. Reference Source\n\nAlford S, Cheetham N, Hauser D: Science and Success in Developing Countries: Holistic Programs That Work to Prevent Teen Pregnancy, HIV and Sexually Transmitted Infections. Washington, DC: Advocates for Youth, 2005; 34. Reference Source\n\nCheesbrough S, Ingham R, Massey D: Reducing the rate of teenage conceptions: A review of the international evidence on preventing and reducing teenage conceptions: The United States, Canada, Australia and New Zealand. London: Health Development Agency 2002. Reference Source\n\nKirby D: Do abstinence-only programs delay the initiation of sex among young people and reduce teen pregnancy? The National Campaign for the Prevention of Teen Pregnancy 2002. Reference Source\n\nUnderhill K, Operario D, Montgomery P: Reporting deficiencies in trials of abstinence-only programmes for HIV prevention. AIDS. 2007; 21(2): 266–268. PubMed Abstract | Publisher Full Text\n\nUnderhill K, Operario D, Montgomery P: Abstinence-only programs for HIV infection prevention in high-income countries. Cochrane Database Syst Rev. 2007; 17(4): CD005421. PubMed Abstract | Publisher Full Text\n\nTrenholm C, Devaney B, Fortson K, et al.: Impacts of Four Title V, Section 510 Abstinence Education Programs Final Report. Princeton, NJ: Mathematica Policy Research, Inc. 2007. Reference Source\n\nBruckner H, Bearman P: After the promise: the STD consequences of adolescent virginity pledges. J Adolesc Health. 2005; 36(4): 271–278. PubMed Abstract | Publisher Full Text\n\nThe Alan Guttmacher Institute. Fact in Brief: Sexuality Education, 2002. Reference Source\n\nBennett SE, Assefi NP: School-based teenage pregnancy prevention programs: a systematic review of randomized controlled trials. J Adolesc Health. 2005; 36(1): 72–81. PubMed Abstract | Publisher Full Text\n\nStammers TG: Abstinence under fire. Postgrad Med J. 2003; 79(933): 365–366. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe Physicians Consortium, Sexual Messages in Government-Promoted Programs and Today’s Youth Culture. 2002.\n\nREP. HENRY A. WAXMAN. The Content Of Federally Funded Abstinence-Only Education Programs. United States House Of Representatives Committee On Government Reform Minority Staff Special Investigations Division. 2004. Reference Source\n\nBlake S, Frances G: Just say no to abstinence education. National Children’s Bureau, 2001. Reference Source\n\nBuston K, Wight D, Hart G, et al.: Implementation of a teacher-delivered sex education programme: obstacles and facilitating factors. Health Educ Res. 2002; 17(1): 59–72. PubMed Abstract | Publisher Full Text\n\nForrest S, Strange V, Oakley A, et al.: What do young people want from sex education? The results of a needs assessment from a peer-led sex education programme. Cult Health Sex. 2004; 6(4): 337–354. PubMed Abstract | Publisher Full Text\n\nMeasor L, Tiffin C, Miller K: Young People’s Views on Sex Education: Education, Attitudes and Behaviour. London: Routledge/Falmer 2000; 193. Reference Source\n\nPaton D: The economics of family planning and underage conceptions. J Health Econ. 2002; 21(2): 207–225. PubMed Abstract | Publisher Full Text\n\nNational Centre for Health Statistics Marriage and Divorce (data for US). 2004. Reference Source\n\nWellings K, Nanchahal K, Macdowall W, et al.: Sexual behaviour in Britain: early heterosexual experience. Lancet. 2001; 358(9296): 1843–1850. PubMed Abstract | Publisher Full Text\n\nHubert M, Bajos N, Sandfort T: Sexual Behaviour and HIV/AIDS in Europe: Comparisons of National Surveys. London: University College London Press 1998; 464. Reference Source\n\nDfES Sex and Relationships Guidance, HMSO: London 2000. Reference Source\n\nGuttmacher Institute State Policies in Brief. as of September, 2009; Sex and HIV/STI Education. Reference Source\n\nIto KE, Gizlice Z, Owen-O’Dowd J, et al.: Parent opinion of sexuality education in a state with mandated abstinence education: does policy match match parental preference? J Adolesc Health. 2006; 39(5): 634–41. PubMed Abstract | Publisher Full Text\n\nEisenberg ME, Bernat DH, Bearinger LH, et al.: Support for comprehensive sexuality education: perspectives from parents of school-age youth. J Adolesc Health. 2008; 42(4): 352–359. PubMed Abstract | Publisher Full Text\n\nBMRB International Evaluation of the Teenage Pregnancy Strategy. Tracking Survey. Report of results of benchmark wave, January 2001. Reference Source\n\nCollins C, Priya MPP, Alagiri JD, et al.: Monogragh Series: AIDS Policy Research Center and Center for AIDS Prevention Studies. AIDS Research Institute. 2002. Reference Source\n\nSexuality Information and Education Council of the United States (SIECUS). Public Support for Sexuality Education Reaches Highest Level. (Hickman-Brown Public Opinion Research Survey) [Internet]. March 1999. Reference Source\n\nWiner RL, Hughes JP, Feng Q, et al.: Condom use and the risk of genital human papillomavirus infection in young women. N Engl J Med. 2006; 354(25): 2645–54. PubMed Abstract | Publisher Full Text\n\nJones JM, Toffler W, Mohn JK, et al.: The declines in adolescent pregnancy, birth and abortion rates in the 1990s: What factors are responsible? A special report commissioned by The Consortium of State Physicians Resource Councils [Internet] January 7, 1999. Reference Source\n\nJohn JL: New Study Finds Abstinence Education Effective Comprehensive Sex-Ed Flops. 2010. Reference Source\n\nRobert R: The Effectiveness of Abstinence Education Programs in Reducing Sexual Activity Among Youth. Heritage. 8 Apr. 2002. Reference Source\n\nNational Campaign to Prevent Teen Pregnancy. Fact Sheet: Recent trends in teen pregnancy, sexual activity, and contraceptive use [Internet]. August, 2004. Reference Source\n\nEtuk SJ, Ihejiamaizu EC, Etuk IS: Female adolescent sexual behaviour in Calabar, Nigeria. Niger Postgrad Med J. 2004; 11(4): 269–73. PubMed Abstract\n\nBriggs LA: Adolescent Pregnancy in Port Harcourt Local Government Area of Rivers State. Implications for Health Education A PhD Thesis 1995.\n\nFocus on the Family. Take Twelve - The Truth About Abstinence Education. [Internet]. March 14, 2001.\n\nAbstinence Clearinghouse. Data confirms that the abstinence message, not condoms, is responsible for the reduction in births to teens. [Internet] May 17, 1998. Reference Source\n\nMassey M: Values development. Changing Minds. 2011. Reference Source"
}
|
[
{
"id": "877",
"date": "08 Apr 2013",
"name": "Warren Foster",
"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 examined secondary school student perspectives on abstinence-only sexual education through the use of a questionnaire. The sample size is robust, the statistical methods are appropriate, and overall the paper is well written. Unfortunately, the title of the paper is misleading. Specifically, the authors only examined student perspectives and there were no measures of efficacy of abstinence-only sexual education. The data suggests only that students of the youngest age group are more likely to be receptive to abstinence-only sexual education but no evidence is presented on whether the students would or in fact do incorporate these messages into their lives. The authors also suggest that the data shows that there is a significant relationship between religion and student perspective; however, the numbers of students in other religions than Christianity is too small to make meaningful conclusions. Moreover, it is unclear if the students simply identify with a particular religion or actively participate in the religion. Finally, the conclusions reached and the implications for health education practice are overstated. The most that can be concluded from this study is that young adolescents are potentially more receptive to abstinence-only sexual education. It cannot be concluded that abstinence-only sexual education will be more or less effective than no education or alternative education messages in shaping student sexual practices.",
"responses": []
},
{
"id": "971",
"date": "30 May 2013",
"name": "Laxmi Baxi",
"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 gives the impression that it refers to the entire population; however, it is true only for the youngest group - further clarification is needed.The majority of individuals in this study are Christian, so comparison by religion does not appear to be adequate. The authors need to emphasise this and may describe such findings in a single statement.The manuscript is too long, as is the introduction. It should be shortened to ensure the reader does not lose interest.Several text descriptions should be omitted as tables are self explanatory, e.g. table 1.Furthermore, it should be mentioned that the data only refers to a particular group of the population.",
"responses": [
{
"c_id": "471",
"date": "04 Jun 2013",
"name": "Mfrekemfon Inyang",
"role": "Author Response",
"response": "Much thanks to the referee! The title of the work is what was studied. Title of the work is different from findings.These findings are clearly spelt out in the work.The religion aspect has been addressed already. Other observations are all addressed accordingly.Thanks.Dr. M. P. Inyang"
}
]
},
{
"id": "970",
"date": "30 May 2013",
"name": "Luis Bahamondes",
"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 submitted a manuscript in which they assessed the perspectives of Nigerian secondary school students on abstinence-only sexual education. They applied a questionnaire to obtain the information. The sample size is correct and the analysis of the data is well conducted. However, my specific comments are that the title does not reflect what the authors did, as the authors did not evaluate the efficacy of abstinence-only sexual education; in fact, there is no evidence of whether the abstinence-only sexual education changes the attitudes of the students. Furthermore, the comments on religion are inappropriate because most of the interviewed students practiced only one religion.In my opinion, the following statement is strongly speculative and inappropriate because the authors did not obtain any evidence for it: “Preparing the adolescents of today to become knowledgeable parents of tomorrow can assist in creating a subsequent future of reduced cases of teenage pregnancies, deliveries, abortions and sexually transmitted infections. Whatever is wrong today can be corrected through adequately prepared adolescents who are the future of any nation.”",
"responses": [
{
"c_id": "470",
"date": "04 Jun 2013",
"name": "Mfrekemfon Inyang",
"role": "Author Response",
"response": "Much thanks to the referee for the comments. In this study the authors only looked at the Nigerian secondary school adolescents' perspective on abstinence-only sexual education. The purpose of this study is also clearly stated. The authors have not indicated anywhere in the study of evaluating the efficacy of abstinence-only sexual education, but only intend to do that in a follow up study. This study is not an intervention study so it could not have shown if abstinence-only sexual education could change attitude. It is always necessary to clarify needs before following up with appropriate intervention. The intent for a follow up intervention study has been indicated. The aspect of religion has been earlier addressed. The statement is a recommendation to educate the adolescents with the knowledge of what is right or wrong early in life. Numerous studies have proven the transforming power of information. It has also been established that most of the wrong-doings of adolescence are either due to wrong information or outright ignorance.Thanks.Dr. M. P. Inyang"
}
]
}
] | 1
|
https://f1000research.com/articles/2-86
|
https://f1000research.com/articles/2-263/v1
|
02 Dec 13
|
{
"type": "Case Report",
"title": "Xanthogranulomatous pyelonephritis (XGPN) mimicking a “renal cell carcinoma with renal vein thrombus and paracaval lymphadenopathy”",
"authors": [
"Arvind Ganpule",
"Jitendra Jagtap",
"Sanika Ganpule",
"Amit Bhattu",
"Shailesh Soni",
"Ravindra Sabnis",
"Mahesh Desai",
"Arvind Ganpule",
"Sanika Ganpule",
"Amit Bhattu",
"Shailesh Soni",
"Ravindra Sabnis",
"Mahesh Desai"
],
"abstract": "We present a case of Xanthogranulomatous pyelonephritis mimicking as a renal cell carcinoma. This was an elderly lady who presented with pyonephrosis due to urolithiasis. On evaluation she was found to have a space occupying mass in the right kidney. Further investigations revealed an enhancing tumor with renal vein thrombus and paracaval lymphadenopathy. Subsequent histopathology showed evidence of XGPN with no malignancy. This case report highlights the fact there are a number of imaging and clinical overlaps in the diagnosis, assessment and management of this entity.",
"keywords": [
"A 67 year old Hindu female presented to us in May 2010 with history of right flank pain",
"fever and vomiting. She had raised total leukocyte count: 16600/μL and deranged renal function (serum creatinine: 3.1mg/dL). A non-contrast CT (NCCT) scan revealed moderate hydronephrosis",
"right upper ureteric calculus and a well circumscribed lesion on the medial aspect of the kidney. A percutaneous nephrostomy was performed on account of the deranged renal function. Subsequently",
"the patient underwent a percutaneous nephrolithotomy (PCNL)."
],
"content": "Case presentation\n\nA 67 year old Hindu female presented to us in May 2010 with history of right flank pain, fever and vomiting. She had raised total leukocyte count: 16600/μL and deranged renal function (serum creatinine: 3.1mg/dL). A non-contrast CT (NCCT) scan revealed moderate hydronephrosis, right upper ureteric calculus and a well circumscribed lesion on the medial aspect of the kidney. A percutaneous nephrostomy was performed on account of the deranged renal function. Subsequently, the patient underwent a percutaneous nephrolithotomy (PCNL).\n\nAt one month from presentation and after the serum creatinine improved to 1.47mg/dL, a contrast CT revealed an enhancing mass (enhancement from 33 to 118 Hounsfield units) on the medial aspect of the kidney (Figure 1; a contrast CT not done at initial presentation due to deranged renal function) with evidence of renal vein thrombosis and multiple paracaval lymph nodes. A provisional diagnosis of renal cell carcinoma with renal vein thrombus was made. The clinical stage was T3aN2M0. A laparoscopic radical nephrectomy was done. The gross specimen revealed evidence of renal vein thrombus and Xanthogranulomatous pyelonephritis (XGPN) (Figure 2). On H & E (Hematoxylin & Eosin) microscopic examination, it was composed of foamy macrophages admixed with inflammatory infiltrate (Figure 3). There was no evidence of malignancy. The patient recovered well and was discharged in stable condition after 4 days with a serum creatinine of 1.16mg/dL.\n\n\nDiscussion\n\nXGPN is an uncommon, severe, chronic suppurative renal parenchymal infection characteristically leading to renal destruction. The majority of cases are unilateral and result in a nonfunctioning, massively enlarged kidney associated with obstructive uropathy secondary to urolithiasis. XGPN has been described as a great imitator or a masquerading tumor in adults and pediatric age groups1,2. The etiological factor in this case was the renal calculus with chronic infection. The imaging findings in this case showed a significantly enhancing mass, lymph nodes and a renal vein thrombus. The mass was seen closely abutting the psoas as well. The CT findings mimicked a case of T3N2Mx renal cell carcinoma. Localised XGPN is amenable to partial nephrectomy if diagnosed preoperatively. XGPN has been found to be associated with renal cell carcinoma, papillary transitional cell carcinoma and squamous cell carcinoma and hence nephrectomy should be performed when malignancy cannot be excluded. This case highlights the need to keep XGPN as a differential diagnosis of a renal mass especially in presence of urolithiasis.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nArvind Ganpule and Jitendra Jagtap drafted the manuscript and carried out the literature search. Sanika Ganpule, Amit Bhattu and Shailesh Soni prepared the illustrations and helped to draft the manuscript. Ravindra Sabnis and Mahesh Desai revised the manuscript and did the final proofreading of 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\nReferences\n\nZorzos I, Moutzouris V, Petraki C, et al.: Xanthogranulomatous pyelonephritis--the \"great imitator\" justifies its name. Scand J Urol Nephrol. 2002; 36(1): 74–6. PubMed Abstract | Publisher Full Text\n\nGerber WL, Catalona WJ, Fair WR, et al.: Xanthogranulomatous pyelonephritis masquerading as occult malignancy. Urology. 1978; 11(5): 466–71. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3093",
"date": "15 Jan 2014",
"name": "M. Hammad Ather",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an uncommon clinical situation where XGPN mimicked a renal tumor. XGPN is indeed a rare type of renal infection characterised by granulomatous inflammation with giant cells and foamy histiocytes. It has been shown in many case reports and small case series to mimic an infiltrative malignancy. Renal vein thrombus has also been described in many previous reports.I have few other observations that the authors may like to address. What were the findings on nephroscopy, and did the surgeon take any biopsies of the suspicious lesion? Did you noticed any xanthoma cells in the urine? The lady has a classical presentation of an XGPN (gender, obstructing middle age ... was she diabetic or immunocompromised in any way?) stone until the contrast study. Did the investigators consider a biopsy prior to planning a nephrectomy for a fair functioning kidney?",
"responses": [
{
"c_id": "672",
"date": "20 Jan 2014",
"name": "Jitendra Jagtap",
"role": "Author Response",
"response": "Thank you for your review Dr. M H Ather. Please find below the response to the comments:On nephroscopy during PCNL there were no suspicious lesions noted within the pelvicalyceal system so the surgeon did not take any biopsies. Absence of suspicious findings was double checked by reviewing the intraoperative video of this patient. Urine examination did not reveal the presence of any xanthoma cells. No, the lady was neither diabetic or immunocompromised. Biopsy was not considered, as it would not have altered the further management as the mass had features suggestive of malignancy on radiology namely: significant enhancement from 33 to 118 Hounsfield units, presence of renal vein thrombus, and multiple enlarged lymph nodes obviating the possibility of a nephron sparing procedure. The current roles of renal biopsy as outlined in various guidelines include: confirmation of diagnosis of radiologically indeterminate renal masses; obtaining histology of incidentally detected renal masses in patients who are candidates for nonsurgical treatment (active surveillance, ablative therapies); and selection of the most appropriate targeted therapy for metastatic renal tumours depending upon the histology (Ljungberg B et al., 2013; Novick A et al., 2010; Herts BR & Baker ME, 1995; Campbell SC et al., 1997; Volpe A et al., 2007). Also XGPN has been found to be associated with renal cell carcinoma, papillary transitional cell carcinoma and squamous cell carcinoma, and hence nephrectomy should be performed when malignancy cannot be excluded (Tolia BM et al., 1981; Schoborg TW et al., 1980)."
}
]
},
{
"id": "3601",
"date": "25 Feb 2014",
"name": "Stefanos Kachrilas",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 topic of this case review is not especially novel, and the information provided is unlikely to be useful to other practitioners, as the clinical entity of XGPN is well documented in the existing literature. The background, history, presentation, physical examination, and diagnostic tests are appropriately presented. The authors have not commented on the post-operative management of the patient.",
"responses": []
},
{
"id": "6021",
"date": "04 Sep 2014",
"name": "Daron Smith",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a case of XPN (Xanthogranulomatous Pyelonephritis) that was believed to be a renal cell carcinoma based on imaging. The propensity for XPN to \"imitate\" renal malignancy is well established. Indeed, I wrote a case report many years ago when, in addition to the imaging apparently suggesting a renal cell carcinoma, there was an incidental small RCC in the same kidney! (Smith RD et al., 2000) It would be worth emphasizing what is unique/important about this case, and the learning message that follows. Is it the apparent vascular invasion with thrombus in the vein that the authors wish to highlight?I have a few other suggestions. In the abstract, \"tumour\" should be changed to \"mass\". The patient did not have a renal tumour in the sense of a cancer as this word is often used. “Tumour”, in its most frequently used sense of malignancy, may cause confusion to anyone reading the abstract, believing this was an renal cancer with tumour thrombus.I would prefer to see the creatinine expressed in SI units (umol/L) and an eGFR given as well as the units in mg/dL.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-263
|
https://f1000research.com/articles/2-258/v1
|
25 Nov 13
|
{
"type": "Web Tool",
"title": "C-Sibelia: an easy-to-use and highly accurate tool for bacterial genome comparison",
"authors": [
"Ilya Minkin",
"Hoa Pham",
"Ekaterina Starostina",
"Nikolay Vyahhi",
"Son Pham",
"Ilya Minkin",
"Hoa Pham",
"Ekaterina Starostina",
"Nikolay Vyahhi"
],
"abstract": "We present C-Sibelia, a highly accurate and easy-to-use software tool for comparing two closely related bacterial genomes, which can be presented as either finished sequences or fragmented assemblies. C-Sibelia takes as input two FASTA files and produces: (1) a VCF file containing all identified single nucleotide variations and indels; (2) an XMFA file containing alignment information. The software also produces Circos diagrams visualizing high level genomic architecture for rearrangement analyses. C-Sibelia is a part of the Sibelia comparative genomics suite, which is freely available under the GNU GPL v.2 license at http://sourceforge.net/projects/sibelia-bio. C-Sibelia is compatible with Unix-like operating systems. A web-based version of the software is available at http://etool.me/software/csibelia.",
"keywords": [
"The development of inexpensive genome sequencing technologies and efficient assembly methods has revolutionized the study of bacterial genomes",
"which are being sequenced and assembled on a daily basis. When an assembly is available",
"the most common first task is to compare it against a reference genome (or another assembly",
"if no such genome is available) in order to find genetic differences between the newly assembled and reference genomes. This analysis is critical to understand genetic factors that determine certain phenotypes of the isolates."
],
"content": "Introduction\n\nThe development of inexpensive genome sequencing technologies and efficient assembly methods has revolutionized the study of bacterial genomes, which are being sequenced and assembled on a daily basis. When an assembly is available, the most common first task is to compare it against a reference genome (or another assembly, if no such genome is available) in order to find genetic differences between the newly assembled and reference genomes. This analysis is critical to understand genetic factors that determine certain phenotypes of the isolates.\n\nWe present Comparative Sibelia software (C-Sibelia) for the comparison of two bacterial genomes in the form of complete sequences or draft assemblies. C-Sibelia is able to compare genomes in the presence of rearrangements and duplications. C-Sibelia takes as input two FASTA files (the assembly and reference files; if the reference genome is not available, it can be substituted by another draft assembly) and produces: (1) a VCF file containing all identified single nucleotide variations (SNVs) and indels; (2) annotation of these variants by SnpEff; (3) an XMFA1 file containing alignment information. The web-based version also produces a circular diagram visualizing the rearrangement of synteny blocks in two genomes.\n\nThe performance of C-Sibelia in detecting SNVs and indels is comparable to MUMmer and outperforms Mauve in terms of the false-positive rate. C-Sibelia is a part of the Sibelia comparative genomics suite, which is freely available under the GNU GPL v.2 license at http://sourceforge.net/projects/sibelia-bio. Users are encouraged to use the web-based version of C-Sibelia at http://etool.me/software/csibelia.\n\n\nMethods\n\nThe task of finding SNVs and indels connects closely to the problem of whole-genome alignment. Unlike aligning two short DNA segments, aligning two genomes is more challenging because of the presence of rearrangements and repetitive elements. C-Sibelia addresses this problem by first decomposing genomes into synteny blocks, using the iterative de Bruijn graph algorithm described in Minkin et al.2. This step separates linear operations (indels, substitutions) from non-linear operations (rearrangements) and thus allows us to apply global alignment to multiple instances of each synteny block. C-Sibelia incorporates LAGAN3, a global alignment tool, for aligning different instances of the same synteny block.\n\nFrom alignment to variant calling. C-Sibelia then finds differences between two genomes (indels, SNVs, rearrangements) by analyzing the resulting synteny and alignment blocks. Regions in one genome not covered by synteny blocks are treated as indels. SNVs and small indels that lie within the regions covered by synteny blocks are reported by analyzing the alignment information produced by LAGAN. Identified variants are annotated by using snpEff4. The pipeline of C-Sibelia is described in the following pseudocode.\n\nInput: An assembly and a reference genome (in FASTA format).\n\nAlgorithm:\n\nDecompose the sequences into synteny blocks using Sibelia.\n\nAlign instances of synteny blocks using LAGAN.\n\nAnalyze the synteny block decomposition and alignment information.\n\n– Find indels in non-syntenic regions.\n\n– Find small indels and SNVs in aligned regions (using the alignment information produced by LAGAN).\n\n– Annotate the identified variants using SnpEff.\n\n– Select contigs containing multiple synteny blocks (i.e., rearranged contigs).\n\nOutput:\n\nAll SNVs and indel variants, in a VCF file.\n\nAnnotation of these variants produced by SnpEff4.\n\nA picture in Circos format5 for rearranged contigs and the reference genome.\n\n\nResults\n\nTo evaluate the variant calling feature, we benchmarked C-Sibelia against Mauve6 and MUMmer7 on a simulated dataset, designed as follows.\n\nFrom the complete genome of Staphylococcus aureus (S. aureus) NCTC 8325, we performed 10 deletions of random segments of size 2000 bp, and futher introduced 1000 SNVs in the resulting genome. We then generated five reversals and five translocations of random segments in the genome with size 10,000 bp each to evaluate the capability of these tools to perform an alignment in the presence of rearrangements. We obtained a simulated assembly of this newly simulated genome of 180 contigs; the distribution of contig length was similar to that of the RN4220 assembly reported in Dhanalakshmi et al.8. We further used C-Sibelia, Mauve and MUMmer to find variants in this simulated assembly and the original reference genome (NCTC 8325). Table 1 and Table 2 demonstrate that the performance of C-Sibelia in detecting variants is comparable to MUMmer and improves upon Mauve in terms of the false-positive rate. Figure 1 shows the Circos diagram of the rearranged contigs and the reference genome. The scripts and commands used for this benchmark are available in the Supplementary material.\n\nOnly contigs with multiple synteny blocks rearranged differently in the genome are shown. Green and red bars depict the direction of synteny blocks on the positive and negative strands, respectively.\n\nThe most common approach for comparing an assembly against a reference genome is to first align the assembly against the reference and then write in-house scripts to extract variants. C-Sibelia can achieve this task automatically and with high accuracy. We used C-Sibelia to reproduce the comparison of the S. aureus RN4220 assembly and the reference genome NCTC 8325, reported in Dhanalakshmi et al.8 (the authors used MUMmer and in-house scripts for this comparison). Among 132 single nucleotide variants and four large deletions reported in Dhanalakshmi et al.8, C-Sibelia confirmed 121 SNVs and all four large deletions. C-Sibelia also reported six additional variants, which are also confirmed by BLAST9. The input data as well as the commands for generating these results are available in the Supplementary material.\n\nThe online version of C-Sibelia is available at http://etool.me/software/csibelia. The web form takes as input two FASTA files (one for the assembly and the other for the reference). The web form’s parameters allow users to choose whether or not to annotate variants and display the Circos5 picture for rearrangement analysis (see Figure 1). Results are delivered to registered users by a real time push notification mechanism10,11.\n\n\nDiscussion\n\nIn this application note, we introduced C-Sibelia, a novel software for comparing two closely-related bacterial strains. Performance of C-Sibelia is comparable to MUMmer, and better than Mauve in terms of false positives rate. The web interface of C-Sibelia makes the task of comparing assemblies against a reference genome convenient for microbiologists, who do not want to go to the trouble of downloading and compiling the software. In the future, we plan to extend C-Sibelia to compare multiple genomes or draft assemblies as well as scale the software to larger genomes.",
"appendix": "Author contributions\n\n\n\nSP designed the project. IM, HP, ES, NV performed experiments and developed the computational tool. IM, SP wrote the manuscripts with critical input from NV, ES, HP. 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 partially supported by Russian Foundation for Basic Research RFBR (grant 14-04-31926) and V-P foundation.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank Alla Lapidus, Ekaterina Chernyayeva, Ryan Tewhey, Xiao Li, Harish Nagarajan, Mikhail Kolmogorov, Dhanalakshmi Nair, and David Hernandez for many helpful discussions. We are indebted to Phillip Compeau for for many helpful editorial suggestions.\n\n\nSupplementary material\n\nSupplemenary material can be found online at http://goo.gl/jtLsPl and is permanently available at doi: 10.5281/zenodo.7577.\n\n\nReferences\n\nBrudno M, Poliakov A, Salamov A, et al.: Automated whole-genome multiple alignment of rat mouse, and human. Genome Res. 2004; 14(4): 685–692. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinkin I, Patel A, Kolmogorov M, et al.: A scalable and comprehensive synteny block generation tool for closely related microbial genomes. arXiv preprint arXiv: 1307.7941, 2013. Publisher Full Text\n\nBrudno M, Do CB, Cooper GM, et al.: LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Res. 2003; 13(4): 721–731. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCingolani P, Platts A, Coon M, et al.: A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012; 6(2): 80–92. 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\nDarling AE, Mau B, Perna NT: progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PloS One. 2010; 5(6): e11147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurtz S, Phillippy A, Delcher AL, et al.: Versatile and open software for comparing large genomes. Genome Biol. 2004; 5(2): R12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNair D, Memmi G, Hernandez D, et al.: Wholegenome sequencing of Staphylococcus aureus strain rn4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain. J Bacteriol. 2011; 193(9): 2332–2335. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTatusova TA, Madden TL: BLAST 2 sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett. 1999; 174(2): 247–250. PubMed Abstract | Publisher Full Text\n\nBrandt S, Kristensen A: Web push as an internet notification service. In W3C Workshop on Push Technology, Boston, Massachusetts, 1997. Reference Source\n\nFette I, Melnikov A: The websocket protocol. IETF Internet draft. 2011. Reference Source"
}
|
[
{
"id": "2566",
"date": "27 Nov 2013",
"name": "Jeffrey McLean",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 general, the rationale behind the decision to develop a user friendly tool to compare finished and draft assemblies, to reference genomes, is highly justified. The accurate calling of single nucleotide variations and indels using the comprehensive SnpEff tool will allow a wide variety of users to make use of C-Sibelia. For non-informatic inclined users, the output formats and the optional Circos diagrams visualizing high level genomic architecture for rearrangement analyses, is an excellent addition. The framework for the software is also an interesting choice, making it accessible to social networking style discussions with other users. On the technical side, the authors do demonstrate that the performance of C-Sibelia in detecting variants is comparable to MUMmer and improves upon Mauve in terms of the false-positive rate. These tools are currently highly used and the research community would benefit from a user friendly tool such as C-Sibelia. The approach to decompose the genomes into synteny blocks, using the iterative de Bruijn graph algorithm is novel. In order to validate this tool I have signed in to the web-based version (signing in increases the size of the files you can upload) and tested an assembly against a reference genome and obtained results comparable to MAUVE. An explanation of the common use of the VCF format would help readers further. Visualizations of the SNPs would also be a nice addition in the future. I look forward to further enhancements of this tool.",
"responses": []
},
{
"id": "4953",
"date": "17 Jul 2014",
"name": "Loren J Hauser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nC-Sibelia was created to be a user friendly tool for pairwise genome comparison. The web tool is relatively easy to use for the non-bioinformatics trained and will therefore make these kinds of analysis easier for small groups to perform. This is a big need. I can see the possibility of additional analyses to be added which will enhance the site. However, the output page has some real deficiencies that need to be fixed prior to public use. The web site can’t recognize a fasta file unless named xxx.fasta. This is annoying since all genbank fasta files end in .fna and therefore will have to be renamed before use. This is an easy fix. I tested 2 bacterial genomes over 6 Mb and it took about 5 minutes for the results to come back. This is OK but could be a problem if the site eventually gets a lot of use. The output needs a lot more definitions or explanations: for example 1) the section titled “number of effects by impact”, I have no idea what high, low, moderate or modifier means; 2) there are 10,853 modifiers but there are only 1037 listed in the section “changes by type”; 3) in the section “number of effects by functional class” there are 36 missense and 75 silent mutation listed, but there are 160 non-synonymous_coding and 136 synonymous_coding in the section “number of effect by type and region”; 4) since these were bacterial genomes how are exons defined? There are additional inconsistencies and lack of definitions that I have not pointed out.The best way to fix this is to get somebody who has never used the system and has not been trained in any way to test it in order to point out all of these inconsistencies and lack of definitions. Some of the output was unavailable since in ran off the bottom of the allotted space. This will be a real problem when comparing draft genomes with more than 10 contigs. I did not get to see the circular map or the distribution of changes on the main chromosome because of this. I did not try to download and use the executable, but if it as poorly described as the output it will be hard for novices to implement. Again, get a complete novice to test downloading and installing it to make sure it is easy to use.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-258
|
https://f1000research.com/articles/2-257/v1
|
25 Nov 13
|
{
"type": "Case Report",
"title": "Pulmonary embolism presented by syncope in a low-risk patient: a case report",
"authors": [
"Amr A A Othman",
"Aly M Tohamy",
"Ayman K M Hassan",
"Amr A A Othman",
"Aly M Tohamy"
],
"abstract": "Introduction: Pulmonary embolism, an emergency that can have fatal consequences, can be presented with a common symptom that can be missed, such as syncope.Case presentation: We present a case of a young, low-risk male who presented with attacks of syncope and dyspnea followed by massive pulmonary embolism. We also review the pathophysiology of syncope in pulmonary embolism cases and strategy of how to work up with similar cases.Conclusion: Pulmonary embolism should be considered and excluded in every case of recurrent attacks of syncope.",
"keywords": [
"Pulmonary embolism",
"Syncope",
"Low risk"
],
"content": "Introduction\n\nPulmonary embolism (PE) is a medical emergency that can lead to sudden death. This condition is usually associated with factors that may increase the risk of its occurrence, such as age, high tendency for intravascular coagulation and the presence of certain concomitant conditions such as deep venous thrombosis (DVT) and malignancy1. In this case, we have a presentation of a young, low-risk patient with only recurrent syncope and dyspnea that was eventually diagnosed as a massive PE. It is an uncommon case of pulmonary embolism of unknown cause that deserves attention. Such a common presentation could have led to misdiagnosis of the condition which could have ended with the sudden death of the patient.\n\n\nCase presentation\n\nA 30 year old male patient, a farmer from Upper Egypt, attended the cardiology outpatient clinic at Assiut University Hospitals, Egypt, on June 2012 with repeated attacks of syncope. They took place at irregular intervals, about two or three times per month.\n\nThe attacks of syncope had started eighteen months earlier, with each one lasting about one minute. There was progressive grade III dyspnea between the initial syncope symptoms and presentation at our clinic, but no evidence of orthopnea, paroxysmal nocturnal dyspnea or lower limb swellings.\n\nGeneral examination found that the patient had a body mass index of 28.4 kg/m2. The pulse was 110 beats/min, the respiratory rate was 24 breaths/min and the arterial blood pressure was 110/70 mmHg. There was a raised jugular venous pressure line about 6 centimeters above the level of the sternal angle. There were multiple, soft, painless subcutaneous masses over both thighs and the back of the patient with ill-defined edges. Cardiac examination showed that there was a pansystolic murmur over the tricuspid area which increased with inspiration, ejection systolic murmur over the pulmonary area with accentuated pulmonary component of the second heart sound. Electrocardiography showed sinus tachycardia with inverted T wave in leads II, III, aVF and V1-V6. Chest X-Ray revealed cardiomegaly. 2-D transthoracic echocardiography showed a dilated right side of the heart with a 5.7 cm × 1.4 cm mass in the right atrium that protruded through the tricuspid valve towards the right ventricle. Valvular morphology was normal with moderate tricuspid regurgitation and moderate pulmonary hypertension (Figure 1). Liver and kidney function tests were normal. According to this data, the patient was given a Wells score of 1.5, which gave an indication to a low risk of PE.\n\n2-D transthoracic echocardiography apical 4 chamber view shows a mass in the right atrium that protrudes through the tricuspid valve (arrow).\n\nThe patient was diagnosed initially with a cardiac tumor in the right atrium with suspected positional obstruction of right ventricular inflow track causing syncopal attacks, which was managed accordingly with diuretics, aspirin 150 mg/day and bisoprolol 10 mg/day to control heart rate. He underwent a duplex scan of the deep venous system of both lower limbs, which was found to be normal. While the medical team investigated the patient in hospital, a sudden attack of syncope occurred, with a sudden severe hypotension down to 90/60 mmHg. The Wells score of the patient has sharply increased from the initial 1.5 to 8, which indicated a very high risk of the presence of PE. Urgent Multi-slice CT pulmonary angiography found a massive bilateral pulmonary embolism involving the left and the right pulmonary arteries and most of their segmental branches (Figure 2). A diagnosis of high-risk pulmonary embolism was made.\n\nMulti-slice CT pulmonary angiography shows massive bilateral pulmonary embolism involving the left and the right pulmonary arteries (asterisks).\n\nIn order to save the patient’s life, we put the patient under thrombolytic therapy. A central venous line was set up, and streptokinase was given initially as a loading dose of 250,000 IU over 30 minutes, followed by a dose of 100,000 IU/hour over 24 hours. After the recovery of the patient from the acute stage, anticoagulant therapy was given, initially in the form of enoxaparin sodium at a dose of 90 mg/kg for seven days. In conjunction, warfarin was given at a daily dose of 5 mg until the international normalized ratio (INR) reached 2.8.\n\nThe patient fully recovered from the high-risk pulmonary embolism, and an echocardiogram made 24 hours after recovery showed complete dissolution of the intra-atrial mass with no evidence of any remnants left, suggesting that it was a massive ball thrombus that had a protrusion across the tricuspid valve (thrombus in transit). However, right atrial and ventricular dilation persisted. The patient was discharged after a short period of uneventful monitoring. Right before the discharge of the patient, surgical excisional biopsy of one subcutaneous mass was performed, and pathological assessment revealed superficial subcutaneous lipomas. His brothers also have these lipomas, so we believe that it is of familial origin. Upon taking further history from the patient, it was found that the patient and his brothers had a history of familial hyperlipidemia. Tests for the patient’s hypercoagulable state as assessed by measuring levels of protein C and S and antithrombin III appeared normal. The patient was followed up for 6 months and showed no more syncopal attacks. We do not have details of any further investigations that may lead to a cause of this condition.\n\n\nDiscussion\n\nPE is a serious medical emergency condition, in which obstruction of the pulmonary trunk or any of its branches takes place. It could be partial or complete, which causes sudden obstruction of the blood flow to the lungs, resulting in dyspnea, hypoxia, chest pain and insufficient blood flow to the left side of the heart. PE can be caused by any cause of embolism, including thrombosis, air, fat, talc or amniotic fluid1. About 1–2 people per 1000 may suffer from pulmonary embolism while 1 in every 100 people over the age of 80 may suffer from this condition2.\n\nSigns and symptoms of pulmonary embolism occur suddenly. Dyspnea, tachypnea, chest pain, cough, and hemoptysis take place. In more severe cases, cyanosis, syncope and circulatory instability occur, and sometimes peripheral edema may be present. Sudden death can occur in the most severe cases1. About 25% of PE cases present as sudden death while 15% of all cases of sudden death are attributable to PE2.\n\nThe criteria for diagnosis of PE are put into clinical prediction systems, such as the Wells score, which was initially proposed by Wells et al. in 1995 (Table 1)3. Additional prediction systems to determine the probability of getting a PE are available, such as the Geneva rule, which has a similar manner of prediction, but with some modifications4.\n\nAdapted from Neff MJ (2003)16. DVT: deep vein thrombosis, PE: pulmonary embolism.\n\nSyncope, as a presenting symptom in PE, is not uncommon. While it can have multiple causes, syncope is still an important sign of PE, especially if it is accompanied by dyspnea. A study conducted by Thames et al. in 1977 found that 13% of patients with PE present initially with syncope5. Another study conducted by Calvo-Romero et al. in 2004, in Spain, showed that 9.1% of patients with PE presented with syncope. However, the authors concluded that it was impossible to tell whether syncope could determine the prognosis in such cases6. Apart from these two studies, in 1988, two patients in Israel were reported to have suddenly died of massive PE after having a hip surgery 3 weeks earlier. Several syncopal episodes started in the first week after surgery, and they were considered as a sign of fatal outcome7.\n\nThe mechanisms that cause syncope in PE are variable. One mechanism is that the presence of PE decreases blood flow to the lungs, thus leading to hypoxemia and cerebral hypoxia. Another theory suggests that a vasovagal attack is triggered with the occurrence of PE. A third mechanism suggests that syncope occurs due to the arrhythmias that result from overloading of the right ventricle. A fourth mechanism suggests that PE leads to circulatory obstruction and decreased left ventricular filling, thus leading to decreased cardiac output and ultimately diminished cerebral blood flow8.\n\nA determinant factor in the diagnosis of the cause of syncope is the composition of arterial blood gases. Hypoxemia can indicate the presence of a condition that obstructs normal cardiovascular or respiratory functions. If there is no airway obstruction, PE is highly suspected. Advanced imaging techniques, such as multi-slice CT pulmonary angiography, can give an accurate image of the condition within the pulmonary vascular tree8.\n\nLipomas are common benign tumors formed of adipose tissue. They are the most common soft tissue tumor9. The most common lipoma is the superficial subcutaneous lipoma10. They can be found wherever fat is present under the skin surface. The tendency to develop them is not exclusively hereditary11. Nevertheless, there are familial conditions that may include the presence of lipomas, such as familial multiple lipomatosis12. In 1989, it was reported in Israel that there could be an association between familial combined hyperlipodemia and the presence of nonsymmetric subcutaneous lipomatosis in a family, which is similar to this case13. A growing body of evidence suggests the presence of a relationship between hyperlipidemia and the occurrence of DVT14. Therefore, we feel this co-occurrence is worth noting.\n\nIn this case, the ball thrombus (thrombus in transit), which was present in the right atrium, caused transient obstruction to the tricuspid valve, thus causing a transient obstruction to the outflow of the right atrium. That obstruction caused a transient obstruction to the left ventricular filling that brought about decreased cerebral blood flow. The ball thrombus probably got displaced as the position of the patient changed so that blood flow was restored and the syncopal episode ended. Eventually, the ball thrombus caused further thrombosis and released emboli that were large enough to occlude the pulmonary trunk and its main branches. Fortunately for the patient, the medical team successfully managed to initiate thrombolytic therapy in time, and the ball thrombus, along with the other emboli, were lysed. A similar case in Turkey was reported in 2009, but it was different in that the patient had a high risk due to long-term occupational immobilization and the presence of DVT8. Also a similar case series was reported in 1998 by Wolfe and Allen. They reported three cases that initially presented with syncope and later diagnosed with PE, and one of these cases ended up fatally15. An interesting aspect of the case presented here is that the medical team initially evaluated the patient as low risk according to the Wells score. Duplex scans and tests for hypercoagulable state were normal, unlike the results usually found in most patients with PE. That leaves us with a question as to what could have caused that thrombus to occur. Additional investigations are necessary to find a cause of this condition, such as an undiagnosed malignancy or a familial cause.\n\n\nConclusion\n\nPulmonary embolism is a medical emergency that can be presented by a common symptom such as syncope. Physicians should pay attention to this presentation and check for pulmonary embolism in cases of syncope with dyspnea. Such cases can be easily missed, which may end up with sudden death if not diagnosed and treated promptly.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images.",
"appendix": "Author contributions\n\n\n\nAO was a major contributor to the writing of the manuscript. He also gathered and analyzed the patient’s data about syncope and pulmonary embolism. AT performed the acute treatment to the patient and was a major contributor in writing the manuscript. AH performed and supervised the treatment of the patient and the manuscript writing process. All of the three authors have read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to express our sincere and deepest gratitude towards the Medical Sector Reform Group of Egypt for their continuous support throughout the case report preparation process as a part of the Mentor-Student Research Project. Our special thanks go to Dr. Noha A. Moussa and Omnia M. Omar for their tremendous efforts as coordinators of this project. We are also grateful to Dr. Sameh Nashat for performing echocardiography to the patient and providing us with the results.\n\n\nReferences\n\nGoldhaber S: Pulmonary thromboembolism. In Harrison's Principles of Internal Medicine. 16 edition. Edited by Kasper D, Braunwald E, Fauci A. New York, NY: McGraw-Hill; 2005; 1561–1565.\n\nBeckman MG, Hooper WC, Critchley SE, et al.: Venous thromboembolism: a public health concern. Am J Prev Med. 2010; 38(4 Suppl): S495–501. PubMed Abstract | Publisher Full Text\n\nWells PS, Hirsh J, Anderson DR, et al.: Accuracy of clinical assessment of deep-vein thrombosis. Lancet. 1995; 345(8961): 1326–1330. PubMed Abstract | Publisher Full Text\n\nWicki J, Perneger TV, Junod AF, et al.: Assessing clinical probability of pulmonary embolism in the emergency ward: a simple score. Arch Intern Med. 2001; 161(1): 92–97. PubMed Abstract | Publisher Full Text\n\nThames MD, Alpert JS, Dalen JE: Syncope in patients with pulmonary embolism. JAMA. 1977; 238(23): 2509–2511. PubMed Abstract | Publisher Full Text\n\nCalvo-Romero JM, Perez-Miranda M, Bureo-Dacal P: Syncope in acute pulmonary embolism. Eur J Emerg Med. 2004; 11(4): 208–209. PubMed Abstract\n\nEdelson G, Reis ND, Hettinger E: Syncope as a premonitory sign of fatal pulmonary embolism. Two case reports. Acta Orthop Scand. 1988; 59(1): 71–73. PubMed Abstract | Publisher Full Text\n\nDemircan A, Aygencel G, Keles A, et al.: Pulmonary embolism presenting as syncope: a case report. J Med Case Rep. 2009; 3: 7440. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBancroft LW, Kransdorf MJ, Peterson JJ, et al.: Benign fatty tumors: classification, clinical course, imaging appearance, and treatment. Skeletal Radiol. 2006; 35(10): 719–733. PubMed Abstract | Publisher Full Text\n\nPang D, Zovickian J, Oviedo A: Long-term outcome of total and near-total resection of spinal cord lipomas and radical reconstruction of the neural placode, part II: outcome analysis and preoperative profiling. Neurosurgery. 2010; 66(2): 253–272. discussion 272–273. PubMed Abstract | Publisher Full Text\n\nSalam GA: Lipoma excision. Am Fam Physician. 2002; 65(5): 901–904. PubMed Abstract\n\nLeffell DJ, Braverman IM: Familial multiple lipomatosis. Report of a case and a review of the literature. J Am Acad Dermatol. 1986; 15(2 Pt 1): 275–279. PubMed Abstract | Publisher Full Text\n\nRubinstein A, Goor Y, Gazit E, et al.: Non-symmetric subcutaneous lipomatosis associated with familial combined hyperlipidaemia. Br J Dermatol. 1989; 120(5): 689–694. PubMed Abstract | Publisher Full Text\n\nRay JG, Rosendaal FR: The role of dyslipidemia and statins in venous thromboembolism. Curr Control Trials Cardiovasc Med. 2001; 2(4): 165–170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWolfe TR, Allen TL: Syncope as an emergency department presentation of pulmonary embolism. J Emerg Med. 1998; 16(1): 27–31. PubMed Abstract | Publisher Full Text\n\nNeff MJ: ACEP releases clinical policy on evaluation and management of pulmonary embolism. Am Fam Physician. 2003; 68(4): 759–760. PubMed Abstract"
}
|
[
{
"id": "4035",
"date": "24 Mar 2014",
"name": "Martin Rohacek",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a case of pulmonary embolism (PE) presenting with syncope and hemodynamic instability.Comments:The Wells score is a validated tool to define the pre-test probability of PE before performing diagnostic tests such as D-dimer tests and CTPA (computed tomography pulmonary angiography), but not to predict outcomes such as mortality. The authors state that the Wells score \"sharply increased from 1.5 to 8\" after a drop in the blood pressure (BP). BP drop is not a criterion of a Wells score. Thus, the Wells score did not rise to 8, but remained 1.5, and the patient remained low risk for PE. But, the drop in BP speaks for a hemodynamically relevant PE, which can be treated with thrombolytic agents. The drop in BP worsens the outcome of the patient, but is not a reason to describe this case of PE as \"high-risk pulmonary embolism\". \"In order to save the patient’s life\": Hemodynamically relevant PE is not a 100% killer. Please discuss mortality rates of hemodynamically relevant PE.",
"responses": [
{
"c_id": "746",
"date": "28 Mar 2014",
"name": "Aly Tohamy",
"role": "Reader Comment",
"response": "Dear Dr Martin,We confirm that the patient is low risk for pulmonary embolism, So we state and report the case (as you see in case report title).Although the patient is low risk for pulmonary embolism, he develops hemodynamically relevant pulmonary embolism with huge right atrial mass.Secondly, the in-hospital all-cause case fatality rate was lower in unstable patients who received thrombolytic therapy than those who did not - 15% vs 47% (Stein PD and Matta F (Am J Med. 2012))."
}
]
},
{
"id": "4036",
"date": "02 Apr 2014",
"name": "Massimo Miniati",
"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 clinical case is well-described and, as such, it is worthy of indexation. My major concern is the use of the prediction model introduced by Wells. Even though it has been widely used, it has a very limited value in predicting pulmonary embolism (PE).In the reported case, the Wells score was 1.5 - indicating low clinical probability. In my opinion, instead, the clinical presentation is strongly suggestive of PE.By applying the prediction model we introduced in 2008 (Miniati et al., 2008) the clinical probability of PE would be 86%. Our model allows the estimation of the probability of PE by adding the regression coefficients that apply to a given patient. The probability can be calculated at once by using dedicated software that can be downloaded at www.ifc.cnr.it/pisamodel.I am not saying that the authors should make use of our model, but I think that the availability of other models should be acknowledged in the manuscript.Also, I may say that syncope is a relatively frequent presentation of PE even in young and otherwise healthy subjects. In a survey of 800 patients with an established diagnosis of PE (Miniati et al., 2012) the prevalence of syncope was 24%.In sum, I believe that the manuscript is of interest but requires revision.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-257
|
https://f1000research.com/articles/2-122/v1
|
09 May 13
|
{
"type": "Research Article",
"title": "Eco-physiological adaptation of dominant tree species at two contrasting karst habitats in southwestern China",
"authors": [
"Shouren Zhang",
"Dayong Fan",
"Qian Wu",
"Hui Yan",
"Xinwu Xu",
"Shouren Zhang",
"Qian Wu",
"Hui Yan",
"Xinwu Xu"
],
"abstract": "The purpose of this study was to investigate the eco-physiological adaptation of indigenous woody species to their habitats in karst areas of southwestern China. Two contrasting forest habitats were studied: a degraded habitat in Daxiagu and a well-developed habitat in Tianlongshan, and the eco-physiological characteristics of the trees were measured for three growth seasons. Photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (Tr) of the tree species in Daxiagu were 2-3 times higher than those in Tianlongshan under ambient conditions. However, this habitat effect was not significant when measurements were taken under controlled conditions. Under controlled conditions, Pn, gs, and Tr of the deciduous species were markedly higher than those for the evergreen species. Habitat had no significant effect on water use efficiency (WUE) or photochemical characteristics of PSII. The stomatal sensitivity of woody species in the degraded habitat was much higher than that in the well-developed habitat. Similarly, the leaf total nitrogen (N) and phosphorus (P) contents expressed on the basis of either dry mass or leaf area were also much higher in Daxiagu than they were in Tianlongshan. The mass-based leaf total N content of deciduous species was much higher than that of evergreen species, while leaf area-based total N and P contents of evergreens were significantly higher than those of deciduous species. The photosynthetic nitrogen- and phosphorus-use efficiencies (PNUE and PPUE) of deciduous species were much higher than those of evergreens. Further, the PPUE of the woody species in Tianlongshan was much higher than that of the woody species in Daxiagu.The results from three growth seasons imply that the tree species were able to adapt well to their growth habitats. Furthermore, it seems that so-called “temporary drought stress” may not occur, or may not be severe for most woody plants in karst areas of southwestern China.",
"keywords": [
"karst habitat",
"photosynthesis",
"chlorophyll fluorescence",
"stomatal sensitivity",
"water potential"
],
"content": "Introduction\n\nKarst topography features soluble bedrock, which is usually carbonate rock such as limestone or dolomite1. Karst topography is characterized by a very slow formation of soil from the carbonate rock, leading to low water retention capacity1,2. China’s karst topography is located mainly in the southwestern region, which is also characterized by diversified landscape types and a dense population. Deforestation was once one of the most serious environmental problems in China’s karst region, and was mainly due to fuel wood production, agricultural expansion, and livestock husbandry. This land use has caused the ecosystem of the karst region to degrade beyond its already poor condition. As a result, many woodlands have degraded to rocky desert in many of the karst regions in southwest Guizhou Province. Karst ecosystems are very fragile. If destroyed, the soil regeneration process is slow, and thus karst ecosystems are only restored gradually, if at all. Precipitation is sufficient in this region, but the woody plants are supposedly subjected to temporary water stress. This water stress results from low soil water-holding capability, and the high leakiness of limestone rock2,3. Numerous studies have simulated the above environments, and the data generated has shown that plants are suffering from conditions of temporary water-stress2,3. In most of these studies, potted seedlings were used, and the experiments were conducted under controlled environments (e.g. greenhouses). It is risky to extrapolate the situation for mature tree species in their natural conditions from data from potted seedlings under controlled conditions. So far, reports on woody plant growth and physiological response to temporary water deficiency in the karst field areas have been sparse. This is particularly so for mature woody plants. Investigating how woody plants respond functionally to differently degraded habitats will help understand the adaptive mechanisms that these indigenous species have to their habitats. This will also help in optimizing the selection of tree species for forest ecosystem restoration in karst regions.\n\nFor our study, we selected two types of forests with contrasting karst habitats in the west and southwest of Guizhou Province: a well-developed secondary deciduous and evergreen broad-leaved mixed forest at Tianlongshan Mountain, and a severely degraded forest dominated by spare deciduous woody species at Daxiagu. We made in situ measurements of eco-physiological traits for the dominant tree species in three consecutive growth seasons (2007–2009) to address the following questions:\n\n1) Compared with tree species in the well-developed forest habitat, are the eco-physiological traits down-regulated for the tree species in the degraded karst habitat?\n\n2) Is there a difference in stomatal sensitivity between tree species in the two contrasting forests?\n\n3) How do nutrients affect the eco-physiological characteristics of the tree species in the two contrasting forests?\n\n\nMaterials and methods\n\nFor the purpose of making comparisons of how the tree species adapt to their own different karst habitats, we designed the experiment at two contrasting mature forest habitats. Tianlongshan, located in the west of Guizhou Province, has a relatively well-developed karst secondary forest. Daxiagu, located in the southwest of Guizhou Province, has a severely degraded karst forest.\n\nTianlongshan is located about 10 km south of Puding County in western Guizhou Province (26°15′N, 105°44′E) at an altitude of about 1200 m. This region is dominated by a humid monsoon climate. The mean annual temperature is 15.1°C4. The mean annual precipitation is 1398 mm, and 60–70% of the rainfall events occur during the growth season between May and October4. Tianlongshan has a well-developed secondary evergreen and deciduous broad-leaved mixed forest growing in lime yellow soil. Tree heights range from 3–7 m.\n\nDaxiagu is located 20 km southwest of the town of Huajiang in southwestern Guizhou Province (25°42′N, 105°35′E) at an altitude of about 900 m. This area has a warm temperate climate and a mean annual temperature of 18.4°C4. The mean annual precipitation is 1100 mm, with 83% of this precipitation occurring during the growth season between May and October. Vegetation in this region is characterized by sparsely distributed secondary deciduous trees and shrubs on bare rocks.\n\nField investigations and collections of tree leaf samples in these two sites did not require specific permits. For the purposes of our work, collaboration with local (provincial) universities or research institutes was required. The locations of our field investigations are neither privately-owned nor protected lands. The tree species used in our investigations and sampling were not endangered or protected.\n\nThe dominant tree species in Tianlongshan and Daxiagu found in this study are listed in Table 1.\n\nFor leaf phenology, D represents deciduous and E represents evergreen; For location, T represents Tianlongshan and Dx represents Daxiagu.\n\n3–4 trees for each species were randomly (the nearest one every 20–30 m distance) selected from the study sites, and a detached branch from each tree was obtained from the top or middle sunny side (south-facing) of the canopy. Branches were detached using a pair of pruning shears mounted on a 5 m pole. The detached branch was immediately immersed in a water-filled bucket. The end of each branch was re-cut twice under water, ensuring continuity of the xylem conduit. Photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), and instantaneous water use efficiency (WUE) were measured with new, fully expanded leaves from the detached branches using a LI-6400 photosynthesis system (LI-COR Inc., Lincoln Nebraska, USA). Measurements were conducted around 8:30–13:00 (Beijing Standard Time). Measurement conditions were set at a PAR (photosynthetically active radiation) level of 1000 µmol m-2 s-1. Leaf temperature and humidity were at their ambient conditions during the measurement period of June–July 2007 and 2008. During this time, the leaf temperatures were 22–25°C in Tianlongshan and 30–33°C in Daxiagu.\n\nPlant stomatal sensitivity was obtained using Lohammar’s hyperbolic function method5–7. We measured gs-VPD (leaf-air vapor pressure deficiency) curves in July 2009, in which the VPD was manually set from saturated vapor [over 85% of relative humidity (RH)] to the driest vapor (about 5% of RH). Leaf temperature was held at 30°C, and PAR at 1000 µmol m-2s-1. The gs-VPD curves were fitted using the modified Lohammar’s function: gs = -m × ln D + b, to estimate stomatal sensitivity (m)8. In this equation, gs and D stand for stomatal conductance and VPD, respectively, and m and b are parameters generated in a least square regression analysis.\n\nChlorophyll a fluorescence transients were measured in the leaves from the branches collected as detailed above for photosynthetic gas exchange measurements using a Handy-PEA portable fluorometer (Hansatech Instruments Ltd., Norfolk, UK). Before measurements were taken, the leaves were darkened for at least 30 min using leaf clips (Hansatech Instruments Ltd., Norfolk, UK). The polyphasic chlorophyll a fluorescence transients OJIP [fluorescence levels O: Fo (50 μs); J: FJ (2 ms); I: FI (30 ms), and P: Fp=Fm (tFmax)] were analyzed according to the JIP test procedure9,10. The JIP test procedure has been widely used in studies of eco-physiology and stress physiology10. Several parameters can be derived from the following fluorescence values: 50 μs (Fo, step O), 100 μs (F100), 300 μs (F300), 2ms (step J), 30 ms (step I), and the maximum (Fm, step P), using Biolyzer software (version 3.0.7.2 and available by contacting Dr. Reto Strasser at Reto.Strasser@unige.ch). According to Strasser et al.9,10, the definition of the JIP test parameters used in this study can been summarized as follows: Φ(Po), maximum quantum yield of primary photochemistry; Φ(Eo), quantum yield of electron transport, probability that an absorbed photon will move an electron into electron transport further than QA-; Φ(Do), quantum yield of dissipation; PIabs, performance index on the basis of light energy absorption.\n\nΦ(Po)=1–(Fo/Fm)=Fv/Fm; Fo=F50 μs, minimal fluorescence at O-step (50 μs); Fm=FP, maximal fluorescence at P-step;\n\nΦ(Eo)=(Fv/Fm)Ψ; Ψ=(1–VJ), VJ=(FJ-Fo)/(Fm–Fo), FJ=F2 ms, fluorescence at J-step (2 ms) of O-J-I-P;\n\nΦ(Do)=1–Φ(Po)=Fo/Fm;\n\nPI(abs)=(RC/ABS)×[Φ(Po)/(1–Φ(Po))]×[Ψ/(1-Ψ)]; RC=reaction center, ABS=absorption flux.\n\nThe midday leaf water potential (Ψ) was determined for each species at the two habitats using a PSYPRO Water Potential System (Wescor, Inc., USA). The measurements were carried out between 11:00 and 14:00 hours (Beijing Standard Time) in August 2009 on leaves from the same detached branches from which the photosynthesis/fluorescence measurements were taken.\n\nLeaf total N content and soil total organic N content (%) were determined using the Kjeldahl acid-digestion method (Kjeltec 2200 Auto Distillation Unit, Foss, Denmark). Leaf total phosphorus (P) content and soil total P content (%) were analyzed colorimetrically (UV-visible spectrophotometer, UV-2550, Shimadzu Corporation, Japan).\n\nBefore the Analysis of Variance (ANOVA) was carried out, all data was examined graphically for the normality of distribution (probability plots for residual analysis), and the homogeneity of variance (scatter plots) using Data Desk (version 6.01, Data Description, Inc., 1996). After examination using the above methods, all data except leaf water potential satisfied the assumption for ANOVA for normal distribution and homogeneity of variance. The data for leaf water potential were log-transformed, and the transformed data satisfied the normal distribution assumption for ANOVA. The effects of habitat (i.e. the severely degraded karst habitat vs. well-developed secondary forest habitat) and life form (i.e. deciduous vs. evergreen) were tested using the two-way ANOVA procedure in Data Desk.\n\n\nResults\n\nWhen measured under their respective ambient conditions in 2007 and 2008 (i.e. ambient temperature and humidity, and PAR set at 1000 µmol m-2s-1), the photosynthetic rate (Pn) across all investigated tree species was much higher in Daxiagu (around 2–3 times, P < 0.001) than in Tianlongshan (Figure 1). The gs and Tr of the investigated tree species in Daxiagu were also much higher (P < 0.001) than those in Tianlongshan (Figure 1). However, when measurements were taken under controlled conditions in 2009 (i.e. PAR was set at 1000 µmol m-2s-1, leaf temperature at 30°C, and VPD at 1 KPa), there was no significant habitat effect on Pn (Figure 1). There was a significant difference in Pn between deciduous and evergreen tree species across all investigated tree species in the two habitats. The Pn of deciduous tree species was significantly higher (P < 0.01) than the Pn of evergreen tree species (Figure 1). The gs and Tr of the deciduous tree species were also significantly higher than the gs and Tr of evergreen tree species (P < 0.05 and 0.01, respectively). The habitat effect on gs and Tr was not statistically significant (Figure 1). The effects of leaf phenology (deciduous vs. evergreen) and habitat were not significant for instantaneous water use efficiency (WUE) under either ambient or controlled measurement conditions in either habitat.\n\nEffect of habitat (H), leaf phenology (L), and their interaction (H × L), on Pn, gs, E, and WUE (mean ± SD, n = 3–5) of the tree species in two habitats. T and Dx represent the two study sites Tianlongshan and Daxiagu, respectively. Measurements were conducted under ambient conditions in 2007 and 2008, and under controlled conditions in 2009. The significance levels (*** = P < 0.001, ** = P < 0.01,* = P < 0.05, and ns = P > 0.05) were based on ANOVA results.\n\n\n\nLeaf phenology and habitat had no significant (P > 0.05) effect on chlorophyll fluorescence parameters in 2007 (Figure 2). However, the effect of leaf phenology became significant (P < 0.05) for maximal PSII efficiency (Φ(Po)), quantum yield of PSII electron transport (Φ(Eo)), quantum yield of dissipation (Φ(Do)), and the comprehensive parameter for assessing plant’s vitality: performance index (PI(abs)), for all investigated tree species in 2008 and 2009 (Figure 2). The Φ(Po), Φ(Eo), and PI(abs) of the evergreen tree species were significantly higher than those of the deciduous tree species, while the opposite effect of leaf phenology was seen in Φ(Do).\n\nEffects of habitat (H), leaf phenology (L), and their interaction (H × L), on maximal PSII efficiency (Φ(Po)), quantum yield of PSII electron transport (Φ(Eo)), quantum yield of dissipation Φ(Do), and performance index (PI(abs)) (mean ± SD, n = 3–5) of the tree species in two habitats. The ANOVA for Ψ is based on log-transformed data. See Figure 1 for other explanations.\n\n\n\nHabitat had a significant effect on stomatal sensitivity (m) (P < 0.05), and the stomatal sensitivity (m) was much higher in Daxiagu than in Tianlongshan (Figure 3). There was an interactive effect of habitat and leaf phenology on leaf water potential, and the leaf water potential of deciduous tree species in Daxiagu was significantly lower than the leaf water potential in Tianlongshan (Figure 3). Leaf phenology had a significant effect on the specific leaf area (SLA): the SLA of deciduous species was significantly (P < 0.05) higher than that of evergreen species (Figure 3).\n\nEffects of habitat (H), leaf phenology (L), and their interaction (H × L), on stomatal sensitivity (m), leaf water potential (Ψ) and specific leaf area (SLA) (mean ± SD, n = 3–5) of the tree species in two habitats. The ANOVA for Ψ is based on log-transformed data. See Figure 1 for other explanations.\n\nHabitat had significant effects on leaf total N content expressed on the basis of either dry mass or leaf area (P < 0.01) (Figure 4). Leaf total nitrogen content in Daxiagu was much higher than leaf total nitrogen content in Tianlongshan. Leaf phenology also had a significant effect on leaf total nitrogen content expressed on the basis of either dry mass or leaf area (P < 0.01 and P < 0.05, respectively). The mass-based leaf total N content of the deciduous species was much higher than that of the evergreen species. However, when leaf total nitrogen content was expressed on the basis of leaf area, the leaf total N content of the evergreen species was significantly higher than the leaf total N content of the deciduous species. The habitat had a significant effect on leaf total phosphorus content expressed on the basis of either dry mass or leaf area (P < 0.001). The leaf total phosphorus content in Daxiagu was much higher than leaf total phosphorus content in Tianlongshan (Figure 4). However, when leaf total phosphorus content was expressed on basis of leaf area, the values of the evergreen species were much higher than those of deciduous species, especially in Daxiagu. The habitat also had a significant effect on the ratio of leaf total N to P (N:P) for all measured tree species. The N:P ratio in Tianlongshan was significantly (P < 0.001) higher than the N:P ratio in Daxiagu (Figure 4). Leaf phenology had a significant (P < 0.001) effect on photosynthetic nitrogen use efficiency (PNUE), and the PNUE of deciduous species was much higher than of evergreen species (Figure 5). Both habitat and leaf phenology had significant (P < 0.001) effects on photosynthetic phosphorus use efficiency (PPUE). The PPUE of the woody species in Tianlongshan was much higher than that in Daxiagu (P < 0.001), and the PPUE of deciduous species was much higher than that of evergreen species (P < 0.001) (Figure 5).\n\nEffects of habitat (H), leaf phenology (L), and their interaction (H × L), on leaf area based- and mass-based total nitrogen (N) and phosphorus (P) (mean ± SD, n = 3–5) of the tree species in two habitats. See Figure 1 for other explanations.\n\nEffects of habitat (H), leaf phenology (L), and their interaction (H × L), on photosynthetic N and P use efficiency (PNUE and PPUE, respectively) (mean ± SD, n = 3–5) of the tree species in two habitats. See Figure 1 for other explanations.\n\n\n\n\n\n\nDiscussion\n\nPlants in karst regions are thought to be subject to temporary drought stress in their growth seasons due to the poor water holding capacity of the soil. This is thought to be particularly so in severely degraded karst habitat2,3. However, the data from this study for chlorophyll fluorescence (e.g. (Φ(Po)), Φ(Eo), and PI(abs)) in the three consecutive growth seasons suggests that drought stress does not occur in all of the investigated tree species during their growth seasons. The chlorophyll fluorescence technique has been used as a powerful tool to assess plant vitality in response to environmental stresses10. So-called \"temporary drought stress\" may not occur at all. Further, if such temporary stress really does occur, then it may not be severe for most woody plants in the karst areas of western and southwestern Guizhou Province. Further, the indigenous/native trees adapt well to their habitats, including plants in the most severely degraded karst habitat (Daxiagu in this study). This might be due to karst plants’ adaptive structural strategies (e.g. absorbing water from deep soil through roots penetrating into rock crevasses, according to Zhu2), or the plants’ adaptive functions (e.g. stomatal sensitivity to changed environments). It may also be due to the ample precipitation found in the studied areas (annually around 1000 mm, mainly occurring during the growth season). Liu et al.11 did find that soil water stress (withholding water) affected photosynthesis and growth, and re-watering could remove or alleviate these effects in potted tree seedlings from almost the same karst area as in our study. However, the water stress treatment (withholding water) period in Liu’s et al. experiment was as long as 20 days. According to Zhu et al.12, during the growth season, the most common period between two precipitation events in this region is less than 20 days. Further, the available soil water from a heavy rain event could meet tree transpiration needs for 7–14 days13. This implies that in most cases, temporary soil water stress events would not occur during the growth season of this region. The data for leaf δ13 C value from Yang et al.14 and Fan et al.15 also confirms the above conclusions based on chlorophyll fluorescence analyses. They report that the average leaf δ13 C values of more than 50 tree species in three karst sites across Guizhou are -27.63%14, and -28.14%15. These values are only higher than the values of the tree species of tropic rain forests in Yunnan Province (e.g. -33.11% reported by Qu et al.16), and mostly lower than other areas of China (e.g. -26.24% of temperate forest17 and -27.00% of desert vegetation18). This indicates that the average long-term WUE in tree species in the karst area is low, and that they do not experience severe long-term drought stress.\n\nNevertheless, we did find a difference in photochemical traits of PSII (i.e. Φ(Po), Φ(Eo), and PI(abs)) between the deciduous and evergreen tree species in the 2009 growth season. This is consistent with the results of midday leaf water potential (Figure 3), suggesting that the capacity for maintaining leaf hydro-physiological function in evergreen tree species was higher than that in deciduous trees. Fan et al.15 also confirmed that evergreen tree species had higher water stress tolerance for maintaining branch hydraulic conductivity than did deciduous trees species. This is based on data for the branch’s hydraulic characteristics for roughly the same tree species in the two habitats. In addition, there was a much lower Ψ50 (xylem tensions at 50% of loss in hydraulic conductivity) in evergreen tree species than in deciduous tree species15.\n\nWUE is an intrinsic trait that indicates plant strategies for environmental adaptation, and is understood in terms of a trade-off between carbon gain and water loss. WUE is also a reliable indicator for determining plant survivorship in arid areas19. To adapt to changed environments, in comparison with plants with low VPD, plants with high leaf-atmosphere VPD will generally increase their WUE20,21. That there is no significant variation in the effects of habitat and leaf phenology on WUE also partly confirms that plants adapt well to their environments, even to severely degraded karst habitat. Furthermore, our data on photosynthetic gas exchange showed that the photosynthetic rates (Pn) of the tree species in Daxiagu (the severely degraded habitat) are nearly 2–3 times higher than those of the tree species in Tianlongshan (the well developed secondary forest habitat) under ambient conditions. This indicates that to determine the CO2 assimilation of plants in these areas, it would be more important to take light and temperature into consideration than it would be to consider precipitation. The Daxiagu habitat has much less vegetation coverage than the Tianlongshan habitat. The tree species in Daxiagu are distributed sparsely and most tree canopies receive almost full sunlight. The tree canopies in Tianlongshan are closed and the light environment is lower for the middle and lower canopy leaves. The temperature in Daxiagu in the growth season is clearly higher than in Tianlongshan (with a difference of about 3–5, and -28.14°C). Many studies confirm that leaves exposed to sunlight have higher Rubisco activity, chlorophyll a/b ratio, maximum photosynthetic rate, and light saturation points than do shaded-leaves22–25. In other words, when precipitation (water supply for soil) is guaranteed, full sunlight and higher temperatures improve the photosynthetic capacity of the plant.\n\nIn China, soil phosphorus deficiency occurs more commonly in the southern region than in the northern region. Furthermore, soil total phosphorus density in tropical and subtropical areas is much lower than in other areas26. Soil phosphorus deficiency has a negative effect on plant absorption of nitrogen from the soil; thus phosphorus limitation inhibits plant growth. According to the stoichiometric relationship between N and P, the biomass N:P ratios could be effective indicators of the status of nitrogen and phosphorus in a plant27–29. The average N:P ratio for terrestrial plant species in their natural field habitats is 12–1330–32. Koerselman and Meuleman28 suggest that phosphorus deficiency occurs when the N:P ratio is higher than the critical value of 16. The much higher leaf N:P ratio in Tianlongshan (mean = 20.32) as compared to Daxiagu (mean = 12.26) suggests that there was severe phosphorus limitation in Tianlongshan. Because phosphorus deficiency has negative effects on nitrogen absorption33,34, phosphorus limitation could partly explain why the Pn was lower in Tianlongshan than in Daxiagu, while PPUE was more stimulated in Tianlongshan than in Daxiagu. There is also a marked difference in tree species composition between the two habitats: more than 90% of the tree species measured in Daxiagu are deciduous, while evergreen tree species account for 50% of the measured tree species in Tianlongshan. A great deal of data has shown that the photosynthetic capacity of deciduous species is higher than the photosynthetic capacity of evergreen species25.\n\nThe stomatal sensitivity of woody species in open habitats (i.e. Daxiagu) is much higher than in closed habitats (i.e. Tianlongshan), again suggesting that the light, ambient humidity, and temperature conditions are much more important than precipitation in shaping the stomatal response of woody plants to changed leaf-air VPD. The study of the sensitivity of stomata to changed leaf-air VPD conditions in Ligustrum sinense also confirmed that the relationship between stomatal conductance and stomatal aperture for high-light leaves was more significant than that of low-light leaves35. Higher stomatal sensitivity will help maintain the physiologically required water status of a plant35. This could explain the difference in the stomatal sensitivity of the woody species between the two habitats. This could also explain why there was no difference in photosynthetic instantaneous WUE between the two habitats, although there was a significant difference in water consumption through transpiration in the woody species.",
"appendix": "Author contributions\n\n\n\nSZ and DF conceived and designed the experiment; SZ, DF, QW, HY and XX conducted the field measurements; SZ analyzed the data and wrote the manuscript. DF revised the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the National Natural Science Foundation of China (project Nos. 31070356 and 31170457) and the National Basic Research Program of China (973 Program No. 2006CB403206).\n\n\nReferences\n\nLiu CQ: Biogeochemical Processes and Cycling of Nutrients in the Earth’s Surface: Cycling of Nutrients in Soil–Plant Systems of Karstic Environments, Southwest China. Science Press, Beijing, China. 2009. Reference Source\n\nZhu SQ: Ecological research on karst forest (II). Guizhou Science and Technology Press, Guiyang China. 1997.\n\nZhu SQ: Ecological research on karst forest (I). Guizhou Science and Technology Press, Guiyang China. 1993.\n\nWu JM, Tong BQ, Yang J: The relationship between climate of Karst region in Guizhou province and ecological administration. J Guizhou Meterol. 2003; 5: 25–28.\n\nLohammar T, Larsson S, Linder S, et al.: FAST-simulation models of gaseous exchange in Scots pine. Ecol Bull (Stockholm). 1980; 32: 505–523. Reference Source\n\nLindroth A, Halldin S: Numerical analysis of pine-forest evaporation and surface resistance. Agr Forest Meteorol. 1986; 38(1–3): 59–79. Publisher Full Text\n\nDang QL, Margolis HA, Coyea MR, et al.: Regulation of branch-level gas exchange of boreal trees: roles of shoot water potential and vapor pressure difference. Tree Physiol. 1997; 17(8–9): 521–535. PubMed Abstract | Publisher Full Text\n\nOren R, Sperry JS, Katul GG, et al.: Survey and synthesis of intra- and interspecific variation in stomatal sensitivity to vapour pressure deficit. Plant Cell Environ. 1999; 22(12): 1515–1526. Publisher Full Text\n\nStrasser RJ, Srivastava A, Govindjee: Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem Photobiol. 1995; 61(1): 32–42. Publisher Full Text\n\nStrasser RJ, Srivastava A, Tsimilli-Michael M: Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G and Govindjee (eds) Series: Adv Photosynth Respiration: Chlorophyll Fluorescence, A Signature of Photosynthesis. 2004; 19: 321–362. Publisher Full Text\n\nLiu CC, Liu YG, Guo K, et al.: Influence of drought intensity on the response of six woody karst species subjected to successive cycles of drought and rewatering. Physiol Plant. 2010; 139(1): 39–54. PubMed Abstract | Publisher Full Text\n\nZhu SQ: Ecological research on karst forest (III). Guizhou Science and Technology Press, Guiyang China. 2003.\n\nZhou YC, Pan GX: Adaptation and adjustment of Maolan forest ecosystem to karst environment. Carsologica Sinica. 2001; 20: 47–52. Reference Source\n\nYang C, Liu CQ, Song ZL, et al.: Characteristics of carbon isotopic compositions in karst mountain plants and their indication to water use efficiency - A case study on Yangzhong catchment in Huaxi, Guizhou. Carsologica Sinica. 2007; 26: 105–110. Reference Source\n\nFan DY, Jie SL, Liu CC, et al.: The trade-off between safety and efficiency in hydraulic architecture in 31 woody species in a karst area. Tree Physiol. 2011; 31(8): 865–877. PubMed Abstract | Publisher Full Text\n\nBoyer JS: Plant productivity and environment. Science. 1982; 218(4571): 443–448. PubMed Abstract | Publisher Full Text\n\nQu CM, Han XG, Su B, et al.: The characteristics of foliar δ 13C values of plants and plant water use efficiency indicated by δ 13C Values in two fragmented rainforests in Xishuangbanna, Yunnan. Acta Botanica Sininca. 2001; 43: 186–192. Reference Source\n\nSu B, Han XG, Li LH, et al.: Responses of δ 13C value and water use efficiency of plant species to environmental gradients along the grassland zone of northeast China transect. Acta Phytoecologica Sinica. 2000; 24: 648–655. Reference Source\n\nYan CR, Han XG, Chen LZ: Foliar δ 13C within temperate deciduous forest: spatial, temporal, and species sources of variation. Acta Botanica Sinica. 1998; 40: 835–839.\n\nKnight JD, Livingston NJ, van Kessel C: Carbon isotope discrimination and water-use efficiency of six crops grown under wet and dryland conditions. Plant Cell Environ. 1994; 17(2): 173–179. Publisher Full Text\n\nZhao B, Kondo M, Maeda M, et al.: Water-use efficiency and carbon isotope discrimination in two cultivars of upland rice during different developmental stages under three water regimes. Plant Soil. 2004; 261(1–2): 61–75. Publisher Full Text\n\nBjörkman O, Boardman NK, Anderson JM, et al.: Effect of light intensity during growth of Atriplex patula on the capacity of photosynthetic reactions, chloroplast components and structure. Carnegie Institution of Washington Year book. 1972; 71. : 115–135. Reference Source\n\nBoardman NK, Anderson JM, Thorne SW, et al.: Photochemical reactions of chloroplasts and components of the photosynthetic electron transport chain in two rainforest species. Carnegie Institution of Washington Year book. 1972; 71. : 107–114. Reference Source\n\nPowles SB, Osmond CB: Photoinhibition of intact attached leaves of c(3) plants illuminated in the absence of both carbon dioxide and of photorespiration. Plant Physiol. 1979; 64(6): 982–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarcher W: Physiological Plant Ecology: Ecophysiology and Stress Physiology of Functional Groups (4th Edition). Springer-Verlag Berlin Heidelberg. 2003. Reference Source\n\nWang T, Yang YH, Ma WH: Storage, patterns and environmental controls of soil phosphorus in China. Acta Scientiarum Naturalium Universitatis Pekinensis. 2008; 44(6): 945–952. Reference Source\n\nWoodwell GM, Whittacker RH, Houghton RA: Nutrient concentrations in plants in the Brookhaven oak-pine forest. Ecology. 1975; 56: 318–332. Publisher Full Text\n\nKoerselman W, Meuleman AFM: The vegetation N: P ratio: a new tool to detect the nature of nutrient limitation. J Appl Ecol. 1996; 33: 1441–1450. Publisher Full Text\n\nNiklas KJ: Plant allometry, leaf nitrogen and phosphorus stoichiometry, and interspecific trends in Annual growth rates. Ann Bot. 2006; 97(2): 155–163. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElser JJ, Fagan WF, Denno RF, et al.: Nutritional constraints in terrestrial and freshwater food webs. Nature. 2000; 408(6812): 578–580. PubMed Abstract | Publisher Full Text\n\nGüsewell S, Koerselman W: Variation in nitrogen and phosphorus concentrations of wetland plants. Perspect Ecol Evol Syst. 2002; 5(1): 37–61. Publisher Full Text\n\nKnecht MF, Göransson A: Terrestrial plants require nutrients in similar proportions. Tree Physiol. 2004; 24(4): 447–460. PubMed Abstract | Publisher Full Text\n\nAsner GP, Seastedt TR, Townsend AR: The decoupling of terrestrial carbon and nitrogen cycles. BioScience. 1997; 47(4): 226–234. Publisher Full Text\n\nAber JD, McDowell W, Nadelhoffer K, et al.: Nitrogen saturation in temperate forest ecosystems: hypotheses revisited. BioScience. 1998; 48(11): 921–934. Reference Source\n\nZhang S, Ma K, Chen L: Tempo-Spatial Variations in Stomatal Conductance, Aperture and Density of Ligustrum sinense Acclimated to Different Light Environments. Acta Botanica Sinica. 2002; 44(10): 1225–1232. Reference Source"
}
|
[
{
"id": "968",
"date": "29 May 2013",
"name": "Jian Rang Wang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate for the content of the article. The authors used two contrasting habitats (degraded versus well developed) and leaf phenology (deciduous versus evergreen) across many major tree species to address the ecophysiological adaptation of different tree species to habitat change. The research was well designed and relatively long-term (three year measurements) for ecophysiological study. The information presented in the study will provide significant insight to our understanding of tree species adaptation to climate change and habitat degradation. The conclusions from the study will provide useful guidance for biodiversity management and restoration of degraded sites.",
"responses": []
},
{
"id": "1026",
"date": "26 Jun 2013",
"name": "Jessica Gurevitch",
"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\nScientific study of the ecologically interesting karst forests of southwestern China has been limited, particularly in the English-language literature, making this study potentially valuable. The comparison of in situ with common environment performance was excellent. However, major design flaws make it impossible to reach scientifically valid conclusions from this study. Most importantly, the study compares one degraded forest with one more-intact forest in order to reach conclusions about the ecophysiology of trees in degraded karst forests in comparison to intact forests. This is not possible with one forest of each type; the fallacy in this kind of comparison has been highlighted in the ecological literature for decades (sometimes called “pseudoreplication”). One cannot reach any valid conclusions from comparing trees in a single forest of one type with those of a single forest of a different type. The anova also fails to account for the nested design structure, a very common problem in the ecological literature.",
"responses": [
{
"c_id": "497",
"date": "09 Jul 2013",
"name": "Shouren Zhang",
"role": "Author Response",
"response": "We appreciate the invaluable comments from Prof. Jessica Gurevitch. We agree with Prof. Gurevitch’s comments on the experimental design in our study. However, at least two factors prevented us from selecting more sites for each forest type. Firstly, field acquisition of physiological parameters, such as the photosynthetic rate and maximum photochemical efficiency of PSII, is very time-consuming. Our ability to make the simultaneous measurements needed to compare different sites is therefore limited by resource constraints. Secondly, since these physiological parameters vary significantly throughout the day, they need to be measured at similar times of day to make sense of the comparison. For example, in this study we measured photosynthetic gas exchange and chlorophyll fluorescence from late-morning to noon, which is thought to be the daily based maximal photosynthetic functional period for the sampled trees. These factors make physiological investigations under controlled conditions on multiple sites unpractical. We note, also, that although we compared only two sites, the trees sampled within one site in our study were distributed over a large area (roughly 1 km2), and therefore could be regarded as growing on different sub-sites with quite different local environmental situations. Most importantly, however, the physiological data in our study were collected over three consecutive years in field. Such long-term investigations of physiological processes in field are very scarce in eco-physiological literature."
}
]
},
{
"id": "2396",
"date": "13 Nov 2013",
"name": "Reto Strasser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 read this manuscript several times with great interest. Immediately the reader gets the impression of a carefully conducted and complex investigation. The topic is a case study of ecophysiology as you find it in vivo, in situ in nature.The title alone informs the reader that a complex multiparametric ecological experiment is presented.The areas of this investigation were forests in southwestern China with contrasting habitats, in the Guizhou Province called Tianlongshan and Daxiagu.Some of the main contrasts are listed and compared below:Altitude above sea level: 1200m (Tianlongshan) and 900m (Daxiagu)Karst: Well-developed secondary forest with a humid monsoon climate (Tianlongshan) and severely degraded mixed forest with warm temperatures (Daxiagu)Precipitation: 1398 mm/year (Tianlongshan) and 1100mm/year (Daxiagu)Rain during growth season: 60% to 70% (Tianlongshan) and 83% (Daxiagu)Vegetation: Well developed secondary evergreen and deciduous (Tianlongshan) and secondary deciduous trees and shrubs (Daxiagu)Branches were cut from both places and used for measurements such as gas exchanges (CO2), stomatal conductance, transpiration and water use efficiency (WUE), on new fully expanded leaves. Many in vivo in situ fast fluorescence transients were collected and analyzed according to the equations of the JIP test, which reveals the bioenergetics constellation of the samples.It is seldom that a research team is able to use so many quite different techniques on the same plant material within a short time span and outside in the forest. All techniques are precisely presented in the manuscript and the execution of the measurements appears to have been done very carefully.Several years ago I had the opportunity to accompany a research group of the same institute as a guest for another project, to make such measurements in the forest. I became aware of the high professionalism of experimentation which was invested to get the experimental data recorded. E.g. black socks or sock-shaped towels were used to slip over branches on the trees to guarantee dark adaptation in a non-invasive way during the daytime.In short, I can say that the investigation is technically very complex, however has been executed with a lot of skill and imagination.I have seen the comments of the previous two reviewers. I fully agree with the scientific statements given by the reviewers who have pointed out the high quality of the data. For some logically understandable reasons one reviewer has given the article a ‘Not approved’ rating. Formally that reviewer is right, that some comparisons bypass strict rigor due to the chosen forest constellation which we cannot change. Most probably there is no better place available for performing these experiments in that area. For ecological investigations we are often forced to select a good site, but even so not all parameters may be optimal. Nevertheless the challenge is to make valid deductions of a non-complete or non-perfect data set. My saying is: A Scientist is allowed to do whatever he likes, as long he is saying what he is doing and how he is doing it. Often even incomplete information provides good hints for a better understanding of the topic. With many repetitions of the measurements and the testing of many samples the authors take care regarding the heterogeneity of the plant material, and minimize the risk of making important errors. My impression is that in the given situation the authors are fully aware about the strong and less strong results or conclusions they are giving in the manuscript.It needs courage, skill and perseverance to do these experiments and to present the experimental data in the form given in this article. It’s an excellent investigation, written in a well balanced form, based on solid data. All these facts together are arguments to conclude that the manuscript deserves to be approved but the authors should take into account some modifications proposed by the reviewers.For better understanding I’m proposing some minor changes at the end of the materials and methods section:The JIP test parameters used in this study can be summarized as follows:As flux ratios or quantum yields expressed as maximal trapping flux (TRo) to the reaction center of PS II, or as maximal electron transport flux (ETo) between PS II and PS I, or as maximal heat dissipation flux (DIo) of PSII per the light flux absorbed (ABS) by PS II antenna.As partial and cumulated performances (or so called driving forces DF = log of the performances derived in analogy to the redox potentials.). Please make the frame of Figure 1 taller, so that the labeling of the ordinates on the graphs is better readable and not squashed. The partial yields or probabilities or performances are based on the functional flux-ratios and can be correlated to the following experimental signals:\n\nFPo = TRo /ABS = 1-Fo/Fm = Fv/FmyEo = ET0 /TRo = (1-VJ ) where VJ = (FJ – Fo)/(Fm – Fo) is the relative variable fluorescence at the J-step, and the structural ratio:gRC =\n\nChlRC / Chltotal = ChlRC / Chl(RC+Antenna)\n\nor\n\ngRC /(1-gRC ) = ChlRC / ChlAntenna = RC/ABS = 1/ Antenna size of PS IIQuantum yield (this means: per absorption ABS) for primary photochemistry: FPo = TRo /ABSQuantum yield for electron transport:FEo = FPo * yEo = ETo / ABS as the probability that a photon absorbed by PS II will move an electron into the electron transport chain further than Qa-.Quantum yield for heat dissipation by PS II: FDo = 1 - FPo = DIo /ABS\n\n(please use yEo\n\nto distinguish it from y, which you are using for the midday leaf water potential.)The performance index PIABS, which is responsible for the performance of the electron transport from water to plastoquinone is written as:PIABS = ((gRC /(1-gRC)) x ((FPo / (1-FPo)) x ((yEo /(1- yEo))The multi parametric approach to this realistic field study is appreciated and proves that the authors have a good ecological feeling and understanding.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-122
|
https://f1000research.com/articles/2-255/v1
|
25 Nov 13
|
{
"type": "Research Article",
"title": "A Genome-Wide Association Study of spontaneous preterm birth in a European population",
"authors": [
"Wilfred Wu",
"Erin A S Clark",
"Tracy A Manuck",
"M Sean Esplin",
"Michael W Varner",
"Lynn B Jorde",
"Wilfred Wu",
"Erin A S Clark",
"Tracy A Manuck",
"M Sean Esplin",
"Michael W Varner"
],
"abstract": "Background: Preterm birth is defined as a birth prior to 37 completed weeks’ gestation. It affects more than 10% of all births worldwide, and is the leading cause of neonatal mortality in non-anomalous newborns. Even if the preterm newborn survives, there is an increased risk of lifelong morbidity. Despite the magnitude of this public health problem, the etiology of spontaneous preterm birth is not well understood. Previous studies suggest that genetics is an important contributing factor. We therefore employed a genome-wide association approach to explore possible fetal genetic variants that may be associated with spontaneous preterm birth.Methods: We obtained preterm birth phenotype and genotype data from the National Center for Biotechnology Information Genotypes and Phenotypes Database (study accession phs000103.v1.p1). This dataset contains participants collected by the Danish National Birth Cohort and includes 1000 preterm births and 1000 term births as controls. Whole genomes were genotyped on the Illumina Human660W-Quad_v1_A platform, which contains more than 500,000 markers. After data quality control, we performed genome-wide association studies for the 22 autosomal chromosomes.Results: No single nucleotide polymorphism reached genome-wide significance after Bonferroni correction for multiple testing.Conclusion: We found no evidence of genetic association with spontaneous preterm birth in this European population. Approaches that facilitate detection of both common and rare genetic variants, such as evaluation of high-risk pedigrees and genome sequencing, may be more successful in identifying genes associated with spontaneous preterm birth.",
"keywords": [
"Preterm birth (PTB)",
"defined as birth prior to 37 completed weeks’ gestation",
"is a major public health problem that affects more than 10% of all births worldwide1",
"2. Globally",
"an estimated 15 million babies are born premature each year1",
"2. Despite substantial public health efforts over the past several decades",
"the U.S. PTB rate remained at 11.72% in 20113",
"4."
],
"content": "Introduction\n\nPreterm birth (PTB), defined as birth prior to 37 completed weeks’ gestation, is a major public health problem that affects more than 10% of all births worldwide1,2. Globally, an estimated 15 million babies are born premature each year1,2. Despite substantial public health efforts over the past several decades, the U.S. PTB rate remained at 11.72% in 20113,4.\n\nPTB is the leading cause of neonatal mortality in non-anomalous newborns1,3,5,6. It is also associated with a broad spectrum of lifelong morbidity in surviving preterm infants, including neuro-developmental delay, cerebral palsy, blindness, deafness, and chronic lung disease7–9.\n\nSome PTBs are iatrogenic and can be attributed to obstetric intervention aimed at reducing maternal and/or fetal risk. The remaining PTBs are known as spontaneous PTB (SPTB) and are the focus of research efforts to identify genetic and environmental risk factors. Although SPTB is a pressing health issue, the incomplete understanding of its biology has inhibited development of effective prevention and treatment strategies.\n\nThe etiology of SPTB is complex and multifactorial9–12, although genetic factors are important contributors9–14. In addition, PTB prevalence varies among different population groups15–19. African-American ancestry is consistently associated with an increased PTB risk, even after adjusting for epidemiologic risk factors, such as income, education, lack of prenatal care, and other socioeconomic factors10,20,21.\n\nA candidate gene approach has identified polymorphisms in genes that encode the progesterone receptor, tumor necrosis factor alpha, interleukins 4, 6, and 10, and mannose-binding lectin22–33, that are mildly to modestly associated with SPTB. However, results have been inconsistent29,34–36.\n\nHere, we employed an unbiased, genome-wide approach to search for possible candidate genes associated with SPTB. Genome-wide association studies (GWAS), or whole genome association studies, are a commonly used genetic approach to study a disease or a trait. GWAS compares thousands or even millions of common genetic markers, mainly single nucleotide polymorphisms (SNPs), across individuals with a disease or trait status. There are 1,688 publications identifying 11,299 SNPs that are significant for diseases or traits in 17 different categories37,38. We obtained the SPTB phenotype and genotype data deposited in the National Center for Biotechnology Information (NCBI) Genotypes and Phenotypes Database (dbGaP)39,40, study accession phs000103.v1.p1, to perform a GWAS to explore genetic variants associated with SPTB.\n\n\nMaterials and methods\n\nWe applied to and received approval from dbGaP39,40 for access to a dataset for SPTB phenotype and genotype (study accession phs000103.v1.p1). We followed the Data Use Certification Agreement we signed during the application.\n\nTo access the data, one must apply and agree to the dbGAP terms of usage. A detailed instructions and procedures for application can be obtained at https://dbgap.ncbi.nlm.nih.gov/.\n\nThis dataset contains participants collected by the Danish National Birth Cohort (DNBC)41. DNBC is a prospective cohort that enrolled more than 100,000 pregnant women in the first trimester, before most adverse outcomes occurred, and therefore is free from sampling or collection bias. In this dataset, there are approximately 1000 preterm births and 1000 term births as controls. These study subjects were collected from 1997 to 2003. All are singleton gestations. Each birth has records of mother-child pairs. With the exception of 24 children with one or two grandparents from other Nordic countries, all other children in the dataset had parents and all four grandparents born in Denmark.\n\nThe case (preterm) group contains births delivered before 37 gestational weeks. The control (term) group contains births delivered at approximately 40 weeks’ gestation. In both preterm and term groups, children born with any recognized congenital or genetic abnormality were excluded. Pregnancies with maternal conditions known to be associated with PTB, iatrogenic or spontaneous (placenta previa, placental abruption, hydramnios, isoimmunization, placental insufficiency, pre-eclampsia/eclampsia), were also excluded by DNBC.\n\nThe blood sample (buffy coat) was collected for each mother-child pair. Their whole genomes were genotyped on the Illumina Human660W-Quad_v1_A platform (Illumina, Inc., San Diego, California, USA), which contains more than 500,000 markers. Genotyping was performed by the Johns Hopkins University Center for Inherited Disease Research (Baltimore, Maryland, USA). Further data cleaning and harmonization were done at the GENEVA Coordinating Center at the University of Washington (Seattle, Washington, USA).\n\nIn this study, we focused only on fetal genomes for further analysis. Individuals with missing genotypes greater than 3% were filtered out. In addition, individuals with a heterozygosity rate deviating more than 3 standard deviations were also excluded from further analysis42. After per-individual quality control, we also performed per-SNP filtering. The SNPs that had missing genotypes greater than 3% were excluded42. Those having significantly different genotype missing rate between the case and control groups were also eliminated. A conservative cut-off with p < 1×10-5 was applied42. We also excluded SNPs that significantly deviated from Hardy-Weinberg equilibrium in the control group – those with p < 1×10-5 were filtered out42,43.\n\nAfter data quality control, we performed GWAS for the fetal genomes on 22 autosomal chromosomes using the PLINK software package v1.0744 (http://pngu.mgh.harvard.edu/purcell/plink/). The level of genome-wide significance was set at 9.18×10-8, corresponding to Bonferroni correction for 544,675 multiple independent tests.\n\nQuantile-quantile (QQ) plot was made using STATA Statistical Software, Release 1245. Manhattan plots were generated using Haploview46.\n\n\nResults\n\nWe started with a dataset comprised of 1,900 children. There were 31 children having a missing genotype rate greater than 3%. The average heterozygosity was 0.3238, with standard deviation of 0.0059. There were also 31 children having heterozygosity that deviated more than 3 standard deviations. In fact, there was considerable overlap when applying these two filtering criteria (Figure 1) - 26 individuals were identified by both exclusion criteria. During per-individual quality control, a total of 36 individuals were excluded, resulting in 1864 individuals. However, 66 of them had a missing phenotype, yielding a final total of 849 cases and 949 controls (Table 1).\n\nThe X axis is the missing genotype rate for each individual. The Y axis is the heterozygosity rate for each individual. Each dot represents a person. The vertical dash line is the cut-off for per-individual missing rate: 3%. Individuals with missing rate greater than 3% are excluded. The two horizontal dot lines represent the mean heterozygosity ± 3 standard deviations. People with heterozygosity deviate above 3 standard deviations are also excluded. The two criteria overlap largely at the right lower part of the graph.\n\nsd: standard deviations.\n\nAmong the 560,768 markers, there were 1,933 SNPs that exceeded the missing rate threshold of 3%. There were 367 SNPs that had a significantly different missing rate between the case group and control group (P value < 1×10-5). Further, 885 SNPs significantly deviated from Hardy-Weinberg equilibrium in the control group (P value < 1×10-5). These three criteria also identified some overlapping SNPs; a total of 2,670 SNPs were excluded using all criteria. These quality control steps left 558,098 SNPs remaining in the dataset. Of these SNPs, 544,675 of them are located on 22 autosomes, and were included in the analysis (Table 1).\n\nWe carried out GWAS for these 1,798 individuals, of which 849 are SPTB cases and 949 are term controls, over 544,675 SNPs on 22 autosomal chromosomes. An allelic test was first carried out, and no SNPs reached genome-wide significance after Bonferroni correction for multiple testing (Manhattan plot (Figure 2) and QQ plot (Figure 3)).\n\nThe X axis is the position of each SNP grouped by different chromosomes, and presented with different colors. The Y axis is the P value for each test for each SNP, in –log10 scale. The horizontal blue line indicated the threshold of genome-wide significance after Bonferroni correction. The threshold is at 7.04, which corresponds to –log10 (0.05/544,675) for 544,675 independent tests. No SNP reached genome-wide significance.\n\nThe X axis indicated the expected P value, in –log10 scale. The Y axis indicated the observed P value from allelic test, also in –log10 scale. The red diagonal line is the line of Y=X, where observed equals expected. The genome-wide significance threshold for –log10 (P) scale is at 7.04, which corresponds to –log10 (0.05/544,675) for 544,675 independent tests. No SNP reached genome-wide significance.\n\nWe then performed GWAS with different genetic models. We tested three classical Mendelian inheritance models here. The recessive model assumes that carrying two variant alleles is required to present a different phenotype; while in the dominant model, one variant allele is sufficient to present a different phenotype as carrying two variant alleles. The additive model assumes the heterozygotes present an intermediate phenotype between the two homozygotes and thus consider the three genotypes separately. The Manhattan plot for the additive model (Figure 4), dominant model (Figure 5), and recessive model (Figure 6) are shown. The dominant or recessive models refer to the action of the minor allele. Within these genetic models, no SNP reached genome-wide significance after Bonferroni correction for multiple testing.\n\nThe X axis is the position of each SNP grouped by different chromosomes, and presented with different colors. The Y axis is the P value for each test for each SNP, in –log10 scale. The horizontal blue line indicated the threshold of genome-wide significance after Bonferroni correction. The threshold is at 7.04, which corresponds to –log10 (0.05/544,675) for 544,675 independent tests. No SNP reached genome-wide significance.\n\nThis is the GWAS result assuming the dominant action of minor alleles. The Y axis is the P value for each test for each SNP, in –log10 scale. The horizontal blue line indicated the threshold of genome-wide significance after Bonferroni correction. The threshold is at 7.04, which corresponds to –log10 (0.05/544,675) for 544,675 independent tests. No SNP reached genome-wide significance.\n\nThis is the GWAS result assuming the recessive action of minor alleles. The Y axis is the P value for each test for each SNP, in –log10 scale. The horizontal blue line indicated the threshold of genome-wide significance after Bonferroni correction. The threshold is at 7.04, which corresponds to –log10 (0.05/544,675) for 544,675 independent tests. No SNP reached genome-wide significance.\n\n\nDiscussion\n\nHere we describe a negative result for associations between SPTB and genetic polymorphisms of 22 autosomal chromosomes in a homogeneous European population. Myking et al. reported a GWAS focusing on the X chromosome47 and they incorporated Danish cases and controls from DNBC41,47, in addition to participants enrolled from the Norwegian Mother and Child Cohort Study (MoBa)48. Nevertheless, with a larger sample size (DNBC + MoBa), and fewer independent tests limited to the markers on X chromosomes, no SNP reached genome-wide significance after Bonferroni correction47.\n\nOne way to decrease the probability of a negative result is to increase the sample size, either by recruiting more cases and controls directly, or by combining different studies and conducting a meta-analysis49,50. Instead of sequencing thousands or millions of sporadic cases plus controls, another approach is to study SPTB using a family-based design – i.e., identify high-risk pedigrees in which a genetic mutation is more likely to be present in multiple individuals. Pedigree studies have the additional advantage of reduced phenotypic heterogeneity49. Several loci associated with SPTB have been identified by using family-based linkage studies51,52.\n\nAnother approach is to employ whole genome or whole exome sequencing. This will help to identify rare genetic variants with potentially larger effect sizes.\n\nAnother approach that may increase statistical power is to analyze the SPTB phenotype as a quantitative trait instead of a dichotomous one. The distribution of gestational age in the population is approximately normal53. Therefore, to analyze SPTB as a quantitative trait (i.e., gestational age), samples should be drawn randomly from the population of newborns.\n\n\nConclusion\n\nWe found no evidence of genetic association with SPTB in Danish population using an unbiased genome-wide approach. A family-based design in a high risk pedigree, and whole genome or exome sequencing, may yield higher detection rates of both common and rare variants associated with SPTB.",
"appendix": "Author contributions\n\n\n\nWW conceived of and designed the study. EASC, TAM, MSE, MV, and LBJ provided input to the study design. WW and LBJ applied for data access and conducted the analysis. WW drafted the manuscript. EASC, TAM, MSE, MV, and LBJ provided critical review. 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\nEASC is supported by National Institutes of Health, National Institute of Child Health and Human Development (K23HD061910). We would like to acknowledge DNBC, dbGAP, and NCBI for depositing and hosting the phenotype and genotype data.\n\n\nAcknowledgements\n\nWe would like to acknowledge DNBC, dbGAP, and NCBI for depositing and hosting the phenotype and genotype data. We would like to thank Robert Weiss from Department of Human Genetics, University of Utah, for his detailed consultations for data analysis. We would also like to thank Isabella Johnsen and June Blackburn from University of Utah for their assistances during our application for dbGAP data access.\n\n\nReferences\n\nReich ES: Pre-term births on the rise. Nature. 2012; 485(7396): 20. PubMed Abstract | Publisher Full Text\n\nBlencowe H, Cousens S, Oestergaard MZ, et al.: National, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic analysis and implications. Lancet. 2012; 379(9832): 2162–72. PubMed Abstract | Publisher Full Text\n\nHamilton BE, Hoyert DL, Martin JA, et al.: Annual summary of vital statistics: 2010–2011. Pediatrics. 2013; 131(3): 548–58. PubMed Abstract | Publisher Full Text\n\nRaju TN: Epidemiology of late preterm (near-term) births. Clin Perinatol. 2006; 33(4): 751–763. abstract vii. PubMed Abstract | Publisher Full Text\n\nLiu L, Johnson HL, Cousens S, et al.: Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet. 2012; 379(9832): 2151–61. PubMed Abstract | Publisher Full Text\n\nMathew T, MacDorman M: Infant Mortality Statistics from the 2003 Period Linked Birth/Infant Death Data Set. Natl Vital Stat Rep. 2006; 54(16): 1–29. PubMed Abstract\n\nDamus K: Prevention of preterm birth: a renewed national priority. Curr Opin Obstet Gynecol. 2008; 20(6): 590–6. PubMed Abstract | Publisher Full Text\n\nWood NS, Marlow N, Costeloe K, et al.: Neurologic and developmental disability after extremely preterm birth. EPICure Study Group. N Engl J Med. 2000; 343(6): 378–84. PubMed Abstract | Publisher Full Text\n\nMuglia LJ, Katz M: The enigma of spontaneous preterm birth. N Engl J Med. 2010; 362(6): 529–35. PubMed Abstract | Publisher Full Text\n\nKistka ZA, Palomar L, Lee KA, et al.: Racial disparity in the frequency of recurrence of preterm birth. Am J Obstet Gynecol. 2007; 196(2): 131.e1–6. PubMed Abstract | Publisher Full Text\n\nCollins JW Jr, David RJ, Handler A, et al.: Very low birthweight in African American infants: the role of maternal exposure to interpersonal racial discrimination. Am J Public Health. 2004; 94(12): 2132–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoldenberg RL, Culhane JF, Iams JD, et al.: Epidemiology and causes of preterm birth. Lancet. 2008; 371(9606): 75–84. PubMed Abstract | Publisher Full Text\n\nClausson B, Lichtenstein P, Cnattingius S: Genetic influence on birthweight and gestational length determined by studies in offspring of twins. BJOG. 2000; 107(3): 375–81. PubMed Abstract | Publisher Full Text\n\nTreloar SA, Macones GA, Mitchell LE, et al.: Genetic influences on premature parturition in an Australian twin sample. Twin Res. 2000; 3(2): 80–2. PubMed Abstract | Publisher Full Text\n\nEsplin MS, O’Brien E, Fraser A, et al.: Estimating recurrence of spontaneous preterm delivery. Obstet Gynecol. 2008; 112(3): 516–23. PubMed Abstract | Publisher Full Text\n\nAdams MM, Elam-Evans LD, Wilson HG, et al.: Rates of and factors associated with recurrence of preterm delivery. JAMA. 2000; 283(12): 1591–6. PubMed Abstract | Publisher Full Text\n\nBakketeig LS, Hoffman HJ, Harley EE: The tendency to repeat gestational age and birth weight in successive births. Am J Obstet Gynecol. 1979; 135(8): 1086–103. PubMed Abstract\n\nPorter TF, Fraser AM, Hunter CY, et al.: The risk of preterm birth across generations. Obstet Gynecol. 1997; 90(1): 63–7. PubMed Abstract | Publisher Full Text\n\nWinkvist A, Mogren I, Högberg U: Familial patterns in birth characteristics: impact on individual and population risks. Int J Epidemiol. 1998; 27(2): 248–54. PubMed Abstract | Publisher Full Text\n\nManuck TA, Lai Y, Meis PJ, et al.: Admixture mapping to identify spontaneous preterm birth susceptibility loci in African Americans. Obstet Gynecol. 2011; 117(5): 1078–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsai HJ, Hong X, Chen J, et al.: Role of African ancestry and gene-environment interactions in predicting preterm birth. Obstet Gynecol. 2011; 118(5): 1081–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEhn NL, Cooper ME, Orr K, et al.: Evaluation of fetal and maternal genetic variation in the progesterone receptor gene for contributions to preterm birth. Pediatr Res. 2007; 62(5): 630–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAidoo M, McElroy PD, Kolczak MS, et al.: Tumor necrosis factor-alpha promoter variant 2 (TNF2) is associated with pre-term delivery, infant mortality, and malaria morbidity in western Kenya: Asembo Bay Cohort Project IX. Genet Epidemiol. 2001; 21(3): 201–11. PubMed Abstract | Publisher Full Text\n\nChen D, Hu Y, Wu B, et al.: Tumor necrosis factor-alpha gene G308A polymorphism is associated with the risk of preterm delivery. Beijing Da Xue Xue Bao. 2003; 35(4): 377–81. PubMed Abstract\n\nSimhan HN, Krohn MA, Roberts JM, et al.: Interleukin-6 promoter-174 polymorphism and spontaneous preterm birth. Am J Obstet Gynecol. 2003; 189(4): 915–8. PubMed Abstract | Publisher Full Text\n\nMoore S, Ide M, Randhawa M, et al.: An investigation into the association among preterm birth, cytokine gene polymorphisms and periodontal disease. BJOG. 2004; 111(2): 125–32. PubMed Abstract | Publisher Full Text\n\nKalish RB, Vardhana S, Gupta M, et al.: Interleukin-4 and -10 gene polymorphisms and spontaneous preterm birth in multifetal gestations. Am J Obstet Gynecol. 2004; 190(3): 702–6. PubMed Abstract | Publisher Full Text\n\nMacones GA, Parry S, Elkousy M, et al.: A polymorphism in the promoter region of TNF and bacterial vaginosis: preliminary evidence of gene- environment interaction in the etiology of spontaneous preterm birth. Am J Obstet Gynecol. 2004; 190(6): 1504–1508. discussion 3A. PubMed Abstract | Publisher Full Text\n\nAmory JH, Adams KM, Lin MT, et al.: Adverse outcomes after preterm labor are associated with tumor necrosis factor-alpha polymorphism -863, but not -308, in mother-infant pairs. Am J Obstet Gynecol. 2004; 191(4): 1362–7. PubMed Abstract | Publisher Full Text\n\nAnnells MF, Hart PH, Mullighan CG, et al.: Interleukins-1, -4, -6, -10, tumor necrosis factor, transforming growth factor-beta, FAS and mannose-binding protein C gene polymorphisms in Australian women: Risk of preterm birth. Am J Obstet Gynecol. 2004; 191(6): 2056–67. PubMed Abstract | Publisher Full Text\n\nHärtel C, Finas D, Ahrens P, et al.: Polymorphisms of genes involved in innate immunity: association with preterm delivery. Mol Hum Reprod. 2004; 10(12): 911–5. PubMed Abstract | Publisher Full Text\n\nEngel SAM, Erichsen HC, Savitz DA, et al.: Risk of spontaneous preterm birth is associated with common proinflammatory cytokine polymorphisms. Epidemiology. 2005; 16(4): 469–77. PubMed Abstract | Publisher Full Text\n\nWu W, Clark EAS, Stoddard GJ, et al.: Effect of interleukin-6 polymorphism on risk of preterm birth within population strata: a meta- analysis. BMC Genet. 2013; 14: 30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDiaz-Cueto L, Dominguez-Lopez P, Cantillo-Cabarcas J, et al.: Progesterone receptor gene polymorphisms are not associated with preterm birth in a Hispanic population. Int J Gynaecol Obstet. 2008; 103(2): 153–7. PubMed Abstract | Publisher Full Text\n\nGuoyang Luo, Morgan T, Bahtiyar MO, et al.: Single nucleotide polymorphisms in the human progesterone receptor gene and spontaneous preterm birth. Reprod Sci. 2008; 15(2): 147–55. PubMed Abstract | Publisher Full Text\n\nPlunkett J, Muglia LJ: Genetic contributions to preterm birth: Implications from epidemiological and genetic association studies. Ann Med. 2008; 40(3): 167–79. PubMed Abstract | Publisher Full Text\n\nHindorff LA, Sethupathy P, Junkins HA, et al.: Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A. 2009; 106(23): 9362–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHindorff L, MacArthur J, Morales J, et al.: Catalog of Published Genome-Wide Association Studies [Internet]. [cited 2013 Sep 2]. Reference Source\n\nMailman MD, Feolo M, Jin Y, et al.: The NCBI dbGaP database of genotypes and phenotypes. Nat Genet. 2007; 39(10): 1181–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDatabase of Genotypes and Phenotypes (dbGaP). Bethesda (MD): National Center for Biotechnology Information, National Library of Medicine. [Internet]. Reference Source\n\nOlsen J, Melbye M, Olsen SF, et al.: The Danish National Birth Cohort--its background, structure and aim. Scand J Public Health. 2001; 29(4): 300–7. PubMed Abstract | Publisher Full Text\n\nAnderson CA, Pettersson FH, Clarke GM, et al.: Data quality control in genetic case-control association studies. Nat Protoc. 2010; 5(9): 1564–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalding DJ: A tutorial on statistical methods for population association studies. Nat Rev Genet. 2006; 7(10): 781–91. PubMed Abstract | Publisher Full Text\n\nPurcell S, Neale B, Todd-Brown K, et al.: PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007; 81(3): 559–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStata Corp. LP. STATA Statistical Software: Release 12. College Station, TX; 2011.\n\nBarrett JC, Fry B, Maller J, et al.: Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005; 21(2): 263–5. PubMed Abstract | Publisher Full Text\n\nMyking S, Boyd HA, Myhre R, et al.: X- chromosomal maternal and fetal SNPs and the risk of spontaneous preterm delivery in a Danish/Norwegian genome-wide association study. PLoS One. 2013; 8(4): e61781. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMagnus P, Irgens LM, Haug K, et al.: Cohort profile: the Norwegian Mother and Child Cohort Study (MoBa). Int J Epidemiol. 2006; 35(5): 1146–50. PubMed Abstract | Publisher Full Text\n\nIoannidis JP, Thomas G, Daly MJ: Validating, augmenting and refining genome-wide association signals. Nat Rev Genet. 2009; 10(5): 318–29. PubMed Abstract | Publisher Full Text\n\nEhret GB, Munroe PB, Rice KM, et al.: Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk. International Consortium for Blood Pressure Genome-Wide Association Studies. Nature. 2011; 478(7367): 103–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaataja R, Karjalainen MK, Luukkonen A, et al.: Mapping a new spontaneous preterm birth susceptibility gene, IGF1R, using linkage, haplotype sharing, and association analysis. PLoS Genet. 2011; 7(2): e1001293. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBream EN, Leppellere CR, Cooper ME, et al.: Candidate gene linkage approach to identify DNA variants that predispose to preterm birth. Pediatr Res. 2013; 73(2): 135–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKieler H, Axelsson O, Nilsson S, et al.: The length of human pregnancy as calculated by ultrasonographic measurement of the fetal biparietal diameter. Ultrasound Obstet Gynecol. 1995; 6(5): 353–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3489",
"date": "06 Feb 2014",
"name": "Mohamad Saad",
"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 their paper, “A Genome-Wide Association Study of spontaneous preterm birth in aEuropean population”, the authors perform a GWAS on spontaneous preterm birth in a European population. The GWAS is based on 849 cases (preterm births) and 949 controls (term births), and use a dense genotyping panel that contains over 500,000 SNPs. The paper is nicely written and the analyses are well done. However, I have several issues that need to be taken care of. In its actual form, this paper lacks additional analyses. Below are my non-exhaustive comments: In this study, no SNP reaches the genome-wide significance threshold. However, as we can see in the Manhattan plot (Figure 2), there are several suggestive signals, especially on chromosome 12, 15, and 3. The authors should investigate these signals and try to find the nearby genes. They perform four models and show the corresponding Manhattan plots. First, they do not explain the reason why they perform these four models. Second, the allelic test should give similar results to the additive model test. The authors do not compare between these models, at least for the most significant signals. Otherwise, these analyses seem to be redundant. Providing the Manhattan plots is far from enough. We do not have the ability to see the most significant SNP names. In addition, they should mention if there was a performed GWAS for preterm birth, and if so, they should compare their results with what can be found in the literature. Moreover, they mention that there is an evidence of a candidate gene. They should say how this gene was discovered, and show their SNP results in this gene or its region. In addition, the might consider performing the haplotypic test in this gene or in the region of the most significant SNPs. They should present these results in informative tables. In the introduction, the authors say that there are genetic factors that contribute to SPTB. It is important to mention the heritability estimates or the familial recurrence risk estimate (if there is any) for SPTB. The authors talk about negative results in their GWAS. They should discuss why they did not discover any genome-wide significant SNP (lack of power?). They also need to discuss the weaknesses of this study, and what could be done in the future.",
"responses": []
},
{
"id": "6795",
"date": "24 Nov 2014",
"name": "David Olson",
"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\nFrom Scott Williams:I have now read the paper and as it stands it is ok. I would however recommend several things that may improve it:State that the data generally suggests that most effects are maternal so that this may be a hard task. Note whether any of the candidate genes that have even nominal data fall in regions of statistical significance. Although not proof positive such findings would support an association – albeit not novel. I always like to ask if the cases are in HWE as well as the controls as there are several papers I could cite – including some of our own that indicate this is a good way to detect effects. Lastly, I am concerned that their bonferonni correction is too conservative as the ~550,000 are not all independent. We have paper in press on this point.That said nothing the authors have done is incorrect – only what I would consider to be a too limited exploration of the data. And since this type of study is by nature exploratory I would focus on lowering type 2 errors as opposed to type 1 (e.g., bonferonni corrections). From David Olson: Preterm birth is a very complex and heterogeneous problem; there are many causes, and these can vary according to gestational age. I would have expected the outcomes observed (no significant SNPs) because the authors did not stratify their preterm birth subjects into homogeneous groups. They should have separated single fetus pregnancies from multiple-fetus pregnancies, excluded those with other pre-disposing problems such as pre-eclampsia, preterm premature rupture of membranes, incompetent cervix, etc., and they should have stratified according to age: <28 weeks gestational age, 28-30, 30-34 and 34-37. I would suggest they re-analyze their data according to such stratification in an attempt to create more homogeneous groups.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-255
|
https://f1000research.com/articles/2-254/v1
|
22 Nov 13
|
{
"type": "Opinion Article",
"title": "Are we divorced from the species we study?",
"authors": [
"David Roy Smith"
],
"abstract": "In the past few years my interactions with the species I study have been restricted to strings of nucleotides spread across an LCD screen. Bioinformatics has provided me with an amazing window into some of the most interesting algae on Earth, but it has also made me feel distanced from my research organisms, and biology as a whole. This opinion article touches upon these feelings and asks whether many of us should reconsider our relationship to the taxa we investigate.",
"keywords": [
"Recently",
"I have been infatuated with Dunaliella salina—a fast-swimming",
"unicellular green alga that can flourish in some of the world’s saltiest waters",
"and which",
"given its prodigious lipid content",
"is being hailed as the ultimate biodiesel factory1. My interests lie in Dunaliella’s genomes",
"the ones in its nucleus",
"mitochondrion",
"and chloroplast. For months I have been glued to my laptop computer",
"bioinformatically piecing together Dunaliella genes and measuring genetic diversity among D. salina strains collected from remote regions of the planet. My preliminary findings are exciting",
"I’m uncovering unusual features about this alga’s genomes and how they evolved. But there is one small catch: I have never actually seen",
"grown",
"or worked with Dunaliella",
"either in the lab or in the field. My interaction with this salt-loving unicell have been restricted to reading research papers and scrutinizing long strings of nucleotides stretched across an LCD screen. All of this has made me question my relationship to the project and to Dunaliella",
"and I have started to consider that I may be married to the data but divorced from the species I’m studying."
],
"content": "A long-distance relationship with Dunaliella\n\nRecently, I have been infatuated with Dunaliella salina—a fast-swimming, unicellular green alga that can flourish in some of the world’s saltiest waters, and which, given its prodigious lipid content, is being hailed as the ultimate biodiesel factory1. My interests lie in Dunaliella’s genomes, the ones in its nucleus, mitochondrion, and chloroplast. For months I have been glued to my laptop computer, bioinformatically piecing together Dunaliella genes and measuring genetic diversity among D. salina strains collected from remote regions of the planet. My preliminary findings are exciting; I’m uncovering unusual features about this alga’s genomes and how they evolved. But there is one small catch: I have never actually seen, grown, or worked with Dunaliella, either in the lab or in the field. My interaction with this salt-loving unicell have been restricted to reading research papers and scrutinizing long strings of nucleotides stretched across an LCD screen. All of this has made me question my relationship to the project and to Dunaliella, and I have started to consider that I may be married to the data but divorced from the species I’m studying.\n\nIn many ways my experience with Dunaliella reflects the current scientific research landscape, which is largely built on collaboration, networking, and outsourcing. This landscape has led to extraordinary global research initiatives and achievements, such as the Human Genome Project and ENCODE, but it has also given rise to inordinate bureaucracy, including author lists that fill entire pages of academic papers and email correspondences that can take hours to sort through. Gone are the days of the all-in-one super scientist, of the self-dependent researcher who could single handedly initiate a major project, execute all of the experiments and analyses, and carry them through to the writing, publication, and communication stages. Today’s fast-paced scientific arena is ruled by teams of specialists, by field experts, bench geeks, and lab managers, by statisticians, computer whizzes, and grant connoisseurs, and by CEO scientists and masters of delegation.\n\nMy motivation to explore Dunaliella genomes began over coffee and conversation with a friend, Pierre Durand, during an evolution conference at the University of California, Santa Barbara. “Sounds like a plan, Pierre”! I said, with a mouthful of cookie, as we shook hands and hashed out the details. Pierre’s collaborators in Chile had some rare strains of Dunaliella isolated from a salt pond in northern Chile’s Atacama Desert, which is among the driest places on Earth. The Chilean strains would be shipped to a colleague in South Africa who would isolate their DNA and pass it along to a high-end genome sequencing facility. Much of the bioinformatics would be outsourced, and I would eventually receive the assembled Dunaliella DNA data through email. From there, I would contact other Dunaliella research teams, including the United States Department of Energy Joint Genome Institute, to glean genomic data from additional D. salina geographical isolates, which I could then use to measure genetic diversity and ultimately write a paper.\n\nAs far from the “traditional” research model as this may sound, it is becoming the norm. Science has shifted from a do-it-yourself endeavor to a large, complex, and cosmopolitan affair. That I can sit at my kitchen table in Southern Ontario exploring the inner workings of a green alga from one of the most barren environments on Earth shows just how far science has come, and changed. And overall I think these changes are great. I can certainly make meaningful observations and contributions to Dunaliella research through bioinformatic analyses—whether they are done at a field station in the Atacama Desert or a coffee shop in Toronto should make no difference. Many astronomers never actually see the stars and planets they investigate and, similarly, certain microbiologists can only ever indirectly observe the tiny organisms they study, and in both of these examples the scientific explorations and interactions are often confined to computer screens.\n\nBut a small part of me believes I should have to see, touch, or be near to the thing I am investigating. This may come from a dated and romanticized view of science. As a child I considered biologists to be synonymous with naturalists—Tilley-hatted adventurers, collecting insects, traipsing after birds, and shouting Latin nomenclature at all the exotic plants in their path. Later, when I went into research, I quickly discovered that many biologists, particularly those of the molecular ilk, abhor the outdoors, and an equally large number have never seen the organisms they study in their natural habitat, if they’ve seen them at all. I wonder how many geneticists could name the source of the nucleic acids (i.e., the species and tissue) that Watson, Crick, Franklin, and Wilkins used to decipher the structure of DNA? (The answer is calf thymus).\n\nNot working directly with the species or thing that one is investigating can sometimes lead to a narrow or naïve understanding of that species or thing. The history of science has taught us that great discoveries are often preceded by years of close examination and immersion by a single researcher with his or her study subject. Primatologist Jane Goodall and Nobel laureate and corn geneticist Barbara McClintock immediately come to mind as examples of independent scientists whose devotion and intimate understanding of their study species resulted in major breakthroughs. But in the contemporary research environment, where someone may sequence the genome of a moss one month and a box jellyfish the next, it is hard not to get the sense that certain scientists don’t have the time (or can’t be bothered) to invest years in learning about the taxa whose genes they are so eagerly sequencing.\n\nFor me, not having ever worked with Dunaliella could mean that I am overlooking important aspects of the genetic data. One of the interesting observations I’ve made from analyzing D. salina DNA is that there is a remarkably low level of genetic diversity in the chloroplast genome as compared to the mitochondrial one (unpublished data). If I had a first-hand understanding of Dunaliella—its habitat, its lifecycle, its cellular and physiological quirks—I might be able to link this observation to something substantive about the organism. But in a highly competitive and ever changing research environment, it can be difficult to find the resources and time to develop completely new skillsets for a project that might only last a few months. Moreover, it may not be practical to gain these skillsets when the questions being asked can easily be addressed through collaboration with a world expert in the area.\n\nFor all we have gained from “big science”, we might have lost touch with some of the goals and aspirations that inspired us go into research in the first place—for me it was to explore and understand the remarkable diversity of microbial life. I should likely consider bringing a little more “life” into my own laboratory, which largely sits empty with the buzz of a few high-powered computers. I have heard that the salt lakes in Utah are jammed packed with Dunaliella species, and that when they are in bloom the lakes light up with a deep pink hue from the beta-carotene of the algae. It is probably worth a trip out there to see for myself—that is, as long as those research grant applications come through.\n\nIn fact, maybe all us scientists that are feeling distanced from our research organisms should make an effort to directly interact with them; we certainly could benefit from a more holistic understanding of the amazing species we study, even if we can’t examine each and every aspect in great detail.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nRamos AA, Polle J, Tran D, et al.: The unicellular green alga Dunaliella salina Teod. as a model for abiotic stress tolerance: genetic advances and future perspectives. Algae. 2011; 26(1): 3–20. Publisher Full Text"
}
|
[
{
"id": "2537",
"date": "27 Nov 2013",
"name": "Iñaki Ruiz-Trillo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 opinion article in which the author describes a common problem in biological research, especially in the genomics/genetics field. This problem is a lack of knowledge of the biology and/or ecology of some of the species that we, as researchers, analyze at a genetic level. The author describes why much of the current research is like this and asks whether this may limit us to a narrow understanding of the genome itself. Typically the research is done by combining different expertise, so that some researches will understand the biology and the ecology of the species, while others will unravel the contents of the genome. The manuscript is very well written and will hopefully make a lot of us think twice about the way we perform research nowadays.",
"responses": []
},
{
"id": "2630",
"date": "04 Dec 2013",
"name": "Robert G. Beiko",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting short piece on the disconnect faced by those of us who focus on computational analysis at the expense of experimental biology. The author suggests that experience with an organism can help with the interpretation of the patterns one finds in its genome(s), and that being totally disconnected from the biology of an organism can lead to a certain ennui, even as one gains exciting new insights into an organism's lifestyle via its genes.The article is an interesting read; however, it suggests to me that one might consider the value of contributions made by researchers with different areas of expertise. Few can claim to run the gamut of experimental biology including culture, the generation and interpretation of computational data and development and validation of new computational approaches, whilst also managing a lab and an academic career, with all that this entails. In an age of abundant sequence data, one can perform synthesis as never before without ever leaving your desk, apart from the occasional attention paid to the necessities of life. If one wishes to perform broad synthetic work, it may be impossible to connect oneself with all organisms under investigation anyway. The best comparative genomic, evolutionary and ecological meta-analyses (for example) can provide new insights into big questions, producing hypotheses that can be tested with new data that need not be collected by the initial investigator. While it may be more satisfying to some to carry out the entire chain of experiment - big analysis - further experimentation on their own, it is often the case that all the necessary skills, time, or facilities are not available to one person or one research group.It is true that many of the great biologists (broadly defined) of the past that come immediately to mind, such as Clements, Robert Whittaker, Margaret Dayhoff, Mayr, Baas-Becking, Sanger, and so on, did indeed go on expeditions, work at field stations, grow their own organisms, and purify their own proteins. But if I had one recommendation for this paper, it would be to investigate the matter in more depth, to improve the scholarship by examining the historical contribution of pure theoreticians then and now. Even so, this piece is a worthwhile reflection on the life of those of us who try to make sense of the world without necessarily experiencing it firsthand (except on weekends).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-254
|
https://f1000research.com/articles/2-253/v1
|
22 Nov 13
|
{
"type": "Clinical Practice Article",
"title": "Role of multi-detector computed tomography (MDCT) in management of post percutaneous nephrolithotomy (PCNL) bleeding",
"authors": [
"Arvind P Ganpule",
"Darshan H Shah",
"Sanika A Ganpule",
"Ravindra B Sabnis",
"Mohan M Rajapurkar",
"Mahesh R Desai",
"Darshan H Shah",
"Sanika A Ganpule",
"Ravindra B Sabnis",
"Mohan M Rajapurkar",
"Mahesh R Desai"
],
"abstract": "Objective: To evaluate the role of multidetector computerized tomography (MDCT) angiography in post percutaneous nephrolithotomy (PCNL) bleed and compare findings with conventional angiography (CA).Material and methods: We conducted a retrospective analysis of patients who had post PCNL bleeding and subsequently underwent a MDCT angiography followed by CA. We reviewed eight patients, who presented between January 2009 and January 2013. We performed a MDCT angiography on a 16 slice GE bright speed CT scanner. All angiographies were done by using the Digital Subtraction Angiography Suite. The angioembolisation, if required, was conducted by an interventional nephrologist, specializing in therapeutic embolisation.Results: The mean age of the patients was 42±17 years. Mean time of post PCNL bleed presentation was 10.06±7.9 days. Five patients presented with aneurysm and three presented with an AV fistula with pseudoaneurysm. The right renal unit was involved in six cases and the left in two cases. The lower polar segmental artery was involved in six cases and the upper polar artery in two cases. The CA and MDCT findings matched in all cases and the MDCT helped the clinician to assess and embolise the appropriate arterial tree.Conclusion: MDCT is rapid, reproducible and noninvasive. MDCT angiography performed in the setting of post PCNL bleeding provides an accurate assessment of the site and nature of bleeding. The MDCT angiography matched the CA findings in all patients in the present study.",
"keywords": [
"Bleeding after percutaneous nephrolithotomy (PCNL) occurs in 0.3 to 1% of patients1. The majority of episodes are self limiting and do not require intervention. Life threatening bleeding following percutaneous renal surgery requires accurate imaging",
"which helps the treating physician to make a clinical decision regarding management. Various non-invasive investigative modalities such as \"grayscale\" ultrasonography and Doppler ultrasound have previously been key in the decision-making process. Conventional angiography (CA) can be used in both the diagnosis and treatment of bleeding following PCNL. However",
"CA has limitations and pitfalls including its inability to accurately identify bleeding. In addition",
"although there is a low incidence of access site complications",
"CA requires expertise when used as an intervention2."
],
"content": "Introduction\n\nBleeding after percutaneous nephrolithotomy (PCNL) occurs in 0.3 to 1% of patients1. The majority of episodes are self limiting and do not require intervention. Life threatening bleeding following percutaneous renal surgery requires accurate imaging, which helps the treating physician to make a clinical decision regarding management. Various non-invasive investigative modalities such as \"grayscale\" ultrasonography and Doppler ultrasound have previously been key in the decision-making process. Conventional angiography (CA) can be used in both the diagnosis and treatment of bleeding following PCNL. However, CA has limitations and pitfalls including its inability to accurately identify bleeding. In addition, although there is a low incidence of access site complications, CA requires expertise when used as an intervention2.\n\nMultidetector computerisd tomography (MDCT) is currently being used prior to endourologic intervention, and its role in managing bleeding in the gastrointestinal tract has been assessed in the past3,4. There is a paucity of literature describing its utility in post PCNL bleeding. A MDCT would potentially help in assessing the vascular anatomy, the site of bleeding and hence subsequently aid intervention. In this article, we assess the utility and accuracy of findings of multidetector computerized tomography (MDCT) angiography in patients with post PCNL bleeding. The findings were compared with those seen using CA.\n\n\nMaterial and methods\n\nA retrospective chart review of all patients who presented with post PCNL bleeding was conducted. All patients were admitted prior to the procedure. Among these patients six patients had a PCNL done at our hospital while two were referred from another hospital. Informed written consent regarding the procedure and future use of this data in any form in any meeting or publication was obtained from each patient included in the analysis. Those patients evaluated with MDCT angiography and who subsequently underwent CA and embolisation between January 2009 and January 2013 were included in the analysis. MDCT angiography was conducted on a 16 slice GE Bright SpeedTM CT (GE Healthcare, US) scanner. The findings for MDCT were compared with those with CA. All MDCT angiographies were performed and reported by a single radiologist. All the CAs were performed by an interventional nephrologist.\n\nNo oral or bowel contrast was administered during MDCT angiographies. The contrast agent iopromide (Ultravist), 1.5–2ml/kg body weight was injected in an adequate sized forearm vein using a pressure injector. A triphasic acquisition of the images was performed. The scan ranged from the diaphragm to the inferior pubic ramus. An unenhanced MDCT was obtained at baseline. The locator was positioned over the descending aorta and images were acquired once the contrast was detected in the descending aorta. This constituted the arterial phase. At an interval of 40–60 seconds after contrast injection, the second scan was performed to obtain the images in the venous phase. The delayed scan was acquired after 10 minutes for excretory images. The section thickness was kept at 1mm, the reconstruction thickness was kept at 0.8mm.The gantry rotation time of 0.5 seconds was kept for all the three phases.\n\nThe data was processed with the Maximum intensity projection (MIP) and Volume rendering (VR) techniques. These are methods for 3D reconstruction and image display. Both are post processing techniques with their own advantages and disadvantages5. The parameters noted included the type, size and site of the lesion, the method of embolisation and the conventional and MDCT angiography findings (success being defined as complete arrest of bleeding). The radiologist while reporting the CT scan noted the following parameters:\n\n1) Presence of arteriovenous fistula, pseudoaneurysm or aneurysm.\n\n2) Presence of any residual stones.\n\n3) Presence of perinephric sub capsular hematomas.\n\n4) Presence or absence of clots in the collecting system.\n\n5) Size of the lesion and its relation to primary, secondary or quaternary feeders.\n\nAll angiographies were done on Digital Subtraction Angiography Suite (Medico India Ltd). Prior to embolisation a flush angiogram was performed, following which a selective angiogram was obtained. The angioembolisation, if required, was performed by an interventional nephrologist, specialised in therapeutic embolisation. The vascular system was accessed through the ipsilateral common iliac artery; if access was not possible through a femoral route access was gained through an axillary route. The decision to use a coil or a gel foam stick for embolisation was taken depending on the lesion and the diameter of the lesion to be embolised.\n\n\nResults\n\nThe mean age of the patients was 42±17 years. There were six males and two females. Mean time of post PCNL bleed presentation was 10.06±7.9 days. Five patients presented with aneurysm and three presented with an arteriovenous (AV) fistula with pseudoaneurysm. The right renal artery was involved in six cases and the left in two cases. The lower polar segmental artery was involved in six cases and the upper polar artery in two cases. The CA and MDCT findings matched in all cases. In all cases, MDCT helped the clinician to assess and embolise the appropriate arterial tree. Details of each case are summarized in Table 1.\n\nMDCT: Multidetector computerized tomography, PCNL: Post percutaneous nephrolithotomy, PCS: Pelvicalyceal system.\n\nA 28 year old male presented with gross hematuria with clots, a hemoglobin drop of 3gm/dl following 7 days of PCNL, and required two units of blood transfusion. Upon MDCT evaluation he was found to have right lower pole segmental artery aneurysm. Further it also showed clots in pelvi calyceal system (PCS). There were no residual calculi and hematoma. These findings matched with findings on CA. A coil embolisation was performed, following which the hematuria settled (Figure 1).\n\na) Aneurysmal dilatation seen on MDCT in a branch of the posterior segmental artery (anterior projection; arrow). b) The same lesion seen on posterior projection (arrow). c and d) A platinum coil used for embolisation (arrows).\n\nA 60 year old male presented with gross hematuria with a need for blood transfusion following 6 days of PCNL in a solitary functioning right kidney. A Doppler ultrasound revealed the possibility of an aneurysm. Prior to CA, a MDCT angiography revealed the presence of the aneurysm. Considering the fact that the size of the lesion was more than 1cm and the lesion had multiple feeders, the chance of successful embolisation was guarded. However in view of the solitary kidney status, an attempt of super selective embolisation was done. CA showed AV fistula with pseudoaneurysm, supplied by posterior segmental artery (Figure 2a). The MDCT angiography findings helped the clinician to assess the exact size, location and number of feeding arteries that could be embolised. In addition, the MDCT also revealed the presence of residual stones and the presence of a perinephric hematoma. After diagnostic CA which confirmed findings (Figure 2b) of MDCT embolisation was done.\n\na) A MDCT angiogram showing a large pseudoaneursym from the posterior segmental artery (anteroposterior projection) The arrow shows the feeding vessel. b) A conventional angiogram showing a pseudoaneursym from the branch of the posterior segmental (lateral projection). The arrow shows the feeding vessel.\n\nA 46 year old female patient underwent left PCNL, and on the 8th post operative day developed hematuria. Emergency cystoscopy with clot evacuation with left ureteric and pelvic clots evacuation was done by Fogarty catheter and double J (DJ) stent placed. A therapeutic embolisation with a coil was conducted. MDCT angiography revealed a persistent fistula after embolisation, no residual calculi or hematoma. The MDCT angiogram shows AV fistula of upper polar branch of posterior segmental artery (Figure 3a and 3b). This helped in accurately embolising the concerned vessel (Figure 3c and 3d). Hematuria settled after successful embolisation.\n\na and b) Axial cuts and lateral MIP images showing AV fistula in a branch of the posterior segmental artery. Arrow shows the location of AV fistula. c) Conventional angiography confirms the presence of AV malformation in the branch of the posterior segmental. Arrow shows the location of AV fistula on CA. d). Successful embolisation. Arrow shows the position of the coil.\n\nA 13 year old male developed hematuria and fever on the 5th post operative day following PCNL for a right renal stone. After stabilizing the patient hemodynamically MDCT angiography was done. MDCT showed persistent leakage of contrast from upper pole vessel with clots in PCS and no residual calculi. These findings were confirmed on CA (images not available). Selective angioembolisation of upper pole vessel was done and hematuria settled.\n\nA 63 year old male underwent right PCNL for multiple renal calculi. He had hematuria after 15 days. He needed emergency cystoscopy with clot evacuation and right DJ stenting. A blood transfusion was given. MDCT angiography showed right renal lower segmental artery aneurysm, which was confirmed on CA and embolisation was performed (images not available).\n\nA 54 year old male patient developed hematuria on the 4th day following a right \"Miniperc\" PCNL. MDCT angiography showed right lower pole AV malformation with pseudoaneurysm with clots in PCS. Findings were confirmed on CA and selective angioembolisation was done (images not available).\n\nA 30 year old male patient with no comorbidities underwent right PCNL with DJ stenting for a right renal pelvic stone. On the 12th post operative day he developed hematuria with clot retention. Cystoscopy with clot evacuation was done. MDCT angiography showed extravasation of contrast from lower pole calyces into perinephric collection of 15 × 8cm2 size and aneurysm of the lower pole segmental branch (Figure 4a) of the right upper renal artery. MDCT angiography revealed the presence of two renal arteries, with the AV malformation in the upper artery. He was successfully embolised (Figure 4b and 4c) by CA after confirmation of MDCT findings.\n\na) The MDCT angiogram shows the presence of two renal arteries. In addition it also shows aneurysmal dilatation of lower pole segmental branch of right upper renal artery (arrow). b and c) A conventional angiogram done the catheter is manipulated into the appropriate branch and embolisation done (arrows).\n\n42 year male, known diabetic underwent left PCNL. Procedure was uneventful. On 28th post operative day he had hematuria which increased in amount on the next day. On evaluation found to have left lower pole segmental artery aneurysm on MDCT. CA showed the same findings. He underwent selective angioembolisation following which hematuria settled (Images not available).\n\n\nDiscussion\n\nBleeding after PCNL is a stressful situation for both the patient and the operating surgeon. A clinical decision needs to be made depending on the nature of bleeding (arterial or venous), site of bleeding and the type of vascular anomaly. An ideal imaging modality should be minimally invasive and as non-traumatic as possible. It should have a high sensitivity and specificity and should be able to guide the clinician in the management of the patient. There are multiple factors influencing the ability to visualize active bleeding using CT including the type of bleeding, the type of CT machine used and the experience of the radiologist. If the bleeding is exanguinating, angioembolisation is the treatment of choice. Although there are no \"benchmark\" markers to decide the need for angioembolisation, clinicians decide on the need to embolise depending on the presence of hematuria with clots, hypotension, and/or a serially decreasing hematocrit that does not respond to conservative management6. Richstone and colleagues report in their series the incidence for the need for embolisation to be 1.2% of total PCNL cases7. They concluded that arterial pseudoaneurysms, followed by AV fistulas are the most common findings seen on CA7. The well known limitations of CA are that it reveals the cause of hematuria only in a scenario where there is an active bleed. Second, venous bleeding and bleeding associated with infection may not be readily detected by CA8.\n\nThe advantages of MDCT angiography have been well established in gastrointestinal bleeding2,3. This has led to an increased interest in using the same modality in detecting the cause of bleeding after PCNL. Besides being non invasive and having short scanning times, MDCT provides thinner collimation, greater anatomic coverage, which increases its diagnostic scope2,3. Movement artifacts from respiration and peristalsis hamper CA; this is eliminated with a rapidly performed MDCT. In addition, MDCT does not require bowel preparation.\n\nMDCT in such a setting helps to determine the presence of residual stones and postoperative collections. In addition, it also allows the interventionist to plan how to target the offending vessel3. Although there are plenty of reports describing the utility of CA and selective angiography in post PCNL bleeding, there have only been anecdotal case reports describing the use of MDCT angiography8. Further, as far as the authors are aware, there have been no reports comparing these two modalities.\n\nIn our experience a MDCT angiogram provides a \"roadmap\" for the intervening clinician. Depending on the size and type of lesion, the intervening physician can plan the intervention, including selection of the embolisation material, the endovascular approach to the offending vessel and can even select the most appropriate method of hemostasis (gelfoam/coil etc). The \"roadmap\" has the potential to effectively decrease the number of angiographic series, saving time, radiation exposure and the amount of contrast agent used during intervention. This needs to be assessed objectively in further studies. The second advantage of this approach is in the event that the MDCT angiogram is completely negative it decreases the chance of finding a significant bleeding on interventional angiography and may suggest the need for a more conservative approach for managing the bleeding. Studies have shown that AV fistulas smaller than 3–4mm have a good chance of successful angioembolisation in comparison to larger ones9. The exact nature of the problem can be discerned on a MDCT angiography. The findings should enable the clinician to decide the time, type (gelfoam/platinum coil etc.) and the chances of successful angioembolisation.\n\nWe noted that the MDCT angiogram detected other findings such as residual stones, pelvicalyceal system clots, and perinephric hematomas and collections. MDCT angiograms also reveal the venous anatomy and can help to assess the status of the opposite kidney.\n\nComplications following percutaneous vascular access include hemorrhage, arterial obstruction, pseudoaneurysm and AV fistula at the site of access. In addition there is a risk of non-target organ embolisation as well as contrast induced nephropathy (CIN).\n\nThe MDCT angiography is also limited by the fact that it cannot be used in the presence of renal insufficiency, as there remains a risk of contrast-induced nephropathy10. MDCT angiography is also contraindicated in patients having sensitivity to contrast agents. The limitations of our study include a small sample size. As MDCT and CA findings were interpreted by different individuals in this study there may have been observer’s variations in the findings. The real value of MDCT is in its potential to identify patients who do not have an identifiable lesion, thus avoiding the need for a CA. This aspect was beyond the role of the present study.\n\n\nConclusion\n\nMDCT is rapid, reproducible and noninvasive. MDCT angiography performed in the setting of post PCNL bleeding provides an accurate assessment of the site and nature of bleeding. MDCT angiography findings correlated with the CA findings in all patients in the present study. These findings need to be further validated in larger multicenter comparative studies.",
"appendix": "Author contributions\n\n\n\nAPG, DHS and SAG conceived the study. MMR, SAG carried out the investigations. RBS, MRD contributed to the design of the study. APG and DHS prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMartin X, Murat FJ, Feitosa LC, et al.: Severe bleeding after nephrolithotomy: results of hyperselective embolization. Eur Urol. 2000; 37(2): 136–9. PubMed Abstract | Publisher Full Text\n\nMuller DW, Shamir KJ, Ellis SG, et al.: Peripheral vascular complications after conventional and complex percutaneous coronary interventional procedures. Am J Cardiol. 1992; 69(1): 63–8. PubMed Abstract | Publisher Full Text\n\nAnthony S, Milburn S, Uberoi R: Multi-detecto CT: review of its use in acute GI haemorrhage. Clin Radiol. 2007; 62(10): 938–49. PubMed Abstract | Publisher Full Text\n\nLaing CJ, Tobias T, Rosenblum DI, et al.: Acute Gastrointestinal bleeding: emerging role of Multidetector CT angiography and review of current imaging techniques. Radiographics. 2007; 27(4): 1055–70. PubMed Abstract\n\nFishman EK, Ney DR, Heath DG, et al.: Volume rendering versus maximum intensity projection in CT angiography: what works best, when, and why. Radiographics. 2006; 26(3): 905–22. PubMed Abstract\n\nRastinehad AR, Andonian S, Smith AD, et al.: Management of hemorrhagic complications associated with percutaneous nephrolithotomy. J Endourol. 2009; 23(10): 1763–1767. PubMed Abstract | Publisher Full Text\n\nRichstone L, Reggio E, Ost MC, et al.: First Prize (tie): Hemorrhage following percutaneous renal surgery characterization of angiographic findings. J Endourol. 2008; 22(6): 1129–1135. PubMed Abstract | Publisher Full Text\n\nSivanandam SE, Mathew G, Bhat SH: Emerging role of multi-detector computed tomography in the diagnosis of hematuria following percutaneous nephrolithotomy: A case scenario. Indian J Urol. 2009; 25(3): 392–394. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain V, Ganpule A, Vyas J, et al.: Management of non-neoplastic renal hemorrhage by transarterial embolization. Urology. 2009; 74(3): 522–6. PubMed Abstract | Publisher Full Text\n\nMoos SI, van Vemde DN, Stoker J, et al.: Contrast induced nephropathy in patients undergoing intravenous (IV) contrast enhanced computed tomography (CECT) and the relationship with risk factors: a meta-analysis. Eur J Radiol. 2013; 82(9): e387–99. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2528",
"date": "13 Dec 2013",
"name": "Noor Buchholz",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a few minor comments:The very small number of patients does not allow for the extraction of safe conclusions. The embolisation should be conducted by an interventional radiologist and not an interventional nephrologist, please provide an explanation as to why this occured. The literature results from conventional angiography that the authors provide are excellent, however references regarding the success rates of conventional angiography success rates in post pcnl embolisation are also needed.",
"responses": []
},
{
"id": "2848",
"date": "06 Jan 2014",
"name": "Christian Bach",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a highly interesting and very well written study evaluating the role of MDCT angiography for bleeding post PCNL, based on a case series. This paper is clearly worth publishing and excellent as it stands. I have solely some suggestions:The title should point out that a case series is presented. All patients had bleeding post PCNL – what exactly were the parameters which triggered the angioembolization? Were there patients where MDCT angiography had been performed, and as a results NO embolization had been performed? Otherwise, I join in with the comments of the first reviewer.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-253
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https://f1000research.com/articles/2-188/v1
|
16 Sep 13
|
{
"type": "Research Article",
"title": "Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues",
"authors": [
"Liliana Florea",
"Li Song",
"Steven L Salzberg",
"Li Song",
"Steven L Salzberg"
],
"abstract": "Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. However, despite many efforts, the repertoire of gene splicing variation is still incompletely characterized, even in humans. Here we describe a new computational system, ASprofile, and its application to RNA-seq data from Illumina’s Human Body Map project (>2.5 billion reads). Using the system, we identified putative alternative splicing events in 16 different human tissues, which provide a dynamic picture of splicing variation across the tissues. We detected 26,989 potential exon skipping events representing differences in splicing patterns among the tissues. A large proportion of the events (>60%) were novel, involving new exons (~3000), new introns (~16000), or both. When tracing these events across the sixteen tissues, only a small number (4-7%) appeared to be differentially expressed (‘switched’) between two tissues, while 30-45% showed little variation, and the remaining 50-65% were not present in one or both tissues compared. Novel exon skipping events appeared to be slightly less variable than known events, but were more tissue-specific. Our study represents the first effort to build a comprehensive catalog of alternative splicing in normal human tissues from RNA-seq data, while providing insights into the role of alternative splicing in shaping tissue transcriptome differences. The catalog of events and the ASprofile software are freely available from the Zenodo repository(http://zenodo.org/record/7068; doi:10.5281/zenodo.7068) and from our web site http://ccb.jhu.edu/software/ASprofile.",
"keywords": [
"Alternative splicing is a widespread phenomenon in eukaryotic species",
"and differential regulation of alternative splice variants is gaining recognition as an important mechanism of gene regulation. More than 90% of human genes are estimated to be alternatively spliced1",
"2",
"producing multiple transcripts and (often) different protein sequences from a single locus. The number of variants of a gene ranges from two to potentially thousands3. The resulting proteins may exhibit different and sometimes antagonistic functional and structural properties4",
"and may inhabit the same cell with the resulting phenotype representing a balance between their expression levels5. Defects in splicing have been implicated in human diseases",
"including cancer6–9. Developing a comprehensive catalog of splice variant annotations across a wide range of tissues and conditions is important not only as part of our efforts to create a complete gene list for the human genome",
"but also to serve as a reference for differential expression studies aiming to identify molecular markers of disease."
],
"content": "Background\n\nAlternative splicing is a widespread phenomenon in eukaryotic species, and differential regulation of alternative splice variants is gaining recognition as an important mechanism of gene regulation. More than 90% of human genes are estimated to be alternatively spliced1,2, producing multiple transcripts and (often) different protein sequences from a single locus. The number of variants of a gene ranges from two to potentially thousands3. The resulting proteins may exhibit different and sometimes antagonistic functional and structural properties4, and may inhabit the same cell with the resulting phenotype representing a balance between their expression levels5. Defects in splicing have been implicated in human diseases, including cancer6–9. Developing a comprehensive catalog of splice variant annotations across a wide range of tissues and conditions is important not only as part of our efforts to create a complete gene list for the human genome, but also to serve as a reference for differential expression studies aiming to identify molecular markers of disease.\n\nAnnotation of alternative splicing has traditionally been based on cDNA (expressed sequence tags (EST), mRNA) sequence data from public repositories such as dbEST, RefSeq10, and the Mammalian Gene Collection11. These data sources were compiled over many years, from independent contributions by thousands of investigators working on different genes and systems, and are therefore inconsistent in their coverage of the transcriptome in general and of each gene individually. Because these resources were generated using Sanger sequencing, they were relatively expensive to produce, but despite the cost have insufficient depth to capture the diversity of splicing variations in human cells. RNA-seq technology produces vastly more sequence data in a cost-effective way and in a much shorter amount of time, allowing a deep characterization of the transcriptome in a variety of cells and conditions2,12,13, but so far little has been done to systematically assess its potential14. Starting from one of the most complete sets of RNA-seq data available, the Illumina Human Body Map, we addressed the questions: \"how much alternative splicing do we find?\" and \"how does alternative splicing vary among tissues?\" We used this data set, spanning 16 tissues and containing over 2.5 billion sequences, to build a comprehensive catalog of alternative splicing (AS) within each tissue. We also compared AS profiles across tissue types to derive insights into the role of AS in shaping transcriptome differences.\n\n\nResults\n\nWe analyzed the Illumina Human Body Map RNA-seq set (ArrayExpress accession: E-MTAB-513; http://www.ebi.ac.uk/arrayexpress), consisting of approximately 160 million reads from each of 16 tissues, each from a different individual. This resource is one of the most high-quality and complete to date, and therefore allows us to detect AS events with high accuracy. To determine splicing variations in each tissue, we first mapped reads to the reference genome and assembled them into transcripts or transcript fragments. We then analyzed the transcripts to determine putative alternative splicing events, in particular exon skipping events, within and between samples, and compared them across the tissues. We focused on exon skipping because the alignment evidence for these events is usually clear and unambiguous, and less likely to be confounded by alignment or assembly artifacts. The data support a number of overall findings:\n\n1. Based on a comparison against several annotation databases (Ensembl15, CCDS16, UCSC Genes17 and H-ASDB18), we found that 11–45% of the assembled transcripts in each tissue were unannotated, as well as a majority (65%) of the 26,989 exon skipping events discovered from this data set.\n\n2. These novel events appear to be more tissue-specific than previously annotated (known) events; i.e., they tend to occur in fewer tissue types.\n\n3. When an exon is skipped, it usually occurs in a different tissue from those in which it is present; only 5–23% of events express both forms within the same tissue.\n\n4. Comparing exon skipping profiles across tissues, we found that only 10–20% of the events identified show different splicing ratios between any two given tissues, whereas 50–65% of the cataloged events are not present in either or both tissues.\n\nOverall, our analysis reveals a complex and dynamic picture of alternative splicing across tissue types, where differences among tissue transcriptomes arise from the interplay between constitutive transcription and alternative splicing. Most importantly, we compiled the first large repository of putative exon skipping and other classes of alternative splicing events in normal human tissues detected from RNA-seq data, which will be a valuable resource for studies of regulation and to identify markers of diseases. This catalog and our methods, implemented in the open source software program ASprofile, are freely available from the Zenodo repository (http://zenodo.org/record/7068; doi:10.5281/zenodo.7068) and from our web site http://ccb.jhu.edu/software/ASprofile.\n\nTo determine alternative splicing events and globally characterize alternative splicing within a given tissue, we analyzed 50-bp paired-end sequences from 16 different tissues: adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid and white blood cells. These data are publicly available as the Illumina Human Body Map project (EMBL accession ENA-ERP000546; ArrayExpress accession: E-MTAB-513; http://www.ebi.ac.uk/arrayexpress/browse.html?keywords=E-MTAB-513&expandefo=on). Libraries were made from polyA-selected mRNA with an insert size of 210 bp, independently for each tissue, using a random priming process and unstranded. One run of 2x50 bp paired-end sequencing was performed on the Illumina HiSeq2000 instrument, using one lane per tissue, to produce approximately 80 million pairs of reads (160 million sequences) per tissue. The entire data set comprises ~128 gigabases (GB) of sequence (~8 GB sequence per tissue), making this one of the most complete RNA-seq resources to date and one of very few spanning multiple types of tissues.\n\nWe mapped reads to the human genome with the program TopHat19 (Supplemental Table S1), and then assembled overlapping reads on the genome into transcript fragments using Cufflinks20. Cufflinks represents all reads at a locus as an assembly graph, in which any two reads are connected if they overlap and have compatible splice patterns, and then traverses the graph to produce the minimum number of transcripts that can explain all of the input reads. Because single-exon transcripts, which form the bulk of the assemblies (Figure 1 and Supplemental Table S2), are frequently artifacts of sequencing and mapping, we used only the multi-exon transcripts to measure the gene and transcript content.\n\nSince Cufflinks may break transcripts and genes into multiple fragments when there is insufficient read coverage, we used Cuffcompare to compare transfrags against the Ensembl reference annotations to produce a better estimate for the number of genes and transcripts in the samples. Results in the right panel show the total number of Ensembl annotated as well as novel genes, and respectively transcripts, found in each sample. The number of novel isoforms identified by Cuffcompare is shown in the bottom panel.\n\nAlthough the assemblies produced by Cufflinks can be full-length transcripts, many transcripts can only be assembled into partial fragments (e.g., when the coverage of a transcript contains gaps). We therefore designate all transcript assemblies, complete or otherwise, as transfrags. For each tissue, Cufflinks produced between 23,000–46,000 multi-exon transfrags, clustered into 20,000–30,000 loci. The number of transfrags was greatest in brain and testes, and lowest in liver, colon, white blood cells and skeletal muscle, reflecting the combined effects of the number of expressed genes (Pearson’s r2=0.74) and splicing variation within genes. These findings are consistent with some of the earlier estimates of the sizes of transcriptomes of different tissues1,21–25. To estimate the number of novel splice forms, we compared the assemblies to a known annotation database, Ensembl15, using the program Cuffcompare from the Cufflinks package. This gave us between 35,000–52,000 transfrags per tissue that were associated with 13,000–17,000 Ensembl genes, of which a large fraction (between 5,000–20,000 per tissue, representing 11–45% of the total) appeared to be novel splice forms (Figure 1, Supplemental Figure S3 and Supplemental Table S3). Tissues with large numbers of new splice forms also had a larger fraction of candidate new splice forms.\n\nEven with the best data and software, computational reconstruction of long transcripts from short reads is prone to assembly errors. We therefore focused on classes of alternative splicing events that are most likely to be assembled correctly. Exon skipping events are the most prevalent type of alternative splicing events in the human genome26, and are particularly easy to identify from transcript data and less likely to be mis-assembled. They have been extensively studied and are well represented in the databases. For these reasons, exon skipping events provide an excellent proxy for the number of other types of splicing variants in a sample.\n\nWe define an exon skipping ‘event’ as a pairing between an exon-containing form (‘on’) and an exon-excluding form (‘off’), occurring at the same exon and with the same flanking introns. The same exon (or intron) may be involved in multiple exon skipping events, though the number of such cases is small. To generate a catalog of events for the sixteen tissues, we analyzed transcript assemblies using our software ASprofile and identified differences in exon-intron structures characteristic of the various classes.\n\nWe found over 150,000 candidate alternative splicing events (Supplemental Table S4). Among these, we found 26,989 exon skipping events at 25,017 distinct exons, involving 22,145 distinct introns. Almost all of these events (25,920) were found in comparisons between different tissues, although a significant fraction (16,382) were also found when comparing isoforms within the same tissue. There were 1,069 instances of alternative splicing events that were restricted to a single tissue, most of them in testes (416) and brain (172).\n\nMapping artifacts can create false exon skipping events, due to incorrect or duplicated splice junctions or incorrectly reconstructed exons. To assess the accuracy of the data set and identify potential artifacts for future curation, we first looked for co-located events that showed small variations (≤5 bp) at exon and intron boundaries, which could be caused by imprecise mapping of spliced reads. Such variations could lead to redundancy in reporting the events. For reference, we compared the extent of variation against the ENSEMBL gene annotations. There were 1,822 (6.75%) events in our data set that represented slight variations of other events compared to 427 (1.7%) in the ENSEMBL data, suggesting that up to 5% of events in our data set may be redundant (Supplemental Figure S5 and Supplemental Table S5). However, this figure is likely an overestimate, given that small 5´ and 3´ exon splicing variations are hard to detect with conventional (Sanger) data and are likely underrepresented in the reference gene annotations. We also evaluated the reproducibility of our results when using other transcript assembly methods (IsoCEM27, SLIDE28, and Scripture29), in a second test. We found that 84% (2,471 out of 2,934) of exon skipping events found in the adrenal sample alone were independently discovered when using one of the other transcript assembly methods (Supplemental Table S6). When aggregating data across the sixteen tissues, 92% (24,936) of the introns spanning skipped exons have at least two reads supporting them in the sixteen tissues; although in general exon-skipping introns have fewer supporting reads than other introns (Supplemental Figure S7). Similarly, in 21,469 (80%) of the exon skipping events, the exon was present in two or more tissues. Thus, while some assembly artifacts could still be present, most of the events discovered have strong supporting evidence.\n\nSimply counting the number of transcripts assembled from RNA-seq data is one way to measure the extent of alternative splicing. However, this can be confounded by transcripts that are assembled incompletely or incorrectly. Exon skipping events are discrete and easily counted, although it is worth noting that a given exon might be skipped in multiple distinct transcripts. To avoid the difficulties of counting all splice variants, we used the number of exon skipping events as a surrogate measure of splicing variation.\n\nTo identify which of the splice variants were previously unreported, we searched the 26,989 skipped exon events against four gene annotations databases: CCDS16 (23,353 sequences) (http://genome.ucsc.edu, download May 2011), UCSC Genes17 (73,671) (http://genome.ucsc.edu, download May 2011), Ensembl v.61 (120,122) (http://ensembl.org), and H-DBAS18 (58,609 mRNA and 37,096 fl-cDNA RASVs) (http://h-invitational.jp/h-dbas/, download May 2011). Importantly, these specific data sets and releases had been produced using almost exclusively traditional cDNA (EST, mRNA) resources, and therefore provide a fairly accurate assessment of the potential to discover novel alternative splicing variation in RNA-seq experiments. We found that over 60% (17,442) of the events were novel, even after allowing for slight differences in the annotation of exon boundaries present in the various databases (Supplemental Table S8). New exons, new introns, or both can lead to novel splicing events, but we discovered novel introns much more frequently than novel exons (2,914 exons and 15,958 introns). The majority of novel exons overlap known exons; i.e., one or both exon boundaries are novel, but not the entire exon. 884 exons did not overlap any previously annotated exon. A total of 996 (34.2%) of the novel exons and 3,801 (23.8%) of the novel introns were also supported by EST alignments, which provides independent cDNA evidence for those events.\n\nOne example of a novel event is shown in Figure 2. CHTOP (Chromatin target of Prmt1, synonym FOP) is a small nuclear protein on chromosome 1 characterized by an arginine- and glycine-rich region. It has a role in ligand-dependent activation repression of an estrogen receptor target gene30, and has been shown to be a critical modulator of gamma-globin gene expression31. The 84 bp in-frame exon at chromosome 1 positions 153,611,844–153,611,927, which we observed only in heart tissue, does not overlap any of the annotated structures for this gene and has only weak EST evidence, in the terminal exon of EST DB270513. However, the entire exonic region is highly conserved in placental mammals, strongly suggesting that this region is part of the spliced gene. Further, DNaseI hypersensitive sites lend support to an alternative transcript starting at this exon in thyroid tissue. This alternative transcription start site was also identified by our method, and is also missing from the annotation.\n\nThis novel 84-bp exon, marked with a red circle in the figure, is the 4th exon in one of the transcripts from heart tissue (heart.79763.3), and it appears exclusively in that tissue, although a partial form is present in a skeletal muscle transcript.\n\nAnother novel event occurs in the gene ASB15 (ankyrin repeat and SOCS box containing 15) (Supplemental Figure S9). Human ASB15 is known to be expressed predominantly in skeletal muscle and to participate in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. Bovine ASB15 mRNA was also found in heart and pituitary gland tissue, and rat ASB15 was additionally present in kidney and lung tissue, but the amount in most other tissues analyzed was scarce32. These results are consistent with the Illumina Human Body Map data set. Here, exon chr7:123,257,633–123,257,718 is a novel shorter variant that shares its 5´ end with the annotated exon. Both the exon-containing and the exon skipping form are expressed in heart, and have strong read support in the 16 tissues (136 and 39 reads supporting the flanking introns, and 30 reads spanning the exon). Evidence for the novel splice junction is also present in skeletal muscle tissue. We also found a novel putative intron retention event (chr7:123269489–123270019, 531 bp) whose sequence is conserved across multiple vertebrate species. Overall, our analyses underscore the vast potential for RNA-seq experiments to unearth novel splicing events and isoforms.\n\nWe next sought to characterize the set of exon skipping events within and across the sixteen tissues, which also offers a glimpse into the dynamics of alternative splicing in these tissues. We separately traced the presence of the two forms (‘on’ and ‘off’) to generate an alternative splicing profile for each tissue. For each event, we determined the exon inclusion ratio from the expression levels of isoforms containing the 'on’ and the ‘off’ forms in that tissue: R = FPKMon/(FPKMon+FPKMoff), and then compared the profiles to determine similarities and changes in splicing patterns among tissues. We used the relative inclusion ratio2 to characterize such changes: ∆ = |Ri-Rj|, and classified them based on the size of the difference. We separately trace exon skipping events that show large variation (‘switches’; ∆≥0.5), essentially switching between a minor-form and a major-form, and those that show milder variation. Note that all of these evaluations, based as they are on a single sample from each tissue, provide only a qualitative assessment of variation. Multiple replicates would be required to make any conclusions about the statistical significance of these changes between tissues.\n\nOf the 26,989 exon skipping events, between 10,000–20,000 are present in any given tissue. Most events (77–95%) have only one of the forms expressed in a given tissue, and only 5–23% have both forms present in the same tissue (Figure 3 and Supplemental Table S10). The exon-containing (‘on’) form is generally prevalent (R≥0.5). When comparing the profiles between two tissues (Figure 4), 25–35% of the events show stable splicing patterns (∆<0.1), 5–10% are variable (0.1≤∆<0.5) and only 4–7% appear to switch. These proportions are quite similar among the tissues. Roughly 50–65% of the events are not comparable, with the event not found in either or both tissues. Further examination showed these to be due largely to the gene not being expressed (fragments per kilobase of transcript per million mapped (FPKM)<0.1) or harboring different splice forms, whereas we expect the number of incomparable events caused by computational artifacts to be very low. A significant portion of these genes were expressed at low-to-medium levels (FPKM≤10.0), which makes reconstruction difficult and may cause the event to be missed. (For an example, the comparison between the adrenal and adipose tissues is shown in Supplemental Figure S11). These observations suggest that both transcription and alternative splicing contribute significantly in shaping the transcriptomic differences among tissues, although more complete data sets and experiments are needed to be able to tease apart their specific contributions.\n\nMost events within a given tissue are single variant (top). When both isoforms are present in a tissue, the exon is typically contained in the major form (R ≥ 0.5) (bottom).\n\nThe 255 x 255 matrix shows the dynamics of exon skipping events between a tissue and each of the others. The numbers of similar (blue), variable (green), switch (purple) and not present (red) events between any two tissues are shown along one line.\n\nWe contrasted known and newly found events to determine characteristics that could have made the latter difficult to discover with conventional (Sanger) data and methods, and to derive insights into the types of experiments that can help fill in the gaps in the alternative splicing catalog.\n\nFirst, we analyzed the variability in splicing patterns of events, distinguishing between ‘switches’ and events exhibiting milder variation. There was a slight but statistically significant difference between the distributions of novel and known events (chi-square 274.7; p=0.0; Supplemental Table S12), with switches representing 69% of the known events and only 59% of the novel set.\n\nSecond, we assessed the tissue specificity of known and newly found exons and introns based on the data available (Figure 5). For this test, we binned both the known and the novel features according to the number of tissues in which they were found. Not surprisingly, novel exons and introns were significantly more likely to appear in a small number of tissues compared to their known counterparts, but the prevalence was remarkable for exons. For instance, while novel introns were more likely to belong to a single tissue by a 3.0:1 margin (48% versus 16%), that margin for exons was 5:1 (71% versus 14%). Considering that our search turned out many more novel introns than exons, this observation suggests that targeted studies will be needed in the future to identify these highly tissue-specific exons.\n\n(A) The percentage of exons found in 1, 2, …, 16 tissues are shown as horizontal bars, for the 2,914 novel exons (‘novel-X’) and 24,075 known exons (‘known-X’). (B) Similarly for the 15,958 novel introns (‘novel-I’) and 11,031 known introns (‘known-I’).\n\n\nDiscussion\n\nAlternative splicing is a widely recognized RNA processing mechanism in eukaryotic species, playing a major role in the molecular biology of the cell, and within humans it has been implicated in multiple genetic disorders33. The Human Genome Project created an initial map of splice variation more than a decade ago34,35. However, despite concerted efforts over the following years, this map is still inaccurate and incomplete. The Ensembl annotation15, which is among the most complete to date, currently contains seven variants on average per protein coding gene. This is likely an underestimate, as more variants are added every day. The challenge of cataloging all alternative splice variants is daunting, considering that every tissue and cell type can have a different transcriptome, further differentiated by the condition of the cell at the time it was surveyed.\n\nUnlike traditional methods that have mined heterogeneous cDNA sequences collected over time, RNA-seq experiments can survey the transcriptome of a cell type or tissue at great depth, allowing characterization of alternative splicing in much finer detail than previously. The main drawback to RNA-seq today is that its shorter reads are more challenging to assemble into long isoforms. To avoid some of the uncertainty associated with transcriptome assembly, we focused here on alternative splicing events within a transcript, each of which can be detected with a single read.\n\nWe found over 150,000 candidate alternative splicing events, including roughly 27,000 exon skipping events, most of which (65%) were novel. New introns (15,958) were the main source of novel events in our data set, but we also found a large number of new exons (2,914). A large majority of the new exons appear to be tissue-specific, with 71% present in only one tissue, which may explain why they have not been detected previously. Tissue-specific exons represent a clear and important contribution of alternative splicing to tissue differentiation, hence it is noteworthy that 2,085 (38%) of the 5,520 events in our data set were newly identified in this study. Both novel exons and novel introns were more likely to be tissue-specific than those already in the public annotation sets. This suggests that targeted experiments in different tissues or cellular conditions will be more productive in identifying novel splice forms in the future. This requirement is particularly relevant for identifying new exons, which have already been surveyed quite intensively, whereas even broad range RNA-seq experiments remain a rich source of new introns.\n\nOur analysis of the 27,000 events across the sixteen tissues has also revealed insights into the dynamics of the alternative splicing repertoire and its role in tissue differentiation. With roughly 10–20% of the events showing variation across the tissues and 50–65% incomparable based on the existing data, the picture of alternative splicing contributions to tissue transcriptome differentiation vis-à-vis transcription is shaping up to be significant, albeit incomplete. Indeed, even in a deep and rich data set such as the Illumina Human Body Map, rare splice forms may be poorly represented or can be missed entirely. Also, our analyses here are based on a single experiment per tissue from a single individual, and therefore we cannot rule out polymorphic variation, although we expect its contribution to be small relative to tissue related differences2,14. Of course, experimentally testing the events36 and replication on multiple biological samples, from different individuals, will be essential for full validation.\n\nWhile there are ongoing efforts to incorporate alternative splicing information from RNA-seq data into gene annotation databases37, there is yet no repository specifically for human alternative splicing events. Our analyses have identified thousands of putative alternative splicing events, which we have compiled into a catalog of exon skipping events derived from RNA-seq data from multiple human tissues. This collection will be a valuable resource for investigating the mechanisms and evolution of alternative splicing, and as a complement to existing annotation databases. Although this catalog adds substantially to the list of known alternative splicing events, many more RNA-seq experiments will be needed to fully characterize alternative splicing over the full spectrum of tissue types and cellular conditions. Our methods, as implemented in the ASprofile software, are freely available to allow others to create similar databases for other organisms or experimental systems.\n\n\nMaterials and methods\n\nRNA-seq data for the Illumina Human Body Map Project were downloaded from http://www.ebi.ac.uk/arrayexpress/browse.html?keywords=E-MTAB-513&expandefo=on. For sequencing, samples for each of the 16 tissues (adrenal, adipose, brain, breast, colon, heart, kidney, liver, lung, lymph, ovary, prostate, skeletal muscle, testes, thyroid and white blood cells) were prepared by Illumina using their mRNA-Seq kit (Part #RS-100–0801). In brief, PolyA+ RNA was purified from 100 ng of total RNA with oligo-dT beads, and then fragmented with divalent cations under elevated temperature. First strand synthesis was performed with random hexamer and reverse transcriptase, and second strand synthesis with RNAseH and DNA PolI. Following cDNA synthesis, the double stranded products were end-repaired, a single \"A\" was added and then the Illumina PE adaptors were ligated on to the cDNA products. The ligation products were purified using gel electrophoresis. The target size range for these libraries was ~300 bp, such that the final library for sequencing would have cDNA inserts with sizes of ~200 bp long. One run of 2x50 bp paired-end sequencing was performed on the HiSeq2000 instrument, using one lane per tissue, to produce approximately 80 million read pairs per tissue (160 million sequences) (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/protocols/).\n\nTo determine splice variants within each tissue, we aligned reads to the hg19 genome using TopHat v1.3.3 (parameters ‘-a 6 –F 0.05 –splice_mismatches=1 –max-multihits=10’). To allow TopHat to detect as many splice junctions as possible, we provided an intron database extracted from the UCSC known Genes data set (http://genome.ucsc.edu). Aligned reads were then assembled into transcript fragments using Cufflinks v0.9.3 (parameters ‘-F 0.05’). We used Cuffcompare to compare these transfrags to the Ensembl v.61 annotation, and then Cuffdiff to redistribute reads along a high-confidence set of transcripts obtained after eliminating likely artifacts and assemblies not associated with Ensembl genes. Cuffcompare classifies assembled transcripts into multiple categories in relation to reference transcripts, including equal, contained, new splice isoform, intron-located, pre-mRNA fragment, repeat, etc. We retained only transcripts that were deemed ‘equal’, ‘contained’, or ‘new splice isoforms’ as part of our high-confidence set for each tissue. FPKM expression level values for this set were then re-estimated from the original alignments using Cuffdiff.\n\nTo determine alternative splicing events, we developed a software package, ASprofile, to analyze all pairs of transcripts in the sixteen tissues to determine exons included in one transcript and skipped in the other. We restricted the analysis to Ensembl genes with FPKM≥0.1, re-estimated by Cuffdiff as described above. We define an exon skipping event as a pair between an exon containing (‘on’) splice form and an exon skipping (‘off’) splice form, where the boundaries of the flanking introns are required to match precisely. To determine which events are novel, the exons and spanning introns were compared against several annotation data sets (CCDS, UCSC Genes, Ensembl v.61, H-ASDB and dbEST10), allowing for a small difference (up to W=5 bp) at the endpoints. For comparison against ESTs, spliced alignments of all human dbEST sequences were produced with the program ESTmapper38.\n\nFor each event, we calculated the exon inclusion ratio R = FPKMon/(FPKMon+FPKMoff) for each tissue, similarly to Wang et al.2, where FPKMon is the combined FPKM of all isoforms containing the ‘on’ form, and similarly for FPKMoff. To account for minor differences in the annotation of splice junctions, when calculating the expression level of an event we included contributions from splice forms in which the boundaries of the exon and flanking and spanning introns differed slightly (W≤10) from those of the annotated event. The relative inclusion ratio between two tissues, Δij = | Ri–Rj |, was determined for each event and used to classify events based on the size of the differences: stable (Δ < 0.1), variable (0.1 ≤ Δ < 0.5), ‘switch’ (Δ ≥ 0.5), or incomparable, when the event was not found in one or both tissues. For the tissue-specificity analysis, the largest difference between any two tissues was used to determine ‘switches’ versus ‘non-switches’.\n\nWe implemented the methods in a software package, ASprofile, for discovering alternative splicing events in transcripts predicted from RNA-seq data and then comparing them across multiple conditions. ASprofile consists of programs for extracting (‘extract-as’), quantifying (‘extract-as-fpkm’) and comparing (‘collect-fpkm’) alternative splicing events. ‘Extract-as’ takes as input a GTF transcript file, for instance one produced by a transcript assembly program or a set of gene annotations, and compares all pairs of transcripts within a gene to determine exon-intron structure differences that indicate an AS event. The following classes of events are currently implemented: exon skipping, cassette exons, alternative transcript start and termination, retention of single or multiple introns, and alternative exon ends (Supplemental Figure S13). To determine alternative splicing events among multiple samples, a single input file must be created by concatenating the transcript files of individual samples, with the gene names a priori reconciled across the samples (for instance, by using the program Cuffcompare from the Cufflinks suite). The second program, ‘extract-as-fpkm’, calculates the FPKM of each event from those of transcripts harboring the event in a given sample, allowing for small variations (up to V bp, where V is a user-specified value) at the boundaries of the exons and introns. Lastly, the script ‘collect-fpkm’ collects the FPKM event values for all RNA-seq samples, and calculates and compares splicing ratios across samples, which can be used to observe trends in the dynamics of alternative splicing profiles or to select promising candidates for laboratory testing. The software package is written in C and Perl and is available free of charge from the Zenodo repository (http://zenodo.org/record/7068; doi:10.5281/zenodo.7068) and from our web site at http://ccb.jhu.edu/software/ASprofile.",
"appendix": "Author contributions\n\n\n\nLF and SLS conceived the project and designed the experiments. LF and LS carried out the computational analyses. LF wrote the first draft, and SLS edited the final version of the manuscript. 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 in part by the NSF award ABI-1159078 to LF and NIH grants R01-HG006677 and R01-HG006102 to SLS.\n\n\nAcknowledgments\n\nWe thank Illumina for making available the Human Body Map data set.\n\n\nSupplementary materials\n\n\"Mapped\" refers to the number of reads that had at least one and no more than ten alignments to the genome.\n\nCufflinks assembles short reads aligned to a genome into a set of transcript fragments (‘transfrags’ or ‘transcripts’ or ‘txpts’, below), grouped by locus (‘gene’).\n\n*Genes containing multi-exon transcripts.\n\n†Averaged over multi-exon genes.\n\n‡Averaged over multi-exon transcripts.\n\nCuffcompare compares each predicted transcript’s intron chain against those of the reference transcripts and classifies the transfrag as: ‘equal’ to a reference transcript, if their intron chains are identical; ‘contained’, if included in a reference transcript’s; or as a ‘new splice form’, if the intron chain has at least one splice junction in common with the reference transcripts (n.b., other Cuffcompare codes are not relevant and were omitted).\n\nNovel and known isoforms are determined by comparison with ENSEMBL v.61 gene annotations using the program Cuffcompare (see caption to Table S3 for more details).\n\n*Includes 17,200 ‘default’ TSS (TTS) sites for 17,200 genes.\n\nValues shown are for exon skipping (ES) events derived from the Illumina Human Body Map data (‘Cufflinks’) and from Ensembl v.61 gene annotations, from columns 4 and 8 in Table S5.\n\nOne class of mapping artifacts that can contribute to creating false exon skipping events is that of ‘imprecise splice junctions’, where the same intron in different spliced reads is mapped by the alignment software at slightly different positions, typically within a few bp of each other, thus creating the appearance of several introns at that location. Such variations could lead to redundancies in reporting exon skipping events. To estimate the potential effects of imprecise splice junctions on our data set, we searched the 26,989 events against each other to identify those that are co-located and differ only slightly (by some maximum number of bases, V bp) in their exon-intron boundaries. (Note, however, that such events would also include all true alternative 5’ and 3’ exon ends). At each locus, we designate one event as ‘anchor’ (true), and refer to the co-located events as ‘variations’. We repeated the operation for 25,114 events discovered from the Ensembl v. 61 annotation with our software ASprofile, to create a baseline for comparison. For our data set of 26,989 events, labeled ‘Cufflinks’ in Figure S5 below, small ≤5 bp differences in splice junctions account for the majority of ES variations, beyond which their value as a fraction of the total number of ES events becomes relatively stable. In contrast, the percentage of Ensembl v. 61 event variations continues to increase roughly linearly with the distance from the ‘anchor’ splice junction. Comparing the two curves, we estimate that up to ~1,400 of ES events (5%) in our data set could be redundant (column 10 in the Table S5). We expect this value to be an overestimate, given that true small 5´ and 3´ exon alternative splicing variations are poorly represented in annotation databases.\n\nAll – all events co-located within V bp; Loci – clusters of co-located events (each with a representative ‘anchor’); Var (=All-Loci) – variations (not including ‘anchors’), representing potential mapping artifacts.\n\nTo assess the reproducibility of our event discovery method, we compared the exon skipping events discovered from transcripts assembled with Cufflinks and with three other reference transcript assembly algorithms (Scripture29, IsoCEM27 and SLIDE28). We applied each program to the adrenal sample of the Human Body Map data and used our program ASprofile to extract exon skipping events from the assembled transcripts.\n\nAs the results in Table S6 indicate, when compared to Cufflinks, IsoCEM and SLIDE find roughly half the number of events, whereas Scripture finds roughly 60% more events. (Allowing for small differences, up to V bp, in the coordinates of introns and exons to account for imprecise mapping of splice junctions, has negligible effects on the comparison results). There is generally good agreement among the methods, as most of the events predicted with one method can also be retrieved by at least one of the other methods. In particular, 84% (2,471 out of 2,934) of the events predicted when using Cufflinks can be confirmed by other methods. Additionally, our analysis reveals a number of potentially new events, most of them discovered by Scripture, which could be used to enrich our repository in the future. However, each program has its own characteristics and biases, and a careful analysis has to be performed before incorporating these events into the database.\n\n(A) Diagram of an exon skipping event, illustrating the three types of introns: left – blue, right – red, and intron spanning – green. (B) Read histograms for the three categories of introns. The x-axis represents the number of supporting reads for an intron, grouped in bins, and the y-axis shows the percentage of introns by levels of supporting reads (in bins).\n\nExon and intron boundaries were compared allowing for a small difference (up to V=0, 5 and 10 bp). Values in bold are those reported in the main text.\n\nExon chr7:123,257,633–123,257,718, present in heart (heart.653543.1 and heart.653543.2) and with partial support in skeletal muscle tissue (skel_muscle.206929.2), is novel. Red arrows point to the exon appearing in two isoforms reconstructed in the heart sample, and to the (potentially) partial form in a skeletal muscle transcript for the ASB15 gene. The spanning intron in the heart sample is also novel. Additionally, we found a potentially retained intron (chr7:123,269,489–123,270,019; thyroid.843995.1), circled in red, whose 531 bp sequence is conserved across multiple vertebrates.\n\n2,085 (15.0%) event genes are not expressed (FPKM<0.1), whereas 3,487 (33.7%) have FPKM values less than 10.0 and therefore may be incompletely reconstructed, which can cause a splice form to be missed.\n\nNovel events are depleted in ‘switches’ compared to known events (test of homogeneity, df=2, chi-square 274.7, p = 0.0).\n\n(A) Exon skipping (SKIP) and cassette exons (MSKIP); (B) retention of single (IR) and multiple (MIR) introns; (C) alternative exon ends (AE); (D) alternative transcription start site (TSS); and (E) alternative transcription termination site (TTS). Alternatively spliced features are shown in red.\n\n\nReferences\n\nPan Q, Shai O, Lee LJ, et al.: Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008; 40(12): 1413–1415. PubMed Abstract | Publisher Full Text\n\nWang ET, Sandberg R, Luo S, et al.: Alternative isoform regulation in human tissue transcriptomes. Nature. 2008; 456(7221): 470–476. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGraveley BR: Alternative splicing: increasing diversity in the proteomic world. Trends Genet. 2001; 17(2): 100–107. PubMed Abstract | Publisher Full Text\n\nStamm S, Ben-Ari S, Rafalska I, et al.: Function of alternative splicing. Gene. 2005; 344: 1–20. PubMed Abstract | Publisher Full Text\n\nLorson CL, Hahnen E, Androphy EJ, et al.: A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci U S A. 1999; 96(11): 6307–6311. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNarla G, DiFeo A, Yao S, et al.: Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread. Cancer Res. 2005; 65(13): 5761–5768. PubMed Abstract | Publisher Full Text\n\nGarcia-Blanco MA, Baraniak AP, Lasda EL: Alternative splicing in disease and therapy. Nat Biotechnol. 2004; 22(5): 535–546. PubMed Abstract | Publisher Full Text\n\nDavid CJ, Chen M, Assanah M, et al.: HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature. 2010; 463(7279): 364–368. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHofstetter G, Berger A, Fiegl H, et al.: Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer. Oncogene. 2010; 29(13): 1997–2004. PubMed Abstract | Publisher Full Text\n\nWheeler DL, Barrett T, Benson DA, et al.: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2008; 36(Database issue): D13–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGerhard DS, Wagner L, Feingold EA, et al.: The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome Res. 2004; 14(10B): 2121–2127. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Z, Gerstein M, Snyder M: RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009; 10(1): 57–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMortazavi A, Williams BA, McCue K, et al.: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7): 621–628. PubMed Abstract | Publisher Full Text\n\nGonzalez-Porta M, Calvo M, Sammeth M, et al.: Estimation of alternative splicing variability in human populations. Genome Res. 2012; 22(3): 528–538. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlicek P, Amode MR, Barrell D, et al.: Ensembl 2012. Nucleic Acids Res. 2012; 40(Database issue): D84–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPruitt KD, Harrow J, Harte RA, et al.: The consensus coding sequence (CCDS) project: Identifying a common protein-coding gene set for the human and mouse genomes. Genome Res. 2009; 19(7): 1316–1323. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDreszer TR, Karolchik D, Zweig AS, et al.: The UCSC Genome Browser database: extensions and updates 2011. Nucleic Acids Res. 2012; 40(Database issue): D918–923. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTakeda J, Suzuki Y, Sakate R, et al.: H-DBAS: human-transcriptome database for alternative splicing: update 2010. Nucleic Acids Res. 2010; 38(Database issue): D86–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrapnell C, Pachter L, Salzberg SL: TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009; 25(9): 1105–1111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrapnell C, Williams BA, Pertea G, et al.: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2009; 28(5): 511–515. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClark TA, Schweitzer AC, Chen TX, et al.: Discovery of tissue-specific exons using comprehensive human exon microarrays. Genome Biol. 2007; 8(4): R64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu Q, Modrek B, Lee C: Genome-wide detection of tissue-specific alternative splicing in the human transcriptome. Nucleic Acids Res. 2002; 30(17): 3754–3766. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde la Grange P, Gratadou L, Delord M, et al.: Splicing factor and exon profiling across human tissues. Nucleic Acids Res. 2010; 38(9): 2825–2838. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElliott DJ, Grellscheid SN: Alternative RNA splicing regulation in the testis. Reproduction. 2006; 132(6): 811–819. PubMed Abstract | Publisher Full Text\n\nLi Q, Lee JA, Black DL: Neuronal regulation of alternative pre-mRNA splicing. Nat Rev Neurosci. 2007; 8(11): 819–831. PubMed Abstract | Publisher Full Text\n\nSultan M, Schulz MH, Richard H, et al.: A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science. 2008; 321(5891): 956–960. PubMed Abstract | Publisher Full Text\n\nLi W, Feng J, Jiang T: IsoLasso: a LASSO regression approach to RNA-Seq based transcriptome assembly. J Comput Biol. 2011; 18(11): 1693–1707. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi JJ, Jiang CR, Brown JB, et al.: Sparse linear modeling of next-generation mRNA sequencing (RNA-Seq) data for isoform discovery and abundance estimation. Proc Natl Acad Sci U S A. 2011; 108(50): 19867–19872. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuttman M, Garber M, Levin JZ, et al.: Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat Biotechnol. 2010; 28(5): 503–510. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Dijk TB, Gillemans N, Stein C, et al.: Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases. Mol Cell Biol. 2010; 30(1): 260–272. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Dijk TB, Gillemans N, Pourfarzad F, et al.: Fetal globin expression is regulated by Friend of Prmt1. Blood. 2010; 116(20): 4349–4352. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcDaneld TG, Hancock DL, Moody DE: Altered mRNA abundance of ASB15 and four other genes in skeletal muscle following administration of beta-adrenergic receptor agonists. Physiol Genomics. 2004; 16(2): 275–283. PubMed Abstract | Publisher Full Text\n\nSingh RK, Cooper TA: Pre-mRNA splicing in disease and therapeutics. Trends Mol Med. 2012; 18(8): 472–482. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVenter JC, Adams MD, Myers EW, et al.: The sequence of the human genome. Science. 2001; 291(5507): 1304–1351. PubMed Abstract | Publisher Full Text\n\nLander ES, Linton LM, Birren B, et al.: Initial sequencing and analysis of the human genome. Nature. 2001; 409(6822): 860–921. PubMed Abstract | Publisher Full Text\n\nRichard H, Schulz MH, Sultan M, et al.: Prediction of alternative isoforms from exon expression levels in RNA-Seq experiments. Nucleic Acids Res. 2010; 38(10): e112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDerrien T, Johnson R, Bussotti G, et al.: The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012; 22(9): 1775–1789. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlorea L, Di Francesco V, Miller J, et al.: Gene and alternative splicing annotation with AIR. Genome Res. 2005; 15(1): 54–66. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1828",
"date": "07 Oct 2013",
"name": "Manuel Corpas",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 'Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues' presents a systematic analysis of alternative splicing of the Illumina Body Map RNA-seq set, probably the most complete set of RNA-seq data to date. This data set consists of 160M reads each for 16 tissues, each from a different individual. From the standpoint of a reviewer I consider this article to be of very good quality, and have very much enjoyed reviewing it. There are a few suggestions I hope could help improve this work.P3. pp.2: Has alternative splicing profiling been attempted before? If so, could this please be shown in the background section? If not, how does this technique specifically differ from what has been done before? Some more context would be appreciated. P3. pp.3: What is the effect of comparing tissues from 16 different individuals? I would expect that the genetic variation of each individual would have some effects in the actual AS patterns observed when compared. I see that these samples belong to people with different ethnic backgrounds and ages. I would be interested to know what the authors think about how different results would be if all 16 tissues had been from one individual. I tried to compile ASprofile on a mac and I got this error:libc.h:15:20: error: malloc.h: No such file or directoryI solved this by changing this line with #include <stdlib.h>. Perhaps you might want to add these lines, so that mac users do not encounter the problem:#if !defined(__APPLE__)#include <malloc.h>#endif Although the license for ASprofile is available in the Zenodo page, it might also be a good idea to mention it somewhere in the article. Not mentioning a license may make some users uneasy, even if it says it is open source. In S1, how many of the mapped reads were properly paired? Why did you use such old versions of Ensembl genes (Ensembl 61, February 2011), UCSC Genes, CCDS genes and H-DBAs? I also find that the TopHat (v1.3.3) and Cufflinks (v0.9.3) versions are quite old. The current version for TopHat is 2.0.9 and the current version for Cufflinks is 2.1.1. The current version of Ensembl is version 73, September 2013. The usage of such old versions concerns me a little, particularly those of the gene annotations, given that a lot of the results that are presented in this work rely heavily on the comparison of AS profiles against those annotations. Some of the results might be different if a newer version of the annotations were up-to-date. P5. pp.5: The example of a novel event in CHTOP does not provide clear evidence as to how many reads support the novel exon. Please provide some numbers. P7. pp1: Please could you provide evidence for the novel putative intron retention event located in the chr7:123269489-123270019 region? In Figure S7.B, I would appreciate some text describing the biological significance of having the 100 bin in the x-axis with the greatest bars. Overall I am unable to understand what this figure means.",
"responses": [
{
"c_id": "622",
"date": "18 Nov 2013",
"name": "Liliana Florea",
"role": "Author Response",
"response": "We wish to thank Manuel Corpas for his thorough review and suggestions. In responding to his comments:We would like to point to our answer to question 2 from Reviewer 1, where we more broadly address this and other related questions. An earlier study by Wang et al. (reference 2) also tackled polymorphic splicing differences, finding that these represent a much smaller fraction of the AS variation compared to tissue differences. Therefore, while some polymorphic differences exist, they are not likely to distort the picture we paint in this study. We agree that analyzing data from multiple tissues in the same individual would be highly informative and we are actively looking for suitable data sets, which are not available at the moment. We thank the reviewer for taking the time to test the software. We updated the code and included a note on the license (GNU GPL) in the text (page 3). We have included proper read pairing information for all tissues in Table S1. With respect to the versions of the software, we note that at the time we ran our analyses we used the latest versions of each of these programs and databases, however our paper was caught up in the reviewing process at another journal for over a year. After two rounds of very slow reviews and multiple requests for revisions, the other journal was still not satisfied and therefore we decided to submit to F1000Research, which has far faster publication turnaround. We should emphasize that none of the previous reviewers’ comments questioned the validity of our results, and our revisions did not substantively change any of our conclusions. Nevertheless, we also searched the ‘novel’ events against the aggregate set of Ensembl 73, GENCODE v.17 and RefSeq representing the most recent versions of these collections, and only found 21 exons and 9 introns (note that these did not include the events depicted in Figures 2 and S9). Hence, the results discussed in the manuscript still hold. We have added read support information for the exon skipping event in Figure 2, and for the putative intron retention event in Figure S9. The ‘100’ bin in Figure S7 (now S8) shows the proportion of introns supported by more than 100 reads, for each of the three types of introns. Most events have deep support (>=100 reads) for the flanking introns, and to a lesser extent for the spanning intron, which is consistent with our earlier finding that the exon tends to be skipped in the minor form (Figure 3)."
}
]
},
{
"id": "2236",
"date": "29 Oct 2013",
"name": "Peter N. Robinson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors address the question of how much alternative splicing can be identified in 16 different human tissues, and how much of the alternative splicing is specific for one or another tissue. To do so, they analyzed 50-bp paired-end sequences from 16 different tissue samples sequenced relatively deeply (ca. 80 million read pairs per tissue sample). The data has been made available by Illumina as the Human Body Map project. Computational analysis was then performed with the programs TopHat and Cufflinks. This type of analysis assembles the reads into a set of transcripts that are compatible with the splicing patterns inferred from reads that are split over multiple exons. Between 23,000–46,000 partial or complete transcript assemblies (“transfrags”) were obtained per tissue. Approximately 5,000–20,000 transfrags per tissue, or 11–45% of the total, were identified as potentially novel splice forms. As the authors note, it is still difficult to assemble complete transcripts from relatively short RNA-seq reads in this way, and they thus make the very reasonable decision to concentrate their further analysis on alternative splicing events that are likely to be valid. Thus, it is easier to analyze the spectrum of exon skipping/inclusion events at a particular exon. In general, the article is well done and will be of interest to those involved with RNA-seq or alternative splicing. One particularly interesting, if not entirely surprising, finding of this study is the fact that the majority of the novel exon insertion events showed a high degree of tissue specificity, thus providing an obvious explanation of why the corresponding exons are not yet in public databases. This suggests that efforts should be spent to characterize the full range of splicing in tissues in order to understand their biology. Comments:It would be useful if the authors could provide more discussion of the current state of the art of transcript identification using RNA-seq reads. In particular, the authors discuss three other transcript assembly programs in the supplement (S6), finding that they identify 50-60% of the isoforms called by Cufflinks. Have the authors performed their downstream analysis using the results of one of these programs? How different would the results be? The authors should try to place their results in the context of many other efforts at identifying novel isoforms from the literature and discuss the relative merits of their approach. It would be useful to have a table with quality metrics for this dataset, e.g., Phred scores, percent of mapable reads etc. - table S1 could be extended for this purpose. Although much of the results deal with exon skipping/insertion events, it would be interesting to hear a little about results for other classes of alternative splicing that can be identified by the authors’ software, e.g. alternative transcription start and termination sites. In the results section, it would help the presentation of the material to provide a brief description of the relative inclusion ratio at this portion of the text and to define what i and j are (i.e., take some of the material from the methods section that explains this).",
"responses": [
{
"c_id": "621",
"date": "18 Nov 2013",
"name": "Liliana Florea",
"role": "Author Response",
"response": "We thank Peter Robinson for his insightful comments and suggestions, which we address in the following:Transcriptome assembly represents a complex topic on its own, and is only a tangent to our work, therefore we believe that it would be best addressed in detail elsewhere. We wish to point the reviewer and the readers to a review on current transcript assembly methods that we wrote recently, to appear in IEEE/ACM Transactions in Bioinformatics and Computational Biology journal. We also included a brief evaluation of several transcript assemblers on a simulated data set (new Table S2), showing that Cufflinks is the only program among those tested that can identify alternative splicing events with high enough accuracy (~90%) to allow meaningful downstream analyses, though we will explore combinations of these programs in the future. Minor correction to the statistic quoted by the reviewer: 84% of the Cufflinks-predicted exon skipping events are reproducible by other methods. There is indeed a rich body of work in the area of alternative splicing. Notably, two early studies (references 1 and 2) highlighted the extent of alternative splicing in the human genome based on analyses of RNA-seq data, albeit those data sets were not nearly large enough to be able to characterize it in detail, and a more recent study (Merkin et al., 2012) extended those analyses in the context of multiple species. Similarly, there are now several methods (e.g., MISO, SpliceTrap and MATS) that infer or quantify exon skipping events directly from RNA-seq reads, and therefore are complementary to our approach. Using a very large data set, our work provides a more detailed picture of alternative splicing variation and produces both an easy-to-use tool and a database that we believe will be valuable in studies of alternative splicing mechanism and function. We added percentage of mapped reads and number of properly paired reads to Table S1, as indicators of read alignment quality. We think that analyzing other types of events is indeed a very interesting experiment, which due to scale we plan to address in our future work. The definitions were already in the main text (page 7, section “Characterization of exon skipping events”), but we have clarified i and j."
}
]
}
] | 1
|
https://f1000research.com/articles/2-188
|
https://f1000research.com/articles/2-251/v1
|
20 Nov 13
|
{
"type": "Research Article",
"title": "Merkel-like cells in the murine conjunctival stroma",
"authors": [
"Ting Fu",
"Yunxia Xue",
"Chaoyong Xia",
"Yabing Yang",
"Peng Liu",
"Jun Liu",
"Wanyu Zhang",
"Jiansu Chen",
"Yangqiu Li",
"Dongqing Cai",
"Zhijie Li",
"Ting Fu",
"Yunxia Xue",
"Yabing Yang",
"Peng Liu",
"Jun Liu",
"Wanyu Zhang",
"Jiansu Chen",
"Yangqiu Li",
"Dongqing Cai"
],
"abstract": "Merkel cells, discovered by Friedrich Sigmund Merkel in 1875, are found in multiple regions of the skin and some mucosa and make contact with specialized nerve fibers, participating in the perception of touch. However, Merkel cells have thus far not been described on the ocular surface. The purpose of this study was to investigate the distribution and ultrastructure of Merkel cells on the ocular surface and study changes in their number and ultrastructure after corneal epithelial wounding. Entire mouse ocular surfaces were obtained and stained by antibodies and tracking dye on fixed whole-mount specimens. The distribution and ultrastructure of specific fluorescence-positive cells were analyzed using deconvolution microscopy and transmission electron microscopy (TEM), respectively. The corneal epithelial wound-healing model was employed to observe the ultrastructural changes of these CK8-positive cells. We found that CK8-positive cells and FM1-43-positive cells were mainly located in the stromal layer, but not in the epithelial basal layer, of the forniceal conjunctiva. Our TEM results indicate that these cells possess the unique characteristic structures of Merkel cells, including electron-dense membrane-surrounded granules and spine-like protrusions of variable lengths, and demonstrate the formation of Merkel cell-neurite complexes. After corneal epithelial wounding, these cells exhibited rapid cell shrinkage and nuclear lobulation. Thus, Merkel-like cells were found in the conjunctival stroma of the ocular surface and may play an important role in maintaining the normal physiological function of the ocular surface.",
"keywords": [
"Merkel cells",
"Ultrastructure",
"Conjunctival",
"CK8",
"FM1-43"
],
"content": "Introduction\n\nMerkel cells, first described by Fredrich S. Merkel, are mostly located in the basal layer of the epidermis in distinct areas of hairy mammalian skin1. Merkel cells have also been shown in vertebrates and cyclostomes2 and can be widely found in hairy skin, the eccrine glandular ridges of the glabrous skin (palms and soles), ectoderm-derived mucosal tissues (palatine mucosa), and whisker pads or taste buds3,4. Merkel cells are generally classified into two distinct subpopulations based on whether or not they are associated with nerves. Those that are associated with nerves are referred to as the Merkel cell-nerve complexes and have mechanoreceptive and chemo-sensitive functions. Those that do not associate with nerves possess endocrine functions4–6. Recent studies have shown that Merkel cells are important in light-touch responses and can work as neurotransmitters, it is suggested that Merkel cells are essential for the perception of space experienced by individuals7.\n\nHowever, Merkel cells have, thus far, not been described on the ocular surface. In this study, we investigated the distribution and ultrastructure of Merkel cells on the ocular surface and revealed a morphological basis for further study of the function of Merkel cells on the ocular surface.\n\n\nMaterials and methods\n\nFifty C57BL/6 male mice were approved by the Experimental Animal Center of Sun Yet-Sen University and housed individually in plastic cages (35×25×15 cm) with sawdust as bedding. Animals were kept at ambient temperature of 25°C with 14L/10D light cycle, and were offered mice feed and water available ad libitum when they were not undergoing experimentation. All animal procedures were carried out with ethical permission and in accordance with the guidelines described in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Vision and Ophthalmic Research and the guidelines of the Animal Experimental Committee at Ji Nan University. All of the mice used in this study were 6–8 weeks old and did not suffer from eye disease.\n\nTissue specimens of the entire ocular surfaces, including the cornea and the conjunctiva up to the mucocutaneous junction, were dissected as previously described8. In brief, the intact eye was dissected together with the eyelids and fixed with freshly prepared 2% paraformaldehyde (Beyotime Institute of Biotechnology, China) in PBS. The eyelids were then turned inside out to expose the entire ocular surface. Next, the eye was cut and divided into a superior and an inferior portion. The muscle and other accessory tissues were carefully removed under a stereoscopic microscope (XZ6, Olympus, Japan). Subsequently, the tissue samples were washed in PBS three times, blocked with 2% BSA (GBC, China) in PBS for 15 minutes at room temperature, and permeabilized with 10% Triton X-100 (Guoao, China) in 2% BSA for 15 minutes, prior to incubation with rat anti-mouse Troma-1 antibody (DSHB, USA, Code No. SP2/0) 1:50 for 48 hours at 4°C. The tissue samples were then washed in PBS three times, incubated with the secondary FITC-Dnk Anti-Rat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, Code No. 712-096-150) 1:100 for 30 minutes, and washed three times again prior to visualization. To flatten the specimen, the eyelids were removed and the specimen was placed epithelial side up and mounted with Fluorescent Mounting Media (Beyotime Institute of Biotechnology, China), which contains 4´, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Code No. D9564-10MG). The whole-mounts were imaged using the DeltaVision Core system (Applied Precision, USA).\n\nThe eyes of the mice were dissected as described above, then embedded in optimal cutting temperature (O.C.T) (Sakura Finetek, Japan, Code No. 4583), frozen at -80°C in the refrigerator freezer for 30 minutes, and sliced into 5µm thick sections, using a cryostat microtome (Leica, CM1900). The tissue sections were incubated with Troma-1 and FITC-IgG as previously described, and washed in PBS three times, the tissues were stained with mouse anti-mouse Math1 (DSHB, USA, Code No. Atoh1) 1:800, mouse anti-mouse neurogenin 3 (DSHB, USA, Code No. F25A1B3) 1:100 and beta-tubulin III (R&D, USA, Code No. NL1195R) 1:100 for 24 hours at 4°C, respectively, then washed in PBS, and incubated with Cy3-Dnk Anti-Mouse IgG (Jackson ImmunoResearch, USA, Code No. 715-165-150) 1:500 antibody for 45 minutes. After washing, the section tissues were mounted with Fluorescent Mounting Media (Beyotime Institute of Biotechnology, China) containing 4´, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Code No. D9564-10MG). The sections were imaged by fluorescence microscopy (Olympus BX51, Japan).\n\nThe eyes were dissected together with the eyelids8, placed in PBS, and processed as previously described. After separating the eye into a superior and an inferior portion, samples were cut from the cornea to the conjunctiva in 1 mm segments. A portion of these tissues was fixed in 2.5% glutaraldehyde (Pelco, USA, Code No. 18426 ) for 4 hours, washed in PBS three times (each time 15 minutes), then fixed in 1% osmic acid (Pelco, USA, Code No. 18456) for 15 minutes , after washed in PBS three times, then tissues were dehydrated with a graded ethanol series, consisting of 50%, 70%, 80%, 90% and 100% ethanol at 10 minutes each. Tissues were then incubated in a 1:1 mix of 100% ethanol: Epon812 (Pelco, USA, Code No. 18010) for 2 hours and then rinsed twice with Epon812, for 1 hour each time. Tissues placed onto a mold filled with Epon812. The assembly was inserted into a vacuum chamber and placed in a 60°C oven for 24 hours, then tissues were sliced into ultrathin sections with ultramicrotome (LKB2088, Sweden), as required for transmission electron microscopy (TEM) sample preparation5. The slices were then stained with 1%~2% uranyl acetate (Electron Microscopy Sciences, USA, Code No. 22400) and lead citrate (Sigma, USA, Code No. 6132-04-3). The remaining portion of the tissues was processed according to the method previously described for immunofluorescence, by washing with PBS three times, then similarly processed for TEM but was not stained with uranyl acetate and lead citrate. All of the TEM slices were examined by using a PHILIP5-TECNAI 10 electron microscope (PHILIP5-TECNAI 10, The Netherlands, Philips).\n\nTwenty mice were intraperitoneally injected with 0.5 ml of FM1-43 (Invitrogen, USA, Code No. F-35355) (10 µg/ml) dissolved in high-potassium (20 mM) Hanks’ solution (Beyotime Institute of Biotechnology, China) and then euthanized by cervical dislocation after CO2-induced unconsciousness, after 12 hours. To this end, the animals were placed in sealed chambers where carbon dioxide gas is introduced. Once unconsciousness has been achieved and the animals are restrained in a normal standing position on a firm, flat surface and grasped the base of the tail. To produce the dislocation, quickly push forward and down with the hand or object restraining the head. The effectiveness of dislocation can be verified by feeling for a separation of cervical tissues.\n\nThe entire ocular surface was immediately separated as previously described and whole-mounted without any further treatment. The FM1-43-positive cells were directly observed using the DeltaVision Core system (Applied Precision, USA).\n\nSome studies have shown that the membrane potential of Merkel cells changes under stress (i.e., stimulation or changes in osmotic pressure) and can initiate Ca2+ and K+ signaling9. To determine whether Merkel cells located on the ocular surface respond to stress, we established a corneal epithelial wound model to observe changes in the ultrastructure of these cells. The wound model was applied as previously described10–12. Briefly, ten mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (Beyotime Institute of Biotechnology, China) (25–50 mg/kg). The central corneal epithelium was demarcated with a 2 mm trephine and removed using a diamond blade for refractive surgery (Accutome, Malvern, PA) under a stereoscopic microscope (XZ6, Olympus, Japan). Care was taken to minimize any injury to the epithelial basement membrane and stroma. Twenty-four hours after wounding, the specimen tissues were processed as described above8, fixed, sectioned, stained, and analyzed5. Finally mice were euthanized by cervical dislocation after CO2-induced unconciousness.\n\n\nResults\n\nAs previously reported, Merkel cells express CK 84. In this study, we first identified the location of CK8-positive cells on the ocular surface. The ocular surface whole-mounts show that CK8-positive cells were localized in the fornix and the palpebral conjunctiva, and CK8-positive immunofluorescence was observed in the membrane, nucleus and cytoplasm of specific cells. We also found that the CK8-positive cells exhibited circular nuclei and visible nucleoli, and the cytoplasmic fluorescence revealed reticular and granular shapes (Figure 1). However, we did not find any CK8-immunofluorescence-positive cells in the bulbar conjunctiva.\n\n(A) CK8-positive cells (green). The CK8-positive immunofluorescence was observed in the membrane, nucleus and cytoplasm of specific cells, and the cytoplasmic fluorescence revealed reticular and granular shapes. (B) Merkel cells incorporated FM1-43 dye. The green fluorescent granules were observed in the cytoplasm of Merkel cells.\n\nDouble immunofluorescence stain showed that there were two kinds of CK8-positive cells on the ocular surface. The types that also stained with Math1 (Figure 2) were located in the stromal layer of the fornix and the palpebral conjunctiva, the other in the epithelial basal layer (we also identified that the CK8-positive cells in the epithelial basal layer were goblet cells). So we speculated that the Merkel cells were located in the stromal layer and could express Ngn3 (Figure 3) and beta-tubulin III (Figure 4) in the cytoplasm.\n\nThe CK8-positive cells (green) also express Math-1 (red) in the cytoplasm.\n\nThe CK8-positive cells (green), in the stroma, can express Ngn3 (red) in the cytoplasm, but the CK8-positive cells in the epithelial layer did not express Ngn3.\n\nThe CK8-positive cells (green) in the stroma express beta-tubulin III (red), but the CK8-positive cells in the epithelial layer did not express beta-tubulin III.\n\nThe classical method to identify Merkel cells has been electron microscopy, so we used TEM to analyze the ultrastructure of the conjunctival cells. As shown in Figure 5, there is a class of cells contain electron-dense membrane-surrounded granules, mitochondria and intermediate filaments (Figure 5A). In addition, these cells exhibited spine-like protrusions of variable lengths from the cell surface (Figure 5B) and located in the conjunctival stroma (Figure 5C). These characteristics are similar to the ultrastructural features of Merkel cells4. The granules were not only observed within these cells (Figure 5A) but were also found in the extracellular regions (Figure 5B). We also observed some cells that were associated with nerve terminals, forming Merkel cell-neurite complexes with vesicles at the nerve endings (Figure 5D). Shrinkage of the membrane-surrounded granules and Merkel cells in close proximity to the conjunctival epithelium and nuclear lobulation were investigated after corneal wounding (Figure 5E). CK8-specific Merkel cells were observed in the conjunctival stroma by immuno-electron microscopy (Figure 5F). A small number of CK8-specific particles were observed in the conjunctival epithelium cells under high magnification (Dataset-1).\n\n(A) Merkel cells contain mitochondria (M) and intermediate filaments (indicated by a star); bar:1µm. (B) Merkel cells exhibited spine-like protrusions of variable lengths (indicated by black arrow); bar:2µm, and electron-dense membrane-surrounded granules (indicated by white arrow) can been seen in the extracellular regions; bar:1µm. (C) Merkel cells (indicated by white arrow) and myofibroblast (indicated by black arrow) located in the stroma of the conjunctiva. Conjunctival epithelium was indicated by black star; bar:10µm. (D) Merkel cells associate with nerve terminals (T) forming Merkel cell-nerve complexes (bar:10µm), vesicles (indicated by a bent arrow) can be seen in the nerve endings (bar:0.5µm). (E) Merkel cells are shown near the epidermis after corneal wounding; (bar:2µm). (F) Merkel cells observed by immuno-electron microscopy; (bar:5µm).\n\nIt has been reported that the FM1-43 dye is a useful marker for live Merkel cells13. Therefore, we used FM1-43 dye to identify conjunctival Merkel cells. Twelve hours after intraperitoneal injection of FM1-43, green fluorescent granules were observed in the cytoplasm of Merkel cells, and nuclear labeling was found in the conjunctiva of the stroma using the deconvolution microscopy (Figure 1B).\n\n\n\n\nDiscussion\n\nThe conjunctiva is an important part of the ocular surface. It plays a very important role in the process of mucus secretion and immunological reactions. The conjunctiva can be divided into two parts, the conjunctival epithelium and the stroma. The epithelial cells can be further classified into superficial cells, intermediate cells and basal cells14. The conjunctiva, similar to the skin and some mucosal tissues, is developmentally derived from the ectoderm. Previous studies have shown that Merkel cells are located in the epithelia of the skin and in the oral mucosa, where they participate in endocrine and mechanoreceptor functions3,4. However, their presence on the ocular surface had not yet been confirmed.\n\nThe classical method used to identify Merkel cells is to check for their ultrastructure using TEM. Recently, various studies have confirmed that Merkel cells express specific cytokeratins (CK), such as CK8, CK18, CK19, and CK2015–17, and neuropeptides, such as vasoactive intestinal peptide (VIP)18, substance P19 and calcitonin gene-related peptide (CGRP)20, and other proteins, such as Math1, neurofilament-H, and neurotrophin-33. These bio-markers provide a basis for the identification of Merkel cells using light microscopy. Currently, there are two hypotheses for the origin of Merkel cells: one suggests that Merkel cells differentiate from epidermal keratinocyte-like cells and the other states that Merkel cells arise from stem cells of neural crest origin that migrated during embryogenesis3,4.\n\nIn this study, we observed Merkel cells located at the bottom of the basal adjacent stroma in the fornix and the palpebral conjunctiva and the cells could be stained with CK8, Math1, Ngn3 and beta-tubulin III. This suggests that the Merkel cells in the conjunctival might originate from the neural crest.\n\nThe ultrastructure of Merkel cells in the conjunctival stroma exhibits characteristics similar to those of other tissues, such as an electron-dense core with membrane-surrounded granules and intermediate filaments (Figure 5C). They have spine-like protrusions of variable lengths (Figure 5B)1,4,21, and some Merkel cells make contact with nerve terminals to form Merkel cell-neurite complexes (Figure 5D). The conjunctival Merkel cells, however, also have unique features, as they are distributed in the conjunctival stroma and were not associated with keratinocytes (Figure 5A, B). In addition, their granules can be visualized not only within the cells (Figure 5A) but also in extracellular regions (Figure 5B).\n\nThe technique of marking live Merkel cells with quinacrine has been employed to differentiate these cells from other epidermal cells in vitro and in vivo. However, the quinacrine dye incorporated by Merkel cells is rapidly lost, thus making the fluorescence very difficult to identify22–24. Another important reason that chloroquine is no longer used is due to the agent’s severe toxicity to the optic nerve. Fukuda et al.13 examined the absorption of FM dyes in Merkel cell cultures and found that FM dyes produce a specific fluorescence that remains sufficiently long to allow for clear identification of Merkel cells. The FM1-43 dye exhibited the best effects as compared with other FM dyes. Our results showed that an intraperitoneal injection of FM1-43 could effectively label the conjunctival stromal Merkel cells in vivo, which may be a useful technique for future studies of Merkel cells.\n\nThere is still some controversy regarding the function of Merkel cells. In vitro, neuropeptides secreted from Merkel cells (e.g., VIP and substance P) can promote cell growth and differentiation of keratinocytes and fibroblasts. These neurosecretory substances are released from the Merkel cells and act as neurotransmitters in the Merkel cell-axon complexes4. However, the exact function of Merkel cells in the conjunctiva stroma is still unclear. Thus, we established a corneal abrasion model to observe the response of these cells to stress. In this study, we demonstrated that wounding the corneal epithelium can induce nuclear lobulation, and shrinkage of cell-volume and an increase in the membrane-surrounded granules of Merkel cells (Figure 5E). These results indicate that Merkel cells in the conjunctival stroma might play an important physiological and pathological role in the stress response of corneal epithelium wounding. Further research work about the functions of the cells is ongoing in our laboratory.\n\n\nConclusions\n\nIn conclusion, our data showed that Merkel cells are mainly present in the conjunctival stroma, with the following findings: (1) The cells are distributed in the forniceal conjunctival stroma. (2) The Merkel cells express CK8, Math1, Ngn3 and β-tubulin III. (3) The ultrastructure of the Merkel cells contains an electron-dense core with membrane-surrounded granules, intermediate filaments and mitochondria. (4) Some Merkel cells make contact with nerve terminals to form Merkel cell-neurite complexes. (5) The cells exhibit endocytosis of FM1-43. (6) After corneal epithelial wounding, nuclear lobulation, shrinkage of cell-volume, and an increase in the membrane-surrounded granules of the Merkel cells was also observed. However, further studies are needed to verify the exactly physiological function and role of these cells in specific ocular surface diseases.",
"appendix": "Author contributions\n\n\n\nTF, YX, and CX performed histological analysis, data analysis, imaging and co-wrote the manuscript. TF, YX, YY, PL and JL conducted the animal experiments. WZ, JC, YL, and DC contributed data analysis. ZL designed the study, supervised experiments, analyzed data and wrote the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nZL is funded by National Natural Science Foundation of China grants 39970250, 30772387 and 81070703.\n\n\nReferences\n\nHalata Z, Grim M, Bauman KI: Friedrich Sigmund Merkel and his \"Merkel cell\", morphology, development, and physiology: review and new results. Anat Rec A Discov Mol Cell Evol Biol. 2003; 271(1): 225–39. PubMed Abstract | Publisher Full Text\n\nTachibana T, Nawa T: Recent progress in studies on Merkel cell biology. Anat Sci Int. 2002; 77(1): 26–33. PubMed Abstract | Publisher Full Text\n\nBoulais N, Misery L: Merkel cells. J Am Acad Dermatol. 2007; 57(1): 147–65. PubMed Abstract | Publisher Full Text\n\nMoll I, Roessler M, Brandner JM, et al.: Human Merkel cells--aspects of cell biology, distribution and functions. Eur J Cell Biol. 2005; 84(2–3): 259–71. PubMed Abstract | Publisher Full Text\n\nYin J, Wang H, Racey P, et al.: Distribution and ultrastructure of Merkel cell of the fishing bat (Myotis ricketti). Sci China C Life Sci./Chinese Academy of Sciences. 2009; 52(9): 802–6. PubMed Abstract | Publisher Full Text\n\nLucarz A, Brand G: Current considerations about Merkel cells. Eur J Cell Biol. 2007; 86(5): 243–51. PubMed Abstract | Publisher Full Text\n\nMaricich SM, Wellnitz SA, Nelson AM, et al.: Merkel cells are essential for light-touch responses. Science. 2009; 324(5934): 1580–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagasaki T, Zhao J: Uniform distribution of epithelial stem cells in the bulbar conjunctiva. Invest Ophthalmol Vis Sci. 2005; 46(1): 126–32. PubMed Abstract | Publisher Full Text\n\nBoulais N, Pennec JP, Lebonvallet N, et al.: Rat Merkel cells are mechanoreceptors and osmoreceptors. PLoS One. 2009; 4(11): e7759. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi Z, Burns AR, Miller SB, et al.: CCL20, gammadelta T cells, and IL-22 in corneal epithelial healing. FASEB J. : official publication of the Federation of American Societies for Experimental Biology. 2011; 25(8): 2659–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu Q, Smith CW, Zhang W, et al.: NK cells modulate the inflammatory response to corneal epithelial abrasion and thereby support wound healing. Am J Pathol. 2012; 181(2): 452–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi Z, Burns AR, Han L, et al.: IL-17 and VEGF are necessary for efficient corneal nerve regeneration. Am J Pathol. 2011; 178(3): 1106–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFukuda J, Ishimine H, Masaki Y: Long-term staining of live Merkel cells with FM dyes. Cell Tissue Res. 2003; 311(3): 325–32. PubMed Abstract | Publisher Full Text\n\nZhu WQ, Xu JJ, Sun XH, et al.: [Normal human bulbar conjunctiva on confocal microscopy in vivo]. Zhonghua yan ke za zhi. Chinese journal of ophthalmology. 2009; 45(4): 344–9. PubMed Abstract\n\nMoll R, Moll I, Wiest W: Changes in the pattern of cytokeratin polypeptides in epidermis and hair follicles during skin development in human fetuses. Differentiation; research in biological diversity. 1982; 23(2): 170–8. PubMed Abstract | Publisher Full Text\n\nMoll R, Moll I, Franke WW: Identification of Merkel cells in human skin by specific cytokeratin antibodies: changes of cell density and distribution in fetal and adult plantar epidermis. Differentiation; research in biological diversity. 1984; 28(2): 136–54. PubMed Abstract | Publisher Full Text\n\nSaurat JH, Didierjean L, Skalli O, et al.: The intermediate filament proteins of rabbit normal epidermal Merkel cells are cytokeratins. J Invest Dermatol. 1984; 83(6): 431–5. PubMed Abstract | Publisher Full Text\n\nHartschuh W, Weihe E, Yanaihara N: Immunohistochemical analysis of chromogranin A and multiple peptides in the mammalian Merkel cell: further evidence for its paraneuronal function? Arch Histol Cytol. 1989; 52(Suppl): 423–31. PubMed Abstract | Publisher Full Text\n\nKatayama I, Nishioka K: Substance P augments fibrogenic cytokine-induced fibroblast proliferation: possible involvement of neuropeptide in tissue fibrosis. J Dermatol Sci. 1997; 15(3): 201–6. PubMed Abstract | Publisher Full Text\n\nKvetnoy IM, Reiter RJ, Khavinson VK: Letter to the Editor. Claude Bernard was right: hormones may be produced by \"non-endocrine\" cells. Neuro Endocrinol Lett. 2000; 21(3): 173–4. PubMed Abstract\n\nWinkelmann RK, Breathnach AS: The Merkel cell. J Invest Dermatol. 1973; 60(1): 2–15. PubMed Abstract\n\nNurse CA, Mearow KM, Holmes M, et al.: Merkel cell distribution in the epidermis as determined by quinacrine fluorescence. Cell Tissue Res. 1983; 228(3): 511–24. PubMed Abstract | Publisher Full Text\n\nYamashita Y, Akaike N, Wakamori M, et al.: Voltage-dependent currents in isolated single Merkel cells of rats. J Physiol. 1992; 450: 143–62. PubMed Abstract | Free Full Text\n\nYamashita Y, Toida K, Ogawa H: Observation of Merkel cells with scanning electron microscopy. Neurosci Lett. 1993; 159(1–2): 155–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3465",
"date": "07 Mar 2014",
"name": "Ingrid Moll",
"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 paper deals with Merkel-like cells in the murine conjunctival stroma, claimed to be identified by immunofluorescence microscopy, incorporating TM1-43 dye and by electron microscopy. I have various points of criticism and doubts about the nature of the identified cells:Fig. 1:C(Keratin) 8 immunofluorescence staining is stated to have been located in the membrane, nucleus and cytoplasm of the specific cells. However, CK 8 in murine Merkel cells is restricted to the very typical cytoskeleton within the cytoplasm. Moreover, CK 8 can be found in many organs not restricted to Merkel cells and therefore the more specific staining for CK 20 should be used to identify Merkel cells in general. Fig. 2:The TROMA-1 antibody staining remains unclear, most probably the nucleus is stained (see DAPI), but TROMA-1 should again, decorate the cytoskeleton. Fig. 3:The TROMA-1-antibody decorates a cell layer and this cell layer is not decorated by the Ngn3 antibody. Again, cytokeratin 20 should be used to identify Merkel cells. Fig. 4:The same is true for this figure (see Fig. 3). Electron microscopy: the particles stated as being neuroendocrine granules are up to 1µm/ in diameter. However, the neuroendocrine granules of Merkel cells show a diameter 80–120nm and a typical dense cored structure. Therefore, the particles shown could not represent dense cored neuroendocrine granules and thus the cells are not Merkel cells.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-251
|
https://f1000research.com/articles/2-250/v1
|
19 Nov 13
|
{
"type": "Case Report",
"title": "Traditional medicine: a rare cause of lead poisoning in Western countries",
"authors": [
"Halima Muller",
"Simon Regard",
"Nicole Petriccioli",
"Omar Kherad",
"Halima Muller",
"Simon Regard",
"Nicole Petriccioli"
],
"abstract": "A 42-year-old man from Bhutan was admitted to the emergency department with a 5-day history of abdominal pain, nausea and vomiting. Enhanced abdominal CT scan was found negative, however laboratory tests showed hemolytic anemia and basophilic stippling which are often seen in lead and heavy metal poisoning. Additional tests revealed a high level of lead in blood and urine. The patient was administered a chelator treatment with rapid improvement of the symptoms. A detailed interview revealed that the patient had been taking daily Bhutanese traditional medicines to treat a Bell’s palsy from which he had been suffering for a few months. The analysis of these medicines confirmed the presence of a high level of lead.",
"keywords": [
"Lead poisoning is a multisystemic organ disease which can present with non-specific symptoms such as fatigue",
"anorexia",
"arthralgia",
"myalgia",
"neurological disorders",
"anemia abdominal pain and encephalopathy."
],
"content": "Introduction\n\nLead poisoning is a multisystemic organ disease which can present with non-specific symptoms such as fatigue, anorexia, arthralgia, myalgia, neurological disorders, anemia abdominal pain and encephalopathy.\n\nBasophilic stippling, which refers to the presence of blue granules of various sizes dispersed throughout the cytoplasm of the red blood cell may be seen in blood smears. Burton line, and nephropathy (Fanconi-type syndrome), are also described1. The diagnosis of lead poisoning is based on clinician consideration to assess blood lead level and erythrocitic protoporphyrins. The treatment consists of the elimination of the source of exposure and a chelation therapy with DMSA (dimercaptosuccinic acid) or CaEDTA2,3. Timely diagnosis and identification of the source of exposure are critical in preventing the long-term consequences of lead poisoning.\n\nLead exposure and poisoning is still a major health issue in developing countries4. It was estimated to be responsible for 8,955,000 disability-adjusted life years (DALYs) in 2004, i.e. 0.6% of all DALYs5. Lead poisoning is most commonly caused by occupational exposure (especially in adults)6. Other sources of lead poisoning include exposure to paint (especially in children), water and soil (especially in urban areas), food (including game) and toys (especially in children where toys may have lead-based paints), as well as complementary and alternative medicines (CAM). As a consequence of globalization, lead poisoning cases caused by CAM are increasingly reported in developed countries7.\n\n\nCase presentation\n\nA 42-year-old man from Bhutan with a history of hypertension was admitted on 26th October 2011 to the emergency department of a secondary hospital in Geneva, Switzerland, complaining of epigastric pain with nausea and vomiting over the previous 5 days. A gastroscopy showed esophagitis and the patient was discharged with a proton pump inhibitor treatment. He consulted two days later complaining of persistent symptoms. Examination revealed pain on palpation of the right upper abdominal quadrant with no sign of peritonism.\n\nThe patient resides in Geneva and frequently travels to his home country. His occupation involves no risk of lead exposure.\n\nBlood tests revealed a cholestatic liver abnormality and a hemolytic anemia with the hemoglobin level at 90 g/l. An abdominal ultrasound and a thoraco-abdominal CT scan proved normal. Serologies for HIV, cytomegalovirus, Epstein-Barr virus and hepatitis A, B, C were negative. A blood smear was performed and showed basophilic stippling that was highly evocative of a heavy metal poisoning. Blood and urinary lead levels were high at 80.8 mcg/dl and 208.8 mcg/g of creatinine, respectively. An increase in the urinary coproporphyrin III level up to 155.9 nmol/mmol (n<150nmol/mmol) was also noted.\n\nThe origin of the intoxication was discovered to be due to the patient taking Bhutanese traditional medicines to treat a resolutive Bell’s palsy a few months earlier. These medicines were composed of parchment with ink writing and pellets (Figure 1). The patient thought the drug comprised hair of a deceased local priest with therapeutic virtues. The quantitative analysis performed at the University Centre of Legal Medicine (CURML) showed the presence of high level of lead on the red paint surrounding the pellets (1.4 mg in each pellet) and a negligible level in the parchment with ink writing (<1ug).\n\nThe patient was administered an oral chelation therapy with DMSA (dimercaptosuccinic acid) 10 mg/kg 3 times daily for 5 days, and then 10 mg/kg 2 times daily for 14 days.\n\nThe symptoms rapidly improved and the patient was discharged 5 days later from hospital with outpatient control by his general physician. He was advised to stop taking the poisonous medicines. The case was reported to the Bhutanese health authorities for them to address the issue locally and implement an appropriate health care response.\n\n\nDiscussion\n\nComplementary and alternative medicines have become more frequent causes of lead poisoning in the past few decades. In 2007, a review article7 found 76 case reports dating from 1966 to 2007 that involved traditional medicines from the Middle East, South America and India (Ayurveda). It is estimated that at least 15% of ingested lead is absorbed7. Three main reasons explain the presence of lead in CAM medicines: involuntary contamination (e.g. soil where CAM plants grow), voluntary use of lead for therapeutic purposes and voluntary use of lead to increase the weight of medicines (the heavier the better)7.\n\nAs a consequence of general globalization in medicine, CAM medicines are being increasingly used in Western countries as they become more popular and easily available on the internet8,9,10.\n\nCAM medicines play a very important though underrated role in developing countries where they are more accessible and affordable than pharmaceutical drugs and where they are consequently estimated to be used by between 66–80% of the population11. It must be noted that CAM encompass a broad range of practices with various degrees of popular recognition. In our case, it appeared that the ingested balls were not part of any of the officially recognized practices included in the Bhutanese traditional medicine known as gSo-Ba Rig-Pa12, hence making resolution of the issue more complex. Patients often do not overtly disclose their consumption of CAM drugs for fear of lack of understanding by their doctor13. As a consequence the use and misuse of CAM medicines is certainly underreported.\n\nDespite the body of literature on this topic, lead poisoning through CAM consumption remains a public health problem. Enhancing public awareness about the potential risks of many CAM approaches including exposure to harmful contaminants is a major objective of preventative health care. Meanwhile, it remains the responsibility of the physician to obtain a detailed history of medication use, including in developed countries where lead exposure and poisoning can also be reported.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nHM, SR, NP and OK were involved in the overall study design and protocol development. HM, SR and OK participated in writing the manuscript, which all authors have reviewed and approved.\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\nFlora G, Gupta D, Tiwari A: Toxicity of lead: A review with recent updates. Interdiscip Toxicol. 2012; 5(2): 47–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBradberry S, Vale A: A comparison of sodium calcium edetate (edetate calcium disodium) and succimer (DMSA) in the treatment of inorganic lead poisoning. Clin Toxicol (Phila). 2009; 47(9): 841–58. PubMed Abstract | Publisher Full Text\n\nBradberry S, Vale A: Dimercaptosuccinic acid (succimer; DMSA) in inorganic lead poisoning. Clin Toxicol (Phila). 2009; 47(7): 617–31. PubMed Abstract | Publisher Full Text\n\nPokras MA, Kneeland MR: Lead poisoning: using transdisciplinary approaches to solve an ancient problem. EcoHealth. 2008; 5(3): 379–85. PubMed Abstract | Publisher Full Text\n\nGlobal Health Observatory Data Repository. World Health Organization. 2004. Reference Source\n\nNeedleman H: Lead poisoning. Annu Rev Med. 2004; 55: 209–22. PubMed Abstract | Publisher Full Text\n\nKarri SK, Saper RB, Kales SN: Lead Encephalopathy Due to Traditional Medicines. Curr Drug Saf. 2008; 3(1): 54–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarnes PM, Bloom B, Nahin RL: Complementary and alternative medicine use among adults and children: United States, 2007. Natl Health Stat Report. 2008; (12): 1–23. PubMed Abstract\n\nErnst E: Prevalence of use of complementary/alternative medicine: a systematic review. Bull World Health Organ. 2000;78(2): 252–257. PubMed Abstract | Free Full Text\n\nEisenberg DM, Davis RB, Ettner SL, et al.: Trends in alternative medicine use in the United States, 1990–1997: results of a follow-up national survey. JAMA. 1998; 280(18): 1569–1575. PubMed Abstract | Publisher Full Text\n\nTraditional medicine Factsheet. 2008; (12.01.2012). Reference Source\n\nWangchuk P, Wangchuk D, Aagaard-Hansen J: Traditional Bhutanese medicine (gSo-BA Rig-PA): an integrated part of the formal health care services. Southeast Asian J Trop Med Public Health. 2007; 38(1): 161–7. PubMed Abstract\n\nEisenberg DM, Kessler RC, Van Rompay MI, et al.: Perceptions about complementary therapies relative to conventional therapies among adults who use both: results from a national survey. Ann Intern Med. 2001; 135(5): 344–51. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2492",
"date": "25 Nov 2013",
"name": "Arnaud Perrier",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 interesting case of lead poisoning by a Bhutanese alternative medicine treatment. The interest resides in the frequency of complementary and alternative medicine (CAM) as a source of accidental lead poisoning, a fact most physicians are not aware of. Also, the conclusion that CAM medications should be part of treatment history-taking is important and sound advice.Minor points to be improved:The reference interval for blood and urine lead level should be provided.It would be interesting to know how the follow-up of such a case should be performed. At what intervals should lead measurements be repeated?",
"responses": []
},
{
"id": "2494",
"date": "28 Nov 2013",
"name": "Bruno Mégarbane",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 interesting case of lead poisoning following the administration of traditional remedies. This is a rather well-known situation, with several published cases, mainly occurring in the developing countries where traditional medicines are widely used. The case is well-written and discussed.I have only minor comments:Severe lead poisoning is responsible for abdominal pain which may be associated with nausea and/or vomiting but is generally preceded and associated with constipation. Was this patient constipated?Hemolysis and cytolytic (rather than cholestatic) hepatitis are manifestations of subacute (or acute) lead poisoning; could the authors comment on their finding?The clinical (and biological) course of this lead poisoning case could be better described, regarding the onset of renal complications, course of hemolysis and hepatitis, as well as blood lead concentration one or two weeks after chelation.Arthralgia and myalgia are rare features of lead poisoning, as written in the introduction section. The main implications of lead poisoning are neurological (encephalopathy and peripheral neuropathy), hematological and renal (tubular nephropathy, then tubulo-intersitial and glomerular nephropathy).In adults, digestive absorption of lead is poorer (5-10 %) than indicated in the discussion section.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-250
|
https://f1000research.com/articles/2-139/v1
|
11 Jun 13
|
{
"type": "Research Article",
"title": "Using computation to enhance diagnosis and therapy: a novel mutation operator for real-coded adaptive genetic algorithms",
"authors": [
"Maxinder S Kanwal",
"Avinash S Ramesh",
"Lauren A Huang",
"Avinash S Ramesh",
"Lauren A Huang"
],
"abstract": "The fields of molecular biology and neurobiology have advanced rapidly over the last two decades. These advances have resulted in the development of large proteomic and genetic databases that need to be searched for the prediction, early detection and treatment of neuropathologies and other genetic disorders. This need, in turn, has pushed the development of novel computational algorithms that are critical for searching genetic databases. One successful approach has been to use artificial intelligence and pattern recognition algorithms, such as neural networks and optimization algorithms (e.g. genetic algorithms). The focus of this paper is on optimizing the design of genetic algorithms by using an adaptive mutation rate based on the fitness function of passing generations. We propose a novel pseudo-derivative based mutation rate operator designed to allow a genetic algorithm to escape local optima and successfully continue to the global optimum. Once proven successful, this algorithm can be implemented to solve real problems in neurology and bioinformatics. As a first step towards this goal, we tested our algorithm on two 3-dimensional surfaces with multiple local optima, but only one global optimum, as well as on the N-queens problem, an applied problem in which the function that maps the curve is implicit. For all tests, the adaptive mutation rate allowed the genetic algorithm to find the global optimal solution, performing significantly better than other search methods, including genetic algorithms that implement fixed mutation rates.",
"keywords": [
"genetic algorithms",
"mutation rate",
"artificial intelligence",
"bioinformatics",
"genomics"
],
"content": "Introduction\n\nThe last few years have seen an exponential increase in the size of genomic databases cataloging the genetic basis of various diseases. Computing power has not kept pace with this rapid increase in bioinformatics. Therefore, it has become critical to develop clever algorithms to reduce the time needed to search these databases and arrive at solutions to the treatment of genetically determined diseases1. Genetic algorithms provide new hope to overcome this dilemma. Genetic algorithms attempt to copy the principle of “survival of the fittest”, using Darwin’s theory of evolution to find a satisfactory solution. In essence, a group of random solutions is created and ranked, after which the best solutions are allowed to “interbreed”. During interbreeding, small mutations are allowed to add an element of randomness, and in this way aid the genetic algorithm in finding the optimal solution2.\n\nThe applications of genetic algorithms (GAs) are well known and far ranging3. Because of their versatility, GAs have been widely and successfully used to optimize practical problems such as determining a long sequence of base pairs in a genetic database, scheduling drilling operations in a circuit-board factory, and data mining, among others4. However, one major problem faced by GAs is premature convergence, in which the algorithm becomes trapped in a local optimum and is unable to find the global optimum5.\n\nThis study focuses on the use of mutations in GAs. Normally, the mutations occur at a constant rate, known as the mutation rate. However, use of a fixed mutation rate can produce sub-optimal solutions5. We propose a new, variable mutation rate that uses a pseudo-derivative to take into account the time that a GA can be stuck at a certain point. The longer the algorithm has been stuck at a local optimum, the more likely it is that a mutation will occur. This addition of a greater element of randomness to the algorithm allows it to move from the local optimum and look for better solutions. In this paper, we show that the proposed variable mutation rate outperforms fixed mutation rates and other common search algorithms in the effectiveness of their solutions.\n\n\nBackground\n\nMany real-life problems can be modeled as continuous, nonlinear optimization problems. Within a given search space S on the optimization function f, a global (absolute) optimum is sought. This may take the form of a global maximum or minimum, depending on the original problem. A typical 3-dimensional global optimization problem follows the form:\n\n\n\nThe challenge in solving a global optimization problem is in seeking the global optimum rather than becoming trapped in a local optimum, an issue that will be addressed in more detail later6. These optimization problems can be approached with a variety of techniques. One popular technique is the use of GAs, the focus of this study7.\n\nGAs are population-based optimization techniques favored for their properties of self-learning, self-organization, self-adaptation, and implicit parallelism8. Based on the principles of Charles Darwin’s natural selection and meiotic cell division, GAs involve several components: a population, a measure of fitness, and a method of breeding2. The population forms the basis for the GA and is made up of many individuals, often called chromosomes. Over time, “chromosomes” breed with other “chromosomes” to form “children” that make up a new generation in the GA9.\n\nChromosomes can be made up of binary strings or of real values. Binary-coded GAs (BCGAs) have chromosomes of 0s and 1s. While BCGAs are the more traditional method and are adequate for small- to moderate-size optimization problems, they fail for high-dimensional problems because they require more computational time and memory. Real-coded GAs (RCGAs) utilize real values that allow for both improved computational time and memory as compared to BCGAs, making the optimization of multi-dimensional and high-precision problems more feasible5. Each chromosome (binary-coded or real-coded) has a certain fitness value or f value derived from its binary string or its real values, where the f value of a chromosome refers to its value when inputted into the function described in (1). The calculations for the f value of a chromosome vary by problem. In this respect, each chromosome represents a single solution to the optimization problem10. Following the principles of natural selection, chromosomes with higher fitness values give rise to children with high fitness values, so the GA “selects” for fitter chromosomes by giving them a higher probability of breeding and passing on their genes11.\n\nThe breeding stage involves two processes: crossover and mutation. In true meiotic cell division (the process by which a sperm or an egg cell is created), a crossover occurs when two chromosomes pair with each other and exchange portions of their length, forming two hybrid chromosomes. Similarly, chromosomes in a GA exchange values12. A simple example of crossover is illustrated in Figure 1.\n\nAn example of crossover between a set of two binary-coded chromosomes where half the length of chromosome A is exchanged for half the length of chromosome B, resulting in a hybrid set of chromosomes.\n\nThe second operator of the breeding stage is mutation. Mutation is a naturally-occurring phenomenon that may come into play during any replication of chromosomes. Incorporation of mutation into a GA may be considered an optional step, but has generally been found to increase the diversity of the population13. This allows GAs to explore promising areas of the search space8. The major advantage of implementing mutations in a GA is in avoiding premature convergence, in which the GA becomes trapped in a local optimum14. However, the mutation operator has trade-offs in slowing down the learning process5. Mutations have in the past taken the form of point, frame-shift, and translocation mutations, all of which involve swapping, switching, or shifting binary bits, in the case of binary-coded chromosomes15. Random mutation has also been proposed, in which a gene is replaced with a random value within certain lower and upper bounds16, and may provide interesting insight into the mechanisms of mutation, since a small mutation in nature may cause a gene to take on a vastly different role17. Finally, the rate of mutation, rm, can play a key role in the effectiveness of a given GA7.\n\nA variety of modifications have been made to GAs in the breeding stage. A large amount of attention has been devoted to developing improvements in crossover operators, but there have been fewer studies in improving mutation operators, which make up a very promising although largely unexplored branch5,15. Several studies have implemented adaptive mutation, or a changing mutation rate18,19. Adaptive mutation methods have ranged from individual gene-based mutations to Gaussian mutation operators based on the mean and the standard deviation of the Gaussian distribution20. This study proposes a novel adaptive mutation approach and applies it to two tests: the well-known N-queens problem and the maximization of a 3-dimensional function. This study also compares a GA implementing an adaptive mutation operator with other well-known search algorithms, such as the Nelder-Mead method, hill climbing technique, and random search.\n\nNumerous other search algorithms besides GAs exist for solving similar optimization problems21. A basic method is the random search method whereby solutions are randomly chosen and evaluated for a certain amount of time, and the best solution found during the time span is returned22. A more advanced method implements the hill climbing technique. This method begins at a random point and uses a greedy algorithm to move to the best immediate neighboring solution until no neighboring solution is better than the current solution23. The final, and most robust, non-GA search algorithm being examined in this study is the Nelder-Mead algorithm. The Nelder-Mead method is a heuristic search that relies on approximating a local optimum for a problem with N variables by using a simplex (a polytope consisting of N + 1 vertices)24.\n\nThe N-queens problem is an interesting application for testing the accuracy of genetic algorithms due to the inherent difficulty of optimizing the problem under efficient time and memory constraints25. Given an N × N board, find a set of N queens such that zero pairs of queens are in the same row, column, or diagonal (none of the queens are attacking each other). Although there are several approaches (brute force/recursion, simulated annealing, etc.) to solving this optimal placement problem26, the N-queens problem was chosen in this study as an early practical test of the proposed new mutation rate operator’s accuracy.\n\nFinding the maximum or minimum value of a 3-dimensional surface is a more abstract but more visual problem. A surface with multiple local maxima and minima can simultaneously test a GA’s ability to avoid premature convergence and its ability to handle multidimensional optimization problems. Because GAs solve problems with implicit functions of N-dimensions, it is important that the newly proposed mutation operator can deliver reasonable speed and accuracy in the most primal form of the N-dimensional problem, optimizing a space function. While the N-queens problem is adequate for testing the accuracy of a GA, it is not adequate for testing its speed of convergence because there are multiple solutions. Therefore, maximization of a 3-dimensional surface with multiple local maxima but only one absolute maximum was chosen as a practical test of the new mutation operator’s speed of convergence, or number of generations needed to converge to the optimal solution. Furthermore, minimization of the Rastrigin function was chosen as a simple method for comparing accuracies of various search algorithms. The Rastrigin function is an optimal function to choose for such a comparison as the surface holds a myriad of local minima, but only one global minimum at f (0,0) = 0. The Rastrigin function models a difficult problem to optimize, where a non-robust search algorithm may easily become caught in a local optimum27.\n\n\nMethods\n\nThe research question this study attempted to answer was: what are the effects of an adaptive mutation rate, based on the derivative of the fitness function with respect to generations, on the efficiency and accuracy of a GA? We performed three experiments to answer this question. The first experiment in this study tested the null hypothesis that given a problem, which is not always solvable (to the global optimum) by a fixed-rate GA, a GA with an adaptive mutation rate (based on the derivative of the fitness function with respect to generations) would find an optimal solution significantly more often than one with a constant mutation rate. To reject the null hypothesis, we will need to show that an adaptive mutation rate can find an optimal solution significantly more often than one with a fixed mutation rate. The second experiment tested the null hypothesis that given a problem, which is always solvable (to the global optimum) by a constant mutation rate GA, there would be no significant difference in the efficiency between the constant mutation rate GA and a GA with an adaptive mutation rate, based on the derivative of the fitness function with respect to generation. The third experiment tested the null hypothesis that a GA with an adaptive mutation rate is not significantly more accurate in converging to a global optimum of a 3-dimensional function as compared to other search algorithms (i.e. Nelder-Mead, hill climbing, random).\n\nThe mutation rate function rm was the independent variable in the first study. Accuracy (how often a GA finds the optimum solution) measured in percent (%) with an error value of ±0.001, and efficiency (how long it takes to converge to the solution), measured in generations, were the dependent variables. The experiment was set up with three levels: a constant rm of 0% (no mutation, control), a constant rm of 20% (control), and an adaptive rm derived from a pseudo-derivative and a sigmoid function. The independent variable of the second study was the specific search algorithm being run. The accuracy, measured in percent (%) with an error value of ±0.001, was the dependent variable. The experiment was set up with four levels (search algorithms): a random search, a search implementing the hill climbing technique, a search implementing the Nelder-Mead method, and a search implementing the proposed adaptive GA.\n\nThe adaptive mutation rate operator was tested on three problems: the N-queens problem, the maximization of a 3-dimensional surface, and the minimization of a different 3-dimensional surface (the Rastrigin function). The N-queens problem was used specifically to test the accuracy of the new adaptive mutation rate since a GA implementing a constant mutation rate does not have 100% accuracy in the N-queens problem. The maximization of a 3-dimensional surface problem was used to test the efficiency of the new adaptive mutation rate since a GA implementing the 20% constant mutation rate does have 100% accuracy in the 3-dimensional maximization problem. The minimization of the Rastrigin function was used to determine the robustness of the proposed adaptive GA compared to other well-known search algorithms. The Rastrigin function was selected due to a most diverse topography, including a myriad of local optima by which weak search algorithms may be forced to prematurely converge. In the first study, accuracy was defined to be the rate of successful optimization within 100,000 generations to the nearest ±0.001, in order to save computing power. Python 2.5 was used to create and run GAs for the N-queens and 3-dimensional maximization problems. Python 3.0 was used to create and run an adaptive GA, Nelder-Mead, hill climbing, and random search algorithms for the Rastrigin function minimization problem. A vector class was created to aid in simplifying the code for the latter experiment. Both GAs reported mutation rate, convergence, and best f values for later analysis. For both problems, the mutation rate operator function was a sigmoid function fit to the domain x in [0, ∞) and range y in [0, 1]. The sigmoid function is defined as:\n\n\n\nwith domain x in (-∞, ∞), range y in [0, 1], and sigmoid(0) = 0.5. The function used was thus a fit of the monotonic sigmoid function to the desired domain and range, resulting in\n\n\n\nwhere x is given by:\n\n\n\nwith gc representing the current generation and go representing the oldest generation of the same best f value. x thus is inversely related to the derivative of the fitness function with respect to generation. This causes an increase in mutation rate when the fitness between generations is stagnant. A theoretical basis and further details of the adaptive mutation rate operator is included in the Theory section under the Discussion.\n\nFor the N-queens problem, the specific case of N = 8 was chosen. The fitness function was chosen for this problem to be the number of pairs of queens violating the problem specification. A program was written to run 200 trials of GAs maxed at 100,000 generations or until a solution was found (f = 0). The genetic code for each solution in the solution set was a sequence of eight integers, with each index representing a column and each value representing a row. Crossover locations were determined using random integers. All variables were held constant apart from mutation rates, which varied between constant mutation at 0% (no mutation), constant mutation rate of 20%, and the sigmoidally-determined adaptive mutation. For the full code, see Script 1 in the Supplementary materials.\n\nRegarding the maximization of a 3-dimensional function, the proposed sigmoidal mutation operator was again compared to controls of a constant mutation rate of 20% and of no mutation (0%). The function chosen for maximization was\n\n\n\nwhich represents a function with several local maxima and minima, which can be solved by the 20% mutation rate GA but not the 0% mutation rate GA. This allowed for a test of efficiency of the new sigmoidally-determined adaptation mutation rate GA against the 20% fixed mutation rate GA and a test of accuracy for the sigmoidally-determined adaptation mutation rate GA against the 0% fixed mutation rate (no mutation) GA. A graphical representation of the function is shown as Figure 2. The genetic code for each solution in the solution set for this problem was a sequence of two integers, the x and y coordinates. The chromosomes were implemented as real-coded chromosomes containing the actual coordinates rather than binary strings. For crossover, the x coordinate of one solution was chosen and the y coordinate of another solution was chosen. For mutation, a coordinate was replaced by a random number within the domain of the problem. Once again, all variables, except for mutation rates, were held constant throughout each experiment.\n\nVisualized in MATLAB 6.5 Using the Command peaks(40). The three maximums of the surface were located at f = 8.1165 (global maximum), 3.5507, and 3.4652.\n\nThe minimization of the Rastrigin function, given by:\n\n\n\nwas the final test run, with a goal to learn how the proposed adaptive GA compares to non-GA search algorithms. All of the search algorithms were coded to report the run time to converge and the result of the respective convergence. In order for a result to be considered accurate, the returned answer had to be within 0.001 of the true answer. This ensures that the algorithm is converging to the correct minimum and also aids in simulating a difficult optimization problem. Since the random method does not converge, but rather runs for a given time span, the average run time for each GA trial was obtained and used for the run time of the random method search. This allows for direct comparison of the accuracies of the random method and the adaptive GA, as the run time for each method was identical, and thus, held constant. Each algorithm was run for 1,000 trials in order to obtain enough data for a robust analysis. For the full code, see Scripts 2, 3, and 4 in the Supplementary materials. A graphical representation of the Rastrigin function can be found as Figure 3 and Figure 4.\n\nVisualized in MATLAB 6.5: f = @(x,y) 10*2 + x.^2 + y.^2 – 10*cos(2*pi*x) – 10*cos(2*pi*y). The global minimum of the Rastrigin function is f(0,0) = 0.\n\nVisualized in Mathematica 8: ContourPlot[10*2 + x^2 + y^2 – 10 Cos[2 Pi x] – 10 Cos[2 Pi y], {x, -5.12, 5.12}, {y, -5.12, 5.12}, ImageSize -> 1000, Axes -> False, ColorFunction -> ColorData[\"TemperatureMap\"]].\n\n\nResults\n\nThe results of experimentation are summarized in Table 1, Table 2, Table 5 and Table 7. In the N-queens problem, the proposed sigmoid mutation operator displayed both the fastest convergence and greatest accuracy with convergence after an average of 25,455.4 generations and an accuracy of 95.5%. In the 3-dimensional surface maximization problem, there was no significant difference between the speeds of the successful GAs. Accuracies were tied between 20% constant and adaptive mutation, as both techniques consistently identified the global maximum within the 100,000-generation limit. In the Rastrigin function minimization problem, the proposed sigmoid mutation operator GA performed significantly better than the three other search algorithms in terms of accurately converging to the global minimum. See Figure 5–Figure 7 for a summary of the results and significant differences.\n\nA. Graph of the N-queens problem accuracies among 0% constant mutation, 20% constant mutation, and adaptive mutation. ***p < 0.001. B. Graph of the N-queens problem convergences among 0% constant mutation, 20% constant mutation, and adaptive mutation. ***p < 0.001. mutation (mut.), adaptive (adapt.)\n\nA. This is a graph of the 3D surface maximization problem accuracies among 0% constant mutation, 20% constant mutation, and adaptive mutation. B. This is a graph of the 3D surface maximization problem convergences among 20% constant mutation and adaptive mutation. 0% constant mutation was not statistically analyzed because convergence was not achieved. ***p < 0.001. mutation (mut.), adaptive (adapt.)\n\nGraph of the Rastrigin function minimization problem accuracies among random, Hill climbing, Nelder-Mead, and adaptive GA methods. ***p < 0.001. genetic algorithm (GA).\n\nIn the N-queens problem, a series of unpaired, single-tailed Student’s t-tests were used to test the following null hypothesis at the 0.05 level of significance: the accuracy of the adaptive mutation operator will not be significantly higher than that of 20% constant mutation or that of 0% constant mutation. The null hypothesis was rejected for the 20% constant mutation rate (p = 6.108 × 10-25 < 0.05) and the 0% constant mutation rate (p = 2.572 × 10-214 < 0.05). See Table 3 for a summary of the N-queens statistics.\n\nIn the surface maximization problem, a series of unpaired, single-tailed Student’s t-tests were used to test the following null hypothesis at the 0.05 level of significance: the efficiency of the 20% constant mutation rate GA will be significantly higher than that of the adaptive mutation operator GA. The null hypothesis was rejected (p = 0.448 > 0.05) for the 20% level. Because the constant 0% mutation rate GA does not find the global maximum, it was compared to the adaptive mutation operator on the basis of null hypothesis 1 at the 0.05 level of significance: the accuracy of the adaptive mutation operator will not be significantly higher than that of 0% constant mutation rate. The null hypothesis was rejected (p = 0 < 0.05). See Table 4 for a summary of the surface maximization statistics.\n\nIn the Rastrigin function minimization problem, a series of unpaired, single-tailed Student’s t-tests were used to test the following null hypothesis at the 0.05 level of significance: the accuracy of the adaptive mutation operator GA will not be significantly higher than those of the other search methods (Nelder-Mead, hill climbing, random). The null hypothesis was rejected for random method (p = 2.53 × 10-223 < 0.05), for the hill climbing method (p = 9.61 × 10-268 < 0.05), and for the Nelder-Mead method (p = 3.04 × 10-152 < 0.05). Furthermore, a series of unpaired, single-tailed Student’s t-tests were used to test if there were significant differences in the run times of the hill climbing and Nelder-Mead methods when the algorithm accurately converged and when the same algorithm did not accurately converge. Both the hill climbing method (p = 0.140 > 0.05) and the Nelder-Mead method (p = 0.576 > 0.05) showed no significant differences in run time when accurately converging compared to inaccurately converging. These results point to the possibility that these two methods were only accurate when a fortunate random point was selected to start running the search algorithm from. No extra time being needed to converge correctly versus incorrectly, points towards the idea that no extra computation was needed either. See Table 6 and Table 7 for a summary of the Rastrigin function minimization statistics.\n\n\nDiscussion\n\nThe mutation rate operator presented in this study uses randomness to guide a genetic algorithm (GA) towards the optimal solution. A GA with less randomness leads to faster convergence towards local optimums; however, by limiting randomness it limits the search space, which in turns hinders the search for the global optimum. Conversely, a GA with more randomness hinders progress towards local optimums, but allows for a wider search space, aiding the search for the global optimum8,28. Therefore, randomness should be inversely related to the derivative of the fitness function f. Because f is implicit in most problems, the proposed heuristic attempted to look at the derivative of f with respect to generation, creating a pseudo, inversely related function to the derivative that could be used to calculate an adaptive mutation rate. The heuristic subtracted the first generation at which the most optimal fitness value appeared from the current generation to obtain a result inversely related to the derivative, and thus directly related to the randomness or mutation rate. Therefore any monotonically increasing function with domain bounded by x in [0, ∞) and y in [0, 1] should provide an appropriate amount of randomness to either find a local optimum or increase the search space from the local optima in order to find the global optimum.\n\nThe implications of a new, pseudo-derivative-based adaptive mutation rate are considerable. This new genetic algorithm technique could allow for increased accuracy in optimization of all GA problems, such as discovering novel therapeutic drug combinations for complex diseases, hence limiting the risk and expense of clinical trials29. The results found in this study agree with results found in other articles regarding adaptive mutation accuracy and efficiency5,30; this method also has the added benefit of low computational complexity while still being able to guide the GA out of local optimums and towards the global optimum. Studies in adaptive mutation have ranged widely from individual gene-based mutations, to Gaussian operators, to polynomial operators20. However, to the knowledge of the authors, past adaptive mutation techniques have never used the derivative of f with respect to generation to determine a changing mutation rate. This new method of implementing adaptive mutation may open up entirely new areas in which improvement can be made to GAs.\n\n\nConclusion\n\nGAs are powerful tools that can optimize overwhelmingly complex real-life problems, including speedy diagnoses of complex diseases. Previous GAs faced a trade-off between speed and accuracy, as more random GAs sacrificed speed in exchange for a better chance of optimization or less random ones gave up accuracy for speed. The solution of an adaptive mutation operator based on the derivative of f with respect to generation allows for increased accuracy without the loss of speed. Suggestions for research that would expand upon current findings include determining an optimal monotonically increasing function for the mutation, such as a sigmoid, inverse tangent, or scaled linear function.",
"appendix": "Author contributions\n\n\n\nASR conceived the study; MSK and ASR designed the experiments; MSK and ASR performed the research; MSK and LAH analyzed the data; and MSK, ASR, and LAH wrote the paper and agreed with the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe wish to thank Dr. Shane Torbert and Arjun Ramesh for their support and participation in early discussions about formulating the idea of this study, Jessleen K. Kanwal for assisting with Figure 7 and statistical analyses, and Dr. Jagmeet S. Kanwal for providing guidance in writing and submitting this manuscript.\n\n\nSupplementary materials\n\n\n\n4 Script Files\n\n\nReferences\n\nLoh PR, Baym M, Berger B: Compressive genomics. Nat Biotechnol. 2012; 30(7): 627–630. PubMed Abstract | Publisher Full Text\n\nForrest S: Genetic algorithms: principles of natural selection applied to computation. Science. 1993; 261(5123): 872–878. PubMed Abstract | Publisher Full Text\n\nRoss P, Corne D: Applications of genetic algorithms. AISB Quaterly on Evolutionary Computation. 1994; 89: 23–30. Reference Source\n\nBingul Z: Adaptive genetic algorithms applied to dynamic multiobjective problems. Appl Soft Comput. 2007; 7(3): 791–799. Publisher Full Text\n\nTang PH, Tseng MH: Adaptive directed mutation for real-coded genetic algorithms. Appl Soft Comput. 2013; 13(1): 600–614. Publisher Full Text\n\nStorn R, Price K: Differential evolution–a simple and efficient heuristic for global optimization over continuous spaces. Journal of Global Optimization. 1997; 11(4): 341–359. Publisher Full Text\n\nDeep K, Thakur M: A new mutation operator for real coded genetic algorithms. Appl Math and Comput. 2007; 193(1): 211–230. Publisher Full Text\n\nKorejo I, Yang S, Li C: A directed mutation operator for real coded genetic algorithms. Evo Applications. 2010; 6024: 491–500. Publisher Full Text\n\nMühlenbein H: How genetic algorithms really work: I. mutation and hillclimbing. Parallel Problem Solving From Nature. 1992; 2: 15–26. Reference Source\n\nZang H, Zhang S, Hapeshi K: A Review of Nature-Inspired Algorithms. J Bionic Eng. 2010; 7(Suppl): S232–S237. Publisher Full Text\n\nRudolph G: Convergence analysis of canonical genetic algorithms. IEEE Trans Neural Netw. 1994; 5(1): 96–101. PubMed Abstract | Publisher Full Text\n\nGoldberg DE: Genetic algorithms in search, optimization, and machine learning. 1989. Reference Source\n\nLeung Y, Gao Y, Xu ZB: Degree of population diversity-a perspective on premature convergence in genetic algorithms and its markov chain analysis. IEEE Trans Neural Netw. 1997; 8(5): 1165–1176. PubMed Abstract | Publisher Full Text\n\nSrinivas M, Patnaik LM: Adaptive probabilities of crossover and mutation in genetic algorithms. IEEE Trans Syst Man Cybem. 1994; 24(4): 656–667. Publisher Full Text\n\nDe Falco I, Della Cioppa A, Tarantino E: Mutation-based genetic algorithm: performance evaluation. Appl Soft Comput. 2002; 1(4): 285–299. Publisher Full Text\n\nMichalewicz Z: Genetic Algorithms + Data Structures = Evolution Programs. Springer-Verlag, New York, 1992. Publisher Full Text\n\nLee HC, Yang CW, Chen CY, et al.: Single point mutation of microRNA may cause butterfly effect on alteration of global gene expression. Biochem Biophys Res Commun. 2011; 404(4): 1065–1069. PubMed Abstract | Publisher Full Text\n\nCobb HG: An investigation into the use of hypermutation as an adaptive operator in genetic algorithms having continuous, time-dependent nonstationary environments (No. NRL-MR-6760) NAVAL RESEARCH LAB WASHINGTON DC. 1990. Reference Source\n\nBäck T, Schütz M: Intelligent mutation rate control in canonical genetic algorithms. Foundations of Intelligent Systems. 1996; 1079: 158–167 Springer Berlin Heidelberg. Publisher Full Text\n\nUyar Ş, Sariel S, Eryiğit G: A gene based adaptive mutation strategy for genetic algorithms. Genetic and Evolutionary Computation GECCO. 2004; 3103: 271–281. Publisher Full Text\n\nRussell SJ, Norvig P, Davis E, et al.: Artificial intelligence: a modern approach (Vol. 2). Englewood Cliffs: Prentice hall. 2010.\n\nSolis FJ, Wets RJ: Minimization by random search techniques. Mathematics of Operations Research. 1981; 6(1): 19–30. Publisher Full Text\n\nSkalak DB: Prototype and feature selection by sampling and random mutation hill climbing algorithms. In Proceedings of the eleventh international conference on machine learning. 1994; pp. 293–301. Reference Source\n\nNelder JA, Mead R: A simplex method for function minimization. Comput J. 1965; 7(4): 308–313. Publisher Full Text\n\nStone HS, Stone JM: Efficient search techniques–an empirical study of the N-Queens problem. IBM Journal of Research and Development. 1987; 31(4): 464–474. Publisher Full Text\n\nBell J, Stevens B: A survey of known results and research areas for n-queens. Discrete Math. 2009; 309(1): 1–31. Publisher Full Text\n\nKaraboga D, Basturk B: A powerful and efficient algorithm for numerical function optimization: artificial bee colony (ABC) algorithm. J Global Optim. 2007; 39(3): 459–471. Publisher Full Text\n\nMitchell M, Holland JH, Forrest S: When will a genetic algorithm outperform hill climbing? Adv Neural Inf Process Syst. 1993; 51–58. Reference Source\n\nCalzolari D, Bruschi S, Coquin L, et al.: Search algorithms as a framework for the optimization of drug combinations. PLoS Comput Biol. 2008; 4(12): e1000249. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang JM, Kao CY: Integrating adaptive mutations and family competition into genetic algorithms as function optimizer. Soft Computing. 2000; 4(2): 89–102. Publisher Full Text"
}
|
[
{
"id": "1057",
"date": "12 Jul 2013",
"name": "Christos Ouzounis",
"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\nGenetic algorithms (GAs) represent an adaptive computation technique with applications to various optimization problems, borrowing ideas from biological systems – with solution populations and operators that mimic DNA mutation and selection. As the authors of this report mention, GAs can suffer from issues of convergence to local optima, as all optimization algorithms do. It is proposed that a variable mutation rate (as opposed to the more classical approach of a constant mutation rate) can contribute towards better optimization performance, away from local optima: the longer the GA remains at a local optimum, the higher the mutation rate can become. This is the key idea that is addressed in this work.The study is interesting as it focuses on the mutation operator which has not been researched sufficiently, compared to the crossover operator which has been studied quite extensively. The proposed solution - namely a variable mutation rate as an independent variable, at least in one case - is compared to other optimization techniques, such as random search / hill climbing / Nelder-Mead. Experiments are performed for well-established, highly controlled optimization problems, such as the N-Queens problem, maximization of a 3D surface and the Rastrigin function. Performance in terms of both accuracy of solution and efficiency of resource consumption was monitored for all experiments. Interestingly, while adaptive mutation has a two-fold performance in both terms above for the N-Queens problem, there is no significant difference for the 3D surface problem.This is interesting work, but relevance to computational biology and real-world solutions is somewhat peripheral. I am sure the authors are investigating applications of their approach to larger problems, as alluded in their title - which unfortunately is somewhat misleading: without the terms ‘diagnosis’ and ‘therapy’, this would have been a fine, technical report on an exciting subject. Hopefully, we will see some further development towards that direction in the near future.",
"responses": [
{
"c_id": "627",
"date": "19 Nov 2013",
"name": "Maxinder Kanwal",
"role": "Author Response",
"response": "We agree with the reviewer’s point. Our main objective was to show the advantage variable-rate mutation operators in genetic algorithms had over fixed-rate ones when applied correctly. While some of our immediate visions for the algorithm were for applying it towards solving problems in bioinformatics, we realize that the algorithm is more of a general search method that can be applied to a number of problems not just within biology. In order to more accurately portray the content of this article, the title and abstract have been modified to avoid emphasizing a minor aspect of the article. In addition, the Implications section of the Discussion have been expanded on to more comprehensively explain how this algorithm can be applied in the medical field as an example of one of its many applications."
}
]
},
{
"id": "1062",
"date": "15 Jul 2013",
"name": "Mark A. Ragan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGenetic algorithms offer a biologically motivated approach to nonlinear optimisation. In the so-called breeding stage of a genetic algorithm, the diversity of candidate solutions is increased by operations analogous to crossover between chromosomes, and point mutations. If parameterised appropriately, the mutation operator helps the genetic algorithm explore the fitness landscape, in particular by making it less likely to become trapped in a local optimum. Mutation operators are typically of fixed rate, although there is some prior art in the use of variable-rate operators. Here Kanwal, Ramesh and Huang, senior students at Thomas Jefferson High School for Science and Technology in Alexandria, Virginia, develop some theory on the form of adaptive-rate mutation operators, and investigate the performance of a new heuristic motivated by this theory. They report that this heuristic delivers much-improved accuracy in finding the global optimum, at no cost in speed, in two reasonably difficult optimisation scenarios, and describe bounds on the mutation function.The research is clearly of publishable quality. The content explained clearly and in sufficient detail to allow the work to be replicated, and the conclusions are balanced and justified on the basis of the results presented. The writing style is exemplary. However, the manuscript does not deliver against the “diagnosis and therapy” promised in the title and abstract: indeed, the authors rightly allude to much broader contexts. In the absence of a specific case study in the biological application domain, the title might focus solely on the algorithmic contribution. It would be interesting to compare the performance of genetic algorithms implementing their new mutation operator with, for example, simulated annealing or multi-chain MCMC, although this would presumably require a broader comparison framework than was within-scope for the work reported here.Figures 3 and 4 are very similar to those in Wikipedia (http://en.wikipedia.org/wiki/Rastrigin_function, accessed 13 July 2013); does this arise from use of a generic parameterisation?I would prefer “optima” rather than “optimums”. There is a misspelling in the legend of Figure 3.",
"responses": [
{
"c_id": "626",
"date": "19 Nov 2013",
"name": "Maxinder Kanwal",
"role": "Author Response",
"response": "We agree with the reviewer’s point. Our main objective was to show the advantage variable-rate mutation operators in genetic algorithms had over fixed-rate ones when applied correctly. While some of our immediate visions for the algorithm were for applying it towards solving problems in bioinformatics, we realize that the algorithm is more of a general search method that can be applied to a number of problems not just within biology. In order to more accurately portray the content of this article, the title and abstract have been modified to avoid emphasizing a minor aspect of the article. In addition, the Implications section of the Discussion have been expanded on to more comprehensively explain how this algorithm can be applied in the medical field as an example of one of its many applications.In regards to Figures 3 and 4 being similar to those on Wikipedia, the reason for this is because the code given below the images was almost the same code used to generate our figures in the paper with only slight modifications to make the figures more readable. We were aware of the Wikipedia images before generating our own; we simply liked the parameters set in the Wikipedia code, and so we generated our images using that code (along with some additional style changes).The mistakes pointed out have been fixed as well."
}
]
},
{
"id": "1179",
"date": "22 Jul 2013",
"name": "Panayiotis Benos",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article primarily focuses on a very interesting computational problem, namely whether the variable-rate mutation operators in a genetic algorithm offers an advantage over the fixed-rate ones. The main idea is that variable-rate operators will help algorithms escape the local optima and identify the global optimum more efficiently. The authors indeed show that this is the case in at least some of the examples they investigated. I think this is a very promising article that will generate a lot of interest in this area.However, I am a bit unsure about the method’s relevance to biology. There are some biological examples that I can think of where their methodology can make an impact but I fail to see how it can be used for “diagnosis” and “therapy” as the title states. I wish they had provided some more examples towards that direction.Finally, I would like to bring up a point that is frequently overseen in computational studies. Although in many non-biological problems finding the optimal solution is the ultimate goal, many biological problems are better served by finding a set of optimal+ sub-optimal solutions. Examples include protein and RNA folding, and microRNA and transcription factor binding sites. Perhaps the authors would consider extending their methodology towards that direction in the future.",
"responses": [
{
"c_id": "625",
"date": "19 Nov 2013",
"name": "Maxinder Kanwal",
"role": "Author Response",
"response": "We agree with the reviewer’s point. Our main objective was to show the advantage variable-rate mutation operators in genetic algorithms had over fixed-rate ones when applied correctly. While some of our immediate visions for the algorithm were for applying it towards solving problems in bioinformatics, we realize that the algorithm is more of a general search method that can be applied to a number of problems not just within biology. In order to more accurately portray the content of this article, the title and abstract have been modified to avoid emphasizing a minor aspect of the article. In addition, the Implications section of the Discussion have been expanded on to more comprehensively explain how this algorithm can be applied in the medical field as an example of one of its many applications.In regards to your comment, you bring up a very good point. We will certainly look into modifying this algorithm to make it a technique that better serves attempting to solve problems in biology by having it search for a set of optimal and suboptimal solutions."
}
]
}
] | 1
|
https://f1000research.com/articles/2-139
|
https://f1000research.com/articles/2-249/v1
|
18 Nov 13
|
{
"type": "Case Report",
"title": "Misleading hepatitis B testing in the setting of intravenous immunoglobulin",
"authors": [
"Christelle M Ilboudo",
"Erin M Guest",
"Angela M Ferguson",
"Uttam Garg",
"Mary Anne Jackson",
"Erin M Guest",
"Angela M Ferguson",
"Uttam Garg",
"Mary Anne Jackson"
],
"abstract": "Intravenous immunoglobulin (IVIG) is commonly used for a wide range of diagnoses, by multiple pediatric subspecialists. We report two cases of hepatitis B screening results post IVIG infusion, where positive anti-Hepatitis B core antigen serology tests indicated possible occult hepatitis infection, leading to a delay in care. However, serial antibody testing showed results consistent with the passive transfer of antibodies.",
"keywords": [
"Viral serologies",
"Blood product",
"Hepatitis B",
"Intravenous Immunoglobulin"
],
"content": "Introduction\n\nIntravenous immunoglobulin (IVIG) is a blood product prepared by pooling plasma from 3000–10,000 healthy blood donors. Adverse events are reported in 1–15% of treated patients, and most clinicians are aware of common events such as infusion reactions, and more serious reactions that follow IVIG infusion including renal dysfunction (a US Boxed warning), thrombotic events, anti-globulin hemolysis, and aseptic meningitis syndrome1.\n\nSince IVIG is a passive antibody transfer, it can result in transiently positive anti-viral serology tests. We report two cases where screening hepatitis B testing resulted in an unusual pattern of immunoglobulin positivity after IVIG therapy prompting additional laboratory testing and delayed treatment in one of the children. Based on the known half-life of IVIG products of 21 days, we used serial testing of sera to confirm degradation of antibody over time. Clinicians should be aware that passive transfer of antibodies is expected and serologic screening should be performed pre-treatment if IVIG therapy is anticipated.\n\n\nCase reports\n\nA 6 year old African-American female presented with several weeks of bruising and epistaxis. A diagnosis of Evans syndrome and systemic lupus erythematosus was confirmed and she received IVIG in the form of Gamunex™ on days 1, 9, 10 and 18 at doses of 1 gram/kg each time. On day 19, hepatitis screening (Ortho-Clinical Diagnostics VITROS 5600 system Raritan, NJ) was performed in anticipation of possible initiation of rituximab therapy. The results were positive for anti-Hepatitis B core antigen (anti-HBc) and negative for Hepatitis B surface antigen (HBsAg) (Table 1). She had received 3 doses of Hepatitis B vaccine as an infant, had no prior transfusions and her liver function testing was normal. She was born in the US, had never traveled outside of the US and had no other exposures or risk factors for hepatitis B. Specific hepatitis B testing consisting of HBsAg, anti-HBc which is the total immunoglobulin level against the core antigen, anti-Hepatitis B core antigen specific IgM (IgM anti-HBc), hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) was performed on day 23 post-IVIG. The testing was positive for anti-HBc and anti-Hepatitis B e-antigen (anti-HBe), but negative for IgM anti-HBc. The IgM anti-HBc is important to determine whether the total anti-HBc result is consistent with an acute infection or reflective of a chronic or resolved infection (Table 2). The combination of positive anti-HBc and anti-HBe raised the concern of acute, resolved or chronic hepatitis B infection. As a result of the misleading results, the initiation of rituximab therapy was delayed, until further evaluation was completed showing antibody degradation consistent with the passive transfer of antibodies (Table 1). The same hepatitis B specific testing was repeated a month later, since the half-life of IVIG is 21 days. The repeated testing was negative for both anti-HBc and anti-HBe which confirmed the suspicion that her initial positive screen and specific hepatitis B testing were the results of IVIG infusion. Interestingly, her anti-HBs decreased as well over time, further evidence of the IVIG being the source of the immunoglobulins detected.\n\n≠Number of days post IVIG infusion\n\n*Values are in milliInternational Units/ml: > 12 is adequate immunity either from previous infection or vaccines\n\n-Testing not done\n\n*Adapted from: Committee on Infectious Diseases et al. Red Book Online 369–3907. HBV: hepatitis B virus\n\nAn 11 year old Caucasian female with high risk pre B-cell lymphoblastic leukemia failed to enter into remission with standard chemotherapy regimens and was enrolled in the National Cancer Institute study, Anti-CD19 White Blood Cells for Children and Young Adults with B Cell Leukemia or Lymphoma (NCI identifier NCT01593696). In preparation for the cellular therapy, she was given IVIG in the form of Gamunex™ at 0.5 gram/kg once on day 1. Hepatitis B screening (Ortho-Clinical Diagnostics VITROS 5600 system Raritan, NJ) performed in anticipation of bone marrow transplantation, 7 days following IVIG therapy showed a positive anti-HBc with a negative HBsAg, raising the possibility of acute, resolved or chronic hepatitis B infection (Table 1). She had normal liver function at the time of the reactive results. This child was born in the US and had received 3 doses of hepatitis B vaccine as an infant. She had not traveled outside of the US and had no other risk factors for Hepatitis B infection. She had previously received multiple packed red blood cells and platelet transfusions. Additional data confirmed that hepatitis screening had been conducted at another facility, 2 months prior to the IVIG administration and that test result was negative. Table 1 shows serial results of antibody testing consistent with passive antibody degradation. The serial testing consisted of specific hepatitis B testing, using the same laboratory system, on day 8 post-IVIG and 3 months later which revealed a negative IgM anti-HBc. Interestingly, her anti-HBs decreased as well over time as further evidence of IVIG being the source of the immunoglobulins detected. In this case, given her negative prior screening test and her negative IgM anti-HBc, her transplantation was delayed until the results of the testing on day 8 post–IVIG was available. She did receive her transplantation a month prior to the last hepatitis B specific testing.\n\n\nDiscussion\n\nWe report two instances where an unusual pattern of hepatitis B positivity related to IVIG infusion complicated the care of children with an underlying autoimmune or oncologic diagnosis.\n\nA variety of serologic tests are available to confirm hepatitis B infection, and typically more than one marker is present in acute or chronic infection. The pattern of hepatitis B seropositivity noted in our two patients, where anti-HBc was present, while less common, is compatible with occult hepatitis infection.\n\nThe association of anti-HBc positivity with certain IVIG products was previously noted by Arnold et al. during a rituximab study of patients with refractory immune-mediated thrombocytopenia2. Pre-treatment hepatitis screening was conducted on 24 study patients because of the known risk for hepatitis B reactivation following rituximab. Anti-HBc positivity was found in 45% of the cohort and the investigators noted that IVIG use was common in their patient group. They were able to show seroreversion over time in 10/11 of their IVIG-treated patients, consistent with degradation of passively transferred antibody. They tested five immune globulin preparations and confirmed anti-HBc presence in three, including Gamunex™, the preparation received by our patients. Benton et al. additionally described a patient being considered for rituximab therapy whose care was delayed secondary to misleading hepatitis B testing post IVIG infusion3. A false positive enzyme immunoassay and a Treponema pallidum haemagglutinin assay have also been reported following IVIG infusion4,5. Positive human T-lymphotropic virus (HTLV) testing following IVIG infusion for chemotherapy-induced polyneuropathy has also been reported in a leukemic patient resulting in a delay in his cord blood transplantation6. In all the above cases, repeated testing 4–8 weeks post IVIG revealed lower or negative antibody titers.\n\nThere are 11 immune globulin preparations available in the US and Canada, and specific diagnostic indications, FDA approved ages, dosing recommendations, infusion rates, IgA composition and routes of administration vary by product (Table 3). Based on the number of diagnoses for which IVIG is used, a wide range of subspecialists are involved in prescribing IVIG and education regarding passive antibody transfer therefore needs to be targeted to clinicians in those subspecialties such as neurology, immunology, hepatology, hematology and rheumatology as well as general practitioners.\n\nOur cases serve to remind clinicians of the potential for passive antibody transfer with IVIG products and to define the potential clinical confusion that can arise in situations where infectious serologic screening is not performed pre-infusion. In all situations where IVIG is prescribed, the patient’s underlying diagnosis, clinical indication, the product brand and dose, lot number and specific infusion instructions should be explicitly outlined and all baseline serologic specimens, when indicated, obtained before infusion of the product.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the parent/guardians of the patients.",
"appendix": "Author contributions\n\n\n\nCI and MAJ conceived the study. CI drafted the first draft of the paper. EG provided patient information for the second case. AF and UG provided expertise in clinical laboratory testing as well as reviews of prior cases. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nBonilla FA: Intravenous immunoglobulin: adverse reactions and management. J Allergy Clin Immunol. 2008; 122(6): 1238–1239. PubMed Abstract | Publisher Full Text\n\nArnold DM, Crowther MA, Meyer RM, et al.: Misleading hepatitis B test results due to intravenous immunoglobulin administration: implications for a clinical trial of rituximab in immune thrombocytopenia. Transfusion. 2010; 50(12): 2577–2581. PubMed Abstract | Publisher Full Text\n\nBenton E, Iqbal K, Wade P, et al.: False-positive hepatitis B serology following IVIG therapy: forgotten but not gone!! J Am Acad Dermatol. 2012; 66(3): e123–124. PubMed Abstract | Publisher Full Text\n\nConstable SA, Parry CM, Enevoldson TP, et al.: Positive serological tests for syphilis and administration of intravenous immunoglobulin. Sex Transm Infect. 2007; 83(1): 57–58. PubMed Abstract | Free Full Text\n\nRossi KQ, Nickel JR, Wissel ME, et al.: Passively acquired treponemal antibody from intravenous immunoglobulin therapy in a pregnant patient. Arch Pathol Lab Med. 2002; 126(10): 1237–1238. PubMed Abstract\n\nBelanger SS, Fish D, Kim J, et al.: False-positive human T-lymphotropic virus serology after intravenous immunoglobulin transfusion. CMAJ. 2012; 184(15): 1709–1712. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPediatrics AA: Red book: 2012 Report of the Committee on Infectious Diseases. In. Edited by Pickering LK Baker, C.J., Kimberlin D.W., Long, S.S.: Elk Grove Village, IL American Academy of Pediatrics; 2012; 369–390. Reference Source"
}
|
[
{
"id": "2483",
"date": "12 Dec 2013",
"name": "Adlette Inati",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntravenous immunoglobulin (IVIG) infusions are widely used for various medical indications. Since these products are pooled from thousands of healthy blood donors, it is possible to have passive antibody transfer of certain donor-related infections to the IVIG recipient. In this report Ilboudo et al. present two cases where IVIG infusions led to false positive transient hepatitis B titers and delayed the management of two critical patients. Previous reports have also indicated such an observation. Accordingly, this article serves as a reminder of possible positivity of hepatitis titers post IVIG that would resolve in few weeks. It also underscores the importance of screening for infections including hepatitis B pre IVIG infusion. This article is well written and lists the IVIG products available in the US with significant comments that can be used as a reference.",
"responses": []
},
{
"id": "4223",
"date": "24 Mar 2014",
"name": "Melvin Berger",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 cannot say this better than the authors have: 'Our cases serve to remind clinicians of the potential for passive antibody transfer with IVIG products and to define the potential clinical confusion that can arise in situations where infectious serologic screening is not performed pre-infusion'. It is important to realize that this includes HBcAb, since it is not generally recognized that antibodies to hepatitis B antigens other than HBSAg (HBsAb) are present in US IgG products, as shown in Table 3. It is also important to recognize that passive transfer of antibodies also occurs with subcutaneous IgG treatment. The gold standard for identifying blood borne infection is detection of viral genomes in the patient's blood. Nucleic acid tests such as PCR are done to rule out the presence of infectious agents during the processing of plasma for products like IVIG, and are available in many hospital and commercial reference laboratories. Prompt confirmation of the absence of detectable hepatitis B virus DNA in the patient's blood could obviate the need to wait for catabolism and dropping of the titer of passively transferred antibody, and avoided the delays experienced by the patients described in this paper.I have no conflicts of interest regarding this paper. For the purposes of complete disclosure, I am a salaried employee of CSL Behring, LLC, a manufacturer of fractionated plasma products like IVIG, and a stock-holder.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-249
|
https://f1000research.com/articles/2-170/v1
|
12 Aug 13
|
{
"type": "Research Article",
"title": "The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells",
"authors": [
"Michael Hartmann",
"Andrea Hemmerlin",
"Elisabet Gas-Pascual",
"Esther Gerber",
"Denis Tritsch",
"Michel Rohmer",
"Thomas J Bach",
"Michael Hartmann",
"Andrea Hemmerlin",
"Elisabet Gas-Pascual",
"Esther Gerber",
"Denis Tritsch",
"Michel Rohmer"
],
"abstract": "We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.",
"keywords": [
"In higher plants",
"the synthesis of the general isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is accomplished through two different routes",
"the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway (cf.1)."
],
"content": "Introduction\n\nIn higher plants, the synthesis of the general isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is accomplished through two different routes, the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway (cf.1).\n\nThe MVA pathway supplies the biosynthetic precursors for isoprenoids in the majority of eukaryotes (including all animals, the archea, some eubacteria, fungi and the cytosol/mitochondria of some algae and higher plants). It can be found in several important human parasites, such as Trypanosoma and Leishmania2. In humans, the MVA pathway operates alone and produces a variety of critical end products, including cholesterol, steroid hormones, dolichols and the prenyl moiety of cancer-associated cell signaling proteins like RAS3–6. In plants, the cytosolic IPP provided by the MVA pathway serves as precursor for the synthesis of sterols, brassinosteroids, polyprenols, dolichols and most sesquiterpenes and to some extent as a substrate for protein prenylation (cf.7,8). Moreover, cytosolic IPP is imported into the mitochondria, where it serves as precursor for ubiquinone9–11.\n\nThe alternative pathway (or MEP-pathway) for the synthesis of isoprenoids occurs in eubacteria, cyanobacteria, and the plastids of phototrophic algae and plants12–19. In plants, the precursors, provided by the plastidial MEP pathway are used for the biosynthesis of essential isoprenoids of the photosynthetic apparatus such as carotenoids, the side-chains of chlorophyll and plastoquinone, as well as for isoprene, tocopherols, phylloquinones and the phytohormones ABA and gibberellin20, with ABA being a cleavage product of carotenoids, like the more recently discovered strigolactones21,22. In addition to these ubiquitous compounds in plants, the MEP pathway is the route for the biosynthesis of the vast majority of plant terpenoids, including countless secondary metabolites with defensive, allelopathic or signaling properties11,18,23 (and Hemmerlin et al.24 for review of literature). Furthermore, precursors derived from the MEP pathway are used for the post-translational modification of certain proteins by the covalent addition of a farnesyl-(C15) or geranylgeranyl (C20) residue, a process commonly referred to as protein prenylation (cf. the accompanying paper25 and literature cited therein).\n\nIn preceding studies investigating the differential effect of a series of inhibitors of isoprenoid biosynthesis and function in cell cycle progression in unsynchronized and synchronized BY-2 cells26, it had been observed that blockage of protein farnesylation by chaetomellic acid not only led to a considerable percentage increase in dead cells during the culture period, but also to a specific arrest in the transition from G2 into M phase. By contrast, mevinolin for instance (which inhibits the key-regulatory enzyme HMG-CoA reductase in the cytosolic MVA pathway) arrested cells mainly at the transition from G1 to S phase27. This latter finding is most likely due to the lack of some MVA-derived signal formed at the end of mitotic phase that might be implied in the regulation of the cytoplasmic pH28. These and other observations prompted a series of further studies in which the possibility that the plastidial MEP pathway could complement MVA deficiency was tested. Indeed it was demonstrated that exogenously added deoxyxylulose (DX, the dephosphorylated first product of the MEP pathway) could overcome mevinolin-induced growth inhibition, even more efficiently than exogenous MVA29. To enter the plastidial MEP route, this DX needs conversion into its phosphate (DXP) by a cytosolic xylulose kinase30, followed by translocation into plastids. As a logical follow-up, and in view of early reports that in plants geranylgeranylated proteins seem to be present in higher quantities than farnesylated ones31,32, we embarked on studying this phenomenon more closely. Our interest is not only focused on elucidation of the biosynthetic origin of farnesyl and geranylgeranyl residues, but also on the action of inhibitors on precursor availability and on the specificity of prenyltransferases. For instance, we could demonstrate the incorporation of 14C-DX into proteins in BY-2 cells29.\n\nThe central element of our recent studies was an in vivo-visualization system based on a stably transformed tobacco BY-2 (TBY-2) cell line for monitoring the prenylation status of a GFP fusion protein. Isoprenylation of proteins, which occurs in all eukaryotic cells, involves the covalent attachment of a C15 (farnesyl) or C20 (geranylgeranyl) group to a C-terminal CaaX motif, followed by a series of post-prenylation reactions. This lipidic post-translational modification plays an important role in the correct membrane targeting of certain proteins and in their interactions with other proteins.\n\nThis system consisted of a dexamethasone-inducible cell line that expressed a reporter protein (GFP) fused to the carboxy-terminal basic domain of the rice calmodulin (CaM61), which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). After induction, the prenylated fusion protein predominantly associated with the plasma membrane. By using pathway-specific inhibitors, we demonstrated that inhibition of the MEP pathway with oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), triggered a shift in the localization of the GFP-BD-CVIL protein from the plasma membrane to the nucleus1. By contrast, inhibition of the MVA pathway (by mevinolin) or protein farnesyltransferases did not affect the localization of the chimeric fusion protein. Among other experiments, complementation assays with pathway-specific intermediates were performed and clearly indicated that the precursors for the geranylgeranylation of the fusion protein in tobacco BY-2 cells were predominantly provided by the MEP pathway1.\n\nHowever, at the end of this previous study several questions remained unsolved that will be addressed in more detail in the present work, including the impact of inhibitors of sterol biosynthesis and post-prenylation reactions on the subcellular localization of the His6-tagged GFP-BD-CVIL fusion protein. In order to prove that the present bioassay was not only able to serve as a qualitative approach for the identification of new drug candidates, but also as a statistical tool to compare the potential drug candidates with other known inhibitors in vivo, we also performed a quantitative analysis of the intracellular distribution of GFP-DB-CVIL in response to different concentrations of novel prodrugs, targeting the early steps of the MEP pathway.\n\n\nResults\n\nBy blocking the early steps of sterol biosynthesis with pathway-specific inhibitors we tried to determine the impact of sterol depletion in the plasma membrane as well as of the modulation of the pool of endogenously available FPP on the localization of the H6-GFP-BD-CVIL fusion protein.\n\nFirst of all, squalestatin-1 (SQ), also referred to as zaragozic acid33, was used to specifically inhibit the conversion of farnesyl diphosphate (FPP) to squalene by the first committed enzyme in the sterol/triterpene pathway, squalene synthase (SQS). SQ is a competitive inhibitor of SQS and structurally mimics the reaction intermediate presqualene diphosphate33–37.\n\nSeven-day-old BY-2 cells were diluted at a ratio of 1 to 6 in fresh medium and treated with 1µM SQ for 18–24 h (in different independent experiments). 15 h before observation of the cells by fluorescence microscopy, expression of the prenylable fusion protein H6-GFP-BD-CVIL was induced by addition of 10µM dexamethasone (150 rpm, 26°C, growth in the dark).\n\nThe results of this treatment are shown in Figure 1a. Two general observations can be made. First of all, treatment with SQ resulted in a partial mislocalization of the prenylated fusion protein from the PM to the nuclear compartment, with the strongest signal being emitted by the nucleolus. In addition, the overall morphology of the treated cells was changed. Cells treated with 0.5 or 1µM SQ showed stunted growth with clearly reduced length-to-diameter ratios as compared to control cells. This latter effect has already been described38.\n\nControl: GFP fluorescence is almost exclusively associated with the plasma membrane (PM). SQ: Cells treated with squalestatin-1 (SQ), a specific inhibitor of squalene synthase (SQS) show partial translocation of GFP fluorescence to the nucleus (N) and the nucleolus (No). Tb: Cells treated with terbinafine (Tb), an inhibitor of squalene epoxidase (SQE), showing GFP fluorescence associated with the plasma membrane (very faint fluorescence is also seen in the cytoplasm). Ro: Localization of H6-GFP-BD-CVIL in cells treated with Ro48-8071, a general inhibitor of oxidosqualene cyclases (OSC), such as cycloartenol synthase (CAS) in plants, showing GFP fluorescence associated with the plasma membrane. b) Localization of H6-GFP-BD-CVIL fusion protein in transgenic BY-2 cells after treatment with inhibitors of key enzymes of the MVA and MEP pathways. Control cells were induced 15 h before observation by confocal microscopy as described. Treatment with inhibitors occurred 3 h prior to dexamethasone induction116. Mevinolin (Mev, 5µM) did not cause any detectable effect on the localization of GFP-BD-CVIL fusion protein when compared to the untreated control. GFP fluorescence was predominantly located at the periphery of the cells. By contrast, Fosmidomycin (Fos) as well as oxoclomazone (OC) (here both at 30µM) caused a mislocalization of the fusion protein to the nucleus and in particular to the nucleolus. The same phenotype could also be observed after application of 40µM GGTI-2133, a peptidomimetic inhibitor of protein geranylgeranyl transferase 1 (PGGT1). White bar = 10µm.\n\nIn order to examine whether this effect was due to sterol depletion or a side effect of squalestatin on prenyl transferases in general (cf.39), BY-2 cells were treated with terbinafine (Tb), a specific, non-competitive inhibitor of the fungal squalene epoxidase (SQE). In mammals, however, it acts as a competitive inhibitor of this enzyme40. Its inhibitory efficiency in plant cells has been demonstrated with celery (Apium graveolens) cell suspension cultures41, wheat (Triticum aestivum) seedlings42, cat’s claw (Uncaria tomentosa)43 and with our model system, BY-2 cell suspension cultures38. Treatments with Tb were performed at 30µM up to 24 h (as well as 18 h). The cells treated with Tb showed a very slightly stunted growth as compared to the control cells and displayed faint GFP signals in the cytosol, close to the PM. However, no mislocalization of the prenylable fusion protein to the nuclear compartment could be observed under the chosen experimental conditions (Figure 1a).\n\nFinally, Ro48-8071 (Ro) was used to inhibit one of the key steps of sterol biosynthesis, the conversion of the linear oxidosqualene into cycloartenol, the first cyclic precursor of phytosterols. This reaction is catalyzed by cycloartenol-synthase (CAS) in plants44. Ro is a potent inhibitor of oxidosqualene cyclases (OSC) in general, including lanosterol synthase (LAS) in mammals and fungi45. The structure of Ro48-8071, which is an orally active inhibitor of human hepatic OSC, has been determined in complex with the squalene-hopene cyclase (SHC), the prokaryotic counterpart of OSCs, responsible for the conversion of squalene into cyclic compounds in bacteria, and it is suggested that Ro reacts with the expected binding site for squalene46. Ro treatments were performed at 2µg/ml under the same experimental conditions as described previously for SQ and Tb applications. Cells treated with Ro for 18/24 h looked morphologically like the control cells. The GFP fusion protein was mainly localized at the level of the plasma membrane, although there were also faint signals (small speckles) of fluorescence near the PM like those observed for the previous Tb treatments. However, no mislocalization of the GFP fluorescence to the nucleus/nucleolus was observed (Figure 1a). When compared to treatments like those described by Gerber et al.1 and repeated here (Figure 1b), the effects were slightly less pronounced.\n\nVarious complementation experiments were performed with SQ-treated cells. Whereas DX (0.5mM) and geranylgeraniol (GGol, 20µM) were able to re-establish the membrane localization in 100% of the cells (better than the control), neither geraniol (Gol, 20µM) nor mevalonolactone (MVL, 5mM) and its open carboxylic form mevalonate (MVA, 3mM) could complement the mislocalization under the chosen experimental conditions. Finally, squalene was added at 2–4mM, but did not overcome the SQ-induced effect either (Figure 2).\n\nSqualestatin was added at 1µM throughout the series of experiments. Partial delocalization (from the PM to the nucleus/nucleolus) with pathway intermediates, isoprenols and squalene in an attempt to chemically complement the effect of squalestatin. MVA: mevalonic acid; MVL: mevalonolactone; DX: 1-deoxyxylulose; Gol: geraniol; Fol: farnesol; GGol: geranylgeraniol. White bars = 20µm.\n\nAs mentioned earlier, the purification and analysis of the His6-tagged GFP-BD-CVIL fusion protein revealed that it was geranylgeranylated and carboxyl-methylated in BY-2 cells1. In the past the study of post-prenylation inhibition has become a very attractive topic, as both reactions are essential for the localization of many prenylated proteins by mediating their attachment to membranes and protein-protein interactions.\n\nIn preliminary experiments we tested inhibitors of RAS converting enzyme 1 (RCE1), and of isoprenylcysteine carboxyl methyltransferase (ICMT), but most of them induced cell death when used in commonly cited concentrations and time-scales in our model system. This is most likely due to the efficient uptake and high metabolic activity of BY-2 cells29 and requires adjustment of the experimental conditions. Nonetheless, the results obtained after various treatments indicated that both inhibition of RCE1 and ICMT affected the localization of H6-GFP-BD-CVIL (Figure 3). For instance, short-term treatment 15 h induction, then treatment for 3 h with 200µM of the prenylcysteine analog N-acetyl-S-farnesyl-L-cysteine (AFC) strongly changed the distribution pattern of the H6-GFP-BD-CVIL and besides the PM, both the cytosol as well as the nucleolus displayed strong GFP signals (Figure 3). Interestingly, a similar effect on the localization of the prenylated GFP-CaM53 fusion protein of Petunia was observed by Rodríguez-Concepcíon et al.47 in response to AFC treatment (200µM), using bombarded Petunia leaves. Long-term treatments of BY-2 cells (> 10 h) however led to loss of GFP fluorescence and cell death.\n\nAFC: N-acetyl-S-farnesyl-L-cysteine, an inhibitor of isoprenylcysteine carboxyl methyltransferase; TPCK: N-tosyl-L-phenylalanine chloromethyl ketone, a serine and cysteine proteinase inhibitor. White bars = 20µm.\n\nInhibition of the –AAX-proteolysis by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK48) led to a partial mislocalization of the fusion protein to the nuclear compartment. However, optimal conditions to observe the induced effects still need to be identified.\n\nDXS (1-deoxy-D-xylulose 5-phosphate synthase) catalyzes the first step of the alternative MEP pathway, the condensation of glyceraldehyde 3-phosphate (GAP) and pyruvate, yielding 1-deoxy-D-xylulose 5-phosphate (DXP). DXS enzymes are highly conserved in bacteria and plants and analyses of their sequences revealed a weak homology with other thiamine-dependent enzymes, such as transketolases, and the pyruvate dehydrogenase E1 subunit49–52. These enzymes all catalyze similar biochemical reactions53,54 by using thiamine diphosphate (TPP) as a cofactor and pyruvate as a substrate. In addition to TPP, DXS also requires a divalent cation (Mg2+ or Mn2+) for optimum enzyme activity55,56. More recently, efforts were successful to partially crystallize DXS from E. coli and Deinococcus radiodurans (in complex with TPP), after in situ proteolysis of the purified enzyme by a fungal protease57,58, which led to a better understanding of the catalytic mechanism of DXS and possibly paves the way for the design of novel active inhibitors. As to date, only two inhibitors of this enzyme are known: oxoclomazone (OC), for the plant DXS59 and fluoropyruvate, for the DXS of E. coli60. OC, sometimes also referred to as 5-ketoclomazone, was only recently reported to have exhibited antibacterial activity against a pathogenic bacterium, Haemophilus influenza61.\n\nThe synthesis of different classes of pyruvate analogs was inspired by known inhibitors of pyruvate decarboxylases and pyruvate dehydrogenases (cf.62–64). These compounds were then tested with our bioassay for possible effects on the localization of the H6-GFP-BD-CVIL (Figure 4), thus indicating an inhibitory effect on the biosynthesis of GGPP via the MEP pathway. All compounds (cf. Figure S4) were dissolved in their respective solvent (water or acetonitrile). The only exception was p-hydroxyphenylpyruvate, which did not dissolve but gave a homogenous, yellow suspension.\n\nThe cells were treated for 18 h, in the presence of 100µM of pyruvate analogs, and the next day examined by confocal fluorescence microscopy. None of the tested compounds induced a mislocalization of green fluorescence, as it was observed for the positive control, which was treated with 50µM OC. However, the dibromopyruvate-treated cell culture showed significant cytotoxic effects as over 90% of cells did not display any GFP-related fluorescence, indicative of cell death. The fluorescence in the remaining cells appeared in cytoplasmic strands and in the cytoplasm surrounding the nucleus (Figure 4).\n\nThe molecules (at a final concentration of 100µM) were dissolved and tested under standard conditions (3 h treatment, followed by induction with 10µM dexamethasone and examination by fluorescence microscopy 15 h later). Cells were also treated by 50µM oxoclomazone as described previously (positive control). None of the six tested compounds showed a mislocalization of the GFP fluorescence to the nucleus. The compound TJ150, however, appeared to be cytotoxic at the given concentration as cells displayed typical signs of cell death (over 90% of treated cells did not show any fluorescence).\n\n1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the second enzyme of the MEP pathway and catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP). Part of this transformation is an intramolecular rearrangement, yielding 2-C-methyl-D-erythrose 4-phosphate, which is then reduced to MEP in an NADPH-dependent reaction step65–68. Several studies have revealed detailed information about the structure and biochemical properties of the DXR enzyme from E. coli67–69. Those results – including the three-dimensional structures of DXR in a ternary complex with DXP/fosmidomycin (Fos) and the co-factor NADPH – suggest a physiologically active homodimer, with each subunit consisting of three distinct domains70,71. In addition to NADPH, DXR requires a divalent metal cation for activity, such as Mn2+, Mg2+ or, in vitro, Co2+, which is bound by three highly conserved amino acid residues70,72. This metal cation is chelated by DXP before the first step of the conversion to MEP – an intramolecular rearrangement – takes place. According to the structural data, binding of DXR to its substrate (or to Fos) involves a major conformational rearrangement of the enzyme in the presence of NADPH. Fos acts as a competitive inhibitor, chelating a bivalent cation with its hydroxamate group and binding slowly but very tightly to the catalytic site of DXR70. The substrate binding site of the DXR enzyme can be divided into three distinct regions: first of all, a positively charged pocket which interacts with the phosphonate function of Fos (“phosphate-recognition site”), a hydrophobic region covering the backbone of the molecule, as well as an amphipathic region that binds the hydroxamic acid moiety of the molecule. This results in a conformation with a flexible loop covering the central catalytic site, thus forming a barrier with the surrounding solvent71.\n\nFos is known to inhibit the DXR enzyme from higher plants73,74, and experiments with various plant species have demonstrated its potential as herbicide, as approved by the chlorotic and bleaching phenotypes that have been observed after its application75. In addition, Fos successfully inhibited the isoprenylation of the GFP-BD-CVIL fusion protein in our fluorescent bioassay at concentrations in the micromolar range1. Fos and its methylated derivative FR-90098 are phosphonohydroxamic acids. In both compounds a hydroxamate function is linked to a phosphonic acid function by a propyl chain. Fos and FR-90098 as well as two hydroxamate derivatives 4-(hydroxyamino)-4-oxobutyl-phosphonic acid (LK1) and 4-[hydroxy(methyl)amino]-4-oxobutyl-phosphonic acid (LK276) have been tested on the transgenic BY-2 cell line expressing the H6-GFP-BD-CVIL marker protein using the well-established standard conditions. At 100µM, the majority of fluorescence emitted by the GFP marker protein of the cells treated with Fos, with FR-900098 or LK2 accumulated in the nucleus, indicating an efficient inhibition of the MEP pathway (Figure 5a,b). At this concentration, the mislocalization affected about 95% of the observed cells. The only exception was LK1, which needed considerably higher concentrations to cause a similar effect.\n\na) Confocal images were taken after treatment with different inhibitors of DXR, under standard conditions (3 h pretreatment, followed by 15 h induction with 10µM dexamethasone). Control: Cells were treated with the same volume of solvent (DMSO). Inhibitors: All inhibitors (Fosmidomycin = FOS, FR-900098, LK1 and LK2) were tested at 100µM and caused a significant translocation of the GFP fusion protein to the nucleus/nucleolus. White bars = 10µm. b) Quantitative analysis of H6-GFP-BD-CVIL localization in BY-2 cells treated with inhibitors of DXR. Percentage of cells showing a partial localization of the fluorescence to the nucleus and nucleolus (without taking care of the total intensity of this translocation) and the plasma membrane. For each treatment more than 100 individual cells were counted.\n\nA major disadvantage of phosphonate drugs is that the phosphonate group is highly deprotonated at physiological pH. Because of the resulting high polarity (and/or low lipophilicity), the transport across biological membranes and the general bioavailability are restricted (cf.77). A common strategy to overcome this problem is masking the phosphonic acid moiety by esterification. For instance, double ester drugs of FR-900098 have shown a 2 to 3 fold higher biological activity compared to their unmodified models in experiments with malaria-infected mice78.\n\nSeveral double ester prodrugs based on the original structures of LK1 (derived from fosmidomycin) and LK2 (derived from FR-900098) were recently synthesized79. They will be referred to as SP1 to SP6 in this work (Figure S4). The goal was to enhance the bioavailability of both parent compounds by overcoming barriers, such as poor uptake of the drug by target organisms. After entering the cell by diffusion through the cell wall and transport through the PM, the drug should be released by esterases and chemical hydrolysis. In addition, the drug has to overcome a second barrier within BY-2 cells, the plastid envelope, to reach its target enzyme. The experiments were performed for two major reasons: First of all as a proof of concept to validate the bioassay with yet untested inhibitors. Second, it was desirable to see how the prodrugs will act in comparison to the reference compound Fos or the direct drug “role models”, LK1 and LK2. It was also important to see whether the bioassay may prove to be valuable for a statistical approach. As all inhibitors, with the exception of LK1, were found to be totally inhibitory at 100µM we started testing six prodrug derivatives of LK1 at 50µM79 to detect a first “all-or-nothing” effect. At 50µM, all the six prodrugs were able to induce a mislocalization of the H6-GFP-BD-CVIL fusion protein in at least 50% of treated cells. The cells were counted by the operator of the fluorescence microscope at low magnification (using a 10x apochromatic objective) and divided into three major groups-according to their respective phenotype – to facilitate a statistical evaluation (Figure 6a):\n\n1. Cells in which the fluorescence emitted from the nucleus was clearly dominant;\n\n2. Cells with significant intensity of fluorescence still emitted from the PM (nucleus and membrane were more or less equal in fluorescence intensity);\n\n3. Cells in which the fluorescence was mainly associated with the PM and looking like the untreated control (the important part being that no fluorescence was visible in the nucleolus).\n\nSix molecules have been tested in total. The tested molecules can be divided into two groups. I) Three non-methylesterprodrugs (derived from LK1: SP1, SP3 and SP5). II) Three molecules with a methyl group bound to the N-atom (derived from LK2: SP2, SP4 and SP6). The respective phosphonate groups were masked as acyloxymethyl phosphonate esters79. Cells were pre-treated for 3 h with 50µM of each inhibitor (solvent: methanol) before expression of H6-GFP-DB-CVIL was induced by addition of dexamethasone (10µM final concentration). At the chosen concentration, the majority of all treated cells showed partial or very dominant delocalization of the fluorescence from the plasma membrane to the nucleus/nucleolus. The images are overlays from pictures in transmission light and fluorescence mode and were acquired as described in the main text. Bars = 10µm. b) Statistical approach to compare the impact of six different pro-drugs on the in vivo localization of the prenylable fusion protein H6-GFP-BD-CVIL. The cells (> 100) were treated with 50µM of the respective inhibitor. Cells were counted by view and sorted in three main categories (dominantly membrane localized (PM), nucleus localized and intermediate localization). The number of cells analyzed in each condition is indicated. c) Statistical approach to compare the impact of six different pro-drugs on the in vivo localization of the prenylable fusion protein H6-GFP-BD-CVIL. The cells were treated with 5µM of the respective inhibitor. Cells (> 100) were counted by view and sorted in three main categories as described above.\n\nThe results obtained at a final concentration of 50µM indicated that most of the prodrugs, especially SP2 and SP4, caused a fluorescence shift comparable to that observed after treatment with 50µM Fos (Figure 6b). Among the six prodrugs, SP2 and SP4 appeared to be very active. In both cases, more than 80% of counted cells showed a dominant fluorescent signal from the nuclear region of the cell, indicating a significant inhibition of GGPP biosynthesis through the MEP pathway. The other compounds did not act so efficiently, such as Fos. In summary, a general tendency could be observed, clearly indicating that the methylated analogs of the respective prodrugs were far more efficient than their non-methylated counterparts. This corroborates the results obtained with LK1 and LK2 (cf.76) as well as those obtained with Fos and FR-900098 (cf.78).\n\nHowever, at 50µM it was not possible to determine whether the prodrug SP2 was a better inhibitor than Fos. To address this issue, we therefore conducted a second set of experiments, using the inhibitors at a final concentration of 5µM. At this concentration, two major results are noteworthy (Figure 6c): First of all, the efficiency of Fos to induce a mislocalization of the fusion protein drops dramatically. More than 30% of the cells treated with 5µM Fos did not show any fluorescence translocation to the nucleus and gave a phenotype similar to that of the untreated control cells. In addition, the percentage of cells with a dominant fluorescence signal emitted by the nucleus (among the remaining cells with a partial mislocalization) decreased by more than 50%. In contrast, SP2 as well as SP4 only showed a slightly reduced efficiency to induce a dominant mislocalization of the GFP fusion protein from the PM to the nucleus. For SP2, 72% of the cells were still significantly inhibited by the treatment at 5µM. As for SP4, the fact that over 90% of cells had mislocalization patterns indicates that there was almost no detectable loss of efficiency after a 10-fold dilution from 50 to 5µM.\n\n\nDiscussion\n\nThe inhibition of the sterol biosynthetic pathway was used as a tool to check whether a possible re-direction of the metabolic flux in BY-2 cells, in particular FPP molecules non-incorporated into sterols, could lead to a change in the GFP fluorescence pattern or even contribute to overcome the Fos-induced mislocalization of the H6-GFP-BD-CVIL, reported earlier. The fact that MVA, at high concentrations, was able to rescue the fluorescence at the PM, whereas farnesol (Fol) only partially complemented the inhibition by Fos suggests (among other possibilities) the existence of a bottleneck for the synthesis of GGPP from FPP in the cytosol (no GGPP synthase expression, under our conditions), or the incapacity of FPP to be translocated into the plastid, where the formation of GGPP could take place.\n\nThis bottleneck is perhaps HMG-CoA reductase (HMGR), which is generally considered to catalyze the rate-limiting step in sterol synthesis, for both animals2 and plants7,8,80. This is supported by the fact that tobacco plants over-expressing HMGR were shown to produce higher amounts of sterol intermediates and end products81,82, whereas the over-expression of a FPP synthase in Arabidopsis did not result in an increased production of sterols83.\n\nDue to previous experiments with sterol biosynthesis inhibitors we had some experience on the effective concentrations of SQ and Tb in BY-2 cells38. SQ was shown to be a very efficient inhibitor of SQS (IC50 = 5.5nM), almost completely inhibiting SQS activity after treatment for 24 h at 50nM. In addition, the same concentration of SQ led to a 97% decrease in total radioactivity incorporated into sterols, when radiolabeled [14C] sodium acetate was added for 2 h before cell harvesting. At 0.5µM, the cells showed a significant decrease in cell mass (50%), which correlates very well with the stunted growth observed during this study. In a preceding study it was demonstrated that the presence of SQ led to a growth arrest of BY-2 cells in the early G1 phase, but did not cause any cytotoxic effect or apoptosis26. Inhibition of SQS by SQ was also shown to trigger a stimulation of HMGR expression and activity38.\n\nInhibition by Tb was revealed to cause a significant decrease in the cell sterol content as well as a massive accumulation of squalene, which accumulated in cytosolic lipid bodies. However, the cell growth was not impaired as indicated by the analysis of the cell mass after treatment with 30µM Tb for 24 h. Interestingly, Tb treatment also stimulated HMGR activity, but not the corresponding transcript levels38.\n\nKeeping all this in mind, how can the particular phenotype observed with SQ-treated cells, i.e. the partial mislocalization of the GFP fusion protein be explained? In fact, in a first set of experiments, we added 20µM exogenous Fol to BY-2 cells treated with both Fos and SQ. In cells treated with SQ only (control experiment), a partial mislocalization of the fluorescence was observed in the majority of treated cells (> 85%), whereas neither Tb nor Ro-treated cells displayed a similar effect. In cells treated with both SQ and Fos and complemented with Fol, the same pattern of partial mislocalization of the fluorescence was observed. It needs to be pointed out that this mislocalization was clearly visible, but less pronounced at the level of a single cell, as compared to Fos- or OC-treatments.\n\nHypothetically, due to SQS inhibition, the redirection of the metabolic flux might provide much more FPP to other FPP-metabolizing enzymes, such as GGPP synthase or protein farnesyltransferase. FPP in excess could also be transported to mitochondria (cf.84) or other organelles or converted to Fol, which might become cytotoxic85,86. Feeding experiments with radiolabeled farnesol showed that all these scenarios could occur. However, inhibition with SQ did not lead to an additional incorporation of FPP into the ubiquinone side-chain84.\n\nThus, a protein farnesyltransferase (PFT) might use the excess FPP to non-specifically farnesylate the H6-GFP-BD-CVIlL fusion protein, despite its geranylgeranylation motif. Some in vitro cross-reactivity of PFT from Arabidopsis has been suggested87, however such observations might not reflect the situation in vivo. By the same token, a PGGT-1 might accept FPP as substrate if present at concentrations that are much higher than that of GGPP. When the CVIL motif in our fusion protein was mutated to CVIM, thus transforming it into a potential substrate for PFT, it resulted in a fluorescence pattern with the majority of fluorescence still being associated to the PM of the cell, but also significantly present in and around the nuclear membrane, with less in the nucleus and nucleolus88,89. As the basic domain of the chimeric protein not only serves as a second signal for protein prenylation (besides the CaaX motif), but also contains a putative nuclear localization sequence (NLS), it is possible to imagine that the farnesyl moiety is not sufficient either to guarantee an efficient integration in the PM (because of a shorter hydrophobic domain) or to mediate the transport to the PM (which is still an open question for H6-GFP-BD-CVIL). In addition, the occurrence of several farnesylated, nuclear proteins have been recently reported90. Very recently Chandra et al.91 identified a role for the GDI-like solubilizing factor (GSF) photoreceptor cGMP phosphodiesterase δ subunit δ(PDEδ) in modulating signaling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. In this study it was shown that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm91. This would allow more effective trapping of (depalmitoylated) Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane91.\n\nStudies with SQ in mammalian systems revealed that besides acting as a specific inhibitor of SQS, SQ was able to inhibit mammalian prenyltransferases, in particular protein farnesyl transferase (PFT)39,92. PFT and PGGT-1, purified from bovine brain, were inhibited by SQ in vitro with an IC50 of 216 and 620nM, respectively92. As we applied comparably high concentrations of SQ (500nM to 1µM) to BY-2 cells, it seems quite reasonable to assume that the treatment with SQ might also have affected the PGGT-1 enzyme in vivo, responsible for the prenylation of the H6-GFP-BD-CVIL, even though in vitro data are not yet available. In addition, by using lower concentrations of SQ (50nM already blocked most of SQS activity), the protein prenyltransferase should theoretically not be inhibited, allowing us to more or less rule out the cross-reactivity and the farnesylation scenario. In order to investigate the capability of isoprenols like geraniol (Gol), farnesol (Fol) and geranylgeraniol (GGol) and intermediates of both isoprenoid biosynthetic pathways (MVA-pathway: mevalonolactone (MVL) and its open-acid form MVA; MEP-pathway: DX) to overcome the (partial) mislocalization induced by SQ, different chemical complementation experiments were performed. Interestingly, both DX and GGol completely complemented the SQ-induced effect. As discussed extensively before, the most likely explanation is an efficient uptake and metabolization of both compounds: DX, after being converted to DXP by cytosolic xylulose kinase30 enters the plastid and is incorporated into IPP, GPP and GGPP, which might then be exported towards the cytosol, where it serves as a substrate for prenylation of the H6-GFP-BD-CVIL. For GGol, again, two scenarios are possible: either its import into the plastid, phosphorylation and export to the cytosol, or direct phosphorylation in the cytosol. The existence of a system for phosphorylation of prenyldiphosphates was mentioned previously93.\n\nSQ is known to be an irreversible, competitive inhibitor of squalene synthase (SQS). Therefore, the DX-induced recovery of the PM fluorescence itself cannot result from an effect on SQS. As SQ structurally resembles FPP, it would be more likely that, by increasing the pool of bio-available GGPP, we succeeded in replacing SQ by GGPP at the substrate-binding site of PGGT-1 (or even both prenyltransferases) and thereby restored the correct geranylgeranylation of H6-GFP-BD-CVIL.\n\nInterestingly, mevalonate kinase is down-regulated at the post-transcriptional level by high levels of FPP and GGPP94. Therefore, if the tobacco BY-2 mevalonate-kinase were also sensitive to the feedback-regulation by FPP and GGPP, this would very conveniently explain why externally fed MVA could not complement the SQ-induced phenotype due to a large pool of FPP. Moreover, the resulting decrease in cytosolic IPP due to the block in the MVA pathway could also prevent a sufficient supply of IPP for the putatively cytosolic conversion of FPP to GGPP.\n\nThe fact that Gol does not complement the effects of SQ is in agreement with previous results88 and suggests that Gol is not accepted as a substrate for the phosphorylation system that exists in plants93. Intriguingly, exogenously applied Fol was previously able to restore the PM localization of H6-GFP-BD-CVIL to over 60% in Fos-treated cells88. However, as there are very specific inhibitors that act either on PFT or on PGGT-1, and are thus useful for “calibration”, in a follow-up experiment an unknown inhibitor giving a “hit”, but then applied at much lower concentration and carrying out similar complementation assays, would easily distinguish between inhibition of sterol biosynthesis or protein prenylation.\n\nSome previous studies in our laboratory focused on the intracellular transport of the prenylated GFP-fusion protein from the ER to the PM in BY-2 cells88. However, the transport of GFP-BD-CVIL was not significantly affected by any treatment targeting either components of the cytoskeleton (microtubules: taxol and oryzalin; actin-filaments: cytochalasin D) or the trans-Golgi-network (TGN: brefeldin A88), suggesting that GFP-BD-CVIL was not transported to the PM via the classical secretory pathway. This result was surprising, as this vesicular transport route is used by the majority of identified prenylated proteins95, such as HRAS, NRAS and KRAS4A in mammals. But the correct targeting of Ras proteins is successfully accomplished only when the proteins contain a second “signal” in addition to their prenylation. In the case of HRAS, NRAS and KRAS4A this second signal consists of one or more S-acylation sites (“palmitoylation”)96,97, whereas KRAS4B contains a lysine-rich polybasic (hypervariable) domain, located nearby the prenylation side98–100. The same combination, a polybasic domain with a cluster of basic amino acids, in addition to the CAAX motif is also found in the H6-GFP-BD-CVIL fusion protein.\n\nBesides this structural resemblance, KRAS4B is also insensitive to inhibitors of vesicular transport and transits to the PM by a largely unknown, non-vesicular (Golgi-independent) pathway95,101,102 that requires, at least in yeast, mitochondrial function and vacuolar sorting proteins (VPS)103.\n\nDespite the fact that corresponding experimental efforts with transformed BY-2 cells were not crowned by much success88, the study of molecules that could interfere with the transport of the H6-GFP-BD-CVIL fusion protein might be a promising tool to observe the effects of post-prenylation inhibition in plants in general, and this knowledge might even be transferred to other models.\n\nOverall, two sets of experiments have been conducted in the context of validating the test system. Two groups of novel inhibitor candidates, each targeting a specific enzyme of the MEP pathway, DXS in the first and DXR in the second case, were tested to assess their capacity to inhibit the in vivo isoprenylation (more precisely: geranylgeranylation) of the chimeric H6-GFP-DB-CVIL protein.\n\nFirst, six compounds that were inspired by known inhibitors of pyruvate decarboxylases and pyruvate dehydrogenases were tested. In summary, all tested pyruvate derivatives were inefficient in our in vivo assay, despite the fact that one of them, TJ150, induced cytotoxic effects at extreme concentrations (Figure 4). At this point two scenarios were plausible: either their uptake into the cells and subsequent transport through the plastidial envelope might have been impaired or they did not inhibit their molecular target, DXS. However, in parallel to the tests conducted with the H6-GFP- BD-CVIL cell line, in vitro DXS activity from E. coli was determined in the presence of some of the tested molecules using an end-point assay, in which the remaining pyruvate substrate was converted to lactate by lactate dehydrogenase. The consumption of NADH (and the formation of NAD+) can be measured by spectrometry at 340nm (cf.57). None of the tested compounds exhibited an inhibitory effect, in agreement with the results obtained with our bioassay (D. Tritsch, unpublished observations).\n\nThe second set of experiments focused on inhibitors and novel prodrugs targeting the second enzyme of the plastidial pathway, DXR. Only recently, analogs of Fos and FR-900098 with a rearrangement of the hydroxamate group had been tested in vitro76. Those compounds (here referred to as LK1 and LK2) successfully inhibited E. coli DXR. The IC50 values measured were 170nM and 48nM for LK1 and LK2, respectively. The IC50 value of LK2 was even comparable to that of Fos (32nM). In addition, both molecules inhibited the growth of E. coli, but with a lower efficiency than Fos76. Interestingly, the N-methylated compound (LK2 – derived from FR-900098) also successfully inhibited the growth of an E. coli strain that was cross-resistant to Fos and fosfomycin, which bears some faint structural similarity, but acts on another molecular target, UDP-N-acetylglucosamine enolpyruvyltransferase104. Among other reasons, this could be due to a difference in the uptake or detoxification of these two antibiotics, which are known to share a common import mechanism via the L-3-glycerolphosphate and the glucose 6-phosphate pathway in E. coli105. As the N-methylated compound LK2 nearly equaled Fos in terms of in vitro inhibition of the DXR enzyme76, changing the bioavailibility of this compound was a promising strategy to further enhance the performance of the hydroxamic drugs and provide new active compounds against resistant pathogen strains.\n\nThe in vivo experiments with transformed BY-2 cells were conducted under similar conditions: clonal cells tested under pre-defined standard conditions were counted and examined by visual inspection after different treatments (cf. Figure 7).\n\nGFP expression was induced according to the previously described standard protocol. Both images were taken with a confocal laser scanning microscope (Zeiss LSM510 equipped with an inverted Axiovert 100M). HM-High magnification image taken with a 63x water immersion objective (C-Apochromat 63x/1.2W M27), which is specifically designed for the examination of an aqueous specimen and delivers high resolution in optical thin sections, with satisfying levels of fluorescence brightness. LM-Low magnification image taken with a 10x \"Enhanced Contrast Plan-Neofluar\" universal objective (EC Plan-Neofluar 10x/0.3 M27), adapted for general observations in fluorescence microscopy. If not otherwise stated, the label HM and LM designates the use of these both types of objectives for the image acquisition with the confocal laser scanning microscope. White bars = 20µm. Please, note the different saturation of the images and the significantly higher saturation in the LM image that was used to highlight the homogenous levels of bright fluorescence of the re-selected clonal cell line.\n\nThe test of the six new prodrugs79, here referred to as SP1 to SP6, includes a whole new aspect: for the first time, novel drugs specifically designed on the basis of known pathway inhibitors were tested under standardized conditions and the generated data were exploited in both a qualitative and a quantitative/statistical way. This means that, besides answering the question of whether an inhibitor candidate could efficiently block the MEP pathway, the exploitation of the same set of data would allow a direct comparison of the efficiency of this compound with established MEP pathway inhibitors. By testing the inhibitors at different concentrations, we were able to first define a starting point for the experiments, which consisted in a concentration that inhibited the majority of cells (> 95% of cells showing a dominant mislocalization). The results obtained at 100µM clearly proved that Fos and FR-900098 as well as LK2 were applied at saturating concentrations. We therefore decided to better start our direct comparison at 50µM. At this concentration, Fos, SP2 and SP4 were very active, causing a partial mislocalization in nearly 100% of the cells. The other tested prodrugs did not act that well at this concentration, even though they were able to induce a partial mislocalization in at least 50% of treated cells. At 5µM, only SP2 and SP4 proved to be more efficient than Fos, whereas the other inhibitors only induced a barely detectable effect. At 500nM final concentration, SP2 and SP4 were still able to induce a mislocalization of the GFP fluorescence in more than 50% of the cells (data from a single experiment).\n\nObtaining only two out of the six compounds with a higher efficiency than the respective model drugs might appear deceiving at first view. However, it is a fine example confirming the prodrug concept (Figure S1)106,107. The efficiency of a prodrug in an in vivo assay indeed depends on several parameters, such as transfer through biological membranes and the stability of the prodrug against hydrolysis or esterases.\n\nAll the six prodrugs tested in these experiments were able to induce a localization shift of the prenylable GFP fusion protein to some extent, which indicates that the active drug at least partially entered the plant cell, where it could inhibit the DXR enzyme (located in the plastidial compartment). This point is important to mention, as it indicates that:\n\ni) The prodrugs were soluble in the BY-2 medium;\n\nii) They successfully crossed the plant cell wall;\n\niii) They were imported inside the plant cell across the PM.\n\nHow this transport took place and which transporters were involved remains unclear, even though data from bacteria suggest that a cAMP-dependent glycerol 3-phosphate transporter could be a likely candidate108. Until now, a similar transporter has only been identified in the mitochondrial membrane of plant cells109.\n\nBy relating the results to the structural features of the tested molecules, another important observation can be made. The data obtained for all inhibitors showed that the methylated prodrugs displayed a far better activity than their non-methylated counterparts. This result correlates with observations obtained with Fos, FR-90098, LK1 and LK2 in bacterial systems76,110. Interestingly, the best performing methylated prodrug SP4 also shared the same pro-moieties with the best performing non-methylated prodrug SP3. Although the better performance of the methylated drugs, such FR-900098 or LK2, could be attributed to the hydrophobic interaction of the methyl-group at the catalytic site of the DXR enzyme, the methyl group may also have an influence on the lipophilicity of the prodrug and be responsible for its overall better import rate in tobacco BY-2 cells (Figure S2). Other factors could be the different preferences of endogenous esterases for the three types of substituents used to synthesize the prodrugs or the rate of spontaneous hydrolysis (that may occur either in the extracellular medium or the cytosol).\n\nIt should be noted that the best performing prodrugs in the bioassay with BY-2 cells showed the same order of efficiency when tested on Mycobacterium smegmatis cells, suggesting that knowledge gained from plant cells might be transferred to other organisms79. Nevertheless, keeping in mind the potential of MEP pathway inhibitors as herbicides, these results are very promising and set the course for the development of a chemical drug screen, with both biomedical and agricultural applications, particularly in the light of newly emerging strains of multi-drug resistant bacterial pathogens.\n\nThe experimental approaches used and applied to the specific situation in tobacco BY-2 cells synthesizing isoprenoids via two compartmentalized pathways and expressing the reporter GFP fusion protein are summarized in Figure S3. The scheme indicates also some more hypothetical links to observations in mammalian cells, for instance as to the post-prenylation reactions and the transport of covalently modified proteins to their intracellular destination.\n\n\nMethods and materials\n\nIf not indicated otherwise, chemicals used were provided by Sigma-Aldrich/Fluka. Squalestatin (Sq) was obtained from Glaxo (Greenford, Middelsex, UK) and dissolved in 0.1M Tris-HCl (pH 7.4) to give a 0.1mM stock solution. Terbinafine (Tb) was kindly provided by Dr. N.S. Ryder (Vienna) and dissolved in dimethyl sulfoxide. MVA lactone, Gol, Fol, and GGol were purchased from Sigma-Aldrich. Mevinolin (MV) was a kind gift from Dr. Alfred Alberts (MSD, Rahway, NJ). Before use, the lactones of MV and MVA were converted to their open acid forms. Fosmidomycin (Fos)111 was made available by Robert J. Eilers (Monsanto, St. Louis, MO). The peptidomimetic protein geranylgeranylation inhibitor GGti-2133112 was purchased from Calbiochem (Merck). Oxoclomazone (OC) was kindly supplied by Dr. Klaus Grossmann (BASF, Limburgerhof, Germany). Please see Table 1 for a list of compounds and their final concentration used in this study\n\nThe original untransformed tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cell line used in this work was provided by Professor Toshiyuki Nagata (University of Tokyo, Japan). It was initially isolated by Kato et al.113 and is derived from calli induced from young plants114. The cell suspension was maintained by subculturing the 7-day-old cells (in stationary phase) as described1,28,85.\n\nThe transgenic dexamethasone-inducible Tobacco BY-2 H6-GFP-BD-CVIL cell line has been extensively described by Gerber et al.1 and was maintained as described.\n\nRoutinely, 0.75ml of a 7-day-old culture was diluted into 40ml of modified Murashige and Skoog medium115. The cells were then cultured at 27°C in the dark on a rotary shaker (154 rpm).\n\nThe inducible test system (cf.116) has been adjusted to the 7-day growth cycle of TBY-2 cells. After 6-fold dilution in fresh medium, the cells were treated in commercial multi-well plates. Total volume of treated cells was scaled to 3ml. In general, initial screenings started between 50 and 100µM of a given inhibitor/prodrug and were refined if a delocalization occured. Induction of the fluorescent marker proteins took place 15 to 24 h before observation; the treatments were scheduled 3 to 6 h after the induction. Observation and image acquisition were made by laser scanning microscopy as described (Figure S2).\n\nCell imaging was performed using a LSM510 confocal laser scanning microscope equipped with an inverted Zeiss axiovert 100M microscope (Carl Zeiss, Jena, Germany). For statistical observations, such as counting cells after various treatments, a 10x Zeiss objective (“Plan-Neofluar”) was used. At confocal resolution, images were taken using a 63x, 1.2 numerical aperture water immersion objective (“C-Apochromat”). In both cases, differential interference contrast (DIC) images were recorded.\n\nEpifluorescence microscopy was performed using a Nikon E 800 fluorescence microscope equipped with a Nikon DXM1200 CCD high-resolution color camera (20 x 0.45 objective) and ACT-1 image software. Images were processed with the latest version of the Zeiss LSM Image Browser software (Carl Zeiss, Jena, Germany) and exported as Tiff or JPEG files. Final image analysis and adjustments was performed using either Photoshop version 8.0 (Adobe Systems, San Jose, USA) or the ImageJ software version 1.31 (Wayne Rasband, NIH, USA).",
"appendix": "Author contributions\n\n\n\nMichael Hartmann was responsible for the experimental design and carried out the majority of analyses with confocal microscopy. In addition he transformed, selected and maintained BY-2 cell lines. He generated the figures, wrote major parts of the initial manuscript as part of his PhD thesis and contributed to the final version of the manuscript. Andréa Hemmerlin and Elisabet Gas-Pascual transformed, selected and maintained BY-2 cell lines and carried out complementary experiments. Esther Gerber conducted the initial experiments with transformed BY-2 cells with in the frame of a Ph.D. thesis, which paved the way to the sequel studies described here. Denis Tritsch has carried out enzymatic studies with MEP pathway inhibitors designed by Michel Rohmer and synthesized in his laboratory. Michel Rohmer was instrumental in the interpretation of results and Denis Tritsch selected the best conditions for application of inhibitors to BY-2 cells. Thomas J. Bach initiated and supervised all those studies with the GFP fusion protein expressing BY-2 cells and their use as to the cross-talk between the cyto plasmic MVA and the plastidial MEP pathways. He brought the manuscript into its updated and final form. The project, especially inhibitor testing, represented an essential part of the repeatedly funded collaboration between the research groups of Michel Rohmer and Thomas J. Bach. All authors critically revised the manuscript and agreed the final version for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe are grateful for a Ph.D. fellowship attributed to Michael Hartmann provided by the Région Alsace. The research conducted in the laboratories of T.J. Bach and M. Rohmer was supported by grants from the Agence Nationale de la Recherche (ANR-05-BLAN-0217-01 and -02 TERPENE; ANR-06-BLAN-0291-02 BIOSIS). General support was also provided by the Centre national de la recherché scientifique (CNRS) and the Université de Strasbourg.\n\n\nAcknowledgements\n\nWe wish to thank Prof. Nam-Hai Chua (Rockefeller University) for making available dexamethasone and estrogen inducible expression vectors. We are indebted to Sarah Ponaire, Catherine Zinglé and Catherine Grosdemange-Billiard for the synthesis of MEP pathway inhibitors. Thanks go to Dr. Jérôme Mutterer (Microscopy Platform at the IBMP) for his advices and the maintenance of confocal microscopes and corresponding software.\n\n\nSupplementary figures\n\nThe prodrug is a chemically modified version of the pharmacological active compound. The addition of a covalently linked “promoiety” masks the parent drug and improves for example its physicochemical or pharmacokinetic properties. A) If the parent drug has difficulties overcoming a barrier, such as a biological membrane or is poorly soluble, the addition of a promoiety can be seen as a strategy to overcome this barrier. In an optimal scenario, the prodrug releases the active drug after removal of the promoiety by enzymatic or chemical transformation (“bioactivation”). The drug should ideally be recovered at high rates and the promoiety should be non-toxic. B) In the case of the inhibitors synthesized and described elsewhere79 in the model system BY-2, the prodrugs are ester derivatives of phosphonohydroxamic drugs. After entering the cell by diffusion through the cell wall (CW) and transport through the plasma membrane (PM), the drug is supposed to be released by esterases and/or chemical hydrolysis. In addition, the drug has to overcome a second barrier within the cell, the double membrane of the plastid, to reach its target within the plant cell. Inhibition of DXR activity leads to a dramatic change in the pool of relevant precursors for the geranylgeranylation of H6-BD-GFP-CVIL. This terpenoid precursor, GGPP, is almost completely derived from the plastidial MEP pathway in our test system. Without the prenylation of the C-terminal moiety, the fusion protein is likely to mislocalize from the PM (and to some extent the nuclear membrane) to the nuclear compartment, which is due to the presence of a nuclear localization sequence (NLS) in the truncated peptide derived from OsCaM61 (for further explanation: Gerber et al.1).\n\nThe test system is based on a stably transformed tobacco BY-2 cell line expressing the chimeric, prenylable fusion protein H6-BD-GFP-CVIL. The expression is under the control of a dexamethasone-inducible promoter116. The usual timeline for an experiment consisted of 3 h treatment followed by an induction of protein expression varying between 15–48 h depending on the experimental setup. During that period the cells were grown under constant shaking in the dark at 27°C before the phenotype was analyzed using confocal laser microscopy.\n\nIn previous studies, the following phenotypes were observed after treatments of the transgenic H6-GFP-DB-CVIL cell line with inhibitors of both isoprenoid biosynthetic pathways: Inhibition of the cytosolic MVA pathway by mevinolin (MEV) a specific inhibitor of HMG-CoA reductase (HMGR) did not exhibit any detectable effect on the intracellular localization of the prenylable GFP fusion protein which predominantly associated with the plasma membrane (PM), as observed under control conditions. Specific inhibition of DXS and DXR, key enzymes in the plastidial MEP pathway by oxoclomazone (OC) and fosmidomycin (Fos), respectively, however lead to a dramatic mislocalization of the GFP fluorescence (compare main text for more detailed information) to the nuclear compartment (N/Nu). A similar effect was observed with compounds that interfere with protein geranylgeranyltransferase type I (PGGT-1). This differential localization of the GFP fusion protein in response to inhibitors of the two coexisting routes leading to the synthesis of the universal C5 precursors IPP (and its isomer DMAPP) in plants indicates the suitability of this system for screening of compounds with potential antimicrobial and herbicidal activities. In the present study this potential for drug screening will be addressed by testing novel compounds, specifically designed to inhibit DXS and DXR enzyme activity, thereby validating the test system (yellow box A). In addition, the impact of various inhibitors targeting enzymes involved in the early steps of phytosterol biosynthesis, one of the main routes of cytosolic FPP within the plant cell, will be investigated (yellow box B). Finally, the effect of post-prenylation inhibitors on the localization/transport of H6-GFP-DB-CVIL is addressed, especially in the light of prior experiments aimed at clarifying the details of its yet unknown transport to the PM (yellow box C). These experiments have to be seen in the context of the fate and intracellular transport of typical mammalian isoprenylated proteins, such as members of the RAS family, that play an important role in the development of certain varieties of cancer (compare with discussion in the main text).\n\n\nReferences\n\nGerber E, Hemmerlin A, Hartmann M, et al.: The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells. Plant Cell. 2009; 21(1): 285–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoldstein JL, Brown MS: Regulation of the mevalonate pathway. Nature. 1990; 343(6257): 425–430. PubMed Abstract | Publisher Full Text\n\nEdwards PA, Ericsson J: Sterols and isoprenoids: signaling molecules derived from the cholesterol biosynthetic pathway. Annu Rev Biochem. 1999; 68: 157–185. PubMed Abstract | Publisher Full Text\n\nVeillard NR, Mach F: Statins: the new aspirin? Cell Mol Life Sci. 2002; 59(11): 1771–1786. PubMed Abstract | Publisher Full Text\n\nBuhaescu I, Izzedine H: Mevalonate pathway: A review of clinical and therapeutical implications. Clin Biochem. 2007; 40(9–10): 575–584. PubMed Abstract | Publisher Full Text\n\nMiziorko HM: Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys. 2011; 505(2): 131–143. PubMed Abstract | Publisher Full Text\n\nBach TJ: Hydroxymethylglutaryl-CoA reductase, a key enzyme in phytosterol synthesis? Lipids. 1986; 21(1): 82–88. PubMed Abstract | Publisher Full Text\n\nBach TJ: Some new aspects of isoprenoid biosynthesis in plants – a review. Lipids. 1995; 30(3): 191–202. PubMed Abstract | Publisher Full Text\n\nDisch A, Hemmerlin A, Bach TJ, et al.: Mevalonate-derived isopentenyl diphosphate is the biosynthetic precursor of ubiquinone prenyl side chain in tobacco BY-2 cells. Biochem J. 1998; 331(Pt 2): 615–621. PubMed Abstract | Free Full Text\n\nChappell J: The genetics and molecular genetics of terpene and sterol origami. Curr Opin Plant Biol. 2002; 5(2): 151–157. PubMed Abstract | Publisher Full Text\n\nAi-Xia C, Yong-Gen L, Ying-Bo M, et al.: Plant terpenoids: Biosynthesis and ecological functions. J Integr Plant Biol. 2007; 49(2): 179–186. Publisher Full Text\n\nLichtenthaler HK, Rohmer M, Schwender J: Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants. Physiol Plant. 1997; 101: 643–652. Publisher Full Text\n\nLichtenthaler HK, Schwender J, Disch A, et al.: Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway. FEBS Lett. 1997; 400(3): 271–274. PubMed Abstract | Publisher Full Text\n\nLichtenthaler HK: The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol. 1999; 50: 47–65. PubMed Abstract | Publisher Full Text\n\nRohmer M: The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat Prod Rep. 1999; 16(5): 565–574. PubMed Abstract | Publisher Full Text\n\nRohmer M: Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution. Pure Appl Chem. 2003; 75(2–3): 375–387. Publisher Full Text\n\nRohmer M: From molecular fossils of bacterial hopanoids to the formation of isoprene units: Discovery and elucidation of the methylerythritol phosphate pathway. Lipids. 2008; 43(12): 1095–1107. PubMed Abstract | Publisher Full Text\n\nRodríguez-Concepción M, Boronat A: Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol. 2002; 130(3): 1079–1089. PubMed Abstract | Publisher Full Text\n\nEisenreich W, Bacher A, Arigoni D, et al.: Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell Mol Life Sci. 2004; 61(12): 1401–1426. PubMed Abstract | Publisher Full Text\n\nKasahara H, Hanada A, Kuzuyama T, et al.: Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis. J Biol Chem. 2002; 277(47): 45188–45194. PubMed Abstract | Publisher Full Text\n\nGomez-Roldan V, Fermas S, Brewer PB, et al.: Strigolactone inhibition of shoot branching. Nature. 2008; 455(7210): 189–194. PubMed Abstract | Publisher Full Text\n\nUmehara M, Hanada A, Yoshida S, et al.: Inhibition of shoot branching by new terpenoid plant hormones. Nature. 2008; 455(7210): 195–200. PubMed Abstract | Publisher Full Text\n\nCroteau R, Kutchan T, Lewis N: Natural products (secondary metabolites), Biochemistry and Molecular Biology of Plants. In: Buchanan B, Gruissem W, Jones R, editors. Biochemistry and Molecular Biology of Plants. Rockville, MD: American Society of Plant Biologists. 2000; pp. 1250–1268. Reference Source\n\nHemmerlin A, Harwood JL, Bach TJ: A raison d'être for two distinct pathways in the early steps of plant isoprenoid biosynthesis? Prog Lipid Res. 2012; 51(2): 95–148. PubMed Abstract | Publisher Full Text\n\nHartmann M, Gas-Pascual E, Hemmerlin A, et al.: Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation. 2013.\n\nHemmerlin A, Fischt I, Bach TJ: Differential interaction of branch-specific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells. Physiol Plant. 2000; 110(3): 343–350. Publisher Full Text\n\nHemmerlin A, Bach TJ: Effects of mevinolin on cell cycle progression and viability of tobacco BY-2 cells. Plant J. 1998; 14(1): 65–74. PubMed Abstract | Publisher Full Text\n\nHemmerlin A, Brown SC, Bach TJ: Function of mevalonate in tobacco cell proliferation. Acta Bot Gall. 1999; 146(1): 85–100. Publisher Full Text\n\nHemmerlin A, Hoeffler JF, Meyer O, et al.: Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells. J Biol Chem. 2003; 278(29): 26666–26676. PubMed Abstract | Publisher Full Text\n\nHemmerlin A, Tritsch D, Hartmann M, et al.: A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway. Plant Physiol. 2006; 142(2): 441–457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwiezewska E, Thelin A, Dallner G, et al.: Occurrence of prenylated proteins in plant cells. Biochem Biophys Res Commun. 1993; 192(1): 161–166. PubMed Abstract | Publisher Full Text\n\nShipton CA, Parmryd I, Swiezewska E, et al.: Isoprenylation of plant proteins in vivo. Isoprenylated proteins are abundant in the mitochondria and nuclei of spinach. J Biol Chem. 1995; 270(2): 566–572. PubMed Abstract | Publisher Full Text\n\nBaxter A, Fitzgerald BJ, Hutson JL, et al.: Squalestatin 1, a potent inhibitor of squalene synthase, which lowers serum cholesterol in vivo. J Biol Chem. 1992; 267(17): 11705–11708. PubMed Abstract\n\nBergstrom JD, Kurtz MM, Rew DJ, et al.: Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase. Proc Natl Acad Sci U S A. 1993; 90(1): 80–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nProcopiou PA, Bailey EJ, Bamford MJ, et al.: The squalestatins: novel inhibitors of squalene synthase. Enzyme inhibitory activities and in vivo evaluation of C1–modified analogues. J Med Chem. 1994; 37(20): 3274–3281. PubMed Abstract | Publisher Full Text\n\nThelin A, Peterson E, Hutson JL, et al.: Effect of squalestatin 1 on the biosynthesis of the mevalonate pathway lipids. Biochim Biophys Acta. 1994; 1215(3): 245–249. PubMed Abstract | Publisher Full Text\n\nLiu CI, Jeng WY, Chang WJ, et al.: Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase. J Biol Chem. 2012; 287(22): 18750–18757. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWentzinger LF, Bach TJ, Hartmann MA: Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Physiol. 2002; 130(1): 334–346. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDufresne C, Wilson KE, Singh SB, et al.: Zaragozic acids D and D2: potent inhibitors of squalene synthase and of Ras farnesyl-protein transferase. J Nat Prod. 1993; 56(11): 1923–1929. PubMed Abstract | Publisher Full Text\n\nRyder NS: Terbinafine: mode of action and properties of the squalene epoxidase inhibition. Br J Dermatol. 1992; 126(Suppl 39): 2–7. PubMed Abstract | Publisher Full Text\n\nYates PJ, Haughan PA, Lenton JR, et al.: Effects of terbinafine on growth, squalene and steryl ester content of celery cell suspension culture. Pestic Biochem Phys. 1991; 40: 221–226. Publisher Full Text\n\nSimmen U, Gisi U: Effects of seed treatment with SAN 789F, a homopropargylamine fungicide, on germination and contents of squalene and sterols of wheat seedlings. Pestic Biochem Physiol. 1995; 52: 25–32. Reference Source\n\nFlores-Sánchez IJ, Ortega-López J, del Carmen Montes-Horcasitas M, et al.: Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa. Plant Cell Physiol. 2002; 43(12): 1502–1509. PubMed Abstract | Publisher Full Text\n\nAbe I, Rohmer M, Prestwich GD: Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem Rev. 1993; 93(6): 2189–2206. Publisher Full Text\n\nMorand OH, Aebi JD, Dehmlow H, et al.: Ro 48-8.071, a new 2, 3-oxidosqualene:lanosterol cyclase inhibitor lowering plasma cholesterol in hamsters, squirrel monkeys, and minipigs: comparison to simvastatin. J Lipid Res. 1997; 38(2): 373–390. PubMed Abstract\n\nLenhart A, Weihofen WA, Pleschke AE, et al.: Crystal structure of a squalene cyclase in complex with the potential anticholesteremic drug Ro48-8071. Chem Biol. 2002; 9(5): 639–645. PubMed Abstract | Publisher Full Text\n\nRodríguez-Concepción M, Toledo-Ortiz G, Yalovsky S, et al.: Carboxyl-methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein. Plant J. 2000; 24(6): 775–784. PubMed Abstract | Publisher Full Text\n\nChen Y: Selective inhibition of ras-transformed cell growth by a novel fatty acid-based chloromethyl ketone designed to target Ras endoprotease. Ann N Y Acad Sci. 1999; 886: 103–108. PubMed Abstract | Publisher Full Text\n\nSprenger GA, Schorken U, Wiegert T, et al.: Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc Natl Acad Sci U S A. 1997; 94(24): 12857–12862. PubMed Abstract | Free Full Text\n\nLange BM, Wildung MR, McCaskill D, et al.: A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway. Proc Natl Acad Sci U S A. 1998; 95(5): 2100–2104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLois LM, Campos N, Putra SR, et al.: Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc Natl Acad Sci U S A. 1998; 95(5): 2105–2110. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWalter MH, Hans J, Strack D: Two distantly related genes encoding 1-deoxy-D-xylulose 5-phosphate synthases: differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots. Plant J. 2002; 31(3): 243–254. PubMed Abstract | Publisher Full Text\n\nCostelloe SJ, Ward JM, Dalby PA: Evolutionary analysis of the TPP-dependent enzyme family. J Mol Evol. 2008; 66(1): 36–49. PubMed Abstract | Publisher Full Text\n\nWidmann M, Radloff R, Preiss J: The thiamine diphosphate dependent enzyme engineering database: A tool for the systematic analysis of sequence and structure relations. BMC Biochemistry. 2010; 11: 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuzuyama T, Takagi M, Takahashi S, et al.: Cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase from Streptomyces sp. Strain CL190, which uses both the mevalonate and nonmevalonate pathways for isopentenyl diphosphate biosynthesis. J Bacteriol. 2000; 182(4): 891–897. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHahn FM, Eubanks LM, Testa CA, et al.: 1-deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus. J Bacteriol. 2001; 183(1): 1–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiang S, Usunow G, Lange G, et al.: Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis. J Biol Chem. 2007; 282(4): 2676–2682. PubMed Abstract | Publisher Full Text\n\nXiang S, Usunow G, Lange G, et al.: 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis. In: Bach TJ and Rohmer M (eds) Isoprenoid Synthesis in Plants and Microorganisms: New Concepts and Experimental Approaches. Springer Science + Business Media New York 2013; pp. 17–28. Publisher Full Text\n\nMüller C, Schwender J, Zeidler J, et al.: Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis. Biochem Soc Trans. 2000; 28(6): 792–793. PubMed Abstract\n\nEubanks LM, Poulter CD: Rhodobacter capsulatus 1-deoxy-D-xylulose 5-phosphate synthase: steady-state kinetics and substrate binding. Biochemistry. 2003; 42(4): 1140–1149. PubMed Abstract | Publisher Full Text\n\nMatsue Y, Mizuno H, Tomita T, et al.: The herbicide ketoclomazone inhibits 1-deoxy-D-xylulose 5-phosphate synthase in the 2-C-methyl-D-erythritol 4-phosphate pathway and shows antibacterial activity against Haemophilus influenzae. J Antibiot (Tokyo). 2010; 63(10): 583–588. PubMed Abstract | Publisher Full Text\n\nLowe PN, Perham RN: Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli. Biochemistry. 1984; 23(1): 91–97. PubMed Abstract | Publisher Full Text\n\nMenon-Rudolph S, Nishikawa S, Zeng X, et al.: Rate of decarboxylation, monitored via the key enzyme-bound enamine, of conjugated a-keto acids by pyruvamide activated pyruvate decarboxylase is kinetically competent with turnover. J Am Chem Soc. 1992; 114(26): 10110–10112. Publisher Full Text\n\nBrown A, Nemeria N, Yi J, et al.: 2-Oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex. Biochemistry. 1997; 36(26): 8071–8081. PubMed Abstract | Publisher Full Text\n\nTakahashi S, Kuzuyama T, Watanabe H, et al.: A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis. Proc Natl Acad Sci U S A. 1998; 95(17): 9879–9884. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuzuyama T, Takahashi S, Takagi M, et al.: Characterization of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues. J Biol Chem. 2000; 275(26): 19928–19932. PubMed Abstract | Publisher Full Text\n\nHoeffler JF, Tritsch D, Grosdemange-Billiard C, et al.: Isoprenoid biosynthesis via the methylerythritol phosphate pathway. Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Eur J Biochem. 2002; 269(18): 4446–4457. PubMed Abstract | Publisher Full Text\n\nKoppisch AT, Fox DT, Blagg BS, et al.: E. coli MEP synthase: steady-state kinetic analysis and substrate binding. Biochemistry. 2002; 41(1): 236–243. PubMed Abstract | Publisher Full Text\n\nReuter K, Sanderbrand S, Jomaa H, et al.: Crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, a crucial enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. J Biol Chem. 2002; 277(7): 5378–5384. PubMed Abstract | Publisher Full Text\n\nSteinbacher S, Kaiser J, Eisenreich W, et al.: Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC). Implications for the catalytic mechanism and anti-malaria drug development. J Biol Chem. 2003; 278(20): 18401–18407. PubMed Abstract | Publisher Full Text\n\nMac Sweeney A, Lange R, Fernandes RP, et al.: The crystal structure of E. coli 1-deoxy-D-xylulose-5-phosphate reductoisomerase in a ternary complex with the antimalarial compound fosmidomycin and NADPH reveals a tight-binding closed enzyme conformation. J Mol Biol. 2005; 345(1): 115–127. PubMed Abstract | Publisher Full Text\n\nTakenoya M, Ohtaki A, Noguchi K, et al.: Crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase from the hyperthermophile Thermotoga maritima for insights into the coordination of conformational changes and an inhibitor binding. J Struct Biol. 2010; 170(3): 532–539. PubMed Abstract | Publisher Full Text\n\nZeidler J, Schwender J, Müller C, et al.: Inhibition of the non-mevalonate 1-deoxy-D-xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Z Naturforsch C. 1998; 53: 980–986. Reference Source\n\nFellermeier M, Kis K, Sagner S, et al.: Cell-free conversion of 1-deoxy-D-xylulose 5-phosphate and 2-C-methyl-D-erythritol 4-phosphate into β-carotene in higher plants and its inhibition by fosmidomycin. Tetrahedron Lett. 1999; 40: 2743–2746. Publisher Full Text\n\nKamuro Y, Kawai T, Kakiuchi T: Herbicidal methods and compositions comprising Fosmidomycin. Fujisawa Pharmaceutical Co. Ltd., U.S. Patent Application No. 5,002,602. March 26, 1991. Reference Source\n\nKuntz L, Tritsch D, Grosdemange-Billiard C, et al.: Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase. Biochem J. 2005; 386(Pt 1): 127–135. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaumeister S, Wiesner J, Reichenberg A, et al.: Fosmidomycin uptake into Plasmodium and Babesia-infected erythrocytes is facilitated by parasite-induced new permeability pathways. PLoS One. 2011; 6(5): e19334. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrtmann R, Wiesner J, Reichenberg A, et al.: Alkoxycarbonyloxyethyl ester prodrugs of FR-900098 with improved in vivo antimalarial activity. Arch Pharm (Weinheim). 2005; 338(7): 305–314. PubMed Abstract | Publisher Full Text\n\nPonaire S, Zinglé C, Tritsch D, et al.: Growth inhibition of Mycobacterium smegmatis by prodrugs of deoxyxylulose phosphate reductoisomerase inhibitors, promising anti-mycobacterial agents. Eur J Med Chem. 2012; 51: 277–285. PubMed Abstract | Publisher Full Text\n\nGondet L, Weber T, Maillot-Vernier P, et al.: Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme a reductase in a tobacco mutant that overproduces sterols. Biochem Biophys Res Commun. 1992; 186(2): 888–893. PubMed Abstract | Publisher Full Text\n\nChappell J, Wolf F, Proulx J, et al.: Is the reaction catalyzed by 3-hydroxy-3-methylglutaryl Coenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants? Plant Physiol. 1995; 109(4): 1337–1343. PubMed Abstract | Free Full Text\n\nSchaller H, Grausem B, Benveniste P, et al.: Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 in tobacco results in sterol overproduction. Plant Physiol. 1995; 109(3): 761–770. PubMed Abstract | Free Full Text\n\nMasferrer A, Arró M, Manzano D, et al.: Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels. Plant J. 2002; 30(2): 123–132. PubMed Abstract | Publisher Full Text\n\nHartmann MA, Bach TJ: Incorporation of all-trans-farnesol into sterols and ubiquinone in Nicotiana tabacum L. cv Bright Yellow cell cultures. Tetrahedron Lett. 2001; 42(4): 655–657. Publisher Full Text\n\nHemmerlin A, Bach TJ: Farnesol-induced cell death and stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in tobacco cv bright yellow-2 cells. Plant Physiol. 2000; 123(4): 1257–1268. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHemmerlin A, Reents R, Mutterer J, et al.: Monitoring farnesol-induced toxicity in tobacco BY-2 cells with a fluorescent analog. Arch Biochem Biophys. 2006; 448(1–2): 93–103. PubMed Abstract | Publisher Full Text\n\nAndrews M, Huizinga DH, Crowell DN: The CaaX specificities of Arabidopsis protein prenyltransferases explain era1 and ggb phenotypes. BMC Plant Biol. 2010; 10: 118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGerber E: Localisation cellulaire de protéines fluorescentes isoprénylables dans les cellules de tabac BY-2. Thèse doctorale, Université Louis Pasteur (ULP) Strasbourg, France 2005. Reference Source\n\nGerber E, Hemmerlin A, Bach TJ, et al.: The role of plastids in protein geranylgeranylation in tobacco BY-2 cells. In: The Chloroplast: Basics and Applications, CA Rebeiz et al. (eds), Advances in Photosynthesis and Respiration Vol 31, Springer Science + Business Media B.V., 2010; pp. 127–137. Publisher Full Text\n\nBarth O, Vogt S, Uhlemann R, et al.: Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29. Plant Mol Biol. 2009; 69(1–2): 213–226. PubMed Abstract | Publisher Full Text\n\nChandra A, Grecco HE, Pisupati V, et al.: The GDI-like solubilizing factor PDEd sustains the spatial organization and signalling of Ras family proteins. Nat Cell Biol. 2012; 14(2): 148–158. PubMed Abstract | Publisher Full Text\n\nGibbs JB, Pompliano DL, Mosser SD, et al.: Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo. J Biol Chem. 1993; 268(11): 7617–7620. PubMed Abstract\n\nThai L, Rush JS, Maul JE, et al.: Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions. Proc Natl Acad Sci U S A. 1999; 96(23): 13080–13085. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHinson DD, Chambliss KL, Toth MJ, et al.: Post-translational regulation of mevalonate kinase by intermediates of the cholesterol and nonsterol isoprene biosynthetic pathways. J Lipid Res. 1997; 38(11): 2216–2223. PubMed Abstract\n\nChoy E, Chiu VK, Silletti J, et al.: Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell. 1999; 98(1): 69–80. PubMed Abstract | Publisher Full Text\n\nHancock JF, Magee AI, Childs JE, et al.: All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989; 57(7): 1167–1177. PubMed Abstract | Publisher Full Text\n\nGoodwin JS, Drake KR, Rogers C, et al.: Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway. J Cell Biol. 2005; 170(2): 261–272. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHancock JF, Paterson H, Marshall CJ: A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990; 63(1): 133–139. PubMed Abstract | Publisher Full Text\n\nHancock JF, Cadwallader K, Paterson H, et al.: A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of RAS proteins. EMBO J. 1991; 10(13): 4033–4039. PubMed Abstract | Free Full Text\n\nMeder D, Simons K: Cell biology. Ras on the roundabout. Science. 2005; 307(5716): 1731–1733. PubMed Abstract | Publisher Full Text\n\nApolloni A, Prior IA, Lindsay M, et al.: H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol Cell Biol. 2000; 20(7): 2475–2487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilvius JR, Bhagatji P, Leventis R, et al.: K-ras4B and prenylated proteins lacking \"second signals\" associate dynamically with cellular membranes. Mol Biol Cell. 2006; 17(1): 192–202. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang G, Deschenes RJ: Plasma membrane localization of Ras requires class C Vps proteins and functional mitochondria in Saccharomyces cerevisiae. Mol Cell Biol. 2006; 26(8): 3243–3255. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchönbrunn E, Sack S, Eschenburg S, et al.: Crystal structure of UDP-N-acetylglucosamine enolpyruvyltransferase, the target of the antibiotic fosfomycin. Structure. 1996; 4(9): 1065–1075. PubMed Abstract | Publisher Full Text\n\nSakamoto Y, Furukawa S, Ogihara H, et al.: Fosmidomycin resistance in adenylate cyclase deficient (cya) mutants of Escherichia coli. Biosci Biotechnol Biochem. 2003; 67(9): 2030–2033. PubMed Abstract | Publisher Full Text\n\nAlbert A: Chemical aspects of selective toxicity. Nature. 1958; 182(4633): 421–423. PubMed Abstract | Publisher Full Text\n\nRautio J, Kumpulainen H, Heimbach T, et al.: Prodrugs: design and clinical applications. Nat Rev Drug Discov. 2008; 7(3): 255–270. PubMed Abstract | Publisher Full Text\n\nTakahata S, Ida T, Hiraishi T, et al.: Molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli. Int J Antimicrob Agents. 2010; 35: 333–337. PubMed Abstract | Publisher Full Text\n\nShen W, Wei Y, Dauk M, et al.: Involvement of a glycerol-3-phosphate dehydrogenase in modulating the NADH/NAD+ ratio provides evidence of a mitochondrial glycerol-3-phosphate shuttle in Arabidopsis. Plant Cell. 2006; 18(2): 422–441. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJomaa H, Wiesner J, Sanderbrand S, et al.: Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science. 1999; 285(5433): 1573–1576. PubMed Abstract | Publisher Full Text\n\nIguchi E, Okuhara M, Kohsaka M, et al.: Studies on new phosphonic acid antibiotics. II. Taxonomic studies on producing organisms of the phosphonic acid and related compounds. J Antibiotics (Tokyo). 1980; 33(1): 19–23. PubMed Abstract\n\nVasudevan A, Qian Y, Vogt A, et al.: Potent, highly selective, and non-thiol inhibitors of protein geranylgeranyltransferase-I. J Med Chem. 1999; 42(8): 1333–1340. PubMed Abstract | Publisher Full Text\n\nKato K, Matsumoto T, Koiwai S, et al.: Liquid suspension culture of tobaco cells. In: Terui G, editor. Fermentation Technology Today. Osaka: Society of Fermentation Technology. 1972; pp. 689–695.\n\nNagata T, Nemoto Y, Hasezawa S: Tobacco BY-2 cell line as the \"Hela\" cell in the cell biology of higher plants. Int Rev Cytol. 1992; 132: 1–30. Publisher Full Text\n\nMurashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15(3): 473–497. Publisher Full Text\n\nAoyama T, Chua NH: A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 1997; 11(3): 605–612. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1442",
"date": "15 Aug 2013",
"name": "Ivo Feussner",
"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 by Bach, Rohmer and colleagues describes a new and powerful plant screening system for chemical inhibitors of the MEP or Rohmer pathway. The basic concept as well as the underlying chemistry of the manuscript is excellent, but the presentation of the results and conclusions driven from the biological experiments, as well as a number of paragraphs need carefull revision. A few points that shall help the authors to improve the manuscript are:None of the inhibitors the authors measure show their uptake. This should be mentioned/discussed.The size of the cells should be shown with exactly the same magnification in all figures. Otherwise it is hard to compare the morphology of the cells upon treatment with different inhibitors. To my mind it seems that the treatment does result in changes in cell size and shape i.e. Figure 1, unlike what is stated for Ro in the 7th paragraph of page 4. In Figure 2 only the cells in the top line show stunting; the cells shown in the lines below look normal to me.The authors should carefully check again whether parts/paragraphs can be moved into the method section and vice versa.In Figure 6 fluorescence intensity is given. How was this measured, do the authors not mean distribution?",
"responses": [
{
"c_id": "611",
"date": "09 Nov 2013",
"name": "Thomas J. Bach",
"role": "Author Response",
"response": "This was the first comment to be delivered (thank you), but we waited until now, so as to respond to all reviews at the same time. The criticism on the organization of the paper is certainly justified, but in fact it is the result of frequent exchanges of e-mails and versions with the editorial office of F1000. The initial version was a long manuscript with a very large supplement. Upon the advice received, everything was restructured and finally two separate manuscripts were prepared, and to tell you the truth, what was a major part of the so-called supplement is now this first manuscript that describes the effects of a series of MEP pathway inhibitors and some that putatively interfere with protein isoprenylation and processing of correspondingly modified proteins. The follow-up manuscript will provide information on the establishment of a medium-throughput system and what measures need to be taken to miniaturize the system and to facilitate microscopic analysis of rather large numbers of samples, based on BY-2 cells grown and treated in small volumes (96-well plates). We think that by then all questions that remain open in this paper will become clear, including when it comes to statistics and so forth. Only if a compound is absorbed, meaning that it had passed the thin cell wall and the plasma membrane of BY-2 cells it can exert some effect in the cytoplasm (i.e., protein geranylgeranylation), or beyond after having surmounted the barrier of the double membrane that makes up the plastidial envelope to target the MEP pathway. Thus specific uptake studies, for instance with radiolabeled compounds would go far beyond the scope of this paper, not to speak of costs! Dr. Feussner is right in pointing to the different size of cells as they appear in the figures. But for that reason the (high resolution) photographs made with the confocal microscope are automatically completed with white bars for scaling. We know from earlier studies with BY-2 cells treated with sterol biosynthesis inhibitors that cells become more stunted, but in this series of experiments here we searched for putative effects on the insertion of the isoprenylated GFP reporter protein and found none. (Just as an aside, in a completely different study, sterols were depleted from the membrane by cyclodextrin treatment beyond 30%, without noticeable effect on the viability of cells.) Treatment of BY-2 cells with squalestatin (inhibitor of squalene synthase) and by terbinafin (blocks squalene epoxidase) led to similar morphological adaptations (reference #38): After cell division sterols are needed for cell elongation. However, a clear effect on the GFP reporter protein was seen with squalestatin, but this could be attributed to its direct inhibition of protein isoprenylation, as already described in literature. Indeed, the treatment with Ro at the concentrations applied did not really affect the cell shape (Fig. 1a) and the statement criticized in the main text needs to be weakened.Dr. Feussner is right: In Fig. 6 we mean fluorescence distribution, not intensity. This will be changed accordingly. In Fig. 2 chemical complementation assays are displayed, with 1 µM squalestatin as the inhibitor. (This is already a pretty high concentration of this very efficient ± irreversible inhibitor, but which also needs to penetrate into the cells.) The only compound that really counteracted the apparent side effect on protein geranylgeranylation besides DX was geranylgeraniol (GGol). However, in previous studies we had already demonstrated that under certain conditions, when the cytoplasmic MVA pathway is blocked, in the BY-2 cell system exogenous DX can override the inhibition of sterol biosynthesis, which would result in less stunted cells. GGol, at the concentrations tested might prevent the binding of squalestatin to protein geranylgeranyl transferase. This does not hold true for MVA (open acid or lactone form), which would reconstitute sterol biosynthesis to some extent resulting in some cell elongation, but not entirely the inhibition of PGGT type I. The slight effect of geraniol might be due to its conversion into farnesyl-PP, but that is more speculation. We think we can leave this as it is described and discussed now."
}
]
},
{
"id": "1438",
"date": "04 Sep 2013",
"name": "Michael Pirrung",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 chemical genetics study addressing the parallel biosynthetic pathways for isoprenoids in plants (which are also significant in human pathogens). As such, this work provides a paradigm for the discovery of bioactives that might be applied in several contexts. Of course, the unique characteristics of plant cell walls / membranes, contrasted with bacteria, may make this screen less general than might be desirable. Being based on imaging, this screen is relatively low throughout, but cell-based assays typically have some of this sort of limitation. A few new chemotypes have been investigated and found to have significant activity. This study follows well-established processes for examining the sequence of steps in a biosynthetic pathway, with opposing effects of inhibitors and molecules downstream of the site of inhibition. A slight ambiguity about the chemical structures of the pro-drugs and their active forms hinders understanding of the structure-activity relationships. It should be emphasized that the most active SP compounds have N-methyl groups on their hydroxamate nitrogens. Also, structures presented on p. 20 are in error, in that all but TJ290 are missing a carbonyl group.",
"responses": [
{
"c_id": "617",
"date": "13 Nov 2013",
"name": "Thomas J. Bach",
"role": "Author Response",
"response": "The referee went quite deeply into the details of the manuscript and we are very grateful for his sharp eyes, detecting a mistake in the supplementary figure summarizing some (synthetic) inhibitors checked in our study. The corresponding figure will of course be replaced. The colleague points to some limits of such a system, but we have prepared a sequel manuscript also to be sent to F1000research, in which all has been “miniaturized” to the use of 96-well plates and a semi-automatic analysis by confocal microscopy. It was necessary however, to shown that the reporter system as such, based on this GFP fusion protein works sufficiently well before publishing the second manuscript. As to the efficacy of compounds that interact with the MEP pathway or with downstream reactions of protein isoprenylation the first criteria is to penetrate into the BY-2 cells, and ad extremum to get into the plastids. It is obvious and also a major result that “pro-drugs” with increased lipophilicity enter more easily, like is known for a large number of pesticides and pharmaceuticals. It is somewhat ambiguous to go too deeply into the field of structure-activity relationships, but Dr. Pirrung is right in ascribing the highest activity to a certain group of compounds, like those that have N-methyl groups and their hydroxamate nitrogens. There is no “ideal” system for testing inhibitors, and this concerns those whose research is based around bacteria, as well. Let us take for instance the well-known fosmidomycin that penetrates into E. coli, but not into the humano-pathogenic Mycobacterium. Once an inhibitor has been found efficient in our plant cell-based test system, it is clear that in follow-up experiments one has to investigate whether it acts similarly upon a whole collection of such bacteria. Human pathogens are not even limited to bacteria. Simply pragmatically speaking, a plant cell is somewhat representative of different pathogenic cells: protistans, bacteria and fungi."
}
]
},
{
"id": "1583",
"date": "06 Nov 2013",
"name": "Angelos Kanellis",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 entitled “The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells” by Hartmann et al., describes the use of an in vivo system to study the effectiveness of known inhibitors of MEP and MVA pathways and also to screen for new chemicals that affect these pathways with the hope of eventually being able to use them as herbicides, antibacterial/ant-parasitic drugs and as inhibitors of early sterol biosynthesis and of post-prenylation reactions. The experiments were well designed and executed and showed that this is a promising system that can be used not only for the screening of new inhibitors of the MEP pathway, but also to study the possible channelling of the intermediates to different compartments and/or to follow the interplay between the two pathways. The latter can also help with applying metabolic engineering to terpenoid production in plants or microorganisms.",
"responses": [
{
"c_id": "616",
"date": "13 Nov 2013",
"name": "Thomas J. Bach",
"role": "Author Response",
"response": "We are quite grateful for the very positive comments by this colleague, who has provided a broader and deeper perspective to our article than might at first be obvious by a superficial read of the manuscript. It is clear that he is a specialist in the realm of plant isoprenoid research."
}
]
}
] | 1
|
https://f1000research.com/articles/2-170
|
https://f1000research.com/articles/2-247/v1
|
15 Nov 13
|
{
"type": "Research Article",
"title": "No psychological effect of color context in a low level vision task",
"authors": [
"Adam Pedley",
"Alex R Wade",
"Adam Pedley"
],
"abstract": "Background: A remarkable series of recent papers have shown that colour can influence performance in cognitive tasks. In particular, they suggest that viewing a participant number printed in red ink or other red ancillary stimulus elements improves performance in tasks requiring local processing and impedes performance in tasks requiring global processing whilst the reverse is true for the colour blue. The tasks in these experiments require high level cognitive processing such as analogy solving or remote association tests and the chromatic effect on local vs. global processing is presumed to involve widespread activation of the autonomic nervous system. If this is the case, we might expect to see similar effects on all local vs. global task comparisons. To test this hypothesis, we asked whether chromatic cues also influence performance in tasks involving low level visual feature integration.Methods: Subjects performed either local (contrast detection) or global (form detection) tasks on achromatic dynamic Glass pattern stimuli. Coloured instructions, target frames and fixation points were used to attempt to bias performance to different task types. Based on previous literature, we hypothesised that red cues would improve performance in the (local) contrast detection task but would impede performance in the (global) form detection task. Results: A two-way, repeated measures, analysis of covariance (2×2 ANCOVA) with gender as a covariate, revealed no influence of colour on either task, F(1,29) = 0.289, p = 0.595, partial η2 = 0.002. Additional analysis revealed no significant differences in only the first attempts of the tasks or in the improvement in performance between trials.Discussion: We conclude that motivational processes elicited by colour perception do not influence neuronal signal processing in the early visual system, in stark contrast to their putative effects on processing in higher areas.",
"keywords": [
"colour and cognition",
"low level visual processing",
"colour-in-context theory"
],
"content": "Introduction\n\nThere is now strong evidence that colour influence people’s performance on cognitive tasks. These effects are often demonstrated by pre-cuing a subject in a subtle manner (for example, by changing the colour of the ink or cover page in a written examination) and measuring performance in the exam as a function of cue colour.\n\nThese effects appear to be remarkably robust. For example, Elliot, Maier, Moller, Friedman & Meinhardt1 showed that subjects performing a series of demanding cognitive tests performed worse when they were pre-cued with the colour red than when they were cued with control colours such as green, blue or grey. This effect was found across a wide range of cue conditions, exposure times, subjects, environments and even across two continents1. Similar effects have been demonstrated in a series of other experiments. Mehta and Zhu2 demonstrated that blue and red pre-cues similar to those described above influenced performance in tasks. In particular, blue cues led to better performance in creative-oriented, divergent tasks whilst red cues facilitated superior performance in detail-oriented, convergent tasks.\n\nThe effect can even be driven by colour language. For example, in a series of experiments Lichtenfeld, Maier, Elliot & Pekrun3 showed that processing the word 'red' significantly impaired performance on both analogy and numeric IQ tasks. The most striking aspect of this research is the subtlety with which the colour manipulation took place. In one experiment in Germany, participants were simply assigned to either the 'red' (‘rot’) or 'place' (‘ort’) group without a visual colour exemplar. In another experiment the colour manipulation took the form of potential colour words in answers to a multiple choice-question. The effect has also been found when the colour stimulus was entirely incidental to the task. In one experiment the colour manipulation was the copyright label on a cover which read; 'Hogreffe Series of Tests' followed by either the word 'Red' or 'Grey'. Participants were not instructed to read the text but were required to wait with the page open until instructed to start. It seems that in these experiments only the neural evocation of the concept of a colour is required to influence performance.\n\nThese results are now commonly framed within a theory known as the ‘Colour-in-Context’ (CIC) theory4. This formalizes the idea that colour carries semantic messages but that the behaviour resulting from these messages depends on context. In an ‘achievement context’, for example, many researchers believe that the colour red initiates avoidance behaviours through low-level physiological priming mechanisms. The neural correlates of this avoidance behaviour were identified in the Elliot et al. work1 using electroencephalography (EEG). Conversely, several researchers have proposed that, in an achievement context, the colour blue initiates approach behaviours2.\n\nMaier, Elliot & Lichtenfeld5 proposed that approach/avoidance behaviours can also be presented within a global/local processing framework. Specifically, it has been suggested that the colour red initiates a focusing or constricting of the attentional spotlight that facilitates the processing of local features while, the colour blue has the opposite effect, encouraging ‘big picture’ or ‘global’ feature processing.\n\nThe concepts of local and global processing are not defined rigorously in the context of the high-level cognitive tasks commonly used in the literature. In this paper, we examine claims of chromatically-cued task performance using a classic low-level visual paradigm that contains both local and global features. In all experiments, the nature of the psychophysical stimulus was constant and we examined the effect of ancillary chromatic features in a simple two-by-two factorial design. Specifically, we varied the colour of the fixation cross, instructions and target boundaries. We then examined the effects of these changes on performance in one of two tasks: global or local feature detection. Subjects were placed in an achievement context by encouraging a sense of competitiveness and instructing them that ‘success’ on the task would lead to further opportunities for (paid) research participation.\n\nDespite the use of strong chromatic cues and an achievement context, we found no effect of cue colour on performance. Specifically, neither global nor local feature detection thresholds showed any dependence on cue colour. We also examined the effect of cue colour on learning rates and, similarly, found no effect. We discuss these findings with reference to both low-level visual processing and the CIC theory.\n\n\nMethods\n\nThirty native English speaking subjects (21 ± 2.9 years old, 9 male) were recruited and tested at the University of York, UK. All subjects had self reported normal or corrected to normal vision. Colour vision was assessed using a pseudo-Isochromatic Ishihara plate colour vision test6. One subject could not complete the colour vision test and was therefore omitted from the study. One other subject was unable to complete the task to a satisfactory standard (1300% increase on one standard deviation (SD) in the local task, 679% increase on SD in the global task) and was also omitted from the study. Informed written consent was obtained from all subjects.\n\nWe used dynamic Glass patterns7,8 to generate stimuli with both local and global components. Glass patterns7 have long been used to investigate global and local feature processing9. They are formed by placing two slightly shifted copies of an identical random dot pattern on top of each other, generating oriented pairs of dots termed ‘dipoles’. The type of transformation defines both the orientation and size of the dipoles and in turn, defines the overall global percept. Critically, the local statistics of the dipoles (for example, contrast and dot size) can be varied without affecting the global percept.\n\nWhen individual Glass patterns are presented for long durations, they give rise to a static global percept of structure. However, when individual dipoles are replaced and updated rapidly they convey not only form but also a sense of implicit motion. This motion is perceived despite the fact that the position of the updated dipoles is uncorrelated from frame-to-frame10. Such configurations of sequential brief presentations are termed 'dynamic Glass patterns' (dGP). Ross, Badcock & Hayes8 proposed that the implicit motion generated by dGPs is a result of the form and motion aspects of vision combining. In effect, the circular form of the Glass pattern imposes structure to the otherwise random 'motion' of updated dipoles.\n\nGlass patterns, and variations thereof, have been used to reveal the underlying physiological correlates of global form perception and it is now clear that different Glass pattern discrimination tasks (local vs. global) engage different neuronal populations11. Several models have been proposed that describe the hierarchical process by which Glass patterns are perceived. Wilson & Wilkinson12 proposed that the early local stage of filtering is facilitated by a selection of oriented spatial filters. When these are combined at a later stage the signals from these filters combine to form a global percept. Because of their simplicity and clear long- and short-range structure, Glass patterns are ideal stimuli for studying issues relating to global and local visual processing.\n\nIn the colour-cognition literature that motivated this study, the presence of a red cue has been shown to facilitate local processing and impede global processing, whilst a blue cue has been shown to have the opposite effect2,4. Therefore, if we introduce colour priming cues into Glass pattern threshold experiments we expect to see differences in the performance of tasks requiring either local or global processing. In a red cue condition we would expect the local processing to improve (allowing subjects to make more accurate judgements about low-level features of the individual dipole elements) but the global perception of form (for example, the ability to distinguish structured dipole fields from unstructured ones) should be impeded. In a blue cue condition the reverse would be expected.\n\nWe measured local and global processing in two separate tasks. The global task was the detection of concentric dGP form against a backdrop of random noise. Performance in this task was expressed as the smallest global coherence level that could be detected reliably. The local task required subjects to detect changes in the contrast of the dipoles and performance in this task was expressed as a contrast discrimination threshold. The two conditions were orthogonal in the sense that small changes in contrast have no effect on the detectability of Glass pattern coherence and the presence or absence of global organization has no effect on subject’s ability to perform local contrast modulation tasks. We also note that the two tasks truly require global and local strategies: it is not possible to reliably distinguish structured from unstructured fields based on information from low-level ‘local’ receptive fields13 and because we altered contrast (rather than luminance), pooled responses from very large ‘global’ receptive fields are uninformative about local dipole features.\n\nTo assess the effect of chromatic cue on global and local visual processing we introduced a set of chromatic stimulus components (fixation boxes, textual instructions) that could be either red or blue. Based on the existing literature regarding chromatic influences on neural processing we hypothesised that:\n\n1. Red cues would facilitate local processing which would improve the ability to detect changes in contrast but impede the ability to detect dGP form.\n\n2. Blue cues would have the opposite effect: enabling global processing and improving the ability to detect dGP form but impairing the ability to detect local contrast changes and therefore increasing thresholds on the contrast modulation task.\n\nThe experiment used a 2×2 repeated measures design. The independent variables were the colour manipulation (red vs. blue) and the type of task (local vs. global). The dependant measure was the detection threshold level of the task.\n\n\nMaterials\n\nExperiments were designed using Psykinematix Visual Psychophysics 1.4 software14. The experiments consisted of two squares (6.5° radius, 3.0 point line width, no fill) presented horizontally at 5° of eccentricity. Within each square was a concentric dGP made up of 65 dipoles each with a spatial jitter of 0.25° and a lifetime of 0.005ms before being replaced. Concentric dGPs were used as stimuli as previous research has shown that the visual system is most sensitive to detecting these patterns15. Each trial lasted 500ms before a response was required. The next trial started after a response was given (see Figure 1).\n\nWe used a two-alternative forced-choice procedure, presented spatially, in which participants were required to indicate, using a keyboard, which square contained either dGP form (global task) or dipoles with higher levels of contrast (local task). Feedback for correct/incorrect responses was given through auditory signals of high and low pitch respectively. Both coherence and contrast were controlled by a Bayesian adaptive threshold procedure16 to determine 75% discrimination thresholds.\n\nStimuli were presented on a 20\" cathode ray tube (CRT) monitor (Sony GDM-20E21, with a resolution of 1024×768 at 100Hz) driven by an ATI Radeon HD 5770 graphics card on an Apple Mac Pro (OS X 10.6.8). The display, with a mean luminance of 42cd/m2 was calibrated prior to data collection using a ColourVision Spyder4Pro colour calibration unit and the Psykinematix Visual Psychophysics software. Calibration of the display was verified using a fibre-optic based spectrophotometer (USB2000, OceanOptics, Fl) which was itself calibrated against a NIST-traceable source. The display was situated in a dark, quiet room and subjects sat 80cm from the display.\n\nColours were defined in Macleod-Boynton/DKL colour space17,18 by specifying individual cone contrasts relative to a mean-grey background. In all instances of colour presentation the colours were either red (as defined by LMS cone contrast levels of L = -0.508, M = -0.826, S = 1.00, contrast = 39.12%) or blue (L = -0.920, M = -0.749, S = -1.00, contrast = 74.71%). These coordinates were constrained by several criteria. First, our chromatic stimuli were chosen to represent ‘pure’ exemplars of red and blue hues. This type of ‘unique hue’ setting varies slightly from person to person but when queried, our own subjects agreed on the canonical names of these hues unanimously. We used contrast levels (computed relative to a mean gray background) to allow for good replicability and stability of our stimuli: in particular light adaptation levels are not well defined when colours are presented on a CRT against a black background. Finally, to maximize the potential psychological effects of our cues, we used highly saturated colours that were, necessarily, at the limit of the monitor gamut. These constraints meant that our chromatic cues were not isoluminant: the luminance contrast (as computed from the RMS cone coordinates) of the blue stimulus components was approximately twice that of the red components. This confound was unavoidable given the other constraints. However, in both cases, the luminance contrast was well above detection thresholds so that the cues were highly visible (and therefore the instructions, for example), were legible on the basis of luminance alone.\n\nColour cuing was achieved in three ways. First, each experiment had instructions presented in coloured text that were displayed prior to the experiment starting. These instructions were presented in Arial Bold font, 36pt and were displayed until the subject pressed a key to initiate the experiment (approx 10s). Secondly, the fixation cross in the centre of the screen was either red or blue. Finally, the lines of the squares in which the glass patterns were presented were coloured either red or blue. All colour cues were consistent within an experiment. This resulted in four separate experiments; a red and blue version of both the local and global experiments.\n\nSubjects were shown an example of each experiment (devoid of colour manipulation) and were required to read a set of pre-experiment instructions, printed black ink on white paper. Each subject then completed each of the four experiments twice (eight experiment blocks of eighty trials giving 640 trials in total). The order of the blocks was varied randomly across subjects. After the experiment had finished the subjects completed a verbal de-briefing task to determine if subjects were aware of the colour manipulation. The de-briefing task comprised of nine questions and refers to the purpose of the experiment and the colours employed within.\n\nTo ensure that participants performed the task within a strong achievement context they were informed that failure to attain a certain score would result in them not being able to continue to the second part of the experiment, a four question online survey. After the experiment, all participants were informed that they successfully completed the task and were sent the online survey to complete. The experiment was approved by the University of York ethics committee.\n\nAll statistical analyses were run using IBM SPSS ver. 20.0. Data screening involving the calculation of skewness and kurtosis z-scores, Kolmogorov-Smirnov test and Shapiro-Wilk test confirmed normality of the data. Dependent t-tests were conducted to assess if colour cues (red vs. blue) influenced performance within tasks (local vs. global). A two-way repeated measures analysis of covariance (ANCOVA) was conducted to assess if there were any interactions between the colour cues and the type of task with gender factored as a covariate. All inferential analyses used a p value cut off of <.05 to determine significance.\n\n\nResults\n\nWe first asked if the cue colour influenced performance in either task. Specifically, we wanted to determine if a red cue improved performance (reduced discrimination thresholds) in the local task and impeded performance in the global task and if the opposite effect occurred for the blue cue condition. Unless otherwise stated the values used were from the second trials.\n\nA dependant t-test was conducted (colour condition red vs. blue) on local task detection thresholds (M = 5.36, SD = 1.99). The analysis revealed no significant differences between the red (M = 5.18, SD = 1.86) and blue (M = 5.54, SD = 2.13) colour conditions, t(29) = -1.184, p = 0.246, d = 0.180. See Figure 2 for means of both trials and their average value.\n\nMean values of the 1st and 2nd trials as well as the average value of both trials. Error bars indicate the 95% confidence interval.\n\nA dependant t-test was conducted (colour condition red vs. blue) on global task detection thresholds (M = 66.35, SD = 3.93). The analysis revealed no significant differences between the red (M = 66.43, SD = 3.58) and blue (M = 66.26, SD = 4.37) colour conditions, t(29) = 0.218, p = 0.829, d = 0.043. See Figure 3 for means of both trials and their average value.\n\nMean values of the 1st and 2nd trials as well as the average value of both trials. Error bars indicate the 95% confidence interval.\n\nWe also asked if there were any interactions between the two types of task (local and global) and the two colour conditions (red and blue). As gender has been claimed to affect global vs. local visual processing19 it was included as a covariate in this analysis. We performed a 2-way repeated measures analysis of covariance (ANCOVA) and asked if there was a main effect of colour on task, with gender as a covariate, and if there were any interactions between gender, cue colour and task. None of these effects were significant. We found no effect of colour on task performance, F(1,28) = 0.289, p = 0.595, partial η2 = 0.002, no significant interaction between task and colour, F(1,28) = 0.081, p = 0.77, partial η2 = 0.002 and we found no significant interaction between gender and task performance, F(1,28) = 0.737, p = 0.398, partial η2 = 0.013. Finally, this analysis revealed no significant three way interactions of task, colour and gender, F(1,28) = .014, p = 0.905, partial η2 < 0.001.\n\nThe majority of studies in the literature to date have used a between-subjects design in which subjects completed only one of the tasks1,2. It is possible that the absence of significant effects in our own study could be a result of motivational processes being elicited only when first completing a novel task. To control for this, we performed an analysis which only considered each subject's first trial in the experiment block. Significant results would indicate that the influence of cue colour only impacts performance when a task is novel and the effect diminishes through practice or habituation.\n\nAn independent t-test was conducted (colour condition red vs. blue) of the first attempts of both the local and global task (N = 15). The local task analysis (M = 8.107, SD = 4.79) revealed no significant differences between the red (M = 8.08, SD = 5.89) and blue (M = 8.14, SD = 3.58) colour conditions, t(14) = -0.041, p = 0.968, d = 0.012. See Figure 4 for means of the first attempts at the local task as a function of colour.\n\nError bars indicate the 95% confidence interval.\n\nThe global task analysis (M = 68.80, SD = 4.87) revealed no significant differences between the red (M = 68.58, SD = 5.00) and blue (M = 69.02, SD = 4.90) colour conditions, t(14) = -0.233, p = 0.819, d = 0.088. See Figure 5 for means of the first attempts at the global task as a function of colour.\n\nError bars indicate the 95% confidence interval.\n\nMost subjects achieved slightly lower thresholds on their second trials compared to the first trials. Although no effect of colour was detectable in the thresholds of either the first or second trials examined separately, we wondered if colour might still be influencing the rate of improvement.\n\nWe tested this by comparing the first trials to the second trials of the experiments (see Figure 2 and Figure 3). Dependant t-tests were conducted (1st trial vs. 2nd trial) of the four conditions. The results were; Red local task; 1st trial (M = 6.81, SD = 4.60), 2nd trial (M = 5.18, SD = 1.86), t(29) = 1.964, p = 0.030, d = 0.462. Blue local task; 1st trial (M = 7.37, SD = 3.09), 2nd trial (M = 5.54, SD = 2.13), t(29) = 3.56, p < 0.001, d = 0.693. Red global task; 1st trial (M = 67.20, SD = 5.06), 2nd trial (M = 66.43, SD = 3.58), t(29) = 0.944, p = 0.163, d = 0.190. Blue global task; 1st trial (M = 68.54, SD = 4.50), 2nd trial (M = 66.26, SD = 4.37), t(29) = 2.748, p = 0.05, d = 0.514.\n\nThree of the four comparisons revealed significant differences. However, there was no significant improvement between the first and second trial in the red global task. To further examine these differences in performance, we asked if the colour condition influenced the magnitude of the shift in performance between the first and second task. Any differences here could indicate that colour stimuli influence the learning curve of local or global tasks.\n\nTo visualize these effects, we calculated a 'shift-in-performance' (SIP) parameter by subtracting the threshold of the second trial from the threshold of the first trial. Two dependant t-tests were conducted (colour condition red vs. blue) on the SIP values of both the local tasks (M = 1.73, SD = 3.68) and global tasks (M = 2.63, SD = 8.56). The local task analysis revealed no significant differences between the red (M = 1.63, SD = 4.55) and blue (M = 1.83, SD = 2.82), colour conditions, t(29) = -0.205, p = 0.420, d = 0.053. The global task analysis revealed no significant differences between the red (M = 2.98, SD = 12.56) and blue (M = 2.28, SD = 4.55) colour conditions, t(29) = 0.319, p = 0.376, d = 0.074. See Figure 6 for mean SIP values of the local and global tasks as a function of colour.\n\nError bars indicate the 95% confidence interval. N.B., the error bars for the red cue condition straddle zero as some participants exhibited a negative shift-in-performance.\n\nPrevious research has shown that individuals who perform better in global tasks often perform worse in local tasks and vice versa20. A Pearson's correlation was conducted (colour conditions red vs. blue) which revealed no significant correlations of either the red condition local/global tasks, r = 0.235, p = 0.469, or the blue condition local/global tasks, r = 0.216, p = 0.285. We found no relationship, positive or negative, between the performance in the two tasks.\n\nWe were not subtle in our presentation of colour stimuli. We manipulated the highly-saturated colour of the instructions, fixation cross and on-screen stimuli. Given this overt colour manipulation and in line with previous research1,3 we assessed, through a verbal funnelling task, the subjects' awareness of the colour manipulation and the purpose of the experiment. No participants guessed the aims of the experiment. To assess the awareness of colour in the experiment we analysed responses from two questions in the verbal funnelling task;\n\n'Can you name the colour of the fixation cross?'\n\n'Can you name the colour of the instructions at the beginning of each experiment?'\n\nResults showed that 50% of subjects (N = 15) were aware of the colour manipulation and could report both presented colours. 20% (N = 6) were able to name one of the presented colours. 30% of subjects (N = 9) were unable to recall either of the presented colours. Of these 9 subjects, 8 reported seeing colours that were not presented; black, white and yellow. These results indicate that half of the subjects could name the colours in the experiment but this did not reveal the aims or impact on the validity of the study.\n\n\nDiscussion\n\nOur primary finding is a negative result. Contrary to the existing literature, we found no effect of colour on performance in low level visual tasks set in an achievement context. We found no significant differences in the performance of either the local or global tasks as a function of colour cues. We found no significant differences in the first attempts of either task or in the improvement of performance between trials. Subjects' scores improved significantly between trials in most experiments, presumably due to training effects. However further analysis revealed no relationship between training-dependent improvement and colour. As in previous work (see Elliot and colleagues), the significance of the overt colour manipulation that took place was undetected by participants.\n\nEarlier studies have observed strong effects of colour on performance in global tasks such as anagrams, analogies, number sequence tasks1,3, ‘creative use’ tasks2,21 and in ostensibly local tasks such as proof reading and memorization2. While negative results can arise from trivial causes such as poorly-designed experiments or insufficient statistical power, we do not believe these criticisms apply to the current study. We tested a large number of subjects from a relatively homogeneous population, our experimental design was a direct extrapolation of those used in previous studies and the tasks and our stimuli were well-controlled and commonly used in the vision science literature to examine precisely this type of local vs global processing11.\n\nPrevious publications detail at least 19 experiments (across 5 publications) demonstrating that colour influences performance with some reference to the theoretical framework of the CIC1–3,5,21. The effects have been documented across experiments using various exposure times (2s, 5s and constant exposure) and across a wide array of colour presentation techniques (screen background colour, ink and paper colour or a lexical term). The experiments have been conducted in various institutions across Northern America and Europe in both laboratories and classrooms. We argue not that our work contradicts these findings but rather we have identified an area of task performance that remains unaffected by colour cues. In short, we propose that the motivational processes evoked by colour cues in achievement contexts do not influence low level visual perception.\n\nWhere in the visual system are the global and local aspects of our task performed? While local contrast judgements can be supported by short-range V1 receptive fields, detecting and discriminating dynamic Glass patterns requires long-range integrative mechanisms. Wilson, Wilkinson & Assad22 proposed that the perception of Glass patterns occurs through the ventral stream within the neurophysiological account of visual perception. Initial filtering of the dipoles occurs in V1, followed by a second filtering stage in V2. The global spatial pooling of these local features occurs in V4. These claims are supported by empirical evidence. Smith, Bair & Movshon13 demonstrated that the size, structure and performance of the receptive fields in V1/V2 neurons are particularly well suited to analyse the orientation of dipoles. Separate experiments have demonstrated that V113 and V2 cells23 are largely unable to detect the global form of glass patterns. Where modulation of V1 activity in response to global pattern changes is observed, it is most likely due to feedback from higher areas24.\n\nOn average, V4 neurons have receptive fields 5–7 times larger than those in V1/V225 meaning they are much better suited to integrate across the visual field and detect the global form of Glass patterns. Tse et al.26, examined how V1/V2/V4 neurons responded to different glass patterns in macaques. They found that whilst the response of the V4 neurons varied between different Glass patterns, the responses of V1/V2 cells remained stable regardless of the type of Glass pattern presented. This suggests that global form information is extracted at a later stage than local features such as dipole contrast.\n\nIn short, the local features of dGPs are processed first in visual areas V1 and V2 before the formation of the global percept in ventral and lateral areas such as V4 and the LOC. Both these processes take place before visual information reaches the higher level areas of the ventral stream in the inferior temporal (IT) lobe where semantic value is added to visual information. We therefore propose that while general motivational processes evoked by colour perception do influence performance in some tasks that require high level cognition (i.e. anagram task, creative uses task) they do not influence neuronal signal processing in the early visual system.\n\nWe note that there are well-established examples of higher-level mechanisms affecting responses and psychophysical performance based on low-level visual areas. For example, attention to selective features of a stimulus (for example, its motion or colour) generate activations in cortical regions that are sensitive to those features27,28 and top-down attentional mechanisms have been found to affect the sensitivity, tuning and baseline activity of neurons as early as the LGN and V129–34. However, it appears that chromatic context does not generate a form of top-down modulation that falls into one of these classes.\n\nThis study has practical implications for psychophysical experiments assessing low level vision. The existing body of work2,4 would indicate it is vital that the use of colour in experiments is monitored carefully so as to not bias performance through motivational processes. This work demonstrates that in tasks requiring processing in the early visual system, such as contrast, motion and dGP form detection, no such consideration is required.",
"appendix": "Author contributions\n\n\n\nAdam Pedley conceived, implemented and ran experiments and collaborated in the write-up. Alex Wade assisted in implementation, data analysis, writing and supervised the project.\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\nElliot AJ, Maier MA, Moller AC, et al.: Color and psychological functioning: the effect of red on performance attainment. J Exp Psychol Gen. 2007; 136(1): 154–68. PubMed Abstract | Publisher Full Text\n\nMehta R, Zhu RJ: Blue or red? Exploring the effect of color on cognitive task performances. Science. 2009; 323(5918): 1226–9. PubMed Abstract | Publisher Full Text\n\nLichtenfeld S, Maier MA, Elliot AJ, et al.: The semantic red effect: Processing the word red undermines intellectual performance. J Exp Soc Psychol. Elsevier Inc.; 2009; 45(6): 1273–6. Publisher Full Text\n\nElliot FAJ, Maier MA, Patricia CTI, et al.: Color-in-Context Theory. 1st ed. Adv Exp Soc Psychol. Elsevier Inc.; 2012; 61–125.\n\nMaier MA, Elliot AJ, Lichtenfeld S: Mediation of the negative effect of red on intellectual performance. Pers Soc Psychol Bull. 2008; 34(11): 1530–40. PubMed Abstract | Publisher Full Text\n\nIshihara S: Ishihara’s tests for color-blindness (38 plate ed.). Tokyo/Kyoto, Japan: Kanehara, Shuppan. 1990.\n\nGlass L: Moire effect from random dots. Nature. 1969; 223(5206): 578–580. PubMed Abstract | Publisher Full Text\n\nRoss J, Badcock DR, Hayes A: Coherent global motion in the absence of coherent velocity signals. Curr Biol. 2000; 10(11): 679–82. PubMed Abstract | Publisher Full Text\n\nMandelli MF, Kiper DC: The local and global processing of chromatic Glass patterns. J Vis. 2005; 5(5): 405–16. PubMed Abstract | Publisher Full Text\n\nBurr D: Motion vision: are 'speed lines' used in human visual motion? Curr Biol. 2000; 10(12): R440–3. PubMed Abstract | Publisher Full Text\n\nPalomares M, Ales JM, Wade AR, et al.: Distinct effects of attention on the neural responses to form and motion processing: A SSVEP source-imaging study. J Vis. 2012; 12(10): 1–14. PubMed Abstract | Publisher Full Text\n\nWilson HR, Wilkinson F: Detection of global structure in Glass patterns: implications for form vision. Vision Res. 1998; 38(19): 2933–47. PubMed Abstract | Publisher Full Text\n\nSmith MA, Bair W, Movshon JA: Signals in macaque striate cortical neurons that support the perception of glass patterns. J Neurosci. 2002; 22(18): 8334–45. PubMed Abstract\n\nKybervision. Psykinematix Visual Psychophysics Software. Montreal, Canada; 2011.\n\nNankoo JF, Madan CR, Spetch ML, et al.: Perception of dynamic glass patterns. Vision Res. 2012; 72: 55–62. PubMed Abstract | Publisher Full Text\n\nKontsevich LL, Tyler CW: Bayesian adaptive estimation of psychometric slope and threshold. Vision Res. 1999; 39(16): 2729–37. PubMed Abstract | Publisher Full Text\n\nMacLeod D, Boynton R: Chromaticity diagram showing cone excitation by stimuli of equal luminance. J Opt Soc Am. 1979; 69(8): 1183–1186. PubMed Abstract | Publisher Full Text\n\nDerrington AM, Krauskopf J, Lennie P: Chromatic mechanisms in lateral geniculate nucleus of macaque. J Physiol. 1984; 357: 241–65. PubMed Abstract | Free Full Text\n\nRoalf D, Lowery N, Turetsky BI: Behavioral and physiological findings of gender differences in global-local visual processing. Brain Cogn. 2006; 60(1): 32–42. PubMed Abstract | Publisher Full Text\n\nBasso M, Schefft B, Ris M, et al.: Mood and global-local visual processing. J Int Neuropsychol Soc. 1996; 2(3): 249–55. PubMed Abstract | Publisher Full Text\n\nLichtenfeld S, Elliot AJ, Maier MA, et al.: Fertile green: green facilitates creative performance. Pers Soc Psychol Bull. 2012; 38(6): 784–97. PubMed Abstract | Publisher Full Text\n\nWilson HR, Wilkinson F, Asaad W: Concentric orientation summation in human form vision. Vision Res. 1997; 37(17): 2325–30. PubMed Abstract | Publisher Full Text\n\nSmith M, Kohn A, Movshon J: Glass pattern responses in macaque V2 neurons. J Vis. 2007; 7(3): 1–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAngelucci A, Levitt JB, Walton EJ, et al.: Circuits for Local and Global Signal Integration in Primary Visual Cortex. J Neurosci. 2002; 22(19): 8633–46. PubMed Abstract\n\nLevitt J, Kiper D, Movshon J: Receptive fields and functional architecture of macaque V2. J Neurophysiol. 1994; 71(6): 2517–2542. PubMed Abstract\n\nTse P, Smith MA, Augath M, et al.: Using Glass patterns and fMRI to identify areas that process global form in macaque visual cortex. J Vis. 2002; 2(7): 285–93. Publisher Full Text\n\nChao LL, Martin A: Cortical regions associated with perceiving, naming, and knowing about colors. J Cogn Neurosci. 1999; 11(1): 25–35. PubMed Abstract\n\nWinawer J, Huk AC, Boroditsky L, et al.: Common mechanisms for processing of perceived, inferred, and imagined visual motion. J Vis. 2008; 5(3): 276–83. Publisher Full Text\n\nVerghese P, Kim YJ, Wade AR: Attention selects informative neural populations in human V1. J Neurosci. 2012; 32(46): 16379–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKastner S, Pinsk MA, De Weerd P, et al.: Increased activity in human visual cortex during directed attention in the absence of visual stimulation. Neuron. 1999; 22(4): 751–61. PubMed Abstract | Publisher Full Text\n\nO’Connor DH, Fukui MM, Pinsk MA, et al.: Attention modulates responses in the human lateral geniculate nucleus. Nat Neurosci. 2002; 5(11): 1203–9. PubMed Abstract | Publisher Full Text\n\nHuk AC, Heeger DJ: Task-related modulation of visual cortex. J Neurophysiol. 2000; 83(6): 3525–36. PubMed Abstract\n\nHillyard SA, Vogel EK, Luck SJ: Sensory gain control (amplification) as a mechanism of selective attention: electrophysiological and neuroimaging evidence. Philos Trans R Soc Lond B Biol Sci. 1998; 353(1373): 1257–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReynolds JH, Heeger DJ: The normalization model of attention. Neuron. 2009; 61(2): 168–85. PubMed Abstract | Publisher Full Text | Free Full Text"
}
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[
{
"id": "2457",
"date": "20 Jan 2014",
"name": "Diana M. Brainard",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 conducted study that examines whether cuing effects of color on 'local' and 'global' processing manifest with relatively low-level visual tasks. The authors find no such effects, with reasonably well-powered measurements. Indeed the measurements have sufficient power to reveal a learning effect within a subjects’ replications of the task. The error bars for the global task are particularly small. I have only minor suggestions:Although certainly not for this paper, I would be interested in whether the present authors are able to obtain color cuing effects in an exact replication of some of the published studies that do obtain such effects. This might be a good project for a registered replication attempt. The paper as it stands now is in excellent shape. If the authors choose to revise it, I think a quantitative expansion on the classification of their tasks as local and global would be helpful. For example is the spatial integration area known for each task, for stimuli like those employed by the authors? Direct measurements of the integration area would establish experimentally the locality/globality of each task in a satisfying manner. Another analysis that could be considered would be to look at individual observer data. Do any observers show reliable cuing effects, and if so in what direction?\n\n\"... because we altered contrast (rather than luminance), pooled responses from very large ‘global’ receptive fields are uninformative about local dipole features.\" Although it is easy to believe that contrast discrimination is more local than identifying pattern structure, I don't understand this particular argument. It appears from Figure 1 that the pattern elements were all increments, so that changing contrast must also have changed luminance.\n\nTypo: “To assess the effect of chromatic cue” should be changed to: “To assess the effect of chromatic cues”",
"responses": []
},
{
"id": "4429",
"date": "22 Apr 2014",
"name": "Arnold Wilkins",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigation of the effects of colour on behaviour, as in the investigation of hypnosis, it is difficult to separate from other effects the influence of participants’ prior beliefs and expectations. These are culturally determined, and may play a role in influencing outcomes. Within contemporary culture there are widely held beliefs about the effects that colour may have on behaviour, and these have the potential to alter the participants’ motivation and attitudes towards any study involving colour. Attitudes and motivation are more likely to play a role in those cognitive tasks that have given rise to the Colour in Context theory, than in the low-level visual task used here in which performance is judged using two-alternative Psychophysics.The current study appears to have been carefully designed and conducted, but perhaps slightly less carefully reported. I would have liked to have seen the stimuli described in greater detail. For example:What were the chromaticities of the red and blue colours used? It is unclear what made it impossible to match the luminances. Was the positioning of the square such that the centre was at 5 degrees eccentricity, or was it the nearest margin that was at 5 degrees eccentricity? Was the “radius” of the square measured horizontally or diagonally? I do not see how the lifetime of the dipoles could have been 0.005ms. Even if this is a typo and the lifetime was 0.005s, I am still incredulous because the screen refresh rate was 100Hz, so the frame duration was 0.01s and this is twice the supposed duration of the dipoles!A t-test is not a dependant, even when it is a dependent t-test.I would have liked to have seen some discussion of the possible role of colour in affecting the accommodative lag to the visual stimuli on display. There is data in the literature that could have been used to argue that the effects of colour on image blur would have been negligible (about 0.07 dioptres, at maximum), and in the event any effects of accommodation were minimal because colour had no effect on visual performance of either local or global tasks.Given that about half the participants were aware of the colour manipulation and half were not, it might have been of interest to have separated the data for these two groups to see whether any effects of belief (“Colour in Context”) were more apparent in the group that had awareness. Indeed, in the literature on \"Colour in Context\", it would have been useful to have known what preconceptions regarding the effects of colour the participants brought to the tasks they were asked to perform.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-247
|
https://f1000research.com/articles/2-246/v1
|
15 Nov 13
|
{
"type": "Case Report",
"title": "Case Report: Pregnancy in a patient with recurrent glioblastoma",
"authors": [
"Birgit Flechl",
"Marco Ronald Hassler",
"Gerhard Kopetzky",
"Peter Balcke",
"Christine Kurz",
"Christine Marosi",
"Birgit Flechl",
"Marco Ronald Hassler",
"Gerhard Kopetzky",
"Peter Balcke",
"Christine Kurz"
],
"abstract": "We report the case of a woman with relapsed glioblastoma multiforme (GBM) who recently gave birth. She announced her pregnancy shortly after the sixth cycle of a dense regimen of temozolomide, prescribed for treating the first recurrence of glioblastoma. Three years ago, in April 2008, she had undergone gross total resection of a glioblastoma multiforme in the postcentral region of the right hemisphere and had subsequently received treatment according to the actual standard therapy consisting of radiotherapy up to 60 Gy with concomitant and adjuvant temozolomide. The complete amount of temozolomide given before this pregnancy was 20.9 mg/m2. Nevertheless, she delivered a 1890 g child by caesarean section in the 32/6 week of pregnancy. The child showed no anomalies and is developing normally under close surveillance by paediatricians.",
"keywords": [
"glioblastoma",
"pregnancy under chemotherapy"
],
"content": "Introduction\n\nCases of pregnancy and giving birth are rare in patients with malignant gliomas. Besides the grim prognosis of malignant gliomas, the actual standard therapy of glioblastoma adversely affects fertility1. In fact, radiotherapy aimed at the hypothalamus and the pituitary gland exceeding 45 Gy could impair the synthesis of gonadotropins, while chemotherapy with alkylating agents such as temozolomide is associated with impaired ovarian function leading to premature menopause1–3. However, there are some reports of pregnancies in women with malignant gliomas1,4–11, describing women whose brain tumors were diagnosed during pregnancy, as well as women who already had undergone treatment for malignant gliomas and who became pregnant afterwards. Our recent patient is an example of a patient whose fertility was not suppressed by glioblastoma multiforme (GBM) and its treatment.\n\n\nCase\n\nThe patient is a 37 year old woman, born in Austria. She had two children (13 and 15 years) before her GBM diagnosis. She was diagnosed with GBM in the right frontal lobe in April 2008, and treated according to a current treatment standard consisting of a gross total resection of the tumor, fractionated confocal radiotherapy up to 60 Gy (2 Gy/fraction) and concomitant and adjuvant chemotherapy with temozolomide12. Diagnosis and treatment were performed at the Medical University of Vienna (MUV). Following the concomitant therapy, she experienced amenorrhea for 6 months, followed by irregular menstrual cycles with oligomenorrhea. Two years later, a local recurrence of GBM was diagnosed. She underwent a second resection and followed 6 cycles of dose dense temozolomide, 100 mg/m2 orally for five days per week, with a drug holiday over every weekend. She became pregnant less than three weeks after the last intake of the sixth cycle of temozolomide, after a total dose of temozolomide of 20.9 g/m2 (Table 1).\n\nBoth parents wanted to carry the child to term. They were offered intensified pregnancy monitoring and genetic counselling, but no genetic tests were performed. During pregnancy the child developed normally as followed by close meshed ultrasound controls and the mother was doing well until week 27, when she developed signs of increased intracranial pressure as well as weakness of the left leg. She was admitted to the local general hospital for observance and anti edematous treatment. As the mother’s condition worsened, she received corticosteroids to induce lung maturation of the fetus, and the child was delivered after 32 weeks and 6 days of pregnancy by caesarean section using the Misgav Ladach method13,14. The child showed Apgar scores of 8/9/9, one, five and ten minutes after birth, respectively, weighed 1876 g, cried spontaneously and was neurologically inconspicuous. The infant did not need further respiratory assistance after the third day of life, and was discharged from the neonatal intensive care unit after an uncomplicated stay without any signs of neurological or any other organ deficit, weighing 2.5 kg. The child has shown normal growth and development ever since.\n\nThe mother underwent her third neurosurgical procedure two weeks after delivery, followed by chemotherapy with fotemustine 100 mg/m2 every three weeks for six cycles combined with bevacizumab 10 mg/kg every two weeks 6 weeks later. Eight months after the caesarean section, distal progression in the brain was diagnosed on MRI and she was referred for radiotherapy of the distant recurrence.\n\n\nDiscussion\n\nTemozolomide has been classified to pregnancy category D by the US Food and Drug Administration, which means that animal studies have revealed evidence of embryo lethality induced by the drug. Moreover, malformations have been observed in animals, therefore women are advised to avoid becoming pregnant during temozolomide intake. However, we report here the pregnancy of a woman with GBM, treated with radio/chemotherapy using a protocol described by Stupp et al.12 and further dose dense temozolomide in relapse 5 days of seven, as described by Strik et al.15.\n\nThere are some previous reports on pregnancies in women with malignant glioma. Nadine Johnson retrospectively reported the obstetric outcomes of women with intracranial neoplasms, from Ontario, Canada, covering the experience of 6000 deliveries. In this database, Johnson et al. identified 25 pregnancies in women who had been diagnosed with intracranial neoplasms, including 12 patients with gliomas8. This review discusses neurological deterioration due to the hormonal changes causing fluid retention during pregnancy and the complications and the management of increased cranial pressure at the time of delivery. There are some small series and case reports on the same issues4,7–10,14,16,17.\n\nDeborah Blumenthal (2008) reported a series of 6 women with malignant glioma with unplanned pregnancies during glioma treatment. Several women even had drug exposure during the first trimester of pregnancy, three with PCV (procarbazine, cyclophosphamide, vincristine), and three with temozolomide17. All gave birth to healthy, full term infants with no evidence of congenital malformations. Sadly, five of the six mothers died within the following 2.5 years due to recurrence of disease17.\n\nAn adverse effect on disease outcome during pregnancy has been observed in six of eight pregnancies in women with low grade gliomas followed by the French low grade glioma (LGG) group, possibly due to the decreased immune surveillance during pregnancy or to the presence of potential hormonal receptors on glioma cells4,16. In a later series on 12 pregnancies of women with LGG followed by the same research group published 2010, the growth rate of the gliomas increased during pregnancy as measured by successive MRI scans16.\n\nHowever, the prolonged survival of our patient 18+ months after the diagnosis of a relapsed glioblastoma appears noteworthy. This case also should bring to mind the necessity of repeated counselling about contraception – even in patients where the probability of persistent fertility is minimal, as in the patient described in this report. In fact, regardless of all the apprehensions of medical professionals about this pregnancy, the patient and her husband were simply happy about it. They wanted to keep this child, even if it would have meant severe adverse complications for the mother. They perceived this pregnancy as a gift of life and enjoyed every moment, even when it was steadily overshadowed by the glioblastoma. They remembered perfectly well that they had been told that pregnancy should be avoided during and after the glioma treatment, and that they even had signed this as a part of the regular consent for treatment; but on the other hand they told us that this pregnancy had been the only happy event for them in all those years.\n\nThey also tried to organize further care for the child, as the possibility of the baby losing its mother during childhood is very high. As the patient and her husband are aware of the grim prognosis of recurrent GBM, they planned to raise the child with multiple psychological parents, including its grown up siblings and the two sisters of the mother.\n\n\nConclusion\n\nThis report shows that pregnancy and the birth of a healthy infant can occur even in women that have been heavily pretreated with alkylating agents and that conception can occur as early as three weeks after the last intake of chemotherapy.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report.",
"appendix": "Author contributions\n\n\n\nBF was a major contributor in writing and interpreting the manuscript. RMH has made substantial contributions to the conception and design of the manuscript. GK and PB delivered essential patient data. CK was involved in revising the report critically for important intellectual content. CM made substantial contributions to the conception and design of the report, coordinated the tasks and timelines and was involved in writing and interpreting the manuscript. All authors read and approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nThe author(s) declare that they have no competing interests.\n\n\nGrant information\n\nThe Medical University of Vienna supported this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nMcGrane J, Bedford T, Kelly S: Successful pregnancy and delivery after concomitant temozolomide and radiotherapy treatment of glioblastoma multiforme. Clin Oncol (R Coll Radiol). 2012; 24(4): 311. PubMed Abstract | Publisher Full Text\n\nSitbon Sitruk L, Sanson M, Prades M, et al.: [Unknown gonadotoxicity chemotherapy and preservation of fertility: example of Temozolomide]. Gynecol Obstet Fertil. 2010; 38(11): 660–2. PubMed Abstract | Publisher Full Text\n\nPreusser M, Seywald S, Elandt K, et al.: Pilot study on sex hormone levels and fertility in women with malignant gliomas. J Neurooncol. 2012; 107(2): 387–94. PubMed Abstract | Publisher Full Text\n\nPallud J, Duffau H, Razak RA, et al.: Influence of pregnancy in the behavior of diffuse gliomas: clinical cases of a French glioma study group. J Neurol. 2009; 256(12): 2014–20. PubMed Abstract | Publisher Full Text\n\nHaba Y, Twyman N, Thomas SJ, et al.: Radiotherapy for glioma during pregnancy: fetal dose estimates, risk assessment and clinical management. Clin Oncol (R Coll Radiol). 2004; 16(3): 210–4. PubMed Abstract | Publisher Full Text\n\nTopliss DJ: Optic-nerve glioma and pregnancy. Med J Aust. 1989; 150(5): 287–8. PubMed Abstract\n\nDucray F, Colin P, Cartalat-Carel S, et al.: Management of malignant gliomas diagnosed during pregnancy. Rev Neurol (Paris). 2006; 162(3): 322–9. PubMed Abstract\n\nJohnson N, Sermer M, Lausman A, et al.: Obstetric outcomes of women with intracranial neoplasms. Int J Gynaecol Obstet. 2009; 105(1): 56–9. PubMed Abstract | Publisher Full Text\n\nLynch JC, Gouvêa F, Emmerich JC, et al.: Management strategy for brain tumour diagnosed during pregnancy. Br J Neurosurg. 2011; 25(2): 225–30. PubMed Abstract | Publisher Full Text\n\nMackenzie AP, Levine G, Garry D, et al.: Glioblastoma multiforme in pregnancy. J Matern Fetal Neonatal Med. 2005; 17(1): 81–3. PubMed Abstract | Publisher Full Text\n\nSöderström-Anttila V, Salokorpi T, Pihlaja M, et al.: Obstetric and perinatal outcome and preliminary results of development of children born after in vitro maturation of oocytes. Hum Reprod. 2006; 21(6): 1508–13. PubMed Abstract | Publisher Full Text\n\nStupp R, Mason WP, van den Bent MJ, et al.: European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005; 352(10): 987–96. PubMed Abstract | Publisher Full Text\n\nHolmgren G, Sjoholm L, Stark M: The Misgav Ladach method for cesarean section: method description. Acta Obstet Gynecol Scand. 1999; 78(7): 615–21. PubMed Abstract | Publisher Full Text\n\nDarj E, Nordstrom ML: The Misgav Ladach method for cesarean section compared to the Pfannenstiel method. Acta Obstet Gynecol Scand. 1999; 78(1): 37–41. PubMed Abstract | Publisher Full Text\n\nStrik HM, Marosi C, Kaina B, et al.: Temozolomide dosing regimens for glioma patients. Curr Neurol Neurosci Rep. 2012; 12(3): 286–293. PubMed Abstract | Publisher Full Text\n\nPallud J, Mandonnet E, Deroulers C, et al.: Club de Neuro-Oncologie de la Société Française de Neurochirurgie (SFNC); Association des Neuro-Oncologues d'Expression Française (ANOCEF). Pregnancy increases the growth rates of World Health Organization grade II gliomas. Ann Neurol. 2010; 67(3): 398–404. PubMed Abstract | Publisher Full Text\n\nBlumenthal DT, Parreño MG, Batten J, et al.: Management of malignant gliomas during pregnancy: a case series. Cancer. 2008; 113(12): 3349–54. PubMed Abstract | Publisher Full Text\n\nNishio S, Morioka T, Suzuki S, et al.: Primary brain tumours manifesting during pregnancy: presentation of six cases and a review of the literature. J Clin Neurosci. 1996; 3(4): 334–7. PubMed Abstract | Publisher Full Text\n\nRoelvink NC, Kamphorst W, van Alphen HA, et al.: Pregnancy-related primary brain and spinal tumors. Arch Neurol. 1987; 44(2): 209–15. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "3122",
"date": "14 Jan 2014",
"name": "Deborah Blumenthal",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, \"Pregnancy in a patient with recurrent glioblastoma\" illustrates a notable case of a 37 year-old woman with recurrent glioblastoma (GB) who became pregnant within 3 weeks of a (6–cycle) dose-dense temozolomide regimen. The case is notable for preservation of fertility, essentially during alkylating treatment; positive outcome of the child at planned 32 week delivery by C-section and normal growth and development at 18 months; and for survival of the mother with recurrent GB 18+ months from recurrence. The authors mention prior related works in the literature regarding the effects of pregnancy on patients with malignant glioma; a series of patients who conceived (with positive fetal outcome) during active alkylator treatment and documentation of accelerated growth rates of gliomas during pregnancy. This report is encouraging for those patients concerned about fertility issues, but should not be interpreted as a guarantee for either male or female patients that fertility will be preserved after alkylating chemotherapy. Additionally, a disclaimer should be made for the potential (confirmed by animal studies on embryo-lethality with exposure to these drugs) danger to the exposed fetus, despite the reported good outcomes of these children.",
"responses": []
},
{
"id": "4214",
"date": "21 Mar 2014",
"name": "Mari-Paule Thiet",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a single case of pregnancy following a diagnosis and treatment of recurrent glioblastoma. It illustrates that spontaneous pregnancy can be achieved after high doses of chemotherapy. There is a review of the current literature regarding outcomes of pregnancy in patients with brain tumors.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-246
|
https://f1000research.com/articles/2-245/v1
|
15 Nov 13
|
{
"type": "Case Report",
"title": "Bell’s palsy during interferon alpha 2a treatment in a case with Behçet uveitis",
"authors": [
"Fatime Nilüfer Yalçindağ",
"Cem Alay",
"Fatime Nilüfer Yalçindağ"
],
"abstract": "Purpose: To present a case who developed Bell’s palsy while using interferon alpha 2a for Behçet uveitis.Methods: A patient with Behçet disease presented with decreased vision in his right eye. Ophthalmic examination, fundus fluorescein angiography and optical coherence tomography were performed. After developing facial paralysis while on interferon therapy, the patient was referred to our neurology service for differential diagnosis and treatment.Results: Examination of right eye revealed panuveitis with branch retinal vein occlusion, so high dose steroids were prescribed. In three days there was no improvement in terms of vitreous inflammation and so steroids were replaced with interferon. At the seventh month, patient experienced a facial paralysis. After eliminating other causes, including viral infections, trauma, cold exposure and neurological evaluation with cranial MRI, the patient was diagnosed to have Bell’s palsy by a neurologist. Interferon was replaced with mycophenolate mofetil and the Bell’s palsy was treated with oral steroids.Conclusion: It is important to be alert to both common and rare complications while treating with interferon.",
"keywords": [
"Behçet disease",
"interferon alpha 2a",
"Bell’s palsy",
"branch retinal vein occlusion",
"uveitis"
],
"content": "Introduction\n\nInterferon alpha 2a is a recombinant biological agent that is used for the treatment of several diseases including chronic hepatitis C, hairy cell leukemia, Philadelphia chromosome positive chronic myelogenous leukemia (CML) and AIDS-related Kaposi’s sarcoma. It is also used in Behçet uveitis that is refractory to conventional immunosuppressive agents1,2. The central (CNS) and peripheral nervous system (PNS) can be affected while using interferon alpha 2a. There are two existing case reports describing Bell’s palsy associated with interferon treatment for chronic hepatitis C infection3,4. Here we present another case report indicating Bell’s palsy during interferon alpha 2a treatment in Behçet uveitis.\n\n\nCase report\n\nA 27-year old Turkish, male, taxi driver who has Behçet’s Disease was referred to our clinic for a decrease in visual acuity in his right eye for 2 days. At presentation, the best-corrected visual acuity was 20/320 in his right eye and 20/20 in his left eye. On slit lamp examination, there were keratic precipitates, anterior chamber cells and vitreous cells in his right eye. Fundus examination revealed optic disc edema, macular edema and abundant intraretinal hemorrhages in the inferior temporal part of retina. His left eye seemed to be normal. Optical coherence tomography (Cirrus HD-OCT Model 4000, Carl Zeiss Meditec) (OCT) and fluorescein angiography (FA) was then performed. Central macular thickness (CMT) was 595 µm on OCT. In FA, there was mild hyperfluorescence on both optic discs. In addition, hypofluorescence in the inferior retinal areas due to hemorrhages and staining on the vessel walls in right eye was spotted. Macular edema and peripheral ischemia were also evident. A diagnosis of panuveitis with inferior temporal branch retinal vein occlusion due to Behçet’s Disease was made.\n\nFirstly, high dose steroids (1 gram/day methylprednisolone, intravenously) were prescribed to the patient for 3 days. Dexamethasone eye drops every hour during the day and topical cyclopentolate 1% three times a day were also prescribed. On the third day, the anterior chamber reaction seemed to be decreased but no improvement was observed, either in the vitreous inflammation or the macular edema. As such, the initial high dose steroid treatment was stopped and interferon alpha 2a (Roferon-A, Roche Pharmaceuticals, Hoffmann-La Roche Inc., Switzerland) 4.5 million IU (MIU) subcutaneously on alternate days and 10 mg/day oral prednisolone were prescribed to the patient. At that point, we had decided upon interferon treatment because the patient was young and the first ocular presentation of Behçet’s disease was seriously threatening in terms of visual prognosis. In addition, green argon laser photocoagulation was applied to the ischemic areas of the retina.\n\nThe patient was examined weekly for the first month in order to observe the effects of treatment closely. After this, the follow-up was done monthly. In the first month, steroid treatment was tapered slowly and stopped while interferon treatment was continued. In the first 6 months, no adverse effects were observed, best corrected visual acuity improved to 20/32 and the panuveitis and macular edema regressed. CMT was 230 µm after the 6-months of interferon alpha 2a therapy. There were no signs of anterior uveitis or vitreous inflammation. The patient’s oral aphthae, genital ulcers and arthralgia (symptoms of Behçet’s Disease) were also improved. At this stage, the dose of interferon alpha 2a was tapered to 3 MU on alternate days.\n\nOne month after tapering the dose, the patient experienced difficulty in closing his right eyelid and stiffness in the right half of his face. His wife also reported that there had been no movement on the right side of his face for about 15 days. There was no history of trauma, infection, cold exposure or any other predisposing factors that might have explained this situation. The patient was tested for several viral infections including herpes viruses, cytomegalovirus (CMV) and Epstein-Barr virus (EBV). All tests were negative. Afterwards, in order to eliminate neurological problems such as brain tumor, stroke or neuro-Behçet disease, the patient was immediately referred to our neurology service. Cranial MRI was normal and the neurological evaluation revealed no pathological findings apart from facial paralysis. As such the patient was diagnosed with Bell’s palsy. Interferon use was considered as a possible reason for this condition and so interferon treatment was tapered and discontinued. Mycophenolate mofetil (720 mg/day, oral) was prescribed for Behçet’s disease because the patient had previously experienced nausea and vomiting with azathioprine, which had been prescribed before as an initial therapy for Behcet’s disease by a rheumatologist. Oral methylprednisolone 48 mg per day was prescribed by the neurologist and gradually tapered (8 mg per week) over 6 weeks. In addition, a program of exercises for the facial muscles was recommended. All symptoms of Bell’s palsy disappeared within 2 months of onset.\n\n\nDiscussion\n\nThere is a wide variety of adverse effects due to the use of interferon alpha 2a, including flu-like syndrome (unusual tiredness, fever, chills, muscle aches, and joint pain), injection site reaction (redness, pain at the site of injection), proneness to serious infections, new or worsening autoimmune disease5–7, myelosuppression8,9, depression, suicide, suicidal thoughts, and cardiovascular problems10,11 such as hypotension or hypertension, arrhythmia and myocardial infarction. Patients may also suffer from nausea, vomiting, loss of appetite, diarrhea, cough, dry mouth, dizziness, vertigo, sweating, itching, hair loss, fatigue, abdominal pain, constipation, sore throat, insomnia12, anxiety and numbness.\n\nInterferon alpha 2a also affects the CNS and PNS. Difficulty of concentration and aggression are the most common CNS problems. Decrease in mental activity, dysphasia, dysarthria, aphasia, aphonia, amnesia, multiple sclerosis-like disease13, transient ischemic attacks, encephalopathy, confusion, somnolence and coma may also occur during the use of interferon. It is also known that several symptoms such as ataxia, paresthesia, tremor and peripheral neuropathies14–18 can be seen if the PNS is affected. In our patient, we encountered Bell’s palsy, a peripheral neuropathy affecting the facial nerve in the 7th month of interferon therapy. No elucidating factors but interferon were present at the time we diagnosed the patient. Hitherto, there have been two other reports of Bell’s palsy related to the use of interferon3,4. So it was important for us to call attention to our case as an example of a rare complication of interferon use. In conclusion, physicians should always keep an eye on patients who are being treated with interferon alpha 2a. Physicians should be alert to the rare side effects of interferon treatment as well as the more common and well-known side effects of this agent.\n\n\nConsent\n\nWritten informed consent for publication of his clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nDr. Alay participated in drafting the article and Dr. Yalçındağ revised it critically for intellectual content. Dr. Alay and Dr. Yalçındağ were involved in the assessment and treatment of the patient. Both authors gave final approval of the version to be submitted and revised version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nKötter I, Zierhut M, Eckstein AK, et al.: Human recombinant interferon alfa-2a for the treatment of Behçet's disease with sight threatening posterior or panuveitis. Br J Ophthalmol. 2003; 87(4): 423–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYalçindağ FN, Uzun A: Results of Interferon Alpha-2a Therapy in Patients with Behcet's Disease. J Ocul Pharmacol Ther. 2012; 28(4): 439–43.PubMed Abstract | Publisher Full Text\n\nOgundipe O, Smith M: Bell’s palsy during interferon therapy for chronic hepatitis C infection in patients with haemorrhagic disorders. Haemophilia. 2000; 6(2): 110–2. PubMed Abstract | Publisher Full Text\n\nHoare M, Woodall T, Alexander GJ: Bell’s palsy associated with IFN-alpha and ribavirin therapy for hepatitis C virus infection. J Interferon Cytokine Res. 2005; 25(3): 174–6. PubMed Abstract | Publisher Full Text\n\nRizvi R, Hojjati M: Interferon-α induced lupus in a patient with chronic hepatitis C virus. J Clin Rheumatol. 2011; 17(3): 152–3. PubMed Abstract | Publisher Full Text\n\nSavvas SP, Papakostas N, Giannaris M, et al.: Interferon alpha-induced hashimoto thyroiditis followed by transient graves disease in a patient with chronic HCV infection. South Med J. 2010; 103(6): 585–8. PubMed Abstract | Publisher Full Text\n\nYang D, Arkfeld D, Fong TL: Development of anti-CCP-positive rheumatoid arthritis following pegylated interferon-α2a treatment for chronic hepatitis C infection. J Rheumatol. 2010; 37(8): 1777. PubMed Abstract | Publisher Full Text\n\nZizer E, Bommer M, Barth T, et al.: Severe agranulocytosis as a rare side effect of pegylated interferon therapy for chronic hepatitis B. Z Gastroenterol. 2011; 49(5): 596–8. PubMed Abstract | Publisher Full Text\n\nHajder J, Stanisavljević N, Marković O, et al.: Late onset of severe thrombocytopenia during interferon treatment for chronic hepatitis C infection--case report. Srp Arh Celok Lek. 2010; 138(3–4): 240–3. PubMed Abstract | Publisher Full Text\n\nRoche NC, Paule P, Kerebel S, et al.: Complete atrio-ventricular block: a rare complication of interferon alpha therapy. Presse Med. 2011; 40(3): 316–8. PubMed Abstract | Publisher Full Text\n\nVelasco J, Oriñuela I, Sanjuán AZ, et al.: Pericardial effusion associated to interferon in an immunocompetent patient. Enferm Infecc Microbiol Clin. 2010; 28(10): 749–50. PubMed Abstract | Publisher Full Text\n\nRaison CL, Rye DB, Woolwine BJ, et al.: Chronic interferon-alpha administration disrupts sleep continuity and depth in patients with hepatitis C: association with fatigue, motor slowing, and increased evening cortisol. Biol Psychiatry. 2010; 68(10): 942–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatsuo T, Takabatake R: Multiple sclerosis-like disease secondary to alpha interferon. Ocul Immunol Inflamm. 2002; 10(4): 299–304. PubMed Abstract | Publisher Full Text\n\nKnyazer B, Lifshitz T, Marcus M, et al.: Anterior ischemic optic neuropathy in a patient with hepatitis C treated with interferon-alpha and ribavirin. Isr Med Assoc J. 2011; 13(4): 251–3. PubMed Abstract\n\nKhiani V, Kelly T, Shibli A, et al.: Acute inflammatory demyelinating polyneuropathy associated with pegylated interferon alpha 2a therapy for chronic hepatitis C virus infection. World J Gastroenterol. 2008; 14(2): 318–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTunca A, Erbayrak M, Aytaç S, et al.: Axonal neuropathy and hearing loss associated with alpha interferon treatment in chronic hepatitis B: a case report. Turk J Gastroenterol. 2004; 15(2): 97–9. PubMed Abstract\n\nFukumoto Y, Shigemitsu T, Kajii N, et al.: Abducent nerve paralysis during interferon alpha-2a therapy in a case of chronic active hepatitis C. Intern Med. 1994; 33(10): 637–40. PubMed Abstract | Publisher Full Text\n\nBauherz G, Soeur M, Lustman F: Oculomotor nerve paralysis induced by alpha II-interferon. Acta Neurol Belg. 1990; 90(2): 111–4. PubMed Abstract"
}
|
[
{
"id": "2953",
"date": "14 Jan 2014",
"name": "Erkan Alpsoy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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-organised and well-written paper and I am sure that it will be helpful to readers in this field. I only have one minor comment: that the English Language should be checked and grammatical mistakes corrected.",
"responses": []
},
{
"id": "3445",
"date": "10 Feb 2014",
"name": "Sumru Onal",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Yalcindag and Alay reports on a patient with Bell’s palsy that occurred as a possible adverse effect of interferon alfa-2a that was prescribed in an effort to control Behçet uveitis. It is a well-written paper and will contribute to practicing ophthalmologists. My comments are listed below: I would suggest that the authors name the disease as “Behçet disease” rather than “Behçet’s disease” throughout the paper. The intraocular inflammation has already been well addressed as “Behçet uveitis”. There is need for revision of the English grammar and language. I would recommend that the authors include information from the World Health Organization’s causality assessment of suspected adverse drug reactions in the discussion section. The authors should also classify their observations based on this classification. It seems to me that the described finding is a possible adverse effect. The reference would be: “Edwards IR, Biriell C. Harmonisation in pharmacovigilance. Drug Saf 1994;10:93–102.” The classification is as follows:Certain: A clinical event, including laboratory test abnormality, occurring in a plausible time relationship to drug administration that cannot be explained by concurrent disease or other drugs or chemicals. The response to withdrawal of the drug (dechallenge) should be clinically plausible. The event must be definitive pharmacologically or phenomenologically, using satisfactory rechallenge procedure if necessary.Probable/likely: A clinical event, including laboratory test abnormality, with a reasonable time sequence to administration of the drug, unlikely to be attributed to concurrent disease or other drugs or chemicals, which follows a clinically reasonable response on withdrawal (dechallenge). Rechallenge information is not required to fulfil this definition.Possible: A clinical event, including laboratory test abnormality, with a reasonable time sequence to administration of the drug, which could also be explained by concurrent disease, or other drugs or chemicals, or underlying disease provide plausible explanations.Unlikely: A clinical event, including laboratory test abnormality, with a temporal relationship to drug administration that makes a causal relationship improbable and in which other drugs, chemicals, or underlying disease provide plausible explanations.Conditional/unclassified: A clinical event, including laboratory test abnormality, reported as an adverse reaction, about which more data are essential for a proper assessment or the additional data are undergoing examination.Nonassessable/unclassified: A report suggesting an adverse reaction that cannot be judged because information is insufficient or contradictory and that cannot be supplemented or verified.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-245
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https://f1000research.com/articles/2-198/v1
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25 Sep 13
|
{
"type": "Short Research Article",
"title": "Reprogramming diminishes retention of Mycobacterium leprae in Schwann cells and elevates bacterial transfer property to fibroblasts",
"authors": [
"Toshihiro Masaki",
"Aidan McGlinchey",
"Simon R. Tomlinson",
"Jinrong Qu",
"Anura Rambukkana",
"Toshihiro Masaki",
"Aidan McGlinchey",
"Simon R. Tomlinson",
"Jinrong Qu"
],
"abstract": "Background: Bacterial pathogens can manipulate or subvert host tissue cells to their advantage at different stages during infection, from initial colonization in primary host niches to dissemination. Recently, we have shown that Mycobacterium leprae (ML), the causative agent of human leprosy, reprogrammed its preferred host niche de-differentiated adult Schwann cells to progenitor/stem cell-like cells (pSLC) which appear to facilitate bacterial spread. Here, we studied how this cell fate change influences bacterial retention and transfer properties of Schwann cells before and after reprogramming.Results: Using primary fibroblasts as bacterial recipient cells, we showed that non-reprogrammed Schwann cells, which preserve all Schwann cell lineage and differentiation markers, possess high bacterial retention capacity when co-cultured with skin fibroblasts; Schwann cells failed to transfer bacteria to fibroblasts at higher numbers even after co-culture for 5 days. In contrast, pSLCs, which are derived from the same Schwann cells but have lost Schwann cell lineage markers due to reprogramming, efficiently transferred bacteria to fibroblasts within 24 hours.Conclusions: ML-induced reprogramming converts lineage-committed Schwann cells with high bacterial retention capacity to a cell type with pSLC stage with effective bacterial transfer properties. We propose that such changes in cellular properties may be associated with the initial intracellular colonization, which requires long-term bacterial retention within Schwann cells, in order to spread the infection to other tissues, which entails efficient bacterial transfer capacity to cells like fibroblasts which are abundant in many tissues, thereby potentially maximizing bacterial dissemination. These data also suggest how pathogens could take advantage of multiple facets of host cell reprogramming according to their needs during infection.",
"keywords": [
"The peripheral nervous system (PNS) is the preferred residence for one of the oldest bacterial pathogens known to mankind",
"Mycobacterium leprae (ML). It causes human leprosy",
"which is a chronic neurological disease and still remains a public health problem1. The large gaps in our understanding of the infectious process have halted the progress towards developing effective early diagnostics and therapeutics for the management of nerve damage",
"the pathological hallmark of the disease. The distinctive peripheral nerve involvement is directly associated with the remarkable capacity of ML to invade the supporting glial cells of the adult PNS",
"Schwann cells2. Because peripheral nerves are a privileged site and are therefore protected from immune cells due to blood nerve barrier",
"Schwann cells provide a safer niche for ML survival",
"propagation and initial colonization."
],
"content": "Introduction\n\nThe peripheral nervous system (PNS) is the preferred residence for one of the oldest bacterial pathogens known to mankind, Mycobacterium leprae (ML). It causes human leprosy, which is a chronic neurological disease and still remains a public health problem1. The large gaps in our understanding of the infectious process have halted the progress towards developing effective early diagnostics and therapeutics for the management of nerve damage, the pathological hallmark of the disease. The distinctive peripheral nerve involvement is directly associated with the remarkable capacity of ML to invade the supporting glial cells of the adult PNS, Schwann cells2. Because peripheral nerves are a privileged site and are therefore protected from immune cells due to blood nerve barrier, Schwann cells provide a safer niche for ML survival, propagation and initial colonization.\n\nOne of the primary functions of Schwann cells is to synthesize the myelin sheath around axons that deliver rapid nerve conduction3. Despite the terminal differentiation of Schwann cells to a highly sophisticated myelinated phenotype, these mature Schwann cells show unprecedented plasticity; they can switch off their myelin program in response to injury and acquire a de-differentiated state resembling an immature phenotype, but maintain Schwann cell lineage properties4,5. ML appear to take advantage of this natural property and induce myelin damage (demyelination) as an adaptive mechanism to generate similar de-differentiated Schwann cells that are more favourable for bacterial colonization and manipulation6. We have recently shown that once infected these de-differentiated Schwann cells purified from adult nerves, they undergo a reprogramming process and convert to highly immature progenitor/stem cell-like cells (pSLC) that are more suitable for spreading infection to other tissues either by direct re-differentiation or through macrophages7. However, it is not clear if fibroblasts, which are ubiquitous in many tissues, could play an intermediary role by receiving ML and if so how pSLC differ from non-reprogrammed Schwann cells in terms of bacterial transfer.\n\nIn this study, using primary mouse fibroblasts as a model, we assessed the bacterial transfer capacity of Schwann cells before and after reprogramming. We showed that before reprogramming, lineage committed Schwann cells possess a high bacterial retention capacity. However, after reprogramming, which downregulated all Schwann cell lineage markers and myelin markers, Schwann cells lose bacterial retention and acquire an efficient bacterial transfer property to co-cultured fibroblasts. These findings show an example of how a bacterial pathogen could use an induced cell fate change to suit its own ends during different stages of infectious process.\n\n\nMaterials and methods\n\nAdult CD-1 mice (4–6 week old, ICR strain code: 022) and 6–8 week old GFP mice that constitutively express eGFP (strain: C57BL/6-Tg [ACTB-EGFP]1Osb/J, stock: 003291) were purchased from Charles River and Jackson Laboratories (Bar Harbor, ME). Animals were maintained at the Rockefeller University animal facilities in pathogen free cages. Institutional Animal Care and Use Committee (IACUC) at the Rockefeller University approved all animal procedures and ethical issues. For isolating Schwann cells, 6–8 mice were used and cells were then purified using magnetic cell sorting system and FACS sorting using anti-p75 antibody (AB1554, Millipore, USA) as described7.\n\nPurified Schwann cells were grown in collagen coated T25 or T75 flasks (BD Biosciences, NJ, USA) and infected with ML and reprogrammed cells were generated according to our previous protocol7. In vivo-grown viable ML derived from nude-mouse footpads were prepared as described previously8. Briefly, p75+/Sox10+/Sox2+ Schwann cells purified from adult GFP-mice and wild type were infected with ML and maintained in Schwann cell media as described in details7. At day 3 post-infection, Schwann cells maintain p75+/Sox10+/Sox2+ and all other Schwann cell phenotype, and these infected cells were used in this study. In parallel, infected cells were incubated for four weeks and then FACS sorted for p75-cells; their phenotypes were confirmed as p75-/Sox10-/Sox2+, and reprogrammed cells were isolated from this population based on their ability to grow in mesenchymal stem cell media (StemCell Inc, Vancouver, Canada) and were termed as progenitor/stem-like cells (pSLC)7. pSLC were transduced with CopGFP-CDH-MSCV-cG reporter vector9 obtained from System Biosciences (CA) and stable expression of copGFP in pSLC was performed as reported earlier7. GFP+pSLC were re-infected with ML in order to maintain similar bacterial load as with Schwann cells at day 3 post-infection, since reprogramming process that accompanies long incubation and cell proliferation causes bacterial dilution.\n\nNeural fibroblasts were prepared from adult CD-1 male mice (4–6 week old, ICR strain code: 022) at the same time when Schwann cells were prepared from these mice1. In brief, peripheral nerve tissues were isolated in MEM (Invitrogen) and then digested with 0.125% trypsin (Invitrogen)/0.05%EDTA and 0.1 mg/ml collagenase I (Worthington Biochemical) and passed through 100 micron mesh nylon filter (BD Falcon). Cells were collected and seeded on T25 flask cultured in 10% FCS medium. Propagated cells negative for p75NGFR Schwann cell surface marker were separated by magnetic cell sorting system followed by FACS sorting using anti-p75 antibody (AB1554, Millipore, USA). The purified p75-negative cells, which were also negative for Sox2 and Sox10 were used for co-culture experiments. Skin fibroblasts were prepared from adult wild-type mouse skin using a similar protocol described in detail previously10.\n\nDay 3-infected GFP+Schwann cells and re-infected GFP+pSLC that carry >50–100 ML per cell (infection efficiency is >90%) were first washed to remove any extracellular ML and then co-cultured with GFP-negative fibroblasts for 18 hours, 3 and 7 days. Pre-evaluated culture medium containing DMEM with 6% serum (HyClone, USA) that suits short-term co-cultures, Schwann cell-fibroblasts and pSLC-fibroblasts co-culture combinations were selected. Although we used this medium for short-term cultures for quantification, both mesenchymal media without supplements and Schwann cell media were also supported cell growth for a short period with no apparent phenotypic change in Schwann cells and pSLC. Bacterial transfer to non-GFP fibroblasts from GFP+ cells or bacterial retention within GFP+ cells were evaluated by immunolabeling of fixed cultures using ant-PGL-1 antibody7 against GFP+/- detection. PGL-1/ML+ cells, both GFP+ and non-GFP cells, containing >50 ML per cells were quantified 18 hours, 3 and 5 days after addition of fibroblasts. Data were analyzed for statistical significance using Student’s t test or by regression analysis (SigmaPlot). Paired sample analysis with P values <0.01 were considered as significant.\n\nImmunofluorescence was performed as described7. In brief, cells were fixed with 4% formaldehyde (Sigma) for 10 min at room temperature and 100% methanol (Sigma) for 10 minutes at -20ºC. The samples were washed with PBS, blocked with 10% goat serum and then incubated with primary antibody followed by secondary antibody conjugated with Alexa Fluor 488 or 594 (Invitrogen) as described; details of anti-p75 and anti-PGL-1 and secondary antibodies have been described previously7,14. Images were captured with Nikon Eclipse 2100 microscopy.\n\nGene-Expression Analyses were performed using Affymetrix mouse gene chips according to A Affymetrix protocol as described previously7. In brief, total RNA was isolated from uninfected/control Schwann cells, 3 days post-infection and pSLC derived from day 28-infected Schwann cells using RNeasy columns (QIAGEN). Affymetrix Test3 arrays and mouse genome MG-430A2 arrays were probed with the cRNA prepared by reverse-transcription of the total RNA. Microarray data were processed by the Robust Multichip Average (RMA) method11. The R statistical programming language was used (version 2.15.2), in tandem with the Bioconductor Analysis suite (version 2.12)12. The resulting probe mRNA detection data was searched and selected for markers relating to Schwann cell lineage/myelination, based on published literature3–7. This list of probes contained duplicate probes for the same gene symbol and so was trimmed to include only one representative probe per gene symbol, both for clarity and due to space constraints. The data were presented in the form of heatmap representing expression of the genes (absolute log2 expression values) associated with these probes in each replicate at each time point. Genes were clustered by Euclidean distance and average linkage. Individual values and probes are shown in Supplemental table 1. Also, differentially expressed lineage marker genes (from means of replicates) were calculated as relative to control/uninfected cells in both day 3-infected cells and pSLC state.\n\n\nResults\n\nSchwann cells purified from mouse adult peripheral nerves maintained Schwann lineage and myelin markers and are highly susceptible to ML infection7. In this study, we used Schwann cells which were infected with ML for 3 days, and previously described pSLC7 which were derived from reprogrammed Schwann cells after day 28 infection. Both day 3-infected Schwann cells and pSLC showed high level of infection and strict confinement of ML to the cytoplasm (Figure 1A and B). We found no evidence for bacterial leakage into the surrounding media when either cell type is cultured on its own. Supernatants collected from these cells after infection showed no evidence of ML in the media (data not shown). We next determine if ML infection at day 3 changed Schwann cell lineage marker expression when compared with pSLC, which are known to be reprogrammed cells exhibiting the loss of Schwann cell lineage markers7. Comparative analyses revealed that infected Schwann cells at day 3 express a similar profile of Schwann cell lineage/myelination-related genes as compared to uninfected controls (Figure 1C-a). In contrast, pSLC showed a striking downregulation of the same markers. Absolute expression profiles of Schwann cell lineage/myelination-related genes are shown in Figure 1C-a. Differential expression of day 3-infected and pSLC as compared to uninfected control cells further revealed that there is almost no change in Schwann cell identity in cells at day 3 post-infection as compared to the marked downregulation of the same genes in pSLC (Figure 1C-b). Since pSLC, but not cells infected for 3 days, lost Schwann cell identity, we refer to pSLC and infected Schwann cells at day 3 as reprogrammed and non-reprogrammed Schwann cells respectively (Figure 1C-a, -b).\n\n(A) Purified adult de-differentiated Schwann cells infected with ML for 3 days and labelled with antibodies to p75NTR (red) and ML-specific PGL-1 (green), counterstained with DAPI for nuclei (blue). (B) GFP pSCL (green) derived from day 28 infected Schwann cells labeled with anti-PGL1 and counterstained with DAPI (blue). Asterisks denote the absence of ML outside infected cells; ML strictly retain within the cytoplasm and no evidence of bacterial leakage to the surrounding when maintain as monocultures. Magnification: (A-a,b,c to B-a,b,c) 20x. (C) (a) Expression levels of known Schwann cell lineage/myelination genes, inferred by mRNA detection by Affymetrix Mouse microarray, from 2 samples (S1 and S2) each from control/uninfected Schwann cells, infected Schwann cells for 3 days (non-reprogrammed cells preserving Schwann cell identity) and pSLC-derived cells from day 28-infected Schwann cells (reprogrammed cells with loss of Schwann cell identity). Genes are clustered by Euclidean distance and average linkage for clarity. (b) Differential expression (log2 fold change) of known Schwann cell lineage markers, shown as relative to control for both day 3 infected cells (left) and 28-day-derived pSLC-state cells (right). The mean of both replicates for each of the three time points was used when calculating log2 fold change relative to control. Note that both absolute (a) and differentially expressed (b) patterns show a high degree of similarity between control/uninfected and day 3-infected cells, as compared to marked downregulation of Schwann lineage/in pSLC. Robust Multichip Average (RMA) values representing, on a logarithmic scale (base 2), the relative abundance of an mRNA transcript for a given gene, shown as a colour scale from highest (around 10) to lowest (around 4). Colour scale for heatmap ranges between minimum and maximum detection of selected genes, while full array's range was 2.4 to 14.2.\n\nThe difference between non-reprogrammed and reprogrammed Schwann cells at the mRNA level correlated with their capacity to maintain or transfer ML when donor primary fibroblasts were introduced to these cell types. GFP+ Schwann cells purified from GFP mice show high susceptibility to ML infection. When GFP+ Schwann cells at day 3 post-infection were co-cultured with non-GFP fibroblasts we found intracellular ML to be retained within the cytoplasm of Schwann cells after 3 and 5 days (Figure 2A). Bacterial transfer to GFP negative fibroblasts was minimal even after 5 days of co-culture. Identical results were obtained regardless of the tissue type (neural or skin) from which fibroblasts were isolated and the media used for co-culturing the cells. These data suggest that non-reprogrammed Schwann cells, which preserve Schwann cell identity, expressing the full spectrum of Schwann cell lineage functional markers (Figure 1C), retain ML in large numbers.\n\n(A) De-differentiated Schwann cells purified from adult peripheral nerves from GFP mice were infected with ML for 3 days and incubated with fibroblasts (isolated from wild type/non-GFP peripheral nerves) for another 3 (top panel) and 5 (bottom panel) days. Fixed co-cultures were labelled with antibody to ML-specific PGL-1 (red) and nuclei were counter stained with DAPI (blue). White arrows show non-GFP fibroblasts with very few ML transferred from GFP+ Schwann cells. Note that almost all GFP+ Schwann cells carry high number of ML (yellow arrows). Inset shows GFP+ Schwann cells with typical bipolar morphology with content of ML (same as in bottom panel with 5% reduction). (B, C) GFP+ reprogrammed Schwann cells, pSLC effectively transfer ML to exogenously added fibroblasts. Dissociated GFP-pSLC were co-cultured with fibroblasts and fixed after 18h (B) and 3 days (C) and labelled with anti-PGL1 antibody. Note that numerous ML were transferred to fibroblasts within 18h; arrows in bright phase (left) and dark phase (right) images are shown for PGL-1+ ML in non-GFP fibroblasts. Yellow arrows mark the GFP+pSLC (C). Inset shows GFP+pSLC without anti-PGL-1 labelling. Magnification: (A, B, C) 20x. (D) Quantitative analysis of bacterial retention in GFP+ non-reprogrammed Schwann cells as compared to GFP+ reprogrammed Schwann cells/pSLC in the presence of fibroblasts. * < p 0.01.\n\nWhen infected GFP+ pSLC were co-cultured with primary mouse fibroblasts we found that most of the ML present within pSLC were transferred to non-GFP fibroblasts within 24 hours. Figure 2B shows the transfer of PGL-1+ ML to non-GFP fibroblasts when they co-cultured for 18 hours or 3 days. Regardless of the origin of fibroblasts, whether they are neural fibroblasts (isolated from peripheral nerves) or dermal fibroblasts (isolated from adult skin), and regardless of choice of media for co-culture, mesenchymal media, DMEM with 6% serum or Schwann cell media, bacterial transfer assays from pSLC showed similar results. Unlike macrophages, fibroblasts are not professional phagocytic cells equipped with highly potent immune mediators capable of killing bacteria and their host cells, and thus bacterial residence in fibroblasts may provide an immune-evasion strategy for ML with a decayed genome13. As expected, we could not detect any apoptotic GFP+pSLC debris phagocytosed by these fibroblasts in co-cultures. Therefore, ML transfer to fibroblasts from pSLC is unlikely to occur by an apoptosis-mediated mechanism, but mainly by a cell-to-cell transfer mechanism.\n\n\nDiscussion\n\nResults presented in this study show an example of how a bacterial pathogen could use cell fate change of its preferred host cellular niche to its own advantage during different stages of the infectious process, from bacterial colonization to bacterial transfer to fibroblasts that could facilitate the complex process of dissemination of infection. We have frequently observed that primary Schwann cells as mono-cultures retained intracellular ML for a long period within Schwann cells, regardless of their species origin, rat, human or mouse1,14,15. Intriguingly, intracellular ML maintained Schwann cells without causing any apoptosis; this anti-apoptotic property is a defining feature of ML as compared to other pathogenic bacteria15,16. Co-culture of infected GFP+ Schwann cells with high bacterial load with fibroblasts failed to produce a significant bacterial transmission even after 5 days (Figure 2). Such bacterial retention capacity in adult Schwann cells may also be of functional significance during human infection, since Schwann cells in leprosy patients are known to harbour ML for an extensive period, which may be critical for bacterial expansion within this privileged niche3,17. For this purpose, initial bacterial retention within Schwann cells is critical so that ML replication and colonization can be ensured. On the other hand, following sufficient intracellular propagation of ML within Schwann cells, the next step of the infectious process, as in many bacterial infections, is to transfer their progeny to a secure host cell type, which could serve as either mediator cells or vehicle that can spread the infection locally or systemically18. Tissue fibroblasts could serve as non-immune mediator cell types for spreading the infection, as they are ubiquitously present in many tissues whereas macrophages, which come to action following inflammatory responses, are known to serve as a vehicle for bacterial dissemination both locally and systemically19.\n\nOnce colonized, reprogramming of infected Schwann cells may be necessary for the conversion of bacterial retention capacity of parent Schwann cells to a bacterial transfer property of reprogrammed Schwann cells for effective dissemination. We have recently shown that pSLC, but not Schwann cells, effectively transfer ML to macrophages in vivo under inflammatory conditions7. In this study, we showed that non-immune tissue cells like fibroblasts, which are much safer for ML survival than macrophages and are widely distributed (in the absence of inflammation) in peripheral nerves and skin, two preferred tissue niches for ML20, are a likely target for mediating bacterial dissemination. Effective ML transfer to neural fibroblasts is of particular significance, since neural fibroblasts, which are present in the peripheral nerve microenvironment could serve as an immediate target for ML once they colonized Schwann cells and subsequently undergo reprogramming. Thus, the reprogramming of Schwann cells provide ML with ample advantages – first to colonize intact Schwann cells and then to gradually change the fate of Schwann cells to the pSLC stage, promoting transfer of bacteria to fibroblasts or perhaps to other surrounding tissue cell types. Such a strategy suggests the intriguing possibility of effective bacterial spread to a wide range of tissues via pSLC as the reprogrammed form of infected Schwann cells also acquired other essential features such as re-differentiation, and migratory and immunomodulatory properties that are highly advantageous for bacterial dissemination. Therefore, we propose that the effective ML transfer capacity of the reprogrammed form of Schwann cells to fibroblasts could be a functionally-important event during ML dissemination.\n\nThe underlying mechanisms of effective bacterial retention in non-reprogrammed Schwann cells and rapid bacterial transfer capacity of pSLC are currently unknown. Based on the transcriptomic evidence from gene expression data it is possible that preserved Schwann cell identity in non-reprogammed cells and loss of Schwann cell identity in reprogrammed cells are associated with these two distinct functional properties. It is also intriguing that both non-reprogrammed and reprogrammed Schwann cells retain bacteria in the cytoplasm in the absence of exogenously added cells. However, rapid transfer of bacteria to exogenously added fibroblasts occurs only when pSLC interact with fibroblasts or macrophages7, suggesting that signals received from recipient cells or interacting cells following cell-to-cell interaction could trigger the signals necessary for bacterial transfer process. Since apoptotic events are minimal in these culture conditions, bacterial transfer from pSLC to fibroblasts is likely mediated by non-apoptotic and cell-to-cell transfer mechanisms. Although mechanisms involving such cell-to-cell bacterial transfer process appear to be highly complex, identification of details allow for the development of strategies to ablate bacterial spread at the early stage of infection.",
"appendix": "Author contributions\n\n\n\nAR, TM and JQ conceived the study and designed the experiments. JQ and TM carried out the experiments. AM and SRT performed the bioinformatics of the gene expression analyses. AR wrote the manuscript with contribution from all authors. All authors have agreed to the 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 in part by grants from NINDS (NS45187), NIAID (AI45816) and Wellcome Trust Institutional Strategic Support Funds to A.R. We also thank the American Leprosy Missions and the Order of St. Lazarus for the funding that support the provision of M. leprae.\n\n\nAcknowledgements\n\nWe thank R. Truman and R. Lahiri for the provision of M. leprae, Karen Burr for technical assistance and Emil Gotschlich for the encouragement and support during this study.\n\n\nSupplementary table\n\nRobust Multichip Average (RMA) values for selected Schwann cell lineage markers, representing absolute gene expression as calculated across 6 microarrays, from 2 samples (S1 and S2) each from control/uninfected Schwann cells, infected Schwann cells for 3 days (non-reprogrammed cells preserving Schwann cell identity) and pSLC-derived cells from day 28-infected Schwann cells7.\n\n\nReferences\n\nRodrigues LC, Lockwood D: Leprosy now: epidemiology, progress, challenges, and research gaps. Lancet Infect Dis. 2011; 11(6): 464–470. PubMed Abstract | Publisher Full Text\n\nStoner GL: Importance of the neural predilection of Mycobacterium leprae in leprosy. Lancet. 1979; 2(8150): 994–996. PubMed Abstract | Publisher Full Text\n\nPereira JA, Lebrun-Julien F, Suter U: Molecular mechanisms regulating myelination in the peripheral nervous system. Trends Neurosci. 2012; 35(2): 123–134. PubMed Abstract | Publisher Full Text\n\nJessen KR, Mirsky R: Negative regulation of myelination: relevance for development, injury, and demyelinating disease. Glia. 2008; 56(14): 1552–1565. PubMed Abstract | Publisher Full Text\n\nChen ZL, Yu WM, Strickland S: Peripheral regeneration. Annu Rev Neurosci. 2007; 30: 209–233. PubMed Abstract | Publisher Full Text\n\nRambukkana A: Usage of signaling in neurodegeneration and regeneration of peripheral nerves by leprosy bacteria. Prog Neurobiol. 2010; 91(2): 102–107. PubMed Abstract | Publisher Full Text\n\nMasaki T, Qu J, Cholewa-Waclaw J, et al.: Reprogramming adult Schwann cells to stem cell-like cells by leprosy bacilli promotes dissemination of infection. Cell. 2013; 152(1–2): 51–67. PubMed Abstract | Publisher Full Text\n\nTruman RW, Krahenbuhl JL: Viable M. leprae as a research reagent. Int J Lepr Other Mycobact Dis. 2001; 69(1): 1–12. PubMed Abstract\n\nChoi JK, Hoang N, Vilardi AM, et al.: Hybrid HIV/MSCV LTR enhances transgene expression of lentiviral vectors in human CD34 (+) hematopoietic cells. Stem Cells. 2001; 19(3): 236–246. PubMed Abstract | Publisher Full Text\n\nSeluanov A, Vaidya A, Gorbunova V: Establishing primary adult fibroblast cultures from rodents. J Vis Exp. 2010; (44). 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\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCole ST, Eiglmeier K, Parkhill J, et al.: Massive gene decay in the leprosy bacillus. Nature. 2001; 409(6823): 1007–1011. PubMed Abstract | Publisher Full Text\n\nNg V, Zanazzi G, Timpl R, et al.: Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae. Cell. 2000; 103(3): 511–524. PubMed Abstract\n\nTapinos N, Rambukkana A: Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli. Proc Natl Acad Sci U S A. 2005; 102(26): 9188–9193. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLahiri R, Randhawa B, Krahenbuhl JL: Infection of mouse macrophages with viable Mycobacterium leprae does not induce apoptosis. J Infect Dis. 2010; 201(11): 1736–1742. PubMed Abstract | Publisher Full Text\n\nShetty VP, Antia NH, Jacobs JM: The pathology of early leprous neuropathy. J Neurol Sci. 1988; 88(1–3): 115–131. PubMed Abstract | Publisher Full Text\n\nFalkow S: Is persistent bacterial infection good for your health?. Cell. 2006; 124(4): 699–702. PubMed Abstract | Publisher Full Text\n\nRamakrishnan L: Looking within the zebrafish to understand the tuberculous granuloma. Adv Exp Med Biol. 2013; 783: 251–266. PubMed Abstract | Publisher Full Text\n\nRichard L, Topilko P, Magy L, et al.: Endoneurial fibroblast-like cells. J Neuropathol Exp Neurol. 2012; 71(11): 938–947. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1922",
"date": "10 Oct 2013",
"name": "Maximiliano Gutierrez",
"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 reports an intriguing aspect of the complex pathogenesis of the obligatory intracellular bacteria M. leprae (ML). The authors report that ML infecting Schwann cells are not able to disseminate to fibroblasts but the cells re-programmed by the bacteria in stem-cell like cells are efficiently transferred to the same cells in vitro. These observations are very interesting and contribute to our understanding of the phenomenon of mycobacterial cell-to-cell transfer. There are only three technical points I think that authors could consider in this work, to unambiguously claim that the putative cell-to-cell transfer mechanism is a non-apoptotic, non-lytic one:The authors stated as data not shown that there is not evidence of bacteria in supernatants. It would be important to show this information and indicate in the methods how it was performed.The authors mentioned that they could not detect any apoptotic GFP+pSLC debris. It would be necessary to show these experiments as well, to exclude e.g. efferocytosis. They should indicate in the methods how apoptosis was detected (markers, staining etc…).The bacterial transfer to fibroblasts is measured primarily by detection of one lipid, PGL-1. It is known that lipids from M. tuberculosis can be transferred from infected cells to “bystander” cells (Beatty WL et al. 2000). I understand that the system with ML is much more complicated, but would it be possible to confirm that the fluorescent signal represent ‘intact’ bacteria e.g by electron microscopy? A minor comment: the authors refer to “strict confinement of ML to the cytoplasm” (results) or “retain bacteria in the cytoplasm” (discussion). Do they mean intracellular?",
"responses": [
{
"c_id": "586",
"date": "17 Oct 2013",
"name": "Anura Rambukkana",
"role": "Author Response",
"response": "We thank the reviewer for the constructive and valid comments. We note here that because of the short nature of this article we did not describe all negative results and present additional data in the original version. Here we address and clarify these issues: Evidence for the absence of bacteria in the supernatant and how it was performed is now elaborated in the methods section of the revised manuscript. As described in our previous reports (Masaki et al., 2013), (Tapinos and Rambukkana, 2005) apoptosis was measured using TUNNEL assay. It is known that M. leprae infection does not induce apoptosis (Lahiri et al., 2010), and in the present study no increase in M. leprae TUNNEL-positive cells were detected in infected co-cultures as compared to uninfected cells. Phenolic glycolipid-1 (PGL-1), particularly its sugar moieties, is very unique to M. leprae and is present in high quantity in the M. leprae cell wall. The latter permits the detection of intact M. leprae using antibodies against PGL-1. We have used a well-characterized antibody (IgG) against the native sugar/lipid moieties of PGL-1 that has been widely used for detecting whole/intact M. leprae in infected cells and tissues (Ng et al., 2010; Masaki et al., 2013). PGL-1 antibody activity has also been shown to correlate with acid-fast labeling (Fite’s staining) that specifically stains intact mycobacteria in infected tissues (Masaki et al., 2013). Considering the specificity and the detection of rod-shaped bacteria in high numbers in non-GFP fibroblasts co-cultured with pSLC, it was concluded that anti-PGL1 antibody detect the intact M. leprae, but not free lipids. Moreover, high load of M. leprae in non-reprogrammed Schwann cells (Fig. 2A), reflects the potential high content of bacterial lipids in the cytoplasm, but we failed to detect any significant PGL positivity in fibroblasts co-cultured with non-reprogrammed Schwann cells (Fig. 2A). This further suggests a lack of active (or significant) bacterial lipid transfer from one cell to another under these experimental conditions.Nevertheless, to further clarify this point the updated version includes a representative high-resolution fluorescence image of intact rod-shaped bacteria in fibroblasts detected by anti-PGL-1 antibody. In addition, we also include a representative electron micrograph showing the presence of intact M. leprae in fibroblasts (Fig. 2B). It should be noted that infected fibroblasts are distinguishable from pSLC under the electron microscopy, since pSLC, but not fibroblasts, form cell aggregates/clusters (Fig. 1a). Minor comment: here we emphasize that all M. leprae detection is strictly intracellular."
}
]
}
] | 1
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https://f1000research.com/articles/2-198
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https://f1000research.com/articles/2-203/v1
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04 Oct 13
|
{
"type": "Research Article",
"title": "Homologous electron transport components fail to increase fatty acid hydroxylation in transgenic Arabidopsis thaliana",
"authors": [
"Laura L. Wayne",
"John Browse",
"Laura L. Wayne"
],
"abstract": "Ricinoleic acid, a hydroxylated fatty acid (HFA) present in castor (Ricinus communis) seeds, is an important industrial commodity used in products ranging from inks and paints to polymers and fuels. However, due to the deadly toxin ricin and allergens also present in castor, it would be advantageous to produce ricinoleic acid in a different agricultural crop. Unfortunately, repeated efforts at heterologous expression of the castor fatty acid hydroxylase (RcFAH12) in the model plant Arabidopsis thaliana have produced only 17-19% HFA in the seed triacylglycerols (TAG), whereas castor seeds accumulate up to 90% ricinoleic acid in the endosperm TAG. RcFAH12 requires an electron supply from NADH:cytochrome b5 reductase (CBR1) and cytochrome b5 (Cb5) to synthesize ricinoleic acid. Previously, our laboratory found a mutation in the Arabidopsis CBR1 gene, cbr1-1, that caused an 85% decrease in HFA levels in the RcFAH12 Arabidopsis line. These results raise the possibility that electron supply to the heterologous RcFAH12 may limit the production of HFA. Therefore, we hypothesized that by heterologously expressing RcCb5, the reductant supply to RcFAH12 would be improved and lead to increased HFA accumulation in Arabidopsis seeds. Contrary to this proposal, heterologous expression of the top three RcCb5 candidates did not increase HFA accumulation. Furthermore, coexpression of RcCBR1 and RcCb5 in RcFAH12 Arabidopsis also did not increase in HFA levels compared to the parental lines. These results demonstrate that the Arabidopsis electron transfer system is supplying sufficient reductant to RcFAH12 and that there must be other bottlenecks limiting the accumulation of HFA.",
"keywords": [
"Arabidopsis",
"cytochrome b5",
"cytochrome b5 reductase",
"electron transport chain"
],
"content": "Introduction\n\nRicinoleic acid ((9Z,12R)-12-hydroxyoctadec-9-enoic acid), a hydroxylated fatty acid (HFA), is an important industrial feedstock for products such as lubricants, polyamide 11 (Nylon 11), coatings, inks, surfactants, emulsifiers, and biodiesel1. Castor oil is composed of up to 90% ricinoleic acid that is stored in the developing endosperm of the castor plant (Ricinus communis) in the form of triacylglycerol (TAG). However, castor seeds also contain the deadly toxin ricin, as well as a 2S albumin that is a major allergen2,3. These proteins, as well as the poor agronomic characteristics of the plant, make castor unsuitable as a major crop. While United States farmers are reluctant to grow castor, the demand for castor oil is increasing. Currently, industrial demand for castor oil is met from plants grown and manually harvested in India, China, and Brazil. Therefore, it would be advantageous to produce ricinoleic acid in a suitable crop species, such as canola, soybean, or camelina that lack ricin and the 2S albumin and can be widely grown in the United States. Furthermore, the knowledge gained from studying the biosynthesis of HFA in transgenic plants could be used to explore the synthesis of other novel fatty acids, such as epoxy fatty acid or conjugated fatty acids, which are made through similar biosynthetic mechanisms that rely on catalysis by modified desaturase enzymes1.\n\nThe castor fatty acid hydroxylase (RcFAH12) is a diiron, mixed-function oxidase responsible for the synthesis of ricinoleic acid from oleate (18:1) on phosphatidylcholine (PC) in the endoplasmic reticulum membrane4,5. The reaction mechanism requires transfer of two electrons from NADH through cytochrome b5 reductase (CBR1) and cytochrome b5 (Cb5) and then to the catalytic enzyme RcFAH12, which oxidizes 18:1 to 18:1-OH with the reduction of oxygen to water6,7.\n\nRepeated attempts to express a RcFAH12 cDNA in Arabidopsis under the control of seed-specific promoters have yielded a maximum of only 17% HFA in seed oil8,9. Ricinoleic acid has also been produced in tobacco but at a very low yield7. In Arabidopsis, ricinoleic acid can be further desaturated to densipolic acid (18:2-OH) or elongated to lesquerolic acid (20:1-OH) and auricolic acid (20:2-OH) by the fatty acid elongase FAE1 and associated enzymes8. To reduce the range of HFAs produced, we transformed RcFAH12 into the fae1 background10 under the control of the seed-specific phaseolin promoter11,12. One of the lines generated, CL37, has a total HFA accumulation (18:1-OH plus 18:2-OH) of 17–19%13 and has been chosen for experimental investigations aimed at increasing the accumulation of HFA in the seed oil.\n\nIn CL37 plants, we assume that RcFAH12 relies on the endogenous Arabidopsis electron transfer components, but these may not interact as efficiently with the RcFAH12 protein as do the components of the castor electron transfer system. The Arabidopsis fatty acid desaturases FAD2 and FAD3, which convert 18:1 into linoleic acid (18:2) and 18:2 into α-linolenic acid (18:3), respectively, are also diiron proteins that require reductant from CBR1 and Cb514–17. Previously, we have shown that a hypomorphic mutation in the CBR1 gene (cbr1-1) led to an 85% decrease in HFA levels in RcFAH12 Arabidopsis seeds, but much smaller decreases in 18:3 and 18:215. We concluded that, in the cbr1-1 mutant, the very substantial decrease in HFA was caused by an inadequate supply of electrons reaching the hydroxylase via Cb5, demonstrating that the Arabidopsis cytochrome b5 electron supply was critical to the activity of RcFAH12. It is possible that the decreased accumulation of HFA is a result of poor interaction between heterologous RcFAH12 enzyme and the endogenous Arabidopsis Cb5 proteins. Furthermore, in a separate study, we have shown that the activity of a Tetrahymena thermophila desaturase expressed in yeast is limited by weak interaction with the endogenous yeast Cb5. Activity of this desaturase was increased nearly tenfold by coexpression with a T. thermophila Cb5 protein18. Together, these results suggest that productive protein-protein interactions within the endoplasmic-reticulum electron transport chain are critical to supporting hydroxylase and desaturase activities.\n\nTo test the possibility that electron supply to the RcFAH12 enzyme may be a constraint on hydroxylase activity and accumulation of HFA in seeds of the CL37 line, we set out to identify castor genes encoding components of the endoplasmic reticulum electron transport chain and express them in the CL37 line, under control of seed-specific promoters. We tested three RcCb5 genes, and we also expressed each of these alongside the gene encoding castor cytochrome b5 reductase (RcCBR1). None of the gene combinations that we tested in a total of 270 independent transgenic lines provided any substantial increase in seed HFA content. Our results strongly indicate that electron supply is not limiting the activity of the RcFAH12 hydroxylase in these transgenic Arabidopsis lines.\n\n\nMethods\n\nSeeds from Arabidopsis (ecotype Columbia-0) containing the RcFAH12 transgene line CL3713 were stratified for 2–3 d at 4°C and germinated on 1× MS medium (Sigma-Aldrich) supplemented with 1% (w/v) sucrose and 0.75% agar. Seeds were germinated with a 16 h day/8 h night cycle at 22°C with 80 µmol m-2 s-1 light. Ten to 12 day old seedlings were then transferred to soil and grown under 24 h light at 22°C with 200 µmol m-2 s-1 light at rosette height in a growth chamber or under a 16 h day/8 h night cycle at 22°C with greater than 300 µmol m-2 s-1 light in the greenhouse.\n\nRNA was previously extracted by Dr. Chaofu Lu of Montana State University from castor developing endosperm, when the RcFAH12 is most highly expressed19. This RNA was then used to synthesize cDNA using Superscript III (Life Technologies) reverse transcriptase. Quantitative reverse transcription PCR (RT-PCR) was then performed with a MX3005P QPCR System (Stratagene) using the DNA binding fluorescent dye SYBR Green I (Life Technologies) and five sets of primers, amplifying full-length cDNA from each gene, see Supplementary Table 1 for list of primers. The collected data was then normalized to the RcACTIN control gene and the relative fold change was calculated (=1/(2^(experimental-control))).\n\nCastor genes were subcloned from a castor seed cDNA library19 or cloned using castor endosperm RNA extracted by Dr. Lu. Reverse transcriptase (Superscript™ III First-Strand Synthesis System; Life Technologies) was used to make cDNA from the castor RNA. Full-length transcripts were amplified from cDNA with KOD polymerase (Takara). The 5´ start primer contained the CACC sequence for directional topoisomerase cloning into pENTR-D-Topo (Life Technologies) see Supplementary Table 1 for the list of primers. The castor genes were then sequenced with vector primers (M13 forward and M13 reverse, Life Technologies) and compared with the castor genome at The Institute of Genomic Research (TIGR) castor genome database (http://castorbean.tigr.org/)20. These castor genes were renamed as follows: RcCb5-1, 28014.m000117; RcCb5-2, 30213.m000673; RcCb5-3, 29904.m002991; RcCb5-4, 30204.m001761; RcCb5-5, 29912.m005430; and RcCb5-6, 30174.m009087. From the entry vectors, these genes were subcloned into gateway compatible plant transformation vectors containing the seed specific β-phaseolin promoter, pGate-Phas-Basta (pGPB) or pGate-Phas-dsRed (pGPD), using LR Clonase I (Life Technologies). These constructs were then transformed into Agrobacterium GV3101 and used for plant transformations.\n\nDr. Edgar Cahoon (University of Nebraska-Lincoln) graciously provided a dual-gene plant transformation vector (renamed pEC-dsRed) along with a cassette vector pKMS2, which contains the seed-specific oleosin promoter. The pEC-dsRed vector contains two multiple cloning sites and the dsRed selection marker. This pEC-dsRed vector was made gateway compatible at the multiple cloning site containing the storage protein promoter glycinin. For cloning into pEC-dsRed, the RcCBR1 gene was subcloned from pENTR into pCR-Script (Stratagene) with NotI restriction sites on both primers. RcCBR1 gene out of pCR-Script and subclone it into the NotI site of pKMS2, which contains the oleosin promoter. The AscI sites were used to transfer the oleosin-RcCBR1 cassette from pKMS2 and into pEC-dsRed. The pEC-dsRed-RcCBR1 was then used for three individual gateway reactions with each of the three RcCb5 entry vectors using LR Clonase I (Life Technologies), so that the oleosin promoter controlled the RcCBR1 expression and the glycinin promoter controlled the expression of the RcCb5 genes. This cloning resulted in the three binary plant transformation vectors pEC-dsRed-RcCBR1-RcCb5-2, pEC-dsRed-RcCBR1-RcCb5-3, and pEC-dsRed-RcCBR1-RcCb5-4 that were transformed into the Agrobacterium strain GV3101.\n\nLine CL3713 was transformed with constructs mentioned above using the floral dip method21. In brief, a 72 h 500 mL culture of Agrobacterium containing the plasmid was resuspended in 5% sucrose and 0.05% Silwet L-77 (Lehle Seeds), flowers were dipped in the solution for 30 seconds, and plants were covered with plastic wrap over-night. For constructs containing the dsRed selection marker (pGPD and pEC-dsRed), T1 and T2 selection was conducted using a green LED light with a red filter13. For the pGPB constructs, T1 transformants were selected for Basta® (Bayer) resistance on 1× MS-agar supplemented with 1% sucrose, containing 20 µg/mL glufosinate-ammonium.\n\nFatty acid methyl esters were prepared from 20–50 seeds and analyzed by gas chromatography22. Statistical analyses were conducted by an unpaired two-tailed t-test in Excel, with a 95% confidence (P < 0.05).\n\nTen to 12 developing siliques from select lines were frozen in liquid nitrogen and used for RNA extraction according to the protocol described by Onate-Sanchez and Vicente-Carbajosa23, with minor modifications: tissue was ground in liquid nitrogen with a mortar and pestle and transferred into a pre-chilled microcentrifuge tube where 550 µL of extraction buffer (0.4 M LiCl, 0.2 M Tris pH 8, 25 mM EDTA, 1% SDS) and 550 µL of chloroform were added23. The tubes were vortexed and then centrifuged for 3 min at 14,000 × g at 4°C. 500 µL of water-saturated acidic phenol, 200 µl of chloroform, and 8 µL of iso-amyl alcohol were added to the supernatant and the tubes were vortexed followed by centrifugation for 3 min. The supernatant was extracted twice with 500 µL of chloroform and 1/3 of a volume of 8 M LiCl was added to the resulting supernatant. The tubes were incubated at 20°C for 1.5 h and then centrifuged for 30 min at 14,000 × g at 4°C. The pellet was dissolved in 20 µL DEPC-water and subjected to DNaseI treatment according to the DNA-free RNA kit (Zymo). Complementary DNA was synthesized using Superscript III reverse transcriptase (Life Technologies) and the full-length castor genes were amplified using KOD polymerase (Takara), see Supplementary Table 1 for the list of primers.\n\n\nResults\n\nIn Arabidopsis, five cytochrome b5 proteins (AtCb5-A to AtCb5-E) and one AtCb5-like protein receive electrons from NADH:cytochrome b5 reductase encoded by AtCBR124,25. In developing Arabidopsis seeds, the most highly expressed genes encode AtCb5-E (At5g53560) and AtCb5-D (At5g48810); AtCb5-B (At2g32720) is also strongly expressed, but there is only weak expression of the remaining three genes26,27. The AtCb5-E and AtCb5-D genes are also highly expressed in other tissues of the plant. Proteins encoded by these three strongly expressed isoforms are predicted to localize to the endoplasmic reticulum, based on their homology to Cb5 proteins from tung tree (Vernicia fordii) VfCb5-A, VfCb5-B and VfCb5-C that have been shown to be targeted exclusively to the endoplasmic reticulum28.\n\nThe Arabidopsis and Vernicia protein sequences were used to search the castor genome20 and identify likely RcCb5 orthologues. Sequences from all three species were used to derive an unrooted dendrogram (Figure 1) showing the phylogenetic relationships among the proteins. The castor isoforms designated RcCb5-2, RcCb5-3 and RcCb5-4 (see Materials and methods for accession numbers) fall into a distinct clade with the Vernicia and Arabidopsis endoplasmic reticulum Cb5 proteins. Quantitative PCR (qPCR) was performed to measure the expression of RcCb5 genes in developing endosperm of castor seeds during the period of HFA synthesis in this tissue19. We found that RcCb5-2, RcCb5-3 and RcCb5-4 transcripts were all strongly expressed, with highest transcript levels found for RcCb5-2 (Figure 2). By contrast, the RcCb5-1 and RcCb5-6 genes showed very low expression. Data from a transcript profiling database with ~106 sequences covering five stages of endosperm development29 includes 21 reads for RcCb5-2 and 26 reads for RcCb5-4, while RcCb5-3, RcCb5-1 and RcCb5-6 were not represented in this data set. Taken together, these results indicate that RcCb5-2, RcCb5-3 and RcCb5-4 likely have redundant roles in providing electrons to the FAH12 hydroxylase enzyme in the endoplasmic reticulum of endosperm cells of developing castor seeds.\n\nProtein sequences from Arabidopsis thaliana (At), Ricinus communis (Rc), and Vernicia fordii (Vf) were used to build an unrooted dendrogram in Geneious v5.1 (www.geneious.com). Size bar indicates genetic distance between proteins using the Neighbor-Joining method.\n\nTranscripts were normalized to RcACTIN. Error bars represent standard error of the mean of three independent experiments with three replicates for each experiment.\n\nThe cDNAs encoding the three RcCb5 isoforms that are strongly expressed in seeds (RcCb5-2, RcCb5-3 and RcCb5-4) were each cloned under control of the phaseolin promoter in vector pGate-Phas-Basta and transformed into CL37 plants expressing RcFAH1213. Putative T1 transformants were selected for Basta resistance. Analysis of the bulk T2 seed from 35 independent RcCb5-2 CL37 primary transformants showed no significant difference in total HFA (18:1-OH plus 18:2-OH) levels compared to the control CL37 plants grown at the same time, with a mean of 19.5% HFA (P value = 0.5979) for the 35 RcCb5-2 events (Figure 3A). There were also no significant differences in HFA levels for the 82 independent T1 events for RcCb5-3 CL37 compared to untransformed CL37, with a mean of 18.3% HFA (P value = 0.2155) (Figure 3B), nor for the 44 independent T1 events of RcCb5-4 CL37 (mean of 18.6% HFA) compared to untransformed CL37 (P value = 0.2470; Figure 3C).\n\nTotal HFA (18:1-OH+18:2-OH) contents of mature T2 seeds of individual transformants expressing (A) RcCb5-2, (B) RcCb5-3, or (C) RcCb5-4 in the CL37 line. Control at left is mean HFA in untransformed CL37 plants grown alongside T1 plants. For controls, error bars indicate standard deviation; n=28 in A, n=11 in B, and n=15 in C.\n\nThis analysis of T2 seed samples indicates that there is no dramatic increase in HFA accumulation through the co-expression of any of the three RcCb5 isoforms with the RcFAH12 in Arabidopsis. However, gene expression is typically variable across a population of individual T1 transformants and T2 seeds are segregating for the newly introduced transgene. We therefore identified five T1 events that produced seed with relatively high HFA and that contained a single RcCb5 transgene insert, based on segregation of the Basta-resistance marker in the T2 generation. For each of the chosen lines, RcCb5-2 #34, RcCb5-2 #27, RcCb5-2 #33, RcCb5-3 #43, and RcCb5-4 #40, T2 progeny were grown to maturity and nine to 10 plants homozygous for the RcCb5 transgenes, together with four to nine segregants lacking the transgene from each event, were identified by pedigree analysis of T3 progeny for Basta resistance. Comparisons of the total HFA content between these two sets of sibling segregants (Figure 4) failed to identify any statistically significant increase in HFA as a result of RcCb5 expression. Thus, plants from the three lines of RcCb5-2 CL37, #34, #27, and #33, were not significantly different from their segregating untransformed CL37 counterparts, with P = 0.2204, 0.0942, and 0.3965 respectively (Figure 4). The RcCb5-3 CL37 #43 was not significantly different from segregating untransformed CL37 (P = 0.4211), and the RcCb5-4 CL37 #40 was also not significantly different from segregating untransformed CL37 (P = 0.2102) (Figure 4). Although no changes were observed in the total HFA levels from these lines, we did observe some minor changes in the fatty acid profile; there were increases in the level of 18:1 and minor decreases in 18:2 and 18:3 (Supplementary Table 2).\n\nTotal HFA (18:1-OH+18:2-OH) contents of T3 seed of selected homozygous lines expressing RcCb5-2, RcCb5-3, or RcCb5-4 in CL37 were compared with segregating untransformed CL37 siblings. Error bars represent standard error of the mean for four to ten individual plants.\n\nSince it is assumed that the RcFAH12 in the CL37 line is receiving reductant via the endogenous Arabidopsis CBR1 (AtCBR1) and an AtCb5, we attempted to increase reductant supply to the AtCb5 proteins via overexpressing AtCBR1. However, again we found no changes in total HFA in the T2 seeds (Supplementary Figure 1). Taken together, these results indicate that none of the three RcCb5 proteins or any of the AtCBR1 electron acceptors can provide increased RcFAH12 activity in CL37.\n\nWe transformed CL37 plants with RcCb5 genes on the basis that the RcFAH12 hydroxylase enzyme may not interact optimally with endogenous AtCb5 proteins to receive electrons required for the hydroxylation reaction. However, it is also possible that interactions of the RcCb5 isoforms with the Arabidopsis cytochrome b5 reductase (AtCBR1) are weak, resulting in incomplete reduction of the RcCb5 proteins in the transgenic plants. To test this possibility, and to provide a fully compatible electron transport chain from NADH to the hydroxylase, we transformed CL37 plants with expression constructs containing both RcCBR1, encoding the castor cytochrome b5 reductase isozyme and a RcCb5 coding sequence, under control of strong, seed-specific promoters (see Materials and methods for details). The RcCBR1 and each of the top three RcCb5 genes were subcloned into pEC-dsRed plant transformation vector, resulting in three dual-gene vectors expressing RcCBR1+RcCb5-2, RcCBR1+RcCb5-3, and RcCBR1+RcCb5-4. The T1 transformants were selected by screening for dsRed fluorescence in seeds and grown to maturity. Screening of individual transformation events for total HFA accumulation was performed by gas-chromatography of bulk T2 seeds. The 45 RcCBR1+RcCb5-2 CL37 events generated were not statistically different (mean of 18.4% HFA) from the untransformed CL37 control lines (P = 0.6820) (Figure 5). For the 16 RcCBR1+RcCb5-3 CL37 events the mean of 18.5% HFA was not significantly changed in HFA accumulation compared to the CL37 control plants (P = 0.6834). Similarly, the 30 RcCBR1+RcCb5-4 CL37 events (mean of 17.4% HFA) also were not significantly different from the CL37 controls (P = 0.0898).\n\nTotal HFA (18:1-OH+18:2-OH) contents of mature T2 seeds from individual transformants expressing RcCBR1+RcCb5-2, RcCBR1+RcCb5-3 or RcCBR1+RcCb5-4 in the CL37 line. Results are compared with untransformed CL37 controls grown alongside T1 plants. Red inverse triangles indicate events that were further analyzed. Error bars represent standard deviation; n=25 for control CL37, n=45 for RcCBR1+RcCb5-2, n=16 for RcCBR1+RcCb5-3, and n=30 for RcCBR1+RcCb5-4.\n\nThree single-insert lines with relatively high HFA were selected from the T1 events shown in Figure 5. Progeny from each of the three lines were grown to maturity and four to seven plants homozygous for the RcCBR1 and RcCb5 transgenes were identified by the presence of the DsRed marker in their seeds and were grown together with CL37 control plants. Analyses of total HFA content of the homozygous seed showed no statistically significant difference between RcCBR1+RcCb5-2 #24 double transgenic and seed of CL37 lacking these transgenes (P = 0.0571; Figure 6). Similarly, HFA in seed of homozygous plants of lines RcCBR1+RcCb5-3 #12 and RcCBR1+RcCb5-4 #20 were not statistically different from HFA in CL37 controls (P = 0.1388 and P = 0.5774 respectively; Figure 6).\n\nTotal HFA (18:1-OH+18:2-OH) contents of seed from selected lines expressing RcCBR1+RcCb5-2, RcCBR1+RcCb5-3 or RcCBR1+RcCb5-4 in CL37. Plants homozygous for each dual-gene construct were compared with untransformed CL37 controls. Error bars represent standard error of the mean; n=14 for CL37 controls, n=6 for #24, n=7 for #12, and n=4 for #20.\n\nTo confirm that the RcCBR1 and RcCb5 transgenes were being expressed, we isolated RNA from developing seeds of representative plants from these three homozygous transgenic lines, and from the untransformed CL37 siblings. The RNA samples were used as templates for RT-PCR using primers specific for RcCBR1 and each of the RcCb5 isoforms. Bands of the expected full-length sizes were observed for the transgenic plants but were not detected in the untransformed CL37 siblings (Figure 7). Successful amplification of a band using primers to the ACT8 gene indicated that none of the RNA samples was degraded.\n\n(A) RcCBR1+RcCb5-2 #24, (B) RcCBR1+RcCb5-3 #12 and (C) RcCBR1+RcCb5-4 #20. RNA prepared from developing siliques was subject to RT-PCR using gene-specific primers. In each panel, --- indicates no-template control. U indicates untransformed CL37, T indicates homozygous transformant. The ACTIN8 gene (ACT8, At1g49240) was used as a control to test RNA quality.\n\n\nDiscussion\n\nThe endoplasmic reticulum diiron enzymes that catalyze desaturation and hydroxylation of fatty acids and other substrates require electrons from cytochrome b5(Cb5) proteins6,30. In seeds of transgenic Arabidopsis expressing the RcFAH12 hydroxylase from castor, a hypomorphic mutation in the CBR1 gene encoding cytochrome b5 reductase led to an 85% reduction in total HFA, but had smaller effects on 18:1 and 18:2 desaturation, <25% and <60% respectively15. These results indicated that there is competition among the reactions that utilize electrons from reduced Cb5, and raised the possibility that the RcFAH12 enzyme is not able to efficiently accept electrons from the endogenous AtCb5 proteins. In another study, we have shown that the activity of a T. thermophila desaturase expressed in yeast is strongly limited by the failure of the desaturase to properly interact with, and receive electrons from, the yeast Cb5. A nearly ten-fold increase in desaturase activity was achieved by coexpressing T. thermophila Cb5 proteins18.\n\nWhen RcFAH12 is heterologously expressed in Arabidopsis, the reductant supply for this enzyme comes from the endogenous Arabidopsis proteins. Here, we have tested the proposal that, since the castor orthologs of CBR1 and Cb5 have coevolved with RcFAH12, these proteins may be more efficient at supplying reductant to RcFAH12 and lead to a greater accumulation of HFA. The coevolution hypothesis has been shown to be valid for two castor acyltransferases, RcDGAT2 and RcPDAT1A; when these castor enzymes were heterologously expressed with the RcFAH12 in Arabidopsis, the HFA content of seeds increased from 17% to as much as 30% of total fatty acids, due to the increased efficiency of HFA incorporation into TAG22,31. However, contrary to our hypothesis, we found that there was no substantial increase in HFA accumulation when any of three RcCb5 proteins were expressed in CL37 (Figure 3 and Figure 4). These results argue against there being a substantial difference in the effectiveness of interaction of RcFAH12 with RcCb5 versus AtCb5 protein isoforms. Overexpressing the AtCBR1 in CL37 also did not increase HFA levels (Supplementary Figure 1), indicating that reductant supply via AtCBR1 is not limiting to the hydroxylase activity. However, these experiments cannot exclude the possibility that AtCBR1 does not efficiently reduce RcCb5 proteins.\n\nTo reconstitute the complete electron transport chain from NADH to the hydroxylase present in castor seeds, we transformed RcCBR1 and RcCb5 genes into the CL37 line, using a dual-gene plant expression system. The proportion of HFA in seeds of plants expressing RcCBR1+RcCb5-2 CL37, RcCBR1+RcCb5-3 CL37, or RcCBR1+RcCb5-4 CL37 was not significantly increased in comparison to the parental CL37 line. These results suggest that electron transfer to RcFAH12 is not limiting in the CL37 line, and that the endogenous Arabidopsis electron transfer system appears to be as efficient in transferring electrons to RcFAH12 as the transgenic castor electron transfer system in Arabidopsis.\n\nTaken as a whole, our results fail to show any positive effect of the castor electron-transport components on the synthesis and accumulation of HFA in transgenic Arabidopsis expressing the castor RcFAH12 hydroxylase. However, our CL37 transgenics only accumulate 17–19% HFA in the seed oil, compared to almost 90% HFA found in oil from castor22,31. Some of the constraints on HFA synthesis and accumulation have been identified22,32,33. If these constraints can be alleviated, by more efficient shuttling of HFA into triacylglycerol or reducing feedback inhibition of metabolism for example, it is possible that electron supply to the hydroxylase will then become limiting. If so, the constructs and lines generated in this study can be used for transforming and crossing with the elite lines containing higher levels of HFA.",
"appendix": "Author contributions\n\n\n\nLLW and JB conceived the study; LLW collected and analyzed the data; LLW and JB wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Science Foundation grant DBI-0701919 to JB, and by the Agricultural Research Center at Washington State University. LW was partially supported by the National Institutes of Health through Institutional Training Grant award T32-GM008336.\n\n\nAcknowledgements\n\nWe would like to thank Deirdre Fahy for assistance with cloning and the undergraduates in our laboratory for help with growing and harvesting the Arabidopsis plants. Dr. Edgar Cahoon graciously provided the dual-gene plant transformation vector and associated seed-specific promoter cassettes.\n\n\nSupplementary figure\n\nTotal HFA (18:1-OH+18:2-OH) contents of mature T2 seeds of individual transformants expressing AtCBR1 in CL37. Control at left is mean HFA in untransformed CL37 plants grown alongside the T1 plants. For the control, error bar indicates standard deviation, n=12.\n\n\nReferences\n\nDyer JM, Mullen RT: Development and potential of genetically engineered oilseeds. Seed Science Research. 2005; 15(4): 255–267. Publisher Full Text\n\nFodstad O, Johannessen JV, Schjerven L, et al.: Toxicity of abrin and ricin in mice and dogs. J Toxicol Environ Health. 1979; 5(6): 1073–1084. PubMed Abstract | Publisher Full Text\n\nThorpe SC, Kemeny DM, Panzani RC, et al.: Allergy to castor bean. II. Identification of the major allergens in castor bean seeds. J Allergy Clin Immunol. 1988; 82(1): 67–72. PubMed Abstract | Publisher Full Text\n\nBafor M, Smith MA, Jonsson L, et al.: Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean (Ricinus communis) endosperm. Biochem J. 1991; 280(Pt 2): 507–514. PubMed Abstract | Free Full Text\n\nMoreau RA, Stumpf PK: Recent studies of the enzymic synthesis of ricinoleic acid by developing castor beans. Plant Physiol. 1981; 67(4): 672–676. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith MA, Jonsson L, Stymne S, et al.: Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.). Biochem J. 1992; 287(Pt 1): 141–144. PubMed Abstract | Free Full Text\n\nvan de Loo FJ, Broun P, Turner S, et al.: An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog. Proc Natl Acad Sci U S A. 1995; 92(15): 6743–6747. PubMed Abstract | Publisher Full Text\n\nBroun P, Somerville C: Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean. Plant Physiol. 1997; 113(3): 933–942. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith MA, Moon H, Chowrira G, et al.: Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta. 2003; 217(3): 507–516. PubMed Abstract | Publisher Full Text\n\nJames DW, Lim E, Keller J, et al.: Directed Tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) Gene with the Maize Transposon Activator. Plant Cell. 1995; 7(3): 309–319. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSengupta-Gopalan C, Reichert NA, Barker RF, et al.: Developmentally regulated expression of the bean beta-phaseolin gene in tobacco seed. PNAS. 1985; 82(10): 3320–3324. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan der Geest AH, Hall TC: A 68 bp element of the beta-phaseolin promoter functions as a seed-specific enhancer. Plant Mol Biol. 1996; 32(4): 579–588. PubMed Abstract | Publisher Full Text\n\nLu C, Fulda M, Wallis JG, et al.: A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J. 2006; 45(5): 847–856. PubMed Abstract | Publisher Full Text\n\nBrowse J, McConn M, James D, et al.: Mutants of Arabidopsis deficient in the synthesis of alpha-linolenate. Biochemical and genetic characterization of the endoplasmic reticulum linoleoyl desaturase. J Biol Chem. 1993; 268(22): 16345–16351. PubMed Abstract\n\nKumar R, Wallis JG, Skidmore C, et al.: A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation. Plant J. 2006; 48(6): 920–932. PubMed Abstract | Publisher Full Text\n\nOkuley J, Lightner J, Feldmann K, et al.: Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell. 1994; 6(1): 147–158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith MA, Cross AR, Jones OT, et al.: Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons. Biochem J. 1990; 272(1): 23–29. PubMed Abstract | Free Full Text\n\nDahmen JL, Olsen R, Fahy D, et al.: Cytochrome b5 Coexpression Increases Tetrahymena thermophila Δ6 Fatty Acid Desaturase Activity in Saccharomyces cerevisiae. Eukaryot Cell. 2013; 12(6): 923–931. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu C, Wallis JG, Browse J: An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library. BMC Plant Biol. 2007; 7: 42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChan AP, Crabtree J, Zhao Q, et al.: Draft genome sequence of the oilseed species Ricinus communis. Nat Biotechnol. 2010; 28(9): 951–956. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16(6): 735–743. PubMed Abstract | Publisher Full Text\n\nvan Erp H, Bates PD, Burgal J, et al.: Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis. Plant Physiol. 2011; 155(2): 683–693. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOnate-Sanchez L, Vicente-Carbajosa J: DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques. BMC Res Notes. 2008; 1: 93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagano M, Ihara-Ohori Y, Imai H, et al.: Functional association of cell death suppressor, Arabidopsis Bax inhibitor-1, with fatty acid 2-hydroxylation through cytochrome b5. Plant J. 2009; 58(1): 122–134. PubMed Abstract | Publisher Full Text\n\nWayne LL, Wallis JG, Kumar R, et al.: Cytochrome b5 Reductase Encoded by CBR1. is Essential for a Functional Male Gametophyte in Arabidopsis. Plant Cell. 2013; 25(8): 3052–3066. PubMed Abstract | Publisher Full Text\n\nWinter D, Vinegar B, Nahal H, et al.: An \"Electronic Fluorescent Pictograph\" Browser for Exploring and Analyzing Large-Scale Biological Data Sets. PLoS ONE. 2007; 2(8): e718. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimmermann P, Hirsch-Hoffmann M, Hennig L, et al.: GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox. Plant Physiol. 2004; 136(1): 2621–2632. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHwang YT, Pelitire SM, Henderson MP, et al.: Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria. Plant Cell. 2004; 16(11): 3002–3019. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTroncoso-Ponce MA, Kilaru A, Cao X, et al.: Comparative deep transcriptional profiling of four developing oilseeds. Plant J. 2011; 68(6): 1014–1027. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShanklin J, Guy JE, Mishra G, et al.: Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes. J Biol Chem. 2009; 284(28): 18559–18563. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurgal J, Shockey J, Lu C, et al.: Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Biotechnol J. 2008; 6: 819–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBates PD, Browse J: The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. Plant J. 2011; 68(3): 387–399. PubMed Abstract | Publisher Full Text\n\nHu Z, Ren Z, Lu C: The phosphatidylcholine diacylglycerol cholinephosphotransferase is required for efficient hydroxy fatty acid accumulation in transgenic Arabidopsis. Plant Physiol. 2012; 158(4): 1944–1954. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1998",
"date": "07 Oct 2013",
"name": "Daniel Gallie",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 castor fatty acid hydroxylase (RcFAH12) is responsible for the synthesis of ricinoleic acid which has important industrial applications. Heterologous expression of RcFAH12 in Arabidopsis thaliana resulted in only 17-19% hydroxylated fatty acid (HFA) in seed triacylglycerols (TAG). In this work, the authors suggest that electron supply to RcFAH12 in Arabidopsis is limiting due to its heterologous expression and they test this hypothesis by co-expressing castor NADH:cytochrome b5 reductase (RcCBR1) and cytochrome b5 (RcCb5) that supply electrons to RcFAH12.The work is well presented and performed and indicates that the endogenous electron transfer system in Arabidopsis is not rate limiting in HFA production.",
"responses": []
},
{
"id": "2243",
"date": "06 Nov 2013",
"name": "Mark Smith",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFAD2 family proteins in plants require a reductant that is channelled via an electron transport chain comprising of cytochrome b5 (Cb5) and cytochrome b5 reductase (CBR). This study explores the hypothesis that lower than expected levels of hydroxy fatty acid (HFA) accumulation in engineered Arabidopsis could in part be due to inefficient interaction between the expressed castor hydroxylase and the endogenous Cb5 electron transport chain. The authors used a well characterized line of Arabidopsis (CL37) previously engineered to express the castor hydroxylase. Retransformation with a various cDNAs encoding Cb5 or Cb5+CBR1 failed to generate lines with increased HFA content.The study addresses an important and somewhat overlooked aspect of the ER fatty acid modification processes. The experiments are well planned, and the comparison of null and transgenic lines in single insertion lines (Fig.4) adds confidence to the data. My only concern is that although there is evidence of transgene expression by RT-PCR, there is no evidence to show the assembly of a functional electron transport chain containing the expressed proteins, or an increase in electron transport activity resulting from Cb5 and CBR overexpression. A biologically active Cb5 requires the assembly of cytochrome b5 apoprotein and haem to generate the Cb5-holoprotein. Demonstrating over-expression of active Cb5 would require a spectrophotometric assay of microsomal membranes to quantify the amount of Cb5 present and to determine electron transport activity (not easy in Arabidopsis). Alternatively, a Western blot showing increased Cb5 protein and a good argument that haem-protein assembly in developing Arabidopsis seeds is not limiting would help.An added complication from measuring total HFA in mature seed is that changes in HFA production could be masked by bottlenecks in HFA assembly into TAG. Further development of this work will be very interesting; the conclusions would be better supported by characterization of the electron transport chain.",
"responses": [
{
"c_id": "610",
"date": "07 Nov 2013",
"name": "John Browse",
"role": "Author Response",
"response": "Mark Smith is correct in pointing out that in biochemistry (and it life) it is protein function that matters. We have added a caveat on p.10 to acknowledge the possibilities he raises.Unfortunately, microsomal membranes from Arabidopsis seeds are contaminated with chlorophyll-containing thylakoids and the pigment confounds the spectrophotometric measurement of cytochrome b5. Even with the possibility that redox-competent RcCb5 and RcCBR1 are not produced, our conclusion remains the same: Expression of genes encoding homologous electron-transport components does not present itself as a useful route to increasing HFA accumulation in transgenic plants."
}
]
}
] | 1
|
https://f1000research.com/articles/2-203
|
https://f1000research.com/articles/2-237/v1
|
11 Nov 13
|
{
"type": "Case Report",
"title": "Nasal decongestant and chronic headache: a case of naphazoline overuse headache?",
"authors": [
"Cherubino Di Lorenzo",
"Gianluca Coppola",
"Valeria La Salvia",
"Francesco Pierelli",
"Gianluca Coppola",
"Valeria La Salvia",
"Francesco Pierelli"
],
"abstract": "Background: Chronic headache is an incapacitating condition afflicting patients at least for 15 days per month. In the most cases it is developed as a consequence of an excessive use of symptomatic drugs.Case: Here we report the case of a 34 year-old man suffering from chronic headache possibly related to the overuse of naphazoline nitrate nasal decongestant, used to treat a supposed chronic sinusitis. However, the patient did not suffer from sinusitis, but from a medication overuse headache (ICHD-II 8.3; ICD-10 44.41) that appeared to be due to excessive use of naphazoline.Conclusion: The use of naphazoline nitrate may result in an analgesic effect upon first use, through activation of adrenergic and opioidergic systems, followed by a pro-migraine effect via a late induction of an inflammatory cascade, modulated by nitric oxide and arachidonic acid. The observation that naphazoline detoxification relieved the patient’s headache, indicates that prolonged use of naphazoline may cause chronic headaches. Therefore, physicians should ask for details on the use of nasal decongestants in patients complaining of chronic headache, as they could potentially be suffering from a medication-overuse headache.",
"keywords": [
"naphazoline",
"nasal-spray",
"medication-overuse headache (MOH)",
"chronic headache",
"migraine."
],
"content": "Introduction\n\nChronic headache (≥ 15 days/month) is an incapacitating condition affecting the 4–5% of the general population1, and can be primary (in which case the headache is not due to other causes but is the disease per se) or secondary (i.e., the symptom of another condition)2. In 80% of cases, the chronic headache is a secondary effect caused by an excessive intake of drugs, mainly analgesics3. The excessive intake of pain-killers for more than consecutive 3 months can cause medication overuse headache (MOH), a secondary form of chronic headache, widespread among migraineurs2.\n\nHere we report the case of a patient suffering from a chronic headache related to the overuse of naphazoline nitrate, an over the counter nasal decongestant that the patient spontaneously used to treat a self diagnosed chronic sinusitis.\n\n\nCase report\n\nA 34 year-old man came to our Outpatient Headache Clinic in Latina ICOT hospital, (Italy) presenting a daily orbito-frontal bilateral headache, that he had been suffering from since he was 18. The patient had assumed it was a chronic sinusitis.\n\nThe patient had not recently undergone any diagnostic evaluation. The only examination that had been performed for his condition was a brain MRI, performed 8 years ago in a private clinic. This MRI detected no abnormalities, except a soft radio-opacity of the paranasal and zygomatic sinuses, and hypertrophy of the nasal mucosa. The general practitioner (GP) did not advice any specific treatment after this MRI-based diagnosis, and patient continued to use naphazoline.\n\nThe headache experienced by the patient was bilateral, throbbing, sometimes very severe, and associated with nausea, vomiting, photophobia, osmophobia and worsened by head movements.\n\nThe patient had been treated by his GP with medication to relieve the headache (metamizole, rizatriptan, zolmitriptan, acetylsalicylic acid, nimesulide, ibuprofen, naproxen sodium), consumed more than once a day, until the age of 24. Since then, he had stopped the consumption of such painkillers because he found that naphazoline nitrate nasal spray was more effective. He began with 2 shots (0,14 mg of drug for any shot) for each nostril 3 times a day, and at the time of presentation, due to pain recurrence, was using the spray 5–6 times a day, and experiencing immediate, yet temporary, relief of the symptoms every time. When asked, the patient said that he had used naphazoline nitrate nasal spray on an occasional basis since the age of 16, in order to self-medicate for self-diagnosed chronic rhinitis.\n\nDuring the patient’s visit to the clinic, respiratory examination, blood pressure, heart rate, mental status, reflexes, sensory system, cranial nerve, motor system, gait and coordination were normal. Since his headache characteristics were suggestive for a migraine-like headache, further evaluations were required to exclude the diagnosis of an acute sinusitis. Through a CT scan, a relapse of chronic sinusitis was excluded. The patient was then referred to an otolaryngologist, who suggested a detoxification from naphazoline nitrate through the use of aerosol therapy, with mucolytics and steroids, and thermal water spray.\n\nThe chronic headache disappeared following the treatment suggested by otolaryngologist. After three months of headache diary recording and clinical re-evaluation, the clinical picture was dramatically changed: patient presented only sporadic attacks of migraine without aura (ICHD-II 1.1). By an anamnesis re-evaluation it emerged that an episodic headache arose in childhood and worsen over the years, until it became chronic by the age of 18. One year after naphazoline detoxification, the patient has suffered from a few attacks during the year, treated with triptans.\n\n\nDiscussion\n\nThe case we report is suggestive for a chronic headache secondary to naphazoline nitrate overuse, since drug discontinuation interrupted the clinical symptoms. It is not clear however, whether the development of the chronic headache was due to a well-known naphazoline nitrate adverse event (AE) of inducing headache4, or whether it was due to a MOH-like occurance, since the patient consumed this drug to treat his headache. Indeed, patient experienced a relief on his headache by naphazoline, this was the reason he supposed, erroneously, to suffer of chronic sinusitis. The anti-migraine effect of naphazoline on our patient raises a question: is it right to regard naphazoline as a migraine medication?\n\nNaphazoline is a sympathomimetic drug, an imidazolinic derivate with marked alpha-adrenergic activity4. It enhances the release of noradrenaline from adrenergic termination, immediately relieving the nasal congestion thanks to its vasoconstricting action on the vessels of nasal mucosa5. Because of its adrenergic activity, this drug can also produce adverse effects, like rhinitis medicamentosa, hypertension, headache and acute depression of central nervous system with marked sedation4. Moreover, cases of ischemic and hemorrhagic stroke secondary to naphazoline have also been reported, mediated by the alpha1 and alpha2 adrenergic vasoconstrictive effect that is also exerted on brain vessels6,7.\n\nNaphazoline can trigger headaches because of its adrenergic activity (Figure 1). Alpha1 receptors are associated with G-proteins that generate a cascade of events leading to the production of arachidonic acid (AA) and nitric oxide (NO). AA and NO release leads to a late inflammatory vasodilatation8,9 that could induce a migraine attack10. Moreover, it is also possible that the same naphazoline nitrate, consumed by our patient, could have contributed to the production and release of further NO by a chemical reduction of the naphazoline salt. In fact, NO donors are currently used to induce migraine attacks in clinical and experimental settings11.\n\na. Naphazoline activates α1 receptors that are associated with G-proteins that cause the activation of phospholipase C. Phospholipase C cleavesphosphatidyl-inositol 4,5-bisphosphate (PIP2) into two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which in turn cause an increase in the level of calcium and protein kinase C. Diacylglycerol, by DAG lipase, takes part in the creation of arachidonic acid which is a precursor in the production of prostaglandins (PGs), mediated by cyclooxygenase (COX). The higher concentration of intracellular calcium allows an increase in the complex of calcium-calmodulin and therefore the activation of constitutive nitrous oxide synthetase (cNOS) with the generation of nitric oxide8,9. b. Naphazoline nitrate can contribute to the production and release of further NO by a chemical reduction of the naphazoline salt. Prostaglandins and nitric oxide contribute to the activation of nociceptors and the transmission of the pain pulse from the periphery to the centre10. Consequently there is a release of substances such as potassium ions, P-substance, bradykinin, histamine, serotonin and CGRP that keep nociceptors active and result in vasodilatation and extravasation of plasma proteins from the vessels.\n\nHowever, despite the late effect as a migraine trigger factor, naphazoline might also have an early action as an anti-migraine agent acting on alpha receptors of muscle, immune cells, Locus Coeruleus and spinal cord (Figure 2). In fact, alpha2 adrenergic receptors have a peripheral analgesic effect, thanks to the activation of opioidergic receptors, via endogenous opioid release12. Moreover, both alpha1 and alpha2 adrenergic receptors agonists have been proposed to be specifically useful for the treatment of migraine by mechanisms that mediate the early vasoconstrictive effect related to their activation8, similarly to triptans that carry out an analgesic action through the serotoninergic agonism that also mediates vasoconstriction.\n\na. Naphazoline activates alpha1 receptors that are associated with G-proteins that cause the activation of phospholipase C. Phospholipase C cleaves phosphatidyl-inositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3). Consequently there is a smooth muscle contraction due to an increase of intracellular calcium. b. Alpha-Receptors on immune cells release β-endorphins that cause opioid analgesia. c. Activation of alpha2 adrenoceptors in the Locus Coeruleus and spinal cord provokes the depression of nociceptive transmission12.\n\nTaking together these two opposite effects, it is possible to speculate that naphazoline can have a very early analgesic effect, due to the initial adrenergic activation, and a late migraine-inducing effect, that promotes the recurrence of headache by NO and arachidonic acid modulation.\n\nWe could therefore presume that in our patient, in a similar manner to the \"triptans effect\", naphazoline could exert an antimigraine action but also induce a rebound chronic headache due to medication overuse and/or a proinflammatory cytokine-mediated headache induction. Indeed, our patient had experienced such a sudden analgesic effect; otherwise he would not have continued to take naphazoline.\n\nThe observation that naphazoline detoxification leads to the interruption of headache is in line with the guidelines of the International Headache Society that prescribes treatment discontinuation for drugs that can directly induce headache and in cases of MOH3.\n\nIn conclusion, although naphazoline may cause headache as an AE, it may also have an early analgesic effect in migraine, as experienced by our patient. Our observation enlarges the spectrum of the drugs that can cause MOH and suggests that physicians should pay attention to the consumption of nasal decongestants in their chronic headache patients.\n\n\nConsent\n\nWritten informed consent for publication of this case report was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nCDL and VLS made substantial contribution to the study and prepared the first draft of the manuscript. GC collected patient’s clinical data. FP reviewed the article and approved the final version to be published.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank Giorgia Beolchini who provided assistance in writing the manuscript.\n\n\nReferences\n\nNappi G, Perrotta A, Rossi P, et al.: Chronic daily headache. Expert Rev Neurother. 2008; 8(3): 361–84. PubMed Abstract | Publisher Full Text\n\nInternational Classification of Disorders, Committee of the International Headache Society. Classification and diagnosis criteria for headache disorders, cranial neuralgia and facial pain. Cephalalgia. 2004; 24(Suppl 1): 1–150. Reference Source\n\nPascual J, Colás R, Castillo J: Epidemiology of chronic daily headache. Curr Pain Headache Rep. 2001; 5(6): 529–36. PubMed Abstract | Publisher Full Text\n\nRumack BH: POISINDEX(R) Information System Micromedex, Inc., Englewood, CO 2013; CCIS Volume 156, edition expires May 2013. Hall AH & Rumack BH (Eds): TOMES(R) Information System Micromedex, Inc., Englewood, CO 2013; CCIS Volume 156 edition expires May 2013.\n\nJohnson DA, Hricik JG: The pharmacology of alpha-adrenergic decongestants. Pharmacotherapy. 1993; 13(6 Pt 2): 110S–115S- 143S-146S. PubMed Abstract | Publisher Full Text\n\nCostantino G, Ceriani E, Sandrone G, et al.: Ischemic stroke in a man with naphazoline abuse story. Am J Emerg Med. 2007; 25(8): 983.e1–983.e2. PubMed Abstract | Publisher Full Text\n\nZavala JA, Pereira ER, Zétola VH, et al.: Hemorrhagic stroke after naphazoline exposition: case report. Arq Neuropsiquiatr. 2004; 62(3B): 889–89. PubMed Abstract | Publisher Full Text\n\nWillems EW, Valdivia LF, Villalón CM, et al.: Possible role of alpha-adrenoceptor subtypes in acute migraine therapy. Chephalalgia. 2003; 23(4): 245–257. PubMed Abstract | Publisher Full Text\n\nMunaron L: Shuffling the cards in signal transduction: Calcium, arachidonic acid and mechanosensitivity. World J Biol Chem. 2011; 2(4): 59–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSarchielli P, Alberti A, Codini M, et al.: Nitric oxide metabolites, prostaglandins and trigeminal vasoactive peptides in internal jugular vein blood during spontaneous migraine attacks. Cephalalgia. 2000; 20(10): 907–18. PubMed Abstract | Publisher Full Text\n\nSances G, Tassorelli C, Pucci E, et al.: Reliability of the nitroglycerin provocative test in the diagnosis of neurovascular headaches. Cephalalgia. 2004; 24(2): 110–9. PubMed Abstract | Publisher Full Text\n\nRomero TR, de Castro Perez A, de Francischi JN, et al.: Probable involvement of alpha(2C)-adrenoceptor subtype and endogenous opioid peptides in the peripheral antinociceptive effect induced by xylazine. Eur J Pharmacol. 2009; 608(1–3): 23–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2380",
"date": "12 Dec 2013",
"name": "Christian Wöber",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting case report which should be confirmed by further studies.",
"responses": []
},
{
"id": "2801",
"date": "17 Dec 2013",
"name": "Dimos Mitsikostas",
"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 report showing that naphazoline use was associated with chronic headache in one patient. The patient deserved further evaluation to exclude other brain pathologies related to chronic headache (e.g. no brain MRI or MRA was performed). The authors made a long and nice hypothesis regarding the potential pharmacological induction of headache which, however, needs experimental documentation.",
"responses": [
{
"c_id": "650",
"date": "17 Dec 2013",
"name": "Cherubino Di Lorenzo",
"role": "Author Response",
"response": "We thank Dr Mitsikostas for his kind response.The decision to not repeat the MRI (the first, performed eight years before, was negative although the clinical picture was already arisen) was a consequence of the very positive outcome experienced by patient after discontinuation of nasal spray. In fact, both the waiting list for CT scan and otolaryngologist consultation were very little, if compared to the MRI list, thus the ‘ex adiuvantibus’ confirmation of our suspect induced patient to not require another magnetic resonance because he preferred to avoid the discomfort related to the procedure.We agree that our speculations have to be sustained by further experimental confirmations. However, it is interesting to highlight that the information pamphlet of several nasal decongestants reported headache as a common side effect, but we do not find any notice of it in literature. On the other side, never it was suggested a sudden positive effect for naphazoline on migraine headache. We hope that our report will be the cue for further in-depth analysis on the topic."
}
]
},
{
"id": "4464",
"date": "23 Apr 2014",
"name": "Mark Obermann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting case report of chronic headache possibly related to the nasal decongestant naphazoline. They clearly and reasonably argue that naphazoline may have caused headache as a well known side effect of this drug and/or due to a similar mechanism known from other pain medications (analgesics/triptans/opioids) in terms of medication overuse headache. They laboriously explain the possible pharmacological mechanisms of action that may have caused headache over the long run, as well as early pain relief mechanisms that led to the continued intake of this drug in the first place. All this is highly hypothetical as correctly pointed out by the authors and may or may not stress the possibility of many different drugs that may cause medication overuse headache or may contribute to the development of chronic headache in particular patients.This case report provides a nice impulse for further research in this direction and emphasizes the possibility of other medications that may cause medication overuse headache beyond triptans, analgesics, etc. This case should make us clinicians more alert for these matters in order to better treat our patients with chronic headache.Many pre-clinical and clinical studies will be needed before any of the assumptions made by the authors can be confirmed or discarded.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-237
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https://f1000research.com/articles/2-235/v1
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06 Nov 13
|
{
"type": "Review",
"title": "Food sovereignty: an alternative paradigm for poverty reduction and biodiversity conservation in Latin America",
"authors": [
"M Jahi Chappell",
"Hannah Wittman",
"Christopher M Bacon",
"Bruce G Ferguson",
"Luis García Barrios",
"Raúl García Barrios",
"Daniel Jaffee",
"Jefferson Lima",
"V Ernesto Méndez",
"Helda Morales",
"Lorena Soto-Pinto",
"John Vandermeer",
"Ivette Perfecto",
"Hannah Wittman",
"Christopher M Bacon",
"Bruce G Ferguson",
"Luis García Barrios",
"Raúl García Barrios",
"Daniel Jaffee",
"Jefferson Lima",
"V Ernesto Méndez",
"Helda Morales",
"Lorena Soto-Pinto",
"John Vandermeer",
"Ivette Perfecto"
],
"abstract": "Strong feedback between global biodiversity loss and persistent, extreme rural poverty are major challenges in the face of concurrent food, energy, and environmental crises. This paper examines the role of industrial agricultural intensification and market integration as exogenous socio-ecological drivers of biodiversity loss and poverty traps in Latin America. We then analyze the potential of a food sovereignty framework, based on protecting the viability of a diverse agroecological matrix while supporting rural livelihoods and global food production. We review several successful examples of this approach, including ecological land reform in Brazil, agroforestry, milpa, and the uses of wild varieties in smallholder systems in Mexico and Central America. We highlight emergent research directions that will be necessary to assess the potential of the food sovereignty model to promote both biodiversity conservation and poverty reduction.",
"keywords": [
"At the 2012 Rio+20 meetings",
"political leaders acknowledged the mounting challenges to sustainable development",
"reiterating that many of the world’s poor depend on rapidly disappearing and fragile biodiverse ecosystems. In rural areas",
"poverty traps",
"defined as “self-reinforcing mechanisms that cause poverty",
"however measured",
"to persist”1",
"2 often result from linked ecological and socio-political systems that reach a dynamic equilibrium at a low level of human wellbeing. In relation to biodiversity",
"poverty traps raise the question of how to improve socio-economic wellbeing without further increasing the consumption of scarce",
"fragile",
"or overexploited resources. It has been argued",
"for example",
"that sustained improvements in well-being can be accomplished",
"but at a cost to biodiversity",
"or that in some situations conserving biodiversity would mean keeping a group of people at existing levels of poverty. Alternatively",
"there are also theoretical and empirical arguments that “win-win” situations can be found where fighting poverty and inequality may increase sustainability and biodiversity conservation3–6."
],
"content": "Introduction\n\nAt the 2012 Rio+20 meetings, political leaders acknowledged the mounting challenges to sustainable development, reiterating that many of the world’s poor depend on rapidly disappearing and fragile biodiverse ecosystems. In rural areas, poverty traps, defined as “self-reinforcing mechanisms that cause poverty, however measured, to persist”1,2 often result from linked ecological and socio-political systems that reach a dynamic equilibrium at a low level of human wellbeing. In relation to biodiversity, poverty traps raise the question of how to improve socio-economic wellbeing without further increasing the consumption of scarce, fragile, or overexploited resources. It has been argued, for example, that sustained improvements in well-being can be accomplished, but at a cost to biodiversity, or that in some situations conserving biodiversity would mean keeping a group of people at existing levels of poverty. Alternatively, there are also theoretical and empirical arguments that “win-win” situations can be found where fighting poverty and inequality may increase sustainability and biodiversity conservation3–6.\n\nHowever, empirical and theoretical explorations of the relationships between poverty traps and biodiversity loss are largely underdeveloped. Little attention, for example, has been given to the exogenous socio-economic drivers of those poverty trap dynamics. Thus, Maru et al.2 suggest rethinking current approaches, emphasizing the importance of “causes external to the system” in creating and maintaining poverty traps. For example, income improvements due to the rapid agricultural development of the 1960s and 70s did not reach the most impoverished sectors, exacerbating historical inequalities7.\n\nIn this paper, we examine exogenous factors that contribute to poverty traps for smallholders in Latin America. We suggest a reconsideration of the role of neocolonial/neoliberal policies and agro-export models in addressing poverty: in Latin America, 52% of rural people still remain in poverty 8, with significant evidence linking both the maintenance of rural poverty and the environmental degradation at the agricultural frontier (e.g., biodiversity loss, erosion, deforestation) to agricultural intensification and the growing integration of agriculture into world markets9–11. We then re-examine the relationship between biodiversity and diverse small-scale farming systems, and present evidence that small-scale agroecological farms contribute to enhancing farmer's livelihoods and the conservation of biodiversity at local and landscape levels, as well as ecosystem services. We then assess the ability of an alternative food sovereignty framework to address the challenge of reducing poverty, improving food security and conserving biodiversity and other natural resources in Latin America. We suggest a reframing of the biodiversity loss and poverty trap dilemma and provide an approach for moving beyond the narrow land-sparing/land-sharing debate (e.g., Phalan et al. (2011)12 and Tscharntke et al. (2012)13) in the ongoing global search for how best to feed the world and reduce poverty, while protecting essential ecological services, including biodiversity.\n\nDevelopment economics has long emphasized the strong interdependence between natural systems and human wellbeing, especially in rural areas. Conventional approaches have held that poor rural populations are involved in two vicious circles constituting a poverty trap: 1) the poor are unable or unwilling to regulate their numbers, which, on average, leads to surplus labor and further impoverishment; and 2) poverty leads to the depletion of soil organic matter and other forms of “mining the soil”, generating low productivity and deforestation and leaving those who depend on these resources for livelihoods in continued poverty3,14,15. The policy prescriptions that follow are generally directed at stopping further increases in the population/labor surplus and consequently halting the depletion of natural resources. This broadly re-capitulates earlier Neo-Malthusian views, even though more recent work sometimes nods to more sophisticated analyses based in ideas of “upgrading human capital”: providing education and health programs, and direct welfare assistance16. These “upgrades” are proposed as ways to break vicious circles between poverty, population, and environmental degradation, ignoring the fact that the “vicious circle” conceptualization itself is simplistic and problematic16,17.\n\nThus interventions formulated and implemented in this vein have often fallen far short of their desires to transform the rural poor into a sustainably productive sector. This is due to the extremely simplistic view of poverty dynamics represented by points (1) and (2) above. The rural poor in the capitalist world do not exist in a vacuum. Rather, they participate in complex institutional and economic arrangements involving market and non-market transactions at local and trans-local levels. Moreover, redistributive land reform (i.e., “actual net transfer of effective control” of land to poor peasants;18) has important repercussions for rural livelihoods, hunger, poverty alleviation and biodiversity conservation in the region19,20. More technically stated, existing programs have neglected the combination of the lack of physical and human capital and distortions and failures in the product, labor and credit markets in which the rural poor operate, rendering them incapable of investing resources in ecosystem conservation and restoration. They may then become dynamically inefficient and uncompetitive producers, further restricting their capacity to acquire necessary new capital and overcome their economic disadvantages—in other words, caught in a poverty trap.\n\nFurther, as we will argue, rural poverty traps are also the result of exogenous factors, including the legacy of colonialism and the continuation of historical inequalities in agricultural and trade policies21,22. Various authors14,17,23–28 have documented a number of structural biases against poor rural households (as summarized by Taylor and García-Barrios (1999)16:\n\n“…unfavorable economic policies and public investment priorities (especially with the onset of the debt crisis in the 1980s); structural and institutional contexts that are unfavorable to rural development, including inegalitarian land tenure systems and institutional biases against smallholders in the definition of public goods and services and in their access to them; economic policies and technological biases that reduce employment creation in both the non-agricultural sector and in commercial agriculture; household-specific market failure, economic discrimination and adverse selection in the labor, product and credit markets; [government-abetted] monopolistic power in local formal and informal markets [that generates] compulsory transactions which, like usury, lead to the expropriation of their resources; [and] direct private and State coercive violence”.\n\nTaylor and García-Barrios expand on this, arguing that the highly constrained, unfavorable situations facing the poor may compel what are (in these circumstances) economically rational survival strategies. However, these strategies and transactions easily move from being constrained choices to established, involuntary, and compulsory parts of the rural poor habitus (lifestyle, behavior, and worldview), ultimately maintaining or increasing the poor’s conditions of poverty and dependence. For example, peasant “brain drain” and “labor drain” may undermine local institutional arrangements by eroding social norms and capital. The structural conditions that emerge may generate local institutional insufficiency, systemically affecting the capacity of the poor to reorganize endogenously in the face of new challenges16,29,30.\n\nThis broader set of explanations provides an understanding of poverty traps as multiple and embedded (fractal), and shows that traps resulting from actions by other human actors and socio-economic inequalities may be the norm, not the exception2,31,32. As said by the World Bank, “even where poor people degrade the environment, this is often due to the poor being denied their rights to natural resources by wealthier elites and, in many cases, being pushed onto marginal lands more prone to degradation”33, Box 4. This explanation, however, is still insufficient in two ways.\n\nThe first insufficiency is its lack of a clear assessment of the impacts of institutionalized competitive disadvantages on smallholder farmers, including, for example, international financial institution support for export-oriented commodity production and the liberalization of international agricultural trade34. Such neoliberal agricultural development programs have purported to eliminate structural market failures and create favorable conditions for small farmers and their access to global markets35,36. Such policies, however, resulted in the liberalization and opening of Latin American economies, including the agricultural sector, and the dismantling of public services related to agriculture, such as credit for smallholders, technical support, etc.35. But at the same time, beginning in the mid 1970s and extending through the 1980s and 1990s, the World Bank made it clear that their development programs envisioned two options for Latin American smallholders: 1) become commercial, export-oriented, farmers, or 2) disappear32,37,38.\n\nThe results, however, were far from those intended: the smallholder sector in Latin America has not declined, as anticipated by development theorists, but has actually increased8,36. But though the peasant sector has remained, the challenges facing it have deepened: neoliberal agricultural policies have reinforced fractal poverty traps and deepened patterns of rural inequality; international and internal inequalities of market integration were propagated through multiple scales, with largely negative impacts on welfare in rural areas, including widespread rural displacement and cross-border migration19,22,39,40. Further, neoliberal policies resulted in the inequitable distribution of economic growth: despite an increase in GDP of 25% in real terms for the region, poverty and hunger barely improved, especially in the rural areas. In 1980, 60% of the rural population was poor and 33% suffered from hunger; in 2010 the percentages were 52% and 29%, respectively8,19,36. Indeed, rural Latin America has the most unequal rural sector in the world, with Gini coefficients higher than 0.5 for most countries41. Inequalities in land access, an important asset for rural households, are also the worst in the world, with an average (land-ownership-based) Gini coefficient of 0.78 for the region42. Thus poor households that depend on agriculture as their primary source of income have been the most affected by neoliberal policies, with stagnation or deterioration in welfare over the past 20 years43,44.\n\nIn parallel to these dynamics, changes in agricultural technology and trade policies favoring export-oriented production have also been repeatedly tied to environmental degradation9,45–47: the regional shift to export crops grown in monocultures has led to increased water and agrochemical uses, and has had detrimental impacts on biodiversity48–52; dramatic increases in the use of synthetic inputs (i.e., pesticides and fertilizers) have contributed to rapid, but currently tapering, yield increases worldwide53; and agricultural industrialization has corresponded to increasing rates of deforestation, a massive movement of people from rural to urban environments, and an overall loss of biodiversity47,54,55. Further, export-focused agriculture has often displaced land, research, and institutional support for crops grown for regional or national consumption, hurting small farmers’ livelihoods and food security more broadly46,56–58. While it was hypothesized that the higher yields from agricultural intensification would allow less land to be used for agriculture and more land “saved” for biodiversity, evidence is also accumulating that higher yields rarely create this “land-sparing” effect59,60, and in fact may stimulate expansion of agricultural frontiers, including what has come to be known as the global “land grab”. Beyond this, higher yields do not assure increased access to food or decreases in poverty61–64. This approach is nevertheless manifest in the many programs designed to separate agriculture and nature as distinct land uses, a strategy with mixed results for conservation65.\n\nThe second source of insufficiency of the contemporary poverty trap discourse is that it does not explain the substantial variation of agricultural experiences in the region. Small-scale landholders still represent a large percentage of the agricultural landholdings in Latin America. In a study that included 15 Latin American countries, Chiriboga66 estimated that their smallholder sectors were composed of 6 million semi-commercial family farms controlling 42% of the land, plus 11 million subsistence farms controlling 3% of the land. (Corporate farms were estimated to number around half a million and to control ~56% of all agricultural land). Because the smallholder sector is deeply embedded in local economies, their role in feeding the region and conserving the biota should not be underestimated. For example, the World Development Report 2008: Agriculture for Development marked a shift away from the focus on an export-oriented model and a recognition of the importance of small-scale agriculture in poverty reduction20. The authors also recognized for the first time in almost 30 years the critical role of government in overcoming market failure67. However, the report continues to call for deeper liberalization in agriculture, an approach that has repeatedly failed to address the deep poverty and inequality in Latin America (19,20,22,68; for discussions of similar dynamics in other regions, see Moseley et al. (2010)69 and Buckland (2006)70). This connects to the insufficiency of contemporary discourse in that regional and local variations are rarely accounted for within the grand narratives of development discourse—the exact configurations of disadvantage, historical and exogenous drivers, institutional characteristics, interactions with local ecosystems, and therefore possible solutions are likely to vary, possibly immensely, from case to case, creating the need for approaches based in specific contexts of place and space31,71–73. Expressed more technically, rural social dynamics, of which poverty traps are a result, are complex processes that may render multiple attractors and trajectories. The positive (self-reinforcing) but degrading feedback between poverty and land productivity suggests an alternative positive but upgrading feedback: biodiversity benefiting smallholders, and smallholders practicing diversified agroecology that benefits biodiversity. It has been argued that in contrast to heavily consolidated rural landscapes that have resulted from agricultural liberalization and export agriculture5,22, landscapes composed of mosaics of natural habitats and small-scale, diverse farms oriented toward local markets can also stimulate local economic development and reduce poverty in rural areas74–78. This possibility is the main object of analysis of this article, and to that we now turn.\n\n\nRelationships between biodiversity and smallholder agriculture\n\nThe evidence in support of an alternative and upgrading positive feedback loop between peasant production and biodiversity management is strong, although it also suffers from broad generalizations that have often emerged from small-scale (spatial and temporal) experimental studies79. These caveats notwithstanding, the scientific consensus is that biodiversity is essential for agriculture and that agriculture, in turn, impacts biodiversity, both in positive and negative ways depending on the type of agriculture80.\n\nBiodiversity is the basis of agriculture: it is the origin of all crops and domesticated animals from which humans derive their sustenance. Of the ~30,000 species of edible higher plants, it is estimated that ~7,000 have been cultivated. In addition to enabling the production of food across a wide spectrum of environmental conditions, crop diversity (especially fruits and vegetables) contributes to food security, a diversified diet and higher quality nutrition81–83. In addition to the provisioning services associated with crop and animal production, biodiversity can contribute to ecosystem services that benefit agriculture and society more generally. These include higher yield and overall production output through intercropping and agroforestry, regulation of pest and diseases, nutrient cycling though decomposition of organic matter, carbon sequestration, soil water retention, and pollination services. Although the literature on the relationship between biodiversity, ecosystem services, and agriculture is robust, it is not without controversy. For example, there is a strong debate about the relationship between biodiversity and productivity. While the advantages of intercropping are well-documented, in most cases the overyielding of intercrops as compared to monocultures is the result of the combination of a grass and a legume and not biodiversity per se84. Likewise carbon sequestration or pollination services could, in theory, be maximized with the presence of the most efficient carbon sequestering plant or pollinator. However, for smallholder agriculture it is the diversity of crop and animal varieties, crops and animal species and wildlife that provide these ecosystem services under variable and changing environmental conditions13,85. Diverse agroecological systems also buffer the impacts of climate change86–90 and reduce the vulnerability of smallholders to price and market fluctuations91–94.\n\nAgriculture is recognized as one of the major drivers of biodiversity loss80, mostly through habitat destruction, soil erosion, monocultures and the use of agrochemicals95. But not all types of agriculture have the same effects on biodiversity. Diverse agroecological and organic systems have been shown to contribute to biodiversity conservation at the local and landscape level5,96–101. At the local/farm level agroecological and organic systems can benefit biodiversity by eliminating the use of pesticides and other agrochemicals, increasing crop diversification and crop rotations, preserving hedges and other wild vegetation, and through soil conservation measures. Agrobiodiversity encompasses genetic resources, edible plants and crops, and livestock (planned biodiversity), as well as the associated organisms (associated biodiversity) that provide ecosystems services such as maintenance of soil fertility and prevention of pest attacks102. Higher associated biodiversity is strongly correlated to planned biodiversity, meaning more diverse agricultural systems generally maintain greater levels of ecosystem services and landscape diversity (103–105, but see Balmford et al. (2005)106). In a meta-analysis that included 63 publications comparing organic and conventional farms, Bengtsson and colleagues98 reported that, on average, organic farming increases species richness by 30% and organism abundance by 50% over conventional farming. Although the results were variable, and not all organisms responded in the same way, their meta-analysis provides evidence that organic farming generally supports higher levels of species richness, especially of plants, birds and predatory insects, than conventional agriculture98 Other reviews and meta-analyses have arrived at the same conclusion95,107–113. In a more recent synthesis, Kremen and Miles101 suggest that diversified farming systems enhance ecosystem service provisioning including biodiversity conservation, fostering agro-ecosystem resilience and sustainability.\n\nThe benefits to biodiversity of certain agri-environmental schemes in Europe have been questioned114, with many examples where intended or hoped-for biodiversity benefits have not materialized115. However, it has been proposed that these schemes may not have delivered greater biodiversity benefits because they are typically designed for the farm- or field-scale and frequently ignore the surrounding landscape116, and because they may not be using appropriately researched and designed wildlife-friendly methods100. At a larger scale landscape heterogeneity is an important factor in maintaining biodiversity95,103,105,116–124, and can be as or more important than the type of management at the farm level125. The tendency in the conventional agriculture model, however, has been to reduce diversity not only at the farm level but also at the landscape level95. Furthermore, entire landscapes are tending toward homogenization under current policies, which tend to promote larger farms characterized by large monocultural fields with fewer non-cultivated habitats: live fences, non-cultivated field margins, hedge rows, and scattered trees125–128, and thus exacerbate the negative effects of agricultural intensification on biodiversity129.\n\nAt the individual farm scale, researchers are just beginning to examine the effect of farm size per se on biodiversity. A study of farms of various sizes and management types (organic and conventional) in Sweden reported that, although organic farms had higher diversity than conventional farms, the biggest differences were found between small organic and large conventional farms125. The same study also found 56% more bird species in small versus large organic farms, suggesting that landscape level factors were playing an important role for bird diversity and that size matters. At least two other studies have reported that field size is an important factor affecting biodiversity130,131. Landscape configurational heterogeneity (i.e., pattern complexity; see116) can also be important. For example, when Fahrig et al.116 compared fine-grain and coarse-grain landscapes in France (that is, landscapes with smaller fields and shorter distance between hedgerows versus landscapes with larger fields but similar crop types) they found that carabid beetle species richness accumulated faster in the fine-grain landscapes. And as Fahrig et al. point out, similar results were reported for solitary wasps in Germany132.\n\nThere are few comprehensive studies of the impacts of landscape-level agricultural intensification and homogenization (which tends to be accompanied by the loss of smallholder farmers) in Latin America. One review looked at studies conducted in the Argentinian Pampas, where in the late 1980s mixed cattle grazing-cropping systems were replaced by continuous cropping of a few crops. This corresponded to increased use of no-till technology (mostly with genetically modified cultivars) and an increase in field size, decreasing landscape heterogeneity and led to dramatic reductions in biodiversity in the region. Direct evidence of negative effects was found for rodents and crop-associated insects, especially non-herbivorous insects. The authors of the review by Medan et al. suspected that there had been net negative effects for avifauna, but the results to date were mixed128. It was found that the loss of ecological heterogeneity at the landscape level directly affected diversity, abundance and distribution of small mammals, particularly rare species, habitat specialists and those species that needed grassland remnants for nesting and digging shelters. Increased use of pesticides had an indirect negative effect on rodents by reducing the food availability of invertebrate prey, vegetation cover and seeds. However, not all organisms were negatively affected by intensification. The review found higher abundance and richness of pollinators and suggested that native pollinators may have benefitted from resource-rich crops like sunflower and canola128 (and references therein).\n\nIn summary, inherent trade-offs between biodiversity conservation and farm productivity cannot be assumed53,133. A growing body of evidence indicates that landscapes dominated by small-scale and diverse farms (known as “land-sharing” or “wildlife-friendly” models12,134) may more effectively conserve biodiversity than landscapes dominated by large, energy- and input- intensive monocultures19,46,54,55,85,103,135–137.\n\nUp to this point, the evidence that we have presented regarding how small-scale agroecological farms contribute to biodiversity conservation has taken a static approach to biodiversity. Most of the studies measured biodiversity in different types of farm or landscapes and compared them, implicitly assuming that what is there now was there before, and will be there in the future. This static approach would lead us to conclude that a particular system is good for biodiversity simply because a high number of species are recorded in that system, or vice versa. However, some species that are recorded in a particular habitat could be on their way to extinction (i.e., extinction debt;138), and others that are not recorded could eventually get there through migration (i.e. immigration credit;139). Given this, in addition to sampling biodiversity in various types of management systems and landscapes, we need to consider landscape-level dynamics because biodiversity is ultimately determined by dynamic processes such as extinction and immigration54.\n\nLocal extinction is a natural process that occurs even in continuous habitats, therefore we can assume that it is prevalent, even more so, in fragmented habitats140–146. In fragmented habitats, we5,54 and others (e.g., Mendenhall et al. (2011)123, (2012)147) have argued that the biodiversity that can persist in the long term is largely determined by the quality of the matrix. The underlying ecology is grounded in the fact that a good matrix can not only provide habitat for many organisms and sustain high levels of biodiversity within the matrix itself, but also because a good matrix is one that allows movement of organisms among patches of forest and other natural ecosystems5,148. In a recent quantitative review paper Prevedello and Vieira149 concluded that matrix type is important for biodiversity conservation, but that patch size and isolation are the major determinants for species diversity, persistence, population dynamics, and interactions in fragmented landscapes. However, in 91% of the studies that reported isolation as the main effect, incorporating matrix type significantly improved the explanatory power of the models, suggesting that matrix quality can reduce the patch isolation effect. They also concluded that matrix quality increases with increasing structural similarity with habitat patches. In most cases of fragmentation, the matrix is an agricultural matrix. Simulation models suggest that improving the quality of the matrix can offset extinction risk caused by losses of patch habitat of up to 60%150.\n\nIn line with this, it has been suggested that agroforestry systems, such as shaded coffee and cacao, represent a high quality matrix that can facilitate inter-fragment migration among patches of forests in the tropics151–153. A similar argument has been made for Europe’s agri-environmental schemes when considering landscape level improvement154. Unfortunately, few studies have empirically examined the actual movement of organisms in fragmented habitats through various kinds of agricultural matrices. In a study of the impacts of agri-environmental schemes in Europe, Delattre and colleagues155 demonstrated that leaving grassy field margins, one of the features covered by the agri-environmental schemes of the Common Agricultural Policy framework of the European Union, improved inter-fragment migration of the meadow brown butterfly. For a tropical landscape, using mark-recapture techniques, Marin and colleagues156 demonstrated that combined elements from traditional management, such as Acacia woodlots and live fences, have allowed the conservation of a rich butterfly biodiversity in forest fragments embedded in pasture in southern Mexico. A more direct estimate of inter-fragment communication is the genetic relationships of a particular species among various fragments. As far as we know, there are only two studies that have done this for a tropical agricultural landscape. Jha and Dick157,158 used genetic markers and conducted spatial analysis of pollen dispersal across a coffee matrix. Their results demonstrated the importance of a shade coffee matrix for the genetic diversity of the understory tree Miconia affinis.\n\nTaken together, these studies provide strong evidence that diverse agroecological systems and mosaic landscapes of small-scale farms conserve biodiversity both at the local and landscape levels. In turn, other studies have found that biodiversity provides ecosystem services that contribute to agricultural productivity, sustainability and rural livelihoods (e.g. Hooper et al. (2005)84 and Diaz et al. (2010)159). This evidence, in combination with evidence of the failure of the neoliberal export-led model of agricultural development to reduce rural poverty and conserve biodiversity in Latin America, suggests that a new integrative approach is needed to simultaneously conserve biodiversity and eliminate poverty.\n\n\nIntegrated biodiversity conservation and poverty reduction: the food sovereignty framework\n\nAgroecological intensification160 has been shown to produce food and maintain ecological services more efficiently than conventional monocropping systems161. Critiques of the land-sharing approach suggest that smallholder, agroecological and organic farmers are unable to produce enough food to satisfy the growing global demand for food and agro-fuels. However, it may be argued that given the appropriate enabling conditions, including secure access to strategic resources for small landholdings and agricultural supports commensurate with national agricultural systems that support large-scale-industrial-agriculture, small-scale-diverse-agroecological farms can substantially contribute to present and future food needs46,135,162–166. In a review of 91 studies of organic agricultural systems across a range of geographic contexts, Badgley et al.165 present evidence that organic agricultural production methods—while requiring higher labour inputs—can produce enough food to meet current food needs without expanding the agricultural land base, and that the use of a range of alternative agricultural practices could increase global food production by as much as 50%. Though controversial, this number is consistent with moving towards agroecological best practices and taking advantage of areas favourable to organic agriculture167, supported by recent research in Africa162,164,166. Finally, a recent review of the literature on agroecology and the right to food163 suggests that small-scale farmers can double food production within a decade in critical regions by using agroecological production methods, and research consistently indicates that agrobiodiversity based on indigenous farmer knowledge contributes to food security168,169.\n\nFood sovereignty was broadly defined at the World Food Summit in 1996 as the right of local people to control their own regional and national food systems, including markets, natural resources, food cultures and production modes170–172. The framework stands in stark contrast to the agro-export based concept of food security, and argues that negative externalities, including the social welfare costs incurred by rural displacement and the loss of ecological services caused by monocropping are not calculated against the perceived high yields of agricultural industrialization (Table 1). It postulates that small-scale sustainable farming, based on a dense agroecological matrix where communities have greater levels of security and control over the land, resources, and management regimes, has the potential to “feed the world and cool the planet”173,174. The framework elaborates, specifically for food production systems, the conceptual model of linked social and ecological systems5,175. It promotes agroecological production practices that seek to integrate traditional and localized knowledge with modern agricultural and ecological science to increase food production, support rural livelihoods, preserve genetic and cultural diversity, and conserve soil fertility and biodiversity159,176,177. Of concern here are possible corrections to the degrading feedback loops between biodiversity loss and rural poverty traps associated with agricultural industrialization.\n\n\nPromising food sovereignty-based approaches\n\nThe food sovereignty framework has emerged in national constitutions (Ecuador, Bolivia, Nepal, Mali) and in national policies (Brazil, Cuba), building on civil-society and government led initiatives around the right to food, land redistribution, regional food procurement, and promotion of agroecological production methods178. In the examples that follow, we review promising systems that demonstrate mechanisms and practices oriented towards food sovereignty that combine biodiversity conservation, food production and poverty alleviation. These examples present several important facets of food sovereignty, including a peasant-friendly institutional and economic context, secure land tenure for smallholders, interactions between livelihoods and agrobiodiversity, and the use of local and traditional agroecological knowledge and plants. We conclude with a call for focused research based on multi-disciplinary methodologies that uses a social-ecological systems approach to more effectively evaluate the synergies and trade-offs between poverty alleviation, sustainable food production, and ecological management strategies.\n\nIn the last two decades, Brazil’s explosive agricultural growth has exemplified the global tensions between biodiversity conservation, poverty reduction, and food production136,179,180. The expansion of large-scale commercial agriculture—particularly the soy, beef and sugarcane sectors—has been associated with increased social inequality and environmental degradation181–184. In response, based on Brazil’s constitutional provisions for land reform, food sovereignty proponents advocate an “ecological land reform” that supports production for local and national consumption, and incorporates social and environmental goals into community settlement planning185.\n\nBetween 1942 and 2004, Brazil’s agrarian reform program settled almost 800,000 families on smallholder plots across Brazil. While almost two-thirds of these settlements were located in the Amazon region, Pacheco186 estimates that only 13% of Amazonian deforestation up to 2003 was attributable to smallholders in agrarian reform settlements. Since 1985, a growing percentage of settlements have been located in previously settled and deforested areas near urban centers177,187. Settling smallholders on abandoned land on plots averaging 25–50 hectares has resulted in the development of complex land use mosaics186, producing a wide variety of subsistence and market oriented food and fuel crops, as well as ecological restoration activities required under the regulations for protected and reserve areas in agricultural reform settlements. This model has been shown to result in smallholder settlements that tend to be more intensive, include tree crops, and practice rotational cultivation followed by secondary forest fallows188–190. As part of a program to integrate conservation goals with rural poverty reduction, over 10% of the redistributed area was formally designated as forested environmental reserves, while an additional 13% is voluntarily maintained under forest cover by plot recipients191. These areas provide important pockets for biodiversity conservation within agricultural landscapes, while also serving as a source for non-timber forest products. In addition, many settlements have undertaken ecosystem rehabilitation and reforestation activities, covering over 871,000 hectares by 2001192. For example, several agrarian reform settlements bordering protected areas in the threatened Brazilian Atlantic Forest ecosystem have been partners in the strategic protection and reforestation of forest fragments that act as wildlife corridors, facilitating seed dispersal and providing a buffer zone to protected areas179,193,194.\n\nLarge-scale studies of Brazilian agrarian reform suggest that locating smallholder settlements near urban centers rather than in isolated frontier regions can facilitate not only improved environmental performance, but also farmer incomes and standards of living that are higher than the regional average75,195–197. In an attempt to examine the potential trade-offs between food production, poverty alleviation and environmental degradation, Sparovek et al.195,198 conducted a comprehensive study of 4,340 settlements, comprised of 458,000 families, which were created through government-sponsored land redistribution between 1985 and 2001. These land reform settlements demonstrated significant regional variation in environmental quality (measured as a weighted composite of legal reserve preservation, deforestation, soil degradation, and ecological restoration), with the highest indices of degradation found in the northern Amazonian states and the lowest in traditionally settled areas of the south and center-west192.\n\nCoffee and cocoa agroforestry systems also generate ecological, economic, and social benefits through farmers’ management of high levels of agrobiodiversity—key elements of the food sovereignty framework. Correspondingly, when coffee and cacao are produced as perennial monocrops with little or no shade tree canopy, substantially lower levels of agrobiodiversity are observed199. In Central America and Mexico, research on the relationship between livelihoods provision, poverty reduction, and biodiversity conservation has been conducted in resource-poor, small-farmer coffee communities of Matagalpa, northern Nicaragua, Tacuba, Western El Salvador and in Chiapas and Oaxaca, southern Mexico200–202. Study sites in Central America contained a protected forest surrounded by an agroecological matrix dominated by shade coffee with smaller areas of annual crops. Farmers participating in these long-term studies grow coffee as their primary cash crop, along with a variety of food crops for consumption. A recent synthesis of this work shifted focus from biodiversity in coffee plantations themselves to the associated and planned agrobiodiversity that smallholder coffee households manage in the broader landscape202. This approach uses the household as the first unit of analysis and then considers the broader range of plant biodiversity managed and used by each household in coffee plantations as well as food crop plots and home gardens. The livelihoods framework203 was then used to analyze the contributions of plant biodiversity to coffee farm households. Livelihoods are defined as people’s capacities and means of living (e.g. food, income and assets, such as land, education etc.).\n\nSmall, individually managed farms contained significantly higher levels of shade tree diversity than larger plantations in both countries202 and contained a significantly higher number and diversity of fruit and firewood trees200,201. In related studies on shade coffee-based agroforestry systems in Chiapas204–206, no apparent relationship was found between farmer income levels and shade tree abundance or species composition—belying in this case a perceived trade-off between income and biodiversity. Rather, all of the studied farmers managed their plantations to produce a diversity of shade tree products for consumption. That is, a focus on diversified, small-scale agroecological production—tenets of food sovereignty—helped provide both livelihood benefits and benefits to biodiversity.\n\nMexican and Central American smallholder coffee production systems show strong interdependencies connecting rural livelihoods with high levels of agrobiodiversity. Although these livelihoods remain difficult—seasonal hunger is common and monetary incomes are low—agrobiodiversity and dynamic local organizations connected to alternative trade networks have shown themselves to be important factors in buffering vulnerability to external shocks, including hurricanes and crashing coffee prices200,207–209 (similar results were found in Nicaragua:210). Diversity and multiple land use practiced by small farmers guarantee some level of food security through direct production of food products even when commercial production is not profitable. However, despite the benefits offered by such systems, especially as compared to specialized, input-intensive monocultural alternatives, they ultimately cannot be maintained, or their contributions to poverty alleviation improved, unless they are supported by subsidies, investment, higher and stable prices, and reinforcement of local capacities in order to scale up towards local and regional markets5,19,46,209.\n\nGuatemalan and Mexican peasants continue to practice a polyculture system known as milpa (corn intercropped with beans, squash, chillies, and many other edible and useful plants) as they have done for thousands of years. Diversified livelihoods—including the production of a variety of products from diversified agroecosystems for sale and self-consumption—helps them to guarantee food and economic security and stability and preserve non-economic cultural values19,211,212. By preserving their traditional agricultural practices, small-scale farmers conserve not only crop resources, but also many wild varieties associated with their traditional systems, an approach to food sovereignty that emphasizes local values, autonomy, and biodiversity. In the semi-arid Tehuacan-Cuicatlan biosphere reserve in Mexico, researchers found 1,335 wild vascular plant species with one or more uses (e.g., fodder, medicinal, food, ornamental, soil control)213. These species represent over half of the total regional species diversity of vascular plants, and 82% of familial diversity. Blanckaert et al.214 found almost 150 useful weed species in the same region, with fodder weeds, for instance, cutting costs for industrial animal feed purchases and increasing survival of farm animals in times of drought. Similarly, herbs collected from maize fields in Mexico’s Toluca Valley serve nutritional, medicinal and aesthetic purposes, and their use as fodder boosts the economic returns on maize farming by 55%215. In Chiapas, Mexico, Tzeltal Mayans can recognize more than 1200 species of plants, many of which contribute to their livelihoods216. The use of synthetic herbicides puts this diversity at risk and affects food security; in response, farmers may leave parts of their fields unsprayed to permit continued collection of useful “weeds”215. Thus traditional systems using wild varieties constitute another way that food sovereignty both encourages and depends on broad biological diversity, an approach distinct from and at times even in opposition to that encouraged in Latin America for the past 50 years19.\n\nIn Mexico, researchers have examined the reasons for the persistence of cultivation of traditional maize varieties within the milpa by indigenous communities for domestic consumption, despite both the influx of cheaper imported corn from the U.S. under the North American Free Trade Agreement and the availability of less expensive domestic corn. Surveys of Zapotec indigenous households in the state of Oaxaca—an important center of corn genetic diversity—found that despite mean total production costs of more than 400% above the market cost of corn, families continued to plant and consume many traditional varieties instead of (or in addition to) purchasing corn, for reasons that include perceived higher quality, nutritional superiority, and cultural factors209,217. Thus, despite the threats posed by trade liberalization, the persistence of these traditional varieties helps to sustain food sovereignty, local food security, and biodiversity.\n\n\nConclusion\n\nIn Latin America, the claim that there is an all-inclusive trap where economic poverty leads to biodiversity loss is not supported in the cases reviewed here, particularly in view of the higher biodiversity of typical smallholdings relative to large scale monoculture agriculture. Thus, efforts to help the smallholder agriculture sector escape poverty traps while stemming the tide of biodiversity loss, at least in Latin America, will require a strategy acknowledging the historical and continuing exogenous drivers of both problems. In this paper we have argued that these factors include the income and land structural biases and inequalities pervasive in the region, neoliberal policies that focus on the agro-export model and the conventional agricultural intensification that puts smallholders in a competitive but disadvantageous economic environment (paralleling and reinforcing Maru et al.’s 2012 synthesis of poverty traps among indigenous groups;2). Food sovereignty is an approach originating from the rural poor of Latin America (and beyond) that unites efforts to address unbalanced international trade policies, historical legacies and continuation of inequality, and the continuing consolidation of agricultural modernization policies often associated with negative impacts for small-scale farmers and sustainable ecosystems. Latin American smallholders have maintained and adopted diverse strategies, mixing modern and traditional agricultural varieties and supporting significant levels of on-farm biodiversity. The high on-farm biodiversity associated with smallholder agroecological practices has been empirically tied to greater stability in income and recovery from environmental disaster (i.e., resilience)210,218,219, greater food security19, and generally positive effects for associated biodiversity54,135. While the predominant trend has turned to staples produced by industrial agriculture to boost per capita energy consumption, this strategy threatens biodiversity, the livelihoods of small scale farmers and diet quality53,220,221. It also promotes chronic diseases, including diabetes, heart disease and obesity80.\n\nHowever, evidence elucidating the connections between food sovereignty and its emphasis on diverse traditional crops, wild plants and animal species maintained by small-scale farmers with broader economic and health benefits is still accumulating. Although many traditional systems in Latin America have proved their durability in the long term19, researchers face serious methodological challenges inherent in measuring the relationship between biodiversity and food security within a common framework222–224. In emphasizing the collective right of food producers and consumers to decide the characteristics of their food system at local, regional and national levels, food sovereignty contains a crucial ambiguity—that is, the question of how to resolve possible contradictions within these different geographies, from the nation-state to the individual225. This ambiguity arguably reflects both the empirical reality of immense variation between different sustainable and egalitarian institutions, and the conceptual flexibility necessary to create them.\n\nFor example, Ostrom’s decades of work (e.g., Ostrom and Nagendra (2002)65 and Ostrom (2009)226) have shown that local institutions are crucial for the management of the commons. Her work has also consistently emphasized that devolving power to local stakeholders is never a panacea, nor is there a guaranteed formula. However, there are certain patterns that characterize successful local institutions, an empirical observation shared by other researchers who have posited “deep democracy” and strong local control as necessary but not sufficient conditions for sustainability72. We argue that the food sovereignty framework offers a novel methodological opportunity to align the issues of poverty and conservation within a general socio-ecological model. The cases presented here and in the growing literature on food sovereignty correspond to a growing empirical recognition of the significant power of diversified smallholder agricultural systems19,46, with all the tensions regarding institutions at multiple scales that this implies65. But perhaps most crucially, the food sovereignty framework represents an opportunity for those concerned with biodiversity conservation and poverty to work in alliance with millions of small-scale farmers and their supporters.",
"appendix": "Author contributions\n\n\n\nIP and JV conceived the paper. All authors carried out research for the paper. MJC, HW, and IP prepared the first draft of the manuscript. All authors were involved in revisions of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nMany of the authors have been involved with supporting peasant agriculture and working with farmers in Latin America for a number of years. They declare that they are intellectually and personally committed to supporting equitable and sustainable rural systems using academically rigorous research. They have at various points consulted for and worked with rural organizations supporting food sovereignty, including La Vía Campesina, the organization that helped originate the term food sovereignty. This work does not necessarily reflect the views of anyone but the authors, and was conducted independently of any such previous or on-going ties.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nSeveral anonymous reviewers provided feedback on various versions of this manuscript, as did C. B. Barrett and A. J. Travis. Any errors are ours.\n\n\nReferences\n\nAzariadis C, Stachurski J: Handbook of economic growth. P. Aghion, S. N. Durlauf, Eds. (Elsevier, Amsterdam, 2005), vol. 1, Part 1, 295–384. Publisher Full Text\n\nMaru YT, Fletcher CS, Chewings VH, et al.: A Synthesis of Current Approaches to Traps Is Useful But Needs Rethinking for Indigenous Disadvantage and Poverty Research. Ecology and Society. 2012; 17(2): 7. Publisher Full Text\n\nBarrett CB, Travis AJ, Dasgupta P: On biodiversity conservation and poverty traps. Proc Natl Acad Sci U S A. 2011; 108(34): 13907–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMészáros I: The challenge of sustainable development and the culture of substantive equality. Mon Rev. 2001; 53(7): 10. Reference Source\n\nPerfecto I, Vandermeer JH, Wright AL: Nature's matrix: Linking agriculture, conservation and food sovereignty. (Earthscan, London, 2009). Reference Source\n\nHolland TG, Peterson GD, Gonzalez A: A cross-national analysis of how economic inequality predicts biodiversity loss. Conserv Biol. 2009; 23(5): 1304–13. PubMed Abstract | Publisher Full Text\n\nFreebairn DK: Did the Green Revolution Concentrate Incomes? A Quantitative Study of Research Reports. World Dev. 1995; 23(2): 265–279. Publisher Full Text\n\nCEPAL, Panorama Social de América Latina (Economic Commission of Latin America and the Caribbean; United Nations, Santiago, 2011). Reference Source\n\nTilman D, Cassman KG, Matson PA, et al.: Agricultural sustainability and intensive production practices. Nature. 2002; 418(6898): 671–7. PubMed Abstract | Publisher Full Text\n\nTilman D: Global environmental impacts of agricultural expansion: the need for sustainable and efficient practices. Proc Natl Acad Sci U S A. 1999; 96(11): 5995–6000. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcMichael P: Development and Social Change: A global perspective. (Pine Forge Press, Thousand Oaks, ed. 3rd Edition, 2004). Reference Source\n\nPhalan B, Onial M, Balmford A, et al.: Reconciling Food Production and Biodiversity Conservation: Land Sharing and Land Sparing Compared. Science. 2011; 333(6047): 1289–91. PubMed Abstract | Publisher Full Text\n\nTscharntke T, Clough Y, Wanger TC, et al.: Global food security, biodiversity conservation and the future of agricultural intensification. Biol Cons. 2012; 151(1): 53–59. Publisher Full Text\n\nBarrett CB: Economics of poverty, the environment and natural resource use. R. B. Dellink, A. Ruijs, Eds. (Springer, Dordrecht), 2008: VI. : 212. Reference Source\n\nNadkarni MV: Poverty, Environment, Development: A Many-Patterned Nexus. Econ Polit Weekly. 2000; 35(14): 1184–1190. Reference Source\n\nTaylor PJ, García Barrios R: Global environmental economics: Equity and the limits to markets. T. Mount, H. Shue, M. Dore, Eds. (Blackwell, Oxford, UK, 1999). Reference Source\n\nGray LC, Moseley WG: A geographical perspective on poverty-environment interactions. Geogr J. 2005; 171(1): 9–23. Publisher Full Text\n\nBorras SM: Pro-poor land reform: a critique. (University of Ottawa Press, Ottawa, Ontario, 2007). Publisher Full Text\n\nNivia E: Agriculture at a Crossroads: The International Assessment of Agricultural Knowledge, Science and Technology for Development (IAASTD). R. T. Watson, H. R. Herren, J. Wakhungu, Eds. (Island Press, Washington, D.C., 2009). Reference Source\n\nPérez M, Schlesinger S, Wise TA: La Promesa y los Peligros de la Liberalización del Comercio Agrícola: Lecciones de América Latina. (Asociación de Instituciones de Promoción y Educación (AIPE), and the Global Development and Environment Institute (GDAE) of Tufts University, La Paz Bolivia, 2009). Reference Source\n\nBarrett CB, Swallow BM: Fractal poverty traps. World Dev. 2006; 34(1): 1–15. Publisher Full Text\n\nWeis AJ: The global food economy: The battle for the future of farming. (Zed Books, distributed by Palgrave Macmillan, London and New York, 2007). Reference Source\n\nDasgupta P: An inquiry into well-being and destitution. (Clarendon, Oxford, 1993). Reference Source\n\nBerkes F, Folke C: Linking social and ecological systems. (Cambridge University Press, Cambridge, 1998). Reference Source\n\nVan Kooten GC, Bulte EH: The economics of nature: Managing biological assets. (Malden, Blackwell, 2000). Reference Source\n\nWorld Resources Institute (WRI), \"World resources 2005: The wealth of the poor--Managing ecosystems to fight poverty. (World Resources Institute, Washington, D.C., 2005). Reference Source\n\nWorld Bank, Where is the Wealth of Nations? Measuring Capital for the 21st Century. (The World Bank, Washington, 2006). Reference Source\n\nMarenya PP, Barrett CB: State-conditional Fertilizer Yield Response on Western Kenyan Farms. Am J Agr Econ. 2009; 91(4): 991–1006. Publisher Full Text\n\nGarcía Barrios R, García Barrios LE: Environmental and technological degradation in peasant agriculture: A consequence of development in Mexico. World Dev. 1990; 18(11): 1569–1585. Publisher Full Text\n\nOstrom E: Paper presented at the Ford Foundation 50th Anniversary Forum, Axotla. Mexico City, Mexico, 2012.\n\nRobbins P: Political ecology: A critical introduction. Critical introductions to geography (Blackwell Publishing, Malden, MA, 2004). Reference Source\n\nEscobar A: Encountering Development: The Making and Unmaking of the Third World. Princeton studies in culture/power/history (Princeton University Press, Princeton, N.J., 1995); ix 290. Reference Source\n\nDepartment for International Development (DFID), Directorate General for Development-European Commission (EC), United Nations Development Programme (UNDP), The World Bank, Linking poverty reduction and environmental management: Policy challenges and opportunities.Working Paper. (The World Bank, Washington, D.C., 2002). Reference Source\n\nMcMichael P: A food regime genealogy. J Peasant Stud. 2009; 36(1): 139–169. Publisher Full Text\n\nConroy ME, Murray DL, Rosset PM: A cautionary tale: failed US development policy in Central America. (Lynne Rienner Publishers, Boulder, CO and London, 1996). Reference Source\n\nBerdegué JA, Fuentealba R: paper presented at the New Directions for Smallholder Agriculture. Rome, Italy, 2011. Reference Source\n\nKrueger AO, Schiff M, Valdés A: Agricultural incentives in developing countries: Measuring the effect of sectoral and economywide policies. World Bank Econ Rev. 1988; 2(3): 255–271. Publisher Full Text\n\nWorld Bank Rural development: Sector Policy Paper. (The World Bank, Washington, DC, 1975). Reference Source\n\nAraghi F: Peasants and globalisation: political economy, rural transformation and the agrarian question. A. Haroon Akram-Lodhi, C. Kay Eds. (Routledge, London, 2008), 336. Reference Source\n\nOtero G: Neoliberal Globalization, NAFTA and Migration: Mexico's Loss of Food and Labor Sovereignty. J Poverty. 2011; 15(4): 384–402. Publisher Full Text\n\nSchejtman A, Berdegué JA, Florez Victoria, et al.: Trade and poverty in Latin America. P. Giordano, Ed. (Inter-American Development Bank, Washington, D.C., 2009), 249–322. Reference Source\n\nJustino P, Litchfield J, Whitehead L: The impact of inequality in Latin America. Working Paper 21. (University of Sussex, Sussex, UK, 2003), Reference Source\n\nBerdegué JA, Schejtman Alexander, Chiriboga Manuel, et al.: Towards national and global agendas: Latin America and the Caribbean. Background paper for the World Development Report 2008\" (The World Bank and Rimisp-Latin American Center for Rural Development, Santiago, Chile, 2006). Reference Source\n\nModrego F, Charnay R, Jara E, et al.: Small farmers in Developing Countries: Some results of household surveys data analysis. Background paper for the World Development Report 2008\" (The World Bank and Rimisp-Latin American Center for Rural Development, Santiago, Chile, 2006). Reference Source\n\nVandermeer JH, Perfecto I: Breakfast of biodiversity: The political ecology of rain forest destruction. (FoodFirst Books, Oakland, CA ed. 2nd, 2005), Reference Source\n\nInternational Assessment of Agricultural Knowledge Science and Technology for Development (IAASTD). Agriculture at a crossroads: International assessment of agricultural knowledge, science and technology for development. (Island Press, Washington, D.C., 2009). Reference Source\n\nDeFries R, Rudel TK, Uriarte M, et al.: Deforestation driven by urban population growth and agricultural trade in the twenty-first century. Nature Geosci. 2010; 3: 178–181. Publisher Full Text\n\nThrupp LA, Bergeron G, Waters WF: Bittersweet harvests for global supermarkets: Challenges in Latin America's agricultural export boom. (World Resources Institute, Washington, D.C., 1995). Reference Source\n\nWright AL: Rethinking the Circle of Poison: The Politics of Pesticide Poisoning among Mexican Farm Workers. Lat Am Perspect. 1986; 13(4): 26–59. PubMed Abstract | Publisher Full Text\n\nAltieri MA, Rojas A: Ecological Impacts of Chile's Neoliberal Policies, with Special Emphasis on Agroecosystems. Env Dev Sustain. 1999; 1(1): 55–72. Publisher Full Text\n\nDonald PF: Biodiversity impacts of some agricultural commodity production systems. Conserv Biol. 2004; 18(1): 17–38. Publisher Full Text\n\nLiverman DM, Vilas S: Neoliberalism and the environment in Latin America. Annu Rev Environ Resour. 2006; 31: 327–363. Publisher Full Text\n\nFoley JA, Ramankutty N, Brauman KA, et al.: Solutions for a cultivated planet. Nature. 2011; 478(7369): 337–342. PubMed Abstract | Publisher Full Text\n\nPerfecto I, Vandermeer JH: The agroecological matrix as alternative to the land-sparing/agriculture intensification model. Proc Natl Acad Sci U S A. 2010; 107(13): 5786–5791. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGibbs KE, Mackey RL, Currie DJ: Human land use agriculture, pesticides and losses of imperiled species. Diversity Distrib. 2009; 15(2): 242–253. Publisher Full Text\n\nPatnaik U: Food availability and famine: a longer view. J Peasant Stud. 1991; 19(1): 1–25. Publisher Full Text\n\nDavis M: Late Victorian Holocausts: El Niño famines and the making of the Third World. (Verso, London and New York, 2002). Reference Source\n\nWaldman A: Poor in India starve as surplus wheat rots. The New York Times, December 2, 2002; A3. Reference Source\n\nRudel TK, Schneider L, Uriarte M, et al.: Agricultural intensification and changes in cultivated areas, 1970–2005. Proc Natl Acad Sci U S A. 2009; 106(49): 20675–20680. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEwers RM, Scharlemann JPW, Balmford A, et al.: Do increases in agricultural yield spare land for nature? Glob Change Biol. 2009; 15(7): 1716–1726. Publisher Full Text\n\nSen A: Poverty and famines: an essay on entitlement and deprivation. (Oxford University Press, Oxford, UK, 1981). Publisher Full Text\n\nSmith LC, El Obeid AE, Jensen HH: The geography and causes of food insecurity in developing countries. Agr Econ. 2000; 22(2): 199–215. Publisher Full Text\n\nDas RJ: The Green Revolution and poverty: A theoretical and empirical examination of the relation between technology and society. Geoforum. 2002; 33(1): 55–72. Publisher Full Text\n\nBorras SM, Franco JC: Global Land Grabbing and Trajectories of Agrarian Change: A Preliminary Analysis. Journal of Agrarian Change. 2012; 12(1): 34–59. Publisher Full Text\n\nOstrom E, Nagendra H: Insights on linking forests, trees, and people from the air on the ground, and in the laboratory. Proc Natl Acad Sci U S A. 2006; 103(51): 19224–19231. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChiriboga M: El desarrollo sostenible en el Medio Rural. L. Martínez, Ed. (FLASCO, Quito, Ecuador, 1999). Reference Source\n\nWorld Bank, World development report 2008: Agriculture for development. (The World Bank, Washington, D.C., 2007). Publisher Full Text\n\nHolt-Giménez E: Out of AGRA: The Green Revolution returns to Africa. Development. 2008; 51: 464–471. Publisher Full Text\n\nMoseley WG, Carney J, Becker L: Neoliberal policy, rural livelihoods, and urban food security in West Africa: A comparative study of The Gambia, Côte d'Ivoire, and Mali. Proc Natl Acad Sci U S A. 2010; 107(13): 5774–5779. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuckland J: International Obstacles to Rural Development: How Neoliberal Policies Constrain Competitive Markets and Sustainable Agriculture. Rev Can Etud Dev. 2006; 27(1): 9–24. Publisher Full Text\n\nDe Young R, Princen T: The localization reader: Adapting to the coming downshift. R. De Young, T. Princen, Eds. (MIT Press, Cambridge, MA, 2012), xvii–xxvi. Reference Source\n\nPrugh T, Costanza R, Daly HE: The local politics of global sustainability. (Island Press, Washington, D.C., 2000), xvi 173. Reference Source\n\nMarsden T: Third natures? Reconstituting Space through Place-making strategies for sustainability. Int J Sociol Agr Food. 2012; 19(2): 257–274. Reference Source\n\nLyson TA, Torres RJ, Welsh R: Scale of agricultural production, civic engagement, and community welfare. Soc Forces. 2001; 80(1): 311–327. Publisher Full Text\n\nLeite S, Heredia B, Medeiros L, et al.: Impactos dos Assentamentos: Um estudo sobre o meio rural brasileiro. (Editora UNESP, São Paulo, 2004). Reference Source\n\nGoldschmidt W: As you sow: three studies in the social consequences of agribusiness. (Allenheld, Osmun, New York, 1978). Reference Source\n\nBodley JH: The power of scale: A global history approach. (M.E. Sharpe, Armonk, N.Y., 2003). Reference Source\n\nPatel RC: The value of nothing: How to reshape market society and redefine democracy. (Picador, New York, 2009). Reference Source\n\nSwift MJ, Izac AMN, van Noordwijk M: Biodiversity and ecosystem services in agricultural landscapes - are we asking the right questions? Agr Ecosyst Environ. 2004; 104(1): 113–134. Publisher Full Text\n\nMillennium Ecosystem Assessment (MEA), \"Ecosystems and Human Well-Being: Current State and Trends: Findings of the Condition and Trends Working Group\". (Island Press, Washington, D.C., 2005). Reference Source\n\nThrupp LA: Linking agricultural biodiversity and food security: the valuable role of agrobiodiversity for sustainable agriculture. Int Aff. 2000; 76(2): 265–281. PubMed Abstract\n\nRemans R, Flynn DFB, DeClerck F, et al.: Sustainable diets and biodiversity: Directions and solutions for policy, research and action. B. Burlingame, S. Dernini, Eds. (Food and Agriculture Organization of the United Nations, Rome, Italy, 2010), pp. 134–149. Reference Source\n\nGold K, McBurney RPH: Sustainable diets and biodiversity: Directions and solutions for policy, research and action. B. Burlingame, S. Dernini, Eds. (Food and Agriculture Organization of the United Nations, Rome, Italy, 2010), pp. 108–114. Reference Source\n\nHooper DU, Chapin FS, Ewel JJ, et al.: Effects of biodiversity on ecosystem functioning: a consensus of current knowledge. Ecol Monogr. 2005; 75(1): 3–35. Publisher Full Text\n\nJarvis D, Brown AH, Cuong PH, et al.: A global perspective of the richness and evenness of traditional crop-variety diversity maintained by farming communities. Proc Natl Acad Sci U S A. 2008; 105(14): 5326–5331. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEasterling W, Apps M: Assessing the Consequences of Climate Change for Food and forest Resources: A View from the IPCC. Climatic Change. 2005; 70(1–2): 165–189. Publisher Full Text\n\nBeg N, Morlota JC, Davidson O, et al.: Linkages between climate change and sustainable development. Climate Pol. 2002; 2(2–3): 129–144. Publisher Full Text\n\nKassam K: Pluralism, Resilience, and the Ecology of Survival: Case Studies from the Pamir Mountains of Afghanistan. Ecol Soc. 2010; 15(2). Reference Source\n\nMcLaughlin P, Dietz T: Structure, agency and environment: Toward an integrated perspective on vulnerability. Global Environ Chang. 2008; 18(1): 99–111. Publisher Full Text\n\nTomich TP, Brodt S, Ferris H, et al.: Agroecology: A Review from a Global-Change Perspective. Annu Rev Environ Resour. 2011; 36: 193–222. Publisher Full Text\n\nMcDowell JZ, Hess JJ: Accessing adaptation: Multiple stressors on livelihoods in the Bolivian highlands under a changing climate. Global Environ Chang. 2012; 22(2): 342–352. Publisher Full Text\n\nLin BB: Resilience in Agriculture through Crop Diversification: Adaptive Management for Environmental Change. Bioscience. 2011; 61(3): 183–193. Publisher Full Text\n\nDorsey B: Agricultural Intensification, Diversification, and Commercial Production among Smallholder Coffee Growers in Central Kenya. Econ Geogr. 1999; 75(2): 178–195. Publisher Full Text\n\nKasem S, Thapa GB: Crop diversification in Thailand: Status, determinants, and effects on income and use of inputs. Land Use Policy. 2011; 28(3): 618–628. Publisher Full Text\n\nGomiero T, Pimentel D, Paoletti MG: Environmental Impact of Different Agricultural Management Practices: Conventional vs. Organic Agriculture. Crit Rev Plant Sci. 2011; 30(1–2): 95–124. Publisher Full Text\n\nPaoletti MG, Pimentel D, Stinner BR, et al.: Agroecosystem biodiversity: Matching production and conservation biology. Agr Ecosyst Environ. 1992; 40(1–4): 3–23. Publisher Full Text\n\nSchönning M, Richardsdotter-Dirke M: \"Ekologiskt och konventionellt jordbruk: skillnader i biologisk mångfald och livsmedelskvalitet. En litteraturöversikt (Organic and conventional agriculture: differences in biodiversity and food quality: A literature review)\". Rapport 9304. (Svenska Naturskyddsföreningen (The Swedish Society for Nature Conservation), Stockholm, Sweden, 1996). Reference Source\n\nBengtsson J, Ahnström J, Weibull AC: The effects of organic agriculture on biodiversity and abundance: a meta-analysis. J Appl Ecol. 2005; 42(2): 261–269. Publisher Full Text\n\nScherr SJ, McNeely JA: Biodiversity conservation and agricultural sustainability: Towards a new paradigm of 'ecoagriculture' landscapes. Philos Trans R Soc Lond B Biol Sci. 2008; 363(1491): 477–494. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPywell RF, Heard MS, Bradbury RB, et al.: Wildlife-friendly farming benefits rare birds, bees and plants. Biol Lett. 2012; 8(5): 772–775. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKremen C, Miles AF: Comparing biologically diversified with conventional farming systems: What is known about environmental benefits, externalities and tradeoffs among crop productivity and ecosystem services? Ecol Soc. 2012; 17: 40.\n\nAltieri MA: The ecological role of biodiversity in agroecosystems. Agr Ecosyst Environ. 1999; 74(1–3): 19–31. Reference Source\n\nTscharntke T, Klein AM, Kruess A, et al.: Landscape perspectives on agricultural intensification and biodiversity-ecosystem service management. Ecol Lett. 2005; 8(8): 857–874. Publisher Full Text\n\nVandermeer JH, Lawrence D, Symstad A, et al.: Biodiversity and ecosystem functioning: Synthesis and perspectives. M. Loreau, S. Naeem, P. Inchausti, Eds. (Oxford University Press, Oxford, 2002), 221–236.\n\nGabriel D, Sait SM, Hodgson JA, et al.: Scale matters: the impact of organic farming on biodiversity at different spatial scales. Ecol Lett. 2010; 13(7): 858–69. PubMed Abstract | Publisher Full Text\n\nBalmford A, Green RE, Scharlemann JPW: Sparing land for nature: Exploring the potential impact of changes in agricultural yield on the area needed for crop production. Glob Change Biol. 2005; 11(10): 1594–1605. Publisher Full Text\n\nPfiffner L, Häring A, Dabbert S, et al.: Organic Food and Farming: Towards Partnership and Action in Europe. (Dänish Ministry for Food, Agriculture and Fisheries, Copenhagen, Denmark, 2001), 115–123.\n\nFuller RJ, Norton LR, Feber RE, et al.: Benefits of organic farming to biodiversity vary among taxa. Biol Lett. 2005; 1(4): 431–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHole DG, Perkins AJ, Wilson JD, et al.: Does organic farming benefit biodiversity? Biol Cons. 2005; 122(1): 113–130. Publisher Full Text\n\nGibson RH, Pearce S, Morris RJ, et al.: Plant diversity and land use under organic and conventional agriculture: a whole-farm approach. J Appl Ecol. 2007; 44(4): 792–803. Publisher Full Text\n\nLynch DH, Halberg N, Bhatta GD: Environmental impacts of organic agriculture in temperate regions. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition, and Natural Resources. 2012; 7(10): 1–17. Publisher Full Text\n\nMondelaers K, Aertsens J, van Huylenbroeck G: A meta-analysis of the differences in environmental impacts between organic and conventional farming. British Food J. 2009; 111(10): 1098–1119. Publisher Full Text\n\nWinqvist C, Ahnström J, Bengtsson J: Effects of organic farming on biodiversity and ecosystem services: taking landscape complexity into account. Ann NY Acad Sci. 2012; 1249: 191–203. PubMed Abstract | Publisher Full Text\n\nKleijn D, Baquero RA, Clough Y, et al.: Mixed biodiversity benefits of agri-environment schemes in five European countries. Ecol Lett. 2006; 9(3): 243–254. PubMed Abstract | Publisher Full Text\n\nDavey CM, Vickery JA, Boatman ND, et al.: Assessing the impact of Entry Level Stewardship on lowland farmland birds in England. Ibis. 2010; 152(3): 459–474. Publisher Full Text\n\nFahrig L, Baudry J, Brotons L, et al.: Functional landscape heterogeneity and animal biodiversity in agricultural landscapes. Ecol Lett. 2011; 14(2): 101–12. PubMed Abstract | Publisher Full Text\n\nBenton TG, Vickery JA, Wilson JD: Farmland biodiversity: Is habitat heterogeneity the key? Trends in Ecology and Evolution. 2003; 18(4): 182–188. Publisher Full Text\n\nPurtauf T, Roschewitz I, Dauber J, et al.: Landscape context of organic and conventional farms: Influences on carabid beetle diversity. Agr Ecosyst Environ. 2005; 108(2): 165–174. Publisher Full Text\n\nSchmidt MH, Roschewitz I, Thies C, et al.: Differential effects of landscape and management on diversity and density of ground-dwelling farmland spiders. J Appl Ecol. 2005; 42(2): 281–287. Publisher Full Text\n\nRundlöf M, Smith HG: The effect of organic farming on butterfly diversity depends on landscape context. J Appl Ecol. 2006; 43(6): 1121–1127. Publisher Full Text\n\nLe Roux X, Barbault R, Baudry J, et al.: Agriculture et biodiversité. Valoriser les synergies. Expertise scientifique collective, synthèse du rapport. (INRA, Paris, 2008). Reference Source\n\nNorton L, Johnsonb P, Joys A, et al.: Consequences of organic and non-organic farming practices for field, farm and landscape complexity. Agr Ecosyst Environ. 2009; 129(1–3): 221–227. Publisher Full Text\n\nMendenhall CD, Ehrlich PR, Daily GC, et al.: Predictive model for sustaining biodiversity in tropical countryside. Proc Natl Acad Sci U S A. 2011; 108(39): 16313–16316. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarp DS, Rominger AJ, Zook J, et al.: Intensive agriculture erodes β-diversity at large scales. Ecol Lett. 2012; 15(9): 963–70. PubMed Abstract | Publisher Full Text\n\nBelfrage K, Johanna B, Salomonsson L: The Effects of Farm Size and Organic Farming on Diversity of Birds, Pollinators, and Plants in a Swedish Landscape. Ambio. 2005; 34(8): 582–8. PubMed Abstract | Publisher Full Text\n\nBaudry J, Bunce RGH: Land abandonment and its role in conservation: Proceedings of the Zaragoza/Spain seminar. 10–12 December 1989,. (CIHEAM, Paris, 1989).\n\nSirami C, Brotons L, Martin JL: Vegetation and songbird response to land abandonment: from landscape to census plot. Diversity Distrib. 2007; 13: 42.\n\nMedan D, Torretta JP, Hodara K, et al.: Effects of agriculture expansion and intensification on the vertebrate and invertebrate diversity in the Pampas of Argentina. Biodivers Conserv. 2011; 20(13): 3077–3100. Publisher Full Text\n\nMedley KE, Okey BW, Barrett GW, et al.: Landscape change with agricultural intensification in a rural watershed, southwestern Ohio, U.S.A. Landscape Ecology. 1995; 10(3): 161–176. Publisher Full Text\n\nWeibull AC, Bengtsson J, Nohlgren E: Diversity of butterflies in the agricultural landscape: the role of farming system and landscape heterogeneity. Ecography. 2000; 23(6): 743–750. Publisher Full Text\n\nÖstman Ö, Ekbom B, Bengtsson J, et al.: Landscape complexity and farming practice influence the condition of polyphagous carabid beetles. Ecol Appl. 2001; 11(2): 480–488. Publisher Full Text\n\nHolzschuh A, Tscharntke T, Steffan-Dewenter I: How do landscape composition and configuration, organic farming and fallow strips affect the diversity of bees, wasps and their parasitoids? J Anim Ecol. 2010; 79(2): 491–500. PubMed Abstract | Publisher Full Text\n\nFoley JA, Defries R, Asner GP, et al.: Global Consequences of Land Use. Science. 2005; 309(5734): 570–574. PubMed Abstract | Publisher Full Text\n\nFischer J, Brosi B, Daily GC, et al.: Should agricultural policies encourage land sparing or wildlife-friendly farming? Front Ecol Environ. 2008; 6(7): 380–385. Publisher Full Text\n\nChappell MJ, LaValle LA: Food security and biodiversity: can we have both? An agroecological analysis. Agric Human Values. 2011; 28(1): 3–26. Publisher Full Text\n\nFerreira J, Pardini R, Metzger JP, et al.: Towards environmentally sustainable agriculture in Brazil: challenges and opportunities for applied ecological research. J Appl Ecol. 2012; 49(3): 535–541. Publisher Full Text\n\nDallimer M, Tinch D, Acs S, et al.: 100 Years of Change: Examining Agricultural Trends, Habitat Change and Stakeholder Perceptions through the 20th Century. J Appl Ecol. 2009; 46(2): 334–343. Publisher Full Text\n\nKuussaari M, Bommarco R, Heikkinen RK, et al.: Extinction debt: a challenge for biodiversity conservation. Trends Ecol Evol. 2009; 24(10): 564–71. PubMed Abstract | Publisher Full Text\n\nJackson ST, Sax DF: Balancing biodiversity in a changing environment: extinction debt, immigration credit and species turnover. Trends Ecol Evol. 2010; 25(3): 153–60. PubMed Abstract | Publisher Full Text\n\nNewmark WD: Extinction of mammal populations in western North American national parks. Conserv Biol. 1995; 9(3): 512–526. Publisher Full Text\n\nFischer M, Stöcklin J: Local Extinctions of Plants in Remnants of Extensively Used Calcareous Grasslands 1950 –1985. Conserv Biol. 1997; 11(3): 727–737. Reference Source\n\nFoufopoulos J, Ives AR: Reptile Extinctions on Land-Bridge Islands: Life-History Attributes and Vulnerability to Extinction. Am Nat. 1999; 153: 1–25. Publisher Full Text\n\nKéry M: Extinction Rate Estimates for Plant Populations in Revisitation Studies: Importance of Detectability. Conserv Biol. 2004; 18(2): 570–574. Publisher Full Text\n\nMatthies D, Bräuer I, Maibom W, et al.: Population size and the risk of local extinction: empirical evidence from rare plants. Oikos. 2004; 105(3): 481–488. Publisher Full Text\n\nWilliams NSG, Morgan JW, McDonnell MJ, et al.: Plant traits and local extinctions in natural grasslands along an urban–rural gradient. J Ecol. 2005; 93(6): 1203–1213. Publisher Full Text\n\nWilsey BJ, Martin LM, Polley HW: Predicting Plant Extinction Based on Species-Area Curves in Prairie Fragments with High Beta Richness. Conserv Biol. 2005; 19(6): 1835–1841. Publisher Full Text\n\nMendenhall CD, et al.: paper presented at the 97th Annual Meeting of the Ecological Society of America: Life on Earth: Preserving, Utilizing and Sustaining our Ecosystems Portland, OR 2012. Reference Source\n\nRicketts TH: The matrix matters: Effective isolation in fragmented landscapes. Am Nat. 2001; 158(1): 87–99. PubMed Abstract | Publisher Full Text\n\nPrevedello J, Vieira M: Does the type of matrix matter? A quantitative review of the evidence. Biodivers Conserv. 2010; 19(5): 1205–1223. Publisher Full Text\n\nFahrig L: How much habitat is enough? Biol Cons. 2001; 100(1): 65–74. Publisher Full Text\n\nVandermeer JH, Perfecto I: The agricultural matrix and a future paradigm for conservation. Conserv Biol. 2007; 21(1): 274–277. PubMed Abstract | Publisher Full Text\n\nPerfecto I, Vandermeer JH: Biodiversity conservation in tropical agroecosystems: a new conservation paradigm. Ann NY Acad Sci. 2008; 1134: 173–200. PubMed Abstract | Publisher Full Text\n\nCassano CR, Schroth G, Faria D, et al.: Landscape and farm scale management to enhance biodiversity conservation in the cocoa producing region of southern Bahia, Brazil. Biodivers Conserv. 2009; 18(3): 577–603. Publisher Full Text\n\nDonald PF, Evans AD: Habitat connectivity and matrix restoration: the wider implications of agri-environment schemes. J Appl Ecol. 2006; 43(2): 209–218. Publisher Full Text\n\nDelattre T, Vernon P, Burel F: An agri-environmental scheme enhances butterfly dispersal in European agricultural landscapes. Agr Ecosyst Environ. 2013; 166: 102–109. Publisher Full Text\n\nMarín L, León-Cortés JL, Stefanescu C, et al.: The effect of an agro-pasture landscape on diversity and migration patterns of frugivorous butterflies in Chiapas, Mexico. Biodivers Conserv. 2009; 18(4): 919–934. Publisher Full Text\n\nJha S, Dick CW: Shade coffee farms promote genetic diversity of native trees. Curr Biol. 2008; 18(24): R1126–R1128. PubMed Abstract | Publisher Full Text\n\nJha S, Dick CW: Native bees mediate long-distance pollen dispersal in a shade coffee landscape mosaic. Proc Natl Acad Sci U S A. 2010; 107(31): 13760–13764. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDiaz S, Duffy JE: Encyclopedia of Earth. C. J. Cleveland, Ed. (Environmental Information Coalition, National Council for Science and the Environment, Washington, D.C., 2010).\n\nGodfray HC, Beddington JR, Crute IR, et al.: Food Security: The Challenge of Feeding 9 Billion People. Science. 2010; 327(5967): 812–818. PubMed Abstract | Publisher Full Text\n\nJordan N, Boody G, Broussard W, et al.: Environment. Sustainable Development of the Agricultural Bio-Economy. Science. 2007; 316(5831): 1570–1571. PubMed Abstract | Publisher Full Text\n\nHine RE, Pretty JN, Twarog S: \"Organic agriculture and food security in Africa\" JL (United Nations Environment Programme and United Nations Conference on Trade and Development, New York and Geneva, 2008). Reference Source\n\nDe Schutter O: \"Agroecology and the right to food: Report submitted by the Special Rapporteur on the right to food\" Report. (United Nations Human Rights Council, New York, NY 2011).\n\nSnapp SS, Blackie MJ, Gilbert RA, et al.: Biodiversity can support a greener revolution in Africa. Proc Natl Acad Sci U S A. 2010; 107(48): 20840–20845. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBadgley C, Moghtader J, Quintero E, et al.: Organic agriculture and the global food supply. Renew Agr Food Syst. 2007; 22(2): 86–108. Publisher Full Text\n\nPretty JN, Toulmin C, Williams S: Sustainable intensification in African agriculture. Int J Agr Sustain. 2011; 9(1): 5–24. Publisher Full Text\n\nSeufert V, Ramankutty N, Foley JA: Comparing the yields of organic and conventional agriculture. Nature. 2012; 485(7397): 229–232. PubMed Abstract | Publisher Full Text\n\nRerkasem K, Yimyam N, Korsamphan C, et al.: Agrodiversity Lessons in Mountain Land Management. Mt Res Dev. 2002; 22(1): 4–9. Publisher Full Text\n\nKassam KAS: Viewing Change Through the Prism of Indigenous Human Ecology: Findings from the Afghan and Tajik Pamirs. Hum Ecol. 2009; 37(6): 677–690. Publisher Full Text\n\nWittman HK: Food Sovereignty: A new rights framework for food and nature? Env Soc: Adv Res. 2011; 2(1): 87–105. Publisher Full Text\n\nLa Vía Campesina, in II International Conference Of The Via Campesina. (Tlaxcala, Mexico, 1996).\n\nDesmarais AA: La Vía Campesina: Globalization and the power of peasants. (Pluto Press, London, UK; Ann Arbor, MI USA, 2007). Reference Source\n\nLa Via Campesina, Farmers and Social Movements call for a fundamental restructuring of the global food system. Press Release. 2009. Reference Source\n\nAltieri MA: Linking ecologists and traditional farmers in the search for sustainable agriculture. Front Ecol Environ. 2004; 2(1): 35–42. Publisher Full Text\n\nBerkes F, Colding J, Folke C: Navigating social-ecological systems: Building resilience for complexity and change. (Cambridge University Press, Cambridge, 2003). Reference Source\n\nAltieri MA, Nicholls CI: Scaling up Agroecological Approaches for Food Sovereignty in Latin America. Development. 2008; 51: 472–480. Publisher Full Text\n\nWittman HK: Reworking the metabolic rift: La Vía Campesina, agrarian citizenship, and food sovereignty. J Peasant Stud. 2009; 36(4): 805–826. Publisher Full Text\n\nWittman HK, Desmarais AA, Weibe N: Food Sovereignty: Reconnecting Food, Nature and Community. (Fernwood Publishing, Halifex, 2010). Reference Source\n\nCullen L, Alger K, Rambaldi DM: Land Reform and Biodiversity Conservation in Brazil in the 1990s: Conflict and the Articulation of Mutual Interests. Conserv Biol. 2005; 19(3): 747–755.Publisher Full Text\n\nFearnside PM: Deforestation in Brazilian Amazonia: History, rates, and consequences. Conserv Biol. 2005; 19(3): 680–688.Publisher Full Text\n\nMartinelli LA, Naylor R, Vitousek PM, et al.: Agriculture in Brazil: impacts, costs, and opportunities for a sustainable future. Curr Opin Env Sustain. 2010; 2(5–6): 431–438.Publisher Full Text\n\nMartinelli LA, Filoso S: Expansion of Sugarcane Ethanol Production in Brazil: Environmental and Social Challenges. Ecol Appl. 2008; 18(4): 885–898. PubMed Abstract\n\nBrannstrom C, Jepson W, Filippi AM, et al.: Land change in the Brazilian savanna (Cerrado), 1986–2002: Comparative analysis and implications for land-use policy. Land Use Policy. 2008; 25(4): 579–595. Publisher Full Text\n\nBrown JC, Jepson W, Price KP: Expansion of mechanized agriculture and land-cover change in southern Rondônia, Brazil. J Lat Am Geogr. 2004; 3(1): 96–102. Reference Source\n\nWittman HK: Agrarian Reform and the Environment: Fostering Ecological Citizenship in Mato Grosso, Brazil. Can J Dev Stud. 2010; 29(3–4): 281–298. Publisher Full Text\n\nPacheco P: Agrarian reform in the Brazilian Amazon: Its implications for land distribution and deforestation. World Dev. 2009; 37(8): 1337–1347. Publisher Full Text\n\nWittman HK: Reframing agrarian citizenship: Land, life and power in Brazil. J Rural Stud. 2009; 25(1): 120–130. Publisher Full Text\n\nBrowder JO, Pedlowski MA, Summers PM: Land use patterns in the Brazilian Amazon: Comparative farm-level evidence from Rondonia. Hum Ecol. 2004; 32(2): 197–224. Publisher Full Text\n\nPichón F: Colonist Land-Allocation Decisions, Land Use and Deforestation in the Ecuadorian Amazon Frontier. Econ Dev Cultural Change. 1997; 45(4): 707–744. Publisher Full Text\n\nWalker R, Perz S, Caldas M, et al.: Land use and land cover change in forest frontiers: The role of household life cycles. Int Regional Sci Rev. 2002; 25(2): 169–199. Publisher Full Text\n\nInstituto Brasileiro de Geografía e Estatísticas (IBGE). (Brasilia, 2009), vol. 2010.\n\nvan de Steeg JA, Sparovek G, Lima Ranieri SB, et al.: Environmental Impact of the Brazilian Agrarian Reform Process from 1985 to 2001. Sci Agr. 2006; 63(2): 176. Publisher Full Text\n\nCullen L, Schmink M, Padua CV, et al.: Agroforestry benefit zones: A tool for the conservation and management of Atlantic forest fragments, Sao Paulo, Brazil. Nat Area J. 2001; 21(4): 346–356. Reference Source\n\nValladares-Padua C, Padua SM, Cullen L: Within and surrounding the Morro do Diabo State Park: biological value, conflicts, mitigation and sustainable development alternatives. Environ Sci Policy. 2002; 5(1): 69–78. Publisher Full Text\n\nSparovek G: A Qualidade dos Assentamentos da Reforma Agrária Brasileira. (Páginas e Letras Editora, São Paulo, 2003). Reference Source\n\nSparovek G, Barretto AGOP, Maule RF, et al.: Análise Territorial da Produção nos Assentamentos. (Ministerio de Desenvolvimento Agrícola (MDA), Brasília, 2005). Reference Source\n\nSimmons C, Perz S, Aldrich S, et al.: Doing it for Themselves: Direct Action Land Reform in the Brazilian Amazon. World Dev. 2010; 38(3): 429–444. Publisher Full Text\n\nSparovek G, Pereira Barretto AGO, Maule RF, et al.: \"Análise Territorial da Produção nos Assentamentos\" (Ministerio de Desenvolvimento Agrícola (MDA), Brasília, 2005). Reference Source\n\nPerfecto I, Rice RA, Greenberg R, et al.: Shade coffee: A disappearing refuge for biodiversity. BioScience. 1996; 46(8): 598–608. Publisher Full Text\n\nBacon C: Confronting the coffee crisis: can fair trade, organic and specialty coffees reduce small-scale farmer vulnerability in northern Nicaragua. World Dev. 2005; 33(3): 497–511. Publisher Full Text\n\nMéndez VE, Gliessman SR, Gilbert GS: Tree biodiversity in farmer cooperatives of a shade coffee landscape in western El Salvador. Agr Ecosyst Environ. 2007; 119(1–2): 145–159. Publisher Full Text\n\nMéndez VE, Bacon CM, Olson M, et al.: Agrobiodiversity and shade coffee smallholder livelihoods: A review and synthesis of ten years of research in Central America. Professional Geographer. 2010; 62(3): 357–376. Publisher Full Text\n\nScoones I: Livelihoods perspectives and rural development. J Peasant Stud. 2009; 36(1): 171–196. Publisher Full Text\n\nSoto-Pinto L, Perfecto I, Castillo-Hernandez J, et al.: Shade effect on coffee production at the northern Tzeltal zone of the state of Chiapas, Mexico. Agr Ecosyst Environ. 2000; 80(1–2): 61–69. Reference Source\n\nSoto-Pinto L, Villalvazo-López V, Jiménez-Ferrer G, et al.: The role of local knowledge in determining shade composition of multistrata coffee systems in Chiapas, Mexico. Biodivers Conserv. 2007; 16(2): 419–436. Publisher Full Text\n\nSoto-Pinto L, Anzueto M, Mendoza J, et al.: Carbon sequestration through agroforestry in indigenous communities of Chiapas, Mexico. Agroforest Syst. 2010; 78(1): 39–51. Publisher Full Text\n\nEakin H: Institutional change, climate risk, and rural vulnerability: Cases from Central Mexico. World Dev. 2005; 33(11): 1923–1938. Publisher Full Text\n\nPhilpott SM, Lin BB, Jha S, et al.: A multi-scale assessment of hurricane impacts on agricultural landscapes based on land use and topographic features. Agr Ecosyst Environ. 2008; 128(1–2): 12–20. Publisher Full Text\n\nJaffee D: Brewing justice: Fair trade coffee, sustainability, and survival. (University of California Press, Berkeley, CA 2007). Reference Source\n\nHolt-Giménez E: Measuring farmers' agroecological resistance after Hurricane Mitch in Nicaragua: a case study in participatory, sustainable land management impact monitoring. Agr Ecosyst Environ. 2002; 93(1–3): 87–105. Publisher Full Text\n\nIsakson SR: No hay ganancia en la milpa: the agrarian question, food sovereignty, and the on-farm conservation of agrobiodiversity in the Guatemalan highlands. J Peasant Stud. 2009; 36(4): 725–759. Publisher Full Text\n\nde Janvry A, Sadoulet E: The Global Food Crisis and Guatemala: What Crisis and for Whom? World Dev. 2010; 38(9): 1328–1339. Publisher Full Text\n\nLira R, Casas A, Rosas-López R, et al.: Traditional Knowledge and Useful Plant Richness in the Tehuacan-Cuicatlan Valley, Mexico. Econ Bot. 2009; 63(3): 271–287. Publisher Full Text\n\nBlanckaert I, Vancraeynest K, Swennen RL, et al.: Non-crop resources and the role of indigenous knowledge in semi-arid production of Mexico. Agr Ecosyst Environ. 2007; 119(1–2): 39–48. Publisher Full Text\n\nVieyra-Odilon L, Vibrans H: Weeds as crops: the value of maize field weeds in the Valley of Toluca, Mexico. Econ Bot. 2001; 55(3): 426–443. Publisher Full Text\n\nAltieri MA: Biodiversity of Microorganisms and Invertebrates: Its role in Sustainable Agriculture. D. L. Hawksworth, Ed. (CAB International, Wallingford, UK 1991), 165.\n\nAppendini K, García Barrios R, de la Tejera B: Seguridad alimentaria y \"Calidad\" de los alimentos: ¿Una estrategia campesina? Eur Rev La Am Caribbean Stud. 2003; 75: 65–65. Reference Source\n\nPimentel D, Hepperly P, Hanson J, et al.: Environmental, energetic, and economic comparisons of organic and conventional farming systems. BioScience. 2005; 55(7): 573–582. Publisher Full Text\n\nSecretariat of the Convention on Biological Diversity (SCBD), Linking biodiversity conservation and poverty alleviation: A state of knowledge review. CBD Technical Series 55 (Secretariat of the Convention on Biological Diversity (SCBD), Montreal, Canada, 2010). Reference Source\n\nJohns T, Eyzaguirre P: Biofortification, biodiversity and diet: A search for complementary applications against poverty and malnutrition. Food Policy. 2007; 32(1): 1–24. Publisher Full Text\n\nBurlingame B, Dernini S: Sustainable diets and biodiversity. Directions and solutions for policy, research and action. (Food and Agriculture Organization of the United Nations, Rome, Italy, 2010). Reference Source\n\nBèlanger J, Johns T: Biological diversity, dietary diversity, and eye health in developing country populations: establishing the evidence-base. EcoHealth. 2008; 5(3): 244–56. PubMed Abstract | Publisher Full Text\n\nHaughton AJ, Bond AJ, Lovett AA, et al.: A Novel, Integrated Approach to Assessing Social, Economic and Environmental Implications of Changing Rural Land-Use: A Case Study of Perennial Biomass Crops. J Appl Ecol. 2009; 46(2): 315–322. Publisher Full Text\n\nOrmerod SJ, Marshall EJP, Kerby G, et al.: Meeting the Ecological Challenges of Agricultural Change: Editors' Introduction. J Appl Ecol. 2003; 40(6): 939–946. Publisher Full Text\n\nPatel RC: What does food sovereignty look like? J Peasant Stud. 2009; 36(3): 663–706. Publisher Full Text\n\nOstrom E: A diagnostic approach for going beyond panaceas. Proc Natl Acad Sci U S A. 2007; 104(39): 15181–15187. Publisher Full Text\n\nReardon Simón, Pérez RA: Agroecology and the Development of Indicators of Food Sovereignty in Cuban Food Systems. J Sust Agric. 2010; 34(8): 907–922. Publisher Full Text\n\nRosset PM: Food sovereignty: Global rallying cry of farmer movements. Food First Backgrounder. 2003; 9(4): 1. Reference Source"
}
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[
{
"id": "2371",
"date": "08 Nov 2013",
"name": "Frederick Kirschenmann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 one of the most succinct, yet comprehensive analysis of the complex issues surrounding the development of a resilient, socially viable food system for our future I have ever come across. Consistent with the observations made in recent UN studies, “Agriculture at a Crossroads,” “Agroecology and the Right to Food,” “Save and Grow” and “The Future We Want” this brief paper outlines the key issues that must be incorporated into designs for a viable food system for the future. The tile and abstract accurately reflect the content of the paper and its core position. The paper clearly points out that people in their own communities, and especially small-holder farmers, need to have access to fundamental resources to have the right to food and the necessary information and natural and social capital to achieve food sovereignty, escape the poverty traps that capture so many of the very people who can provide secure food systems and restore and maintain the biodiversity necessary for a resilient food system for future generations. This paper presents a brilliant, science-based alternative paradigm to the neo-liberal, global-export-oriented model which is often presented in our current culture as “the only way to feed 9 billion people” when in point of fact, it fails to address some of the key problems of that system: entrenching the poor in poverty traps, eroding the ecological capital of the very communities it purports to feed, and perpetuating many of the social dysfunctions that prevent large populations in poor rural communities from achieving the right to food. This paper and many of those it cites need to be published widely and called to the attention of the public press so that the general public can become more aware of the issues we all need to address. This paper conforms to our highest scientific standards and makes its case persuasively and competently. I highly recommend it for indexing as presented.",
"responses": []
},
{
"id": "2354",
"date": "10 Dec 2013",
"name": "Ryan Isakson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDrawing upon a systematic review of existing literature, this article evaluates the popular claim that the food sovereignty agenda will not only democratize food provisioning, but also alleviate the poverty of food producers and encourage the conservation of biodiversity in agrarian landscapes. In so doing, it represents a valuable contribution to the academic debate and carries important ramifications for envisioning and implementing future agricultures. The authors effectively deploy the concept of poverty traps to challenge the common claim that environmental degradation, including biodiversity loss, is an inevitable consequence of rural poverty alleviation, thereby contributing to a growing literature demonstrating that economic and political democracy can, in fact, promote sustainable agricultural practices (see, for example James Boyce’s work on natural assets). Their survey of the relevant literature also demonstrates how two tenants of the food sovereignty framework, small-scale peasant-based agriculture and agro-ecological practices, are associated with higher levels of biodiversity and more resilient food systems. Overall, it’s an insightful article that effectively and concisely analyzes the relevant literatures in the social and natural sciences. The title and abstract of the article are appropriate and accurately reflect the content of the paper. The article is logically structured and well-written. The analysis is impressive and draws upon a comprehensive review of the relevant literatures. It’s an important contribution and I highly recommend its indexing.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-235
|
https://f1000research.com/articles/2-234/v1
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06 Nov 13
|
{
"type": "Research Article",
"title": "Integrating small population effects on both detection and persistence probabilities in the design and interpretation of presence/absence surveys.",
"authors": [
"Brian R. Hudgens",
"David K. Garcelon",
"David K. Garcelon"
],
"abstract": "Determining whether or not a species has been extirpated from part of its range is one of the most important yet difficult challenges facing ecologists. Presence/absence surveys are a primary method to determine local extinction, but negative results are difficult to interpret, particularly when informal sightings appear to support local persistence of elusive species. Such conflicts are particularly likely for rare species, which are the most difficult to detect during a presence/absence survey due to small population size. However, integrating small population effects on both detection and extinction probabilities resolves such conflicts by reframing occupancy questions from present to near future terms. We developed methods for integrating the simultaneous effects of survey effort and population size on detection and extinction probabilities in the design and interpretation of presence/absence surveys. We applied these methods to the design and interpretation of a wolverine survey in Sequoia-Kings Canyon National Parks, California, USA, to address questions about the appropriateness of translocations from other populations. Ecological applications that can benefit from this type of analysis, simultaneously incorporating small population effects on both detection and population processes, include management of charismatic, yet elusive species, management of disease risk in translocations, and management of nonnative pests.",
"keywords": [
"occupancy",
"population viability analysis",
"extinction",
"range loss",
"presence/absence survey",
"detection probability",
"Gulo gulo",
"Sierra Nevada"
],
"content": "Introduction\n\nDetection of rare species is one of the fundamental problems in conservation biology. A species cannot be managed for unless its presence is known. Consequently, presence-absence surveys are common first steps in guiding management policies. But, while formal surveys are very good at confirming a species presence, determining if a species is absent from a management unit is more problematic. The crux of the problem lies in the omnipresent possibility that nondetection does not indicate a species absence. Clearly, incorporating nondetection probability, through its inverse, detection probability, is a critical component to both the design and interpretation of presence-absence surveys, and there are well-developed methods for doing so [e.g.,1,2].\n\nThe simplest of these methods assume that detection probabilities depend only on survey effort1. Consequently, designing a survey with a small chance of failing to detect a species present in a management unit depends only on knowing detection probabilities, which can be estimated from surveys conducted in areas of known occupancy [e.g.,1,3]. In the absence of reliable information from surveys in known occupied areas, detection probability is often estimated from similar species4 or simulation5,6.\n\nOf course, detection probabilities do not depend only on survey effort. At the very least, the size of the population within the survey area influences detection probabilities; smaller populations are more difficult to detect than larger ones. Moreover, the influence of population size on detection probability is nonlinear such that the loss of an individual has a larger impact on the detection probability of a small population than of a large one7. This relationship has two underappreciated consequences for designing and interpreting presence-absence surveys. First, the effort required to detect a small population increases at an accelerating pace as population size dwindles. Second, determining the survey effort required to have an acceptably small nondetection probability requires specifying the size of the population being looked for (hereafter, target population size).\n\nGiven limited resources, it generally will not make sense to dedicate a lot of resources to ensure a high probability of detecting the last individual of a species within a management unit. In other words, it is inefficient to spend limited resources to detect a species which may technically be present within a management unit but is effectively locally extinct. It would be more efficient to design a survey to detect a population of two individuals, but such a population would still likely require a large effort to detect and would still face a high risk of local extinction in the near term. It would generally be even more efficient to design a survey based on a target population of three individuals, but because increasing population size generally has a diminishing effect on both detection probability and extinction risk (assuming no Allee effect8), the gain in efficiency would not be as large as the gain in efficiency realized from designing for a target population of two vs. one.\n\nIdeally, a survey design avoids two sources of inefficiency: failing to detect a population that persists into the near future and detecting a population that fails to persist into the near future. As such, the target population size should account for the effects of population size on both detection probability and extinction risk (Figure 1). This can be done thinking of survey power not in terms of the probability of failing to detect a population, but in terms of the probability of failing to detect a viable population. The latter is the equivalent of the product of both the probability of failing to detect at least one individual, which is an increasing function of population size, and the probability of a population persisting into the near future, which is a decreasing function of population size. Framing a presence-absence survey in terms of detecting a viable population also changes the interpretation of null survey results. Rather than a null result implying a species absence, a null result implies that if the species is present in a management unit, its population is so small that it is unlikely to persist into the near future.\n\nA) Lines to the right of the Y-axis depict how trap efficiency increases with population size calculated using the methods of Royle and Nichols7 (dashed line) and the approximation presented in this paper (solid line). The line to the left of the Y-axis shows how trap efficiency increases with increasing density. Density values correspond to the population sizes used in the right-hand side of the graph interacting in an 8171 km2 area. Trap efficiencies were calculated assuming an individual capture probability of 0.003 or reference trap efficiency of 0.024 for a reference population size of 8 individuals at a corresponding reference density of 0.0024 animals per square km. The X-axis is plotted on a log10 scale to emphasize small population numbers. For raw data, please see Dataset 1. B) Points indicate the probability that a population starting at the population size on the X-axis persists for 25 years predicted by PVA (see text for details). For raw data, please see Dataset 2.\n\nIn this paper, we develop a framework for survey design which incorporates small population effects on both detection probability and short-term persistence. We then demonstrate the application of this framework to the design and interpretation of a presence-absence survey for wolverines in Sequoia-Kings Canyon National Parks, California, USA. Finally, we discuss the implications of the wolverine survey for wolverine management in California and the circumstances under which the framework developed here is, and is not, appropriate for designing and interpreting presence-absence surveys.\n\n\nMethods\n\nWe developed methods for optimizing survey effort to detect a viable population given that one persists in the survey region, and for a post-hoc analysis of survey power which serves as the basis for evaluating negative survey results in terms of the probability of overlooking a population that will persist into the near future. A good survey has two critical components: 1) an appropriate method to detect the species of interest, and 2) adequate sampling effort to have a low probability of overlooking the species if it is present. Here we focus on how to determine the amount of survey effort required once appropriate methods have been chosen. Although we envision a survey based on baited camera traps, with detections based on photographing an animal visiting a bait station, the methods described work with any survey protocol for which effort can be quantified in discrete units (e.g., trap-nights, kilometers traversed, etc.) and detection probability can be expressed per unit of effort.\n\nAssuming that additional traps do not interfere with one another, the probability of detecting a species (D) for a given amount of effort is:\n\nD=1-(1-E)T (equation 1)\n\nwhere E is the trap efficiency (i.e., the probability of a detection during a single unit of survey effort) and T is the number of trap-days (i.e., total survey effort) in the study. If all individuals within the surveyed population have the same chance of being detected, trap efficiency will be related to population size as E=1-(1-e)N7, where e is the probability that a particular individual is detected and N is the number of individuals in the survey area. After substitution and simplification, Equation 1 can then be rewritten to account for population size as:\n\nD(N)=1-((1-e)N)T (equation 2)\n\nTo calculate the survey effort required assuming a population size N, to achieve the desired detection probability (D(N)), we solve for T(N):\n\nT(N)=ln(1-D(N))/ln((1-e)N) (equation 3)\n\nNote that as the population size of the target species in the survey region approaches zero, the required effort approaches infinity. The nonlinear relationship between T and N as N approaches zero means that surveys designed to detect the lowest possible size (i.e., a single individual within the survey area) are likely to be prohibitively large.\n\nThe next step to optimize survey effort is to consider the effects of population size not only on detection probability, but also on near-term population viability (i.e., the probability that a population persists beyond some time, t, in the near future). There are many ways to determine the relationship between density and population viability using population viability analysis (PVA). Choosing the most appropriate type of PVA depends on what data are available for the species9. Commonly used count-based PVA, which treats changes in the (log) population size as a diffusion process, explicitly assumes a relationship between population size and extinction risk. For more complex population dynamics (e.g., density dependent or age structured populations), the relationship can be determined by simulation9. Here, we assume that there is some relationship between near-term population viability and the current population size (Z(N)) and that this relationship has been determined using PVA (e.g., Figure 1B).\n\nWhen taking population viability into account, the relevant statistic is probability of failing to detect a viable population given that the species inhabits the survey area at a given population size:\n\nP(N)=(1-Z(N))×(1-D(N))\n\nSubstituting for D(N) and simplification yields\n\nP(N)=(1-Z(N))×(((1-e)N)T) (equation 4)\n\nThe maximum value of P(N), as N varies in equation 4, approximates the probability that a survey failed to detect a population that will persist into the near future. The exact probability is equal to the integral of the product of P(N) and the probability that the population being surveyed has N individuals over all possible values of N. However, the latter probability distribution is extremely unlikely to be known or even estimable for populations which are the focus of presence/absence surveys. Approximating this interval at the maximum value of P(N) results in an estimate of P(N) that is biased high, which for most applications of this analysis will be a conservative bias.\n\nBecause most power analyses calculate the probability of success (e.g., that a study will show a significant difference given that observed groups do differ), it is tempting to estimate survey power as 1-P(N). We caution against doing so because this quantity is difficult to interpret; it is the union of the probabilities that a population is detected or goes extinct in the near future, not the probability that a population is detected given that a viable population persists in the survey area, nor the probability that an undetected population goes extinct.\n\nTo determine the optimal trap effort, one must first determine the desired level of risk for overlooking a viable population independent of population size, P(•). Choosing a critical value for P(•)=(Pcrit) and rearranging equation 4 to solve for T yields\n\nT=(ln(Pcrit)-ln(1-Z(N)))/ln((1-e)N) (equation 5)\n\nAt N=0, equation 5 evaluates to infinity, reflecting that no amount of effort can detect an extinct viable population (itself a logical impossibility). As N increases, equation 5 has a maximum value which indicates the optimal survey effort. If Z(N) is a differentiable function, Tmax can be calculated analytically. If Z(N) was determined by simulation then Tmax must be determined by iterating equation 5, but since extinction risk generally decreases sharply with population size, iterations will typically only have to span a small range of N. Note that if population viability and reference detection probabilities are provided in terms of population density rather than population numbers, equation 5 may still be used by defining N as population density.\n\nPre-survey power analysis. We applied the techniques outlined above to design and interpret a presence-absence survey for wolverines in Sequoia-Kings Canyon National Parks (SKCNP) to address concerns related to the impact that a proposed translocation of wolverines into the southern Sierra Nevada would have on a possible remnant population. The concerns arise from a contradiction between management agencies’ — in particular, California Department of Fish and Wildlife and SKCNP — stance that wolverines persist and should be managed for in the Sierra Nevada10,11 and published conclusions that the species has been extirpated from the state12,13. The conflicting stances largely stem from differences in opinion about how to treat unverified reports by citizen observers and a failure of formal surveys to address survey power. Because most carnivore surveys in the Sierra Nevada take place in the summer2,14, when wolverines are not likely to visit bait stations (there are no published records of summertime efforts which have trapped wolverines), it is not unreasonable to suppose that formal surveys may have overlooked a small remnant population in the Sierra Nevada given numerous reported sightings of the species. The recent discovery of an immigrant male wolverine more than 250 km north of SKCNP15 has done little to quell the debate.\n\nIn order to resolve this conflict, we carried out a winter survey for wolverines using baited camera stations in SKCNP designed to have a high power to detect a wolverine population large enough to have a reasonable probability of persisting at least 25 years. Given a 75-year absence of physical evidence of wolverine presence in the area13, we assumed that if wolverines did remain in the parks, they persisted in low densities compared to areas where detection probabilities were available from previous winter surveys. Consequently, it was clear that the effect of population size on detection probability needed to be accounted for.\n\nOnly two published winter carnivore surveys provide estimates of both detection rates from baited camera stations and wolverine density16,17, and none report both the trap efficiency and population estimate within the effective trap area. Consequently, it is not possible to estimate the per capita detection probability (e) of a wolverine. However, when N and e are small, E is closely approximated by (N/N*)E* where E* is the trap efficiency at a reference site with population size N* (Figure 1A). Application of this approximation for E into equation 5 yields:\n\nT=(ln(Pcrit)-ln(1-Z(N)))/ln(1-(N/N*)E*) (equation 6)\n\nAssuming a constant area, changes in density (d) are equivalent to changes in population size, such that N/N* may be replaced by d/d*. The average trap efficiency and density estimates from these studies were E*=0.024 detections per trap-night and d*=0.0098 wolverines per km2. Corresponding trap efficiencies over a range of wolverine densities are shown in Figure 1A.\n\nWe determined how wolverine population size affects extinction risk using the PVA modeling program VORTEX (v.9.7.2, Chicago Zoological Society 2007) parameterized with demographic rates taken from the published literature (see Appendix A). VORTEX is a widely used, flexible, individual based simulation18 that has been found to perform relatively well compared to other PVA packages19. We set initial population size between 2 and 25 animals and simulated population dynamics for 25 years, corresponding to 5 to 8 wolverine generations. We estimated short-term extinction risk at each initial population size as the fraction of 10,000 replicate simulated populations that went extinct before the end of the simulation (Figure 1B).\n\nBecause published detection probabilities are tied to population densities and extinction risk is tied to population size, we had to translate population size to density in the survey area. Our PVA assumed a well-mixed population without spatial structuring. We therefore assumed that the corresponding area allowed a juvenile at the center of the population to interact with all other animals in the population if it covered the average dispersal distance. The average distance covered by a dispersing wolverine is 51 km20, so we divided population size by 8171 km2 to calculate the corresponding density.\n\nMaximizing equation 5 using the density dependent detection and extinction risks shown in Figure 1 and Figure 2, we calculated that 1160 trap-nights would be sufficient to have a less than 5% probability of failing to detect a viable population of wolverines inhabiting SKCNP (Figure 2A). Assuming that baited camera stations would be available for 2.5 months (75 days), this effort would require a minimum of 16 stations.\n\nTop panel A) shows the trap effort required to have 95% probability of detecting a viable wolverine population given that wolverines inhabit the survey area at the density indicated on the X-axis. The apparent dip of the second point from the left reflects a dramatic decrease in population viability but not detection probability for a population of three compared to one of four individuals. The bottom panel (B) shows the realized power of the survey. The grey lines indicate the probability of failing to detect at least one wolverine and the dark lines indicate the probability of failing to detect a member of a viable wolverine population given that wolverines are present in the study region at the density indicated by the X-axis. Probabilities were calculated from equation 2 and equation 4, respectively, assuming a trap effort of 1418 (solid line) or 982 (dashed line) trap-nights, and reference trap efficiency of 0.024 captures per trap-night at a reference density of 0.0098 wolverines per square km. Black arrows indicate maximum values reported in text. For raw data, please see Dataset 3.\n\nSurvey methods. We placed 18 baited camera stations throughout SKCNP between January 26 and 28, 2006. Baited camera stations have been successfully used to detect wolverines outside our survey region15–17,21, and to detect other mesocarnivores in the southern Sierra Nevada12,14. We exceeded the recommended 16 stations so that camera malfunctions or other unforeseen circumstances affecting up to two stations would not reduce our survey power below 95%.\n\nEach station consisted of a 1.2 m × 10 cm × 10 cm wooden post wrapped in barbed wire held 0.46 m from a tree by an aluminum frame. On top of each post was a ~0.5 m × 0.5 cm steel pole holding 1 to 5 kg of meat, bone, and hair cut from a pig carcass and wrapped in hardware cloth. We also attached a perforated can of wet cat food injected with Gusto (a combination of skunk glands and beaver castor oil; Caven’s Quality Animal Lures) to each post to provide a scent lure. The bottom of each station was placed approximately 1 m above snow level so that the bait was approximately 3 m above snow level (approximately 5–6 m above ground). We mounted a heat/motion triggered camera to a second tree 1.5–5 m away from and 0.5–1 m above the bait station. We constructed camera units using PixControl™ control boards equipped with passive infrared (PIR) sensors and Sony P32 digital cameras with 256 MB memory cards. All components were mounted in waterproof plastic boxes.\n\nWe established survey stations by helicopter on 26–28 January 2006 (Table 1). To ensure adequate coverage across the parks, a 20 km × 20 km grid was overlaid on the park and at least one station was placed in suitable wolverine habitat in each cell (Figure 3). This represents one station per average female home range area (400 km2) or two stations per male home range area (800 km2) at low wolverine density. We placed additional stations in cells that contained a high number of alleged wolverine sightings reported in the 25 years prior to the survey.\n\nBlack dots indicate locations of baited survey stations; stars indicate locations of informal wolverine sightings reported to Sequoia-Kings Canyon National Parks (courtesy NPS) since 1980. Map generated by IWS staff using ArcGIS and World Shaded Relief (ESRI 2009).\n\nThe primary determinants of station locations within each grid were elevations within approximately 200 m elevation of treeline (2744–3506 m), the availability of trees for anchoring bait stations and cameras, and proximity to safe helicopter landing sites. We also considered the accessibility of locations to monitoring teams that were to check the stations and the location of reported wolverine sightings within the past 25 years. The two sites located below 2500 m were placed within 1 km of where purported wolverine tracks were photographed during snow surveys in 1979 and 1980 (T. Andrews, unpublished data) and in the Mineral King area where many unsubstantiated sightings have been reported since 1980.\n\nFourteen of the camera stations were checked at least once between 18 February and 1 May 2006 by Institute for Wildlife Studies personnel, California snow survey teams, or park staff. The person checking each station replaced the camera batteries and memory cards. Frequent snow storms and high avalanche risk prevented surveyors from checking four of the most remote stations until they were taken down by helicopter in early May. Stations remained active for up to 105 days. We reviewed all pictures taken from each camera after memory cards were removed from the cameras during mid-survey checks, and at the end of the survey. We recorded all animals photographed by each camera, days during which photos indicated a camera was obscured by snowfall, how many days the station was available (see results), and the date the last photo was taken.\n\n\nResults\n\nCamera performance. Overall, the PIR-triggered cameras worked very well under a wide range of conditions. We collected 2939 pictures during our survey excluding pictures taken while surveyors set up, maintained, and took down the stations. Pictures were taken during all hours of the day and night throughout the entire survey period. Animals were visible in 602 pictures (Figure 4); most remaining photographs were probably triggered by rapid changes in ambient temperature or by wind-blown branches. An additional 400 pictures were used to identify periods when cameras were operable. These included pictures taken during and immediately following snow events which obscured the camera lens, subsequent pictures indicating that the lens had been cleared, and pictures of surveyors approaching the camera before switching out memory cards or taking down survey stations.\n\nPanels show four carnivores identified at four stations: a) black bear (Ursus americanus), b) coyote (Canis latrans), c) fisher (Martes pennanti), and d) marten (Martes americana).\n\nIn total, cameras were considered operable for 1418 survey-days from 27 January through 11 May. Individual stations were available for between 20 and 105 days (Table 1). A station was judged to be operable from the day it was set up to the day the last photograph of the bait station was taken, excluding days when snow accumulation either obscured the camera lens or buried the bait station. The camera lenses at five stations were temporarily obscured by snowfall accumulating on the camera case and were judged unavailable until the time of the next unobscured photo. This period ranged from 10 minutes to four days. There were no photos taken more than six hours after the first obscured photo that remained obstructed by snow accumulation. Any station known to have been obscured for more than one hour was deemed unavailable for at least one day. Stations were also judged to be unavailable when the bait was buried by snow or was missing.\n\na Date last picture was taken.\n\nb Number of days between 27 January and date of last picture minus the number of days closed.\n\nc Date first picture of any mesocarnivore taken at the station.\n\nWe detected no wolverines. Species detected included nine mammals and four birds. Martens (Martes americana Turton) were recorded in 419 pictures from 14 stations (Dataset 4). Other mesocarnivore species detected include coyote (Canis latran, Say), fisher (Martes pennanti Erxleben), and black bear (Ursus americanus Pallas). We also recorded northern flying squirrels (Glaucomys sabrinus Shaw), Douglas’ squirrels (Tamiasciurus douglasi Bachman), golden-mantle ground squirrel (Spermophilus lateralis Say), deer mouse (Peromyscus maniculatus Wagner), white-tailed jackrabbit (Lepus townsendii Bachman), Clark’s nutcrackers (Nucifraga columbiana Wilson), dark-eyed juncos (Junco hyemalis Linnaeus), green-tailed towhee (Pipilo chlorurus Audubon), and white-crowned sparrow (Zonotrichia leucophrys Forster).\n\nPost-hoc power analysis. Assuming 1418 trap-nights and the density dependent detection and persistence probabilities from Figure 1 and Figure 2, maximizing equation 4 yields an estimated probability for failing to detect a viable wolverine population of P=0.039 (Figure 2B). Because most wolverine surveys are conducted following a pre-bait period, we also calculated the power of our survey assuming that survey stations were not effective until the average date of first visitation by mesocarnivores to the 17 stations that were visited by at least one mesocarnivore (February 21). This reduced the effective survey time to 982 trap-days, increasing the estimated probability of failing to detect a viable wolverine population to P=0.064 (Figure 2B).\n\n\n\n\nDiscussion\n\nA growing literature is devoted to inferring true occupancy from spatially replicated surveys1, and incorporating density effects on detection rates to infer extinction probabilities from temporally replicated surveys2,7 and population size from detection patterns22. The framework presented here extends the application of these methods to the special but important case where replicated surveys fail to detect a species on any occasion. We accomplish this by extending the analyses to simultaneously consider the effects of population size on detection and population processes (i.e., trap efficiency and persistence probability). This extension makes two important advances to the design and interpretation of presence-absence surveys. First, explicitly acknowledging that extremely small populations are very difficult to detect but unlikely to persist facilitates efficient survey designs with enough power to detect species which need to be managed without wasting effort on effectively extinct populations. Second, the framework presented here shifts the interpretation of a failure to detect a species from the conclusion that zero individuals remain within the survey area to the conclusion that no more than a very small number could be within the survey area.\n\nChoosing a target population density based on the effects of small populations on both detection and persistence probabilities was critical to design of an efficient survey. If only a single wolverine inhabited the park, the survey would require 3730 trap days to have 5% failure probability. In contrast, consideration of small population effects on persistence led to the choice of target density that required less than one-third the effort, and expense, to have the same power to detect a viable population.\n\nA consequence of choosing a target population size greater than one is that while the lack of detections during our survey does provide strong evidence that there is not a viable wolverine population within SKCNP, it does not necessarily mean that recent informal sightings are false (somebody could have seen the last remaining individual). We see this as a useful uncertainty when making management decisions. Evaluating null results in terms of a viable population rather than simple occupancy provides a basis for making management decisions that are scientifically justified regardless of the credibility of informally reported observations. When survey results indicate no viable population is present based on density dependent detection and persistence probabilities, informal sightings do not bear on estimates of future occupancy because 1) the possibility that one or a few individuals inhabit the region, and their potential to contribute to future occupancy, has been accounted for, and 2) it is generally not possible to quantify accurately the effort that could lead to an informal observation. Informal sightings often suffer the additional weakness that the probability of misidentification cannot be quantified.\n\nWolverines are but one of many charismatic, yet elusive species, for which informal sightings contradict current published range limits distributions inferred from formal surveys. Other notable examples include several heavily managed species such as pumas (Puma concolor)23, fisher (Martes pennant)24, Iberian lynx (Lynx pardinus)25, or koalas (Phascolarctos cinereus)26. At the core of this conflict are the different types of errors possibly committed when interpreting informal sightings and unsuccessful formal surveys. The interpretation of a lack of verifiable evidence from formal surveys as evidence for extinction may lead to the error of accepting a false negative (i.e., the species is present but undetected). Alternatively, interpretation of informal sightings, particularly sightings unaccompanied by verifiable physical evidence, as confirmation of an extant population despite negative results from formal surveys may lead to the error of rejecting a true negative (i.e., the species was undetected by surveys because it is truly absent). The variety of classification schemes and the evidentiary value of different types of observations in the published literature highlight the range of willingness among biologists to accept one type of error over the other13,23,24. Differences in opinion about which of these errors is more egregious are not merely academic, as the allocation of limited resources often depends on the presence or absence of charismatic species24.\n\nAppropriate application of the methods presented in this paper depends on choosing the proper time horizon to evaluate population viability. Density dependent persistence for time horizons near or below the species’ generation time or for thousands of generations will rarely vary (from near 100% and 0%, respectively) and therefore contribute little to enhancing survey design or interpreting negative results. Worse, using persistence probabilities estimated for time periods beyond the management horizon may yield misleading results. Incorporating small population effects on both detection and population processes to shift analyses from present to future occupancy should only be done if the latter is pertinent to management goals. For example, when the population in question comprises the entirety of a species, even the last pair has high conservation value.\n\nA management challenge particularly suited to the methods developed in this paper is evaluating the appropriateness of translocations as a management strategy. Translocations are powerful tools to reintroduce extirpated species or to prevent local extinction when populations decline to levels highly susceptible to demographic, environmental, and genetic stochastic events or suffer from Allee effects. However, translocations may have the unintended effects of eroding genetic diversity when relatively large numbers are introduced into small but viable populations. For example, the historic Sierra Nevada wolverine population was highly differentiated from other North American populations evidenced by alleles not known from any other population27. Uncertainty about the status of wolverines in the Sierra Nevada caused by numerous informal sightings in SKCNP weighs against translocating animals into the region as a strategy to restore healthy populations to the area, while translocations would be the only recourse if the species truly is extirpated from the region. By evaluating formal survey efforts in terms of near future occupancy, we can conclude from our negative result that even if a small number of wolverines inhabit the parks, their persistence is so tenuous that any contribution they may make to species-wide genetic diversity will most likely be lost to extinction in the absence of translocations. Consequently, translocations may be viewed as the most appropriate strategy to restore a healthy wolverine population to the southern Sierra Nevada regardless of the validity of informal sightings.\n\nIncorporating the concurrent effects of small populations on multiple processes, such as detection and persistence probabilities, has utility for many ecological applications. For example, fish stocking programs face the problem of preventing disease transfer to stocked populations. Disease monitoring of source populations must balance concerns about overlooking a pathogen against logistical constraints, especially because destructive sampling is often necessary for pathogen detection. The detection effort required to achieve some certainty of pathogen detection depends on pathogen prevalence28, which also determines the risk of disease transfer if pathogens are present but not detected. Disease prevalence is often unknown, and assumed at an arbitrary level29. A better strategy would be to determine sampling effort based on the product of prevalence dependent detection and transfer probabilities. Similar problems are faced in determining the success of nonnative eradication efforts. For example, optimal efforts to remove golden eagles (Aquila chrysaetos) from the California Channel Islands because they threaten endemic fox populations would account for increasing costs to find eagles and reduced impact of eagles on foxes as eagle density on the islands approaches zero30. Perhaps the most important benefit of considering the simultaneous effects of population size on detection and population processes is that doing so frames detection studies and their interpretation in terms directly related to their management application and away from the onerous task of proving something does not exist.",
"appendix": "Author contributions\n\n\n\nBH and DG conceived the study and collected the field data. BH conducted the analysis and prepared the manuscript. Both 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\nAcknowledgments\n\nWe thank Sequoia-Kings Canyon National Parks staff, particularly D. Graber, H. Werner, and R. Mazur. We are grateful to C. Rall, who headed IWS monitoring efforts, with C. Miles and B. Czibesz, J. Nelson, and K. Thompson, and to J. King and the California Snow Surveyors who checked stations for us and D. Johnson, M. Potter, R. Rall, and G. Schmidt. Comments from K. Aubry, R. Powell, N. Haddad, and K. Pollock substantially improved this paper.\n\n\nReferences\n\nMacKenzie DI, Royle JA, Brown JA, et al.: Occupancy estimation and modeling for rare and elusive populations. In: Thompson WL, editor. Sampling rare or elusive species: Concepts, designs, and techniques for estimating population parameters. Washington, D.C.: Island Press. 2004; pp. 149–172. Reference Source\n\nDorazio RM: On the choice of statistical models for estimating occurrence and extinction from animal surveys. Ecology. 2007; 88(11): 2773–2782. PubMed Abstract | Publisher Full Text\n\nPeterson JT, Bayley PB: A Bayesian approach to estimating presence when a species is undetected. In: Thompson WL editor. Sampling rare or elusive species: Concepts, designs, and techniques for estimating population parameters. Washington, D.C.: Island Press. 2004; pp. 173–188. Reference Source\n\nReed MJ: Using statistical probability to increase confidence of inferring species extinction. Conserv Biol. 1996; 10(4): 1283–1285. Publisher Full Text\n\nChoquenot D, Ruscoe WA, Murphy E: Colonisation of new areas by stoats: time to establishment and requirements for detection. N Z J Ecol. 2001; 25: 83–88. Reference Source\n\nConn PB, Bailey LL, Sauer JR: Indexes as surrogates to abundance for low abundance species. In: Thompson WL editor. Sampling rare or elusive species: Concepts, designs, and techniques for estimating population parameters. Washington, D.C.: Island Press. 2004; pp. 59–74. Reference Source\n\nRoyle JA, Nichols JD: Estimating abundance from repeated presence-absence data or point counts. Ecology. 2003; 84: 777–790. Publisher Full Text\n\nAllee WC: Animal aggregations: a study in general sociology. AMS Press. 1978. Reference Source\n\nMorris WF, Doak DF: Quantitative conservation biology: Theory and practice of population viability analysis. Sunderland: Sinauer and Associates. 2002; 480. Reference Source\n\nGraber DM: Status of terrestrial vertebrates. In: Sierra Nevada Ecosystem Project: Finalreport to Congress, vol. II, Assessments and scientific basis for management options. Davis: University of California, Centers for Walter and Wildland Resources. 1996. Reference Source\n\nCalifornia Natural Diversity Database. 2012. Reference Source\n\nZielinski WJ, Truex RL, Schlexer FV, et al.: Historical and contemporary distributions of carnivores in forests of the Sierra Nevada, California, USA. J Biogeogr. 2005; 32(8): 1385–1407. Publisher Full Text\n\nAubry KB, McKelvey KS, Copeland JP: Distribution and broadscale habitat relations of the wolverine in the contiguous United States. J Wildl Manage. 2007; 71(7): 2147–2158. Publisher Full Text\n\nGreen R: Distribution of American marten, fisher, and other carnivores in Sequoia and Kings Canyon National Parks. Master's thesis, Humboldt State University. 2007.\n\nMoriarty KM, Zielinski WJ, Gonzales AG, et al.: Wolverine confirmation in California after nearly a century: Native or long-distance immigrant? Northwest Sci. 2009; 83(2): 154–162. Publisher Full Text\n\nLofroth EC, Krebs J: The abundance and distribution of wolverines in British Columbia, Canada. J Wildl Manage. 2007; 71(7): 2159–2169. Publisher Full Text\n\nMulders R, Boulanger J, Paetkau D: Estimation of population size for wolverines Gulo gulo at Daring Lake, Northwest Territories, using DNA based mark-recapture methods. Wildl Biol. 2007; 13(sp2): 38–51. Publisher Full Text\n\nLacy RC: Structure of the VORTEX simulation model for population viability analysis. Ecol Bull. 2000; 48: 191–203. Reference Source\n\nLindenmayer DB, Possingham HP, Lacy RC, et al.: How accurate are population models? Lessons from landscape-scale tests in a fragmented system. Ecol Lett. 2003; 6(1): 41–47. Publisher Full Text\n\nVangen KM, Persson J, Landa A, et al.: Characteristics of dispersal in wolverines. Can J Zool. 2001; 79(9): 1641–1649. Publisher Full Text\n\nBanci V: Wolverine. In: Ruggiero LF, Aubry KB, Buskirk SW, Lyon LJ, Zielinski WJ, editors. The scientific basis for conserving forest carnivores: American marten, fisher, lynx and wolverine in the western United States. Fort Collins: Rocky Mountain Research Station. 1994; pp. 99–127. Reference Source\n\nZhou S, Griffiths SP: Estimating abundance from detection-nondetection data for randomly distributed or aggregated elusive populations. Ecography. 2007; 30(4): 537–549. Publisher Full Text\n\nClark DW, White SC, Bowers AK, et al.: A survey of recent accounts of the mountain lion (Puma concolor) in Arkansas. Southeast Nat. 2002; 1(3): 269–278. Publisher Full Text\n\nMcKelvey KS, Aubry KB, Schwartz MK: Using anecdotal occurrence data for rare or elusive species: The illusion of reality and a call for evidentiary standards. BioScience. 2008; 58(6): 549–555. Reference Source\n\nSarmento P, Cruz J, Monterroso P, et al.: Status survey of the critically endangered Iberian lynx Lynx pardinus in Portugal. Eur J Wildl Res. 2009; 55(3): 247–253. Publisher Full Text\n\nCurtin A, Lunney D, Matthews A: A survey of a low-density koala population in a major reserve system, near Sydney, New South Wales. Aust Mammal. 2001; 23(2): 135–144. Publisher Full Text\n\nSchwartz MK, Aubry KB, McKelvey KS, et al.: Inferring geographic isolation of wolverines in California Using Historical DNA. J Wildl Manage. 2007; 71(7): 2170–2179. Publisher Full Text\n\nDell'Omodarme M, Prati MC: The probability of failing in detecting an infectious disease at entry points into a country. Stat Med. 2005; 24(17): 2669–2679. PubMed Abstract | Publisher Full Text\n\nFenichel EP, Tsao JL, Jones M, et al.: Fish pathogen screening and its influence on the likelihood of accidental pathogen introduction during fish translocation. J Aquat Anim Health. 2008; 20(1): 19–28. PubMed Abstract | Publisher Full Text\n\nBakker VJ, Doak DF, Roemer GW, et al.: Incorporating ecological drivers and uncertainty into a demographic population viability analysis for the island fox. Ecol Monogr. 2009; 79: 77–108. Publisher Full Text\n\nKrebs J, Lofroth E, Copeland J, et al.: Synthesis of survival rates and causes of mortality in North American wolverines. J Wildlife Man. 2004; 68(3): 493–502. Publisher Full Text\n\nPersson J, Landa A, Andersen R, et al.: Reproductive characteristics of female wolverines (Gulo gulo) in Scandinavia. J Mammal. 2006; 87(1): 75–79. Reference Source\n\nCarroll C, Noss RF, Schumaker NH, et al.: Is the return of the wolf, wolverine, and grizzly bear to Oregon and California biologically feasible. In: Maehr, DS, Noss RF, Larkin JL. editors. Large Mammal Restoration. Island Press, Washington. 2001; pp. 25–46. Reference Source\n\n\nAPPENDIX A\n\na Average of two populations (50% male in population 1, 40% male in population 2).\n\nb Persson et al.32 report 53% of females ≥ 3 reproduce with a mean annual birth rate of 0.74 kits/female. We assumed 0.5 females breeding * 1.5 kit/breeding female =0.75 kits/female. This assumption approximates the 0.375 female kits/female adult/year reproductive rate assumed by Krebs et al.31.\n\nc Persson et al.32 report litter sizes between 1–4, averaging 1.88 kits/litter and a 95% CI 1.68–2.07, corresponding to a std of 0.1.\n\nd Mortality rates were calculated as an average of mortality from natural causes of radio-tracked wolverines in montane habitats from both trapped and untrapped sites, weighted by effort in each site type31.\n\ne Simulations assuming 100% male breeding yielded same results."
}
|
[
{
"id": "3623",
"date": "11 Mar 2014",
"name": "Jonathan Rhodes",
"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 paper presents an interesting idea, but I believe there are potentially some technical problems with it. My main concern is with Equation (4). You state that P(N) in Equation (4) is the probability of failing to detect a viable population, given that the species inhabits the survey area at a given population size, N. However, since (1 – Z(N)) is the probability that the population is not viable and (1 – D(N)) is the probability that the species is not detected, then (1 – Z(N)) x (1 – D(N)) is the probability of that the population is not viable and is not detected. I suspect what you want is the probability that the population is viable and missed, which would be Z(N) x (1 – D(N)). It is also not clear to me why you don’t integrate over N. If you assume that N has a Poisson distribution, then this integral should not be too hard, or at least it will be possible to approximate it (see Royle, 2004). I think some broader discussion of this approach in relation to statistical methods for estimating detection error and estimating population parameters in the presence of detection errors would be useful. One thing that I think needs discussion is why here you focus on estimating the detection error and use that to estimating a suitable sample size. This is quite different to many recently developed approaches where the focus is on inferring the underlying population parameters from imperfect observation data. Here your focus is on the detection error itself and it is not clear why. I think some discussion of this in relation to how the data is intended to be used is important.\n\nFinally, you talk about maximising Equation (5). As far as I understand, you are not maximising this equation, you are solving it for a value of Pcrit.",
"responses": []
},
{
"id": "5733",
"date": "07 Aug 2014",
"name": "Michael McCarthy",
"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 takes an interesting approach to determining survey effort by focusing on the probability of detecting a population that will persist into the future. Rather than setting survey effort based on current occupancy, it assumes the population is present and projects persistence (as a function of current abundance) into the future. The probabilities of extinction and failed detection both decline with abundance, so the probability of failing to detect a viable population is greatest for an intermediate abundance.The authors take the approach of setting survey effort such that the probability of failing to detect a viable population is at least smaller than a particular threshold. Because population size is unknown, the authors find the population size where the required search effort to meet the target detection probability is largest. Then for all other population sizes the target detection probability will be achieved.The authors present this result as an approximation to an integral over the uncertainty in abundance. However, the quality of this approximation is not demonstrated and seems unnecessary. Depending on the distribution that might represent uncertainty in population size, the approximation might be very poor. However, the need to frame the solution as an approximation to an integral is unnecessary if instead it is framed in terms of Wald’s maximin approach (which is what I did above).The paper contains some minor errors of expression and fact, it seems to overlook some key literature, and discussion about the suitability of the chosen objective could be more nuanced. I have documented these in a PDF, which can be found here. A transcript is also available below, with bold text added for emphasis. None of these minor issues invalidate the substantive findings of the paper.Transcript of PDF: Introduction, 1st paragraph: “Clearly, incorporating nondetection probability, through its inverse, detection probability...” It is not the inverse – I think you mean the complement. ‘Inverse’ means something else mathematically. Introduction, 3rd paragraph: “First, the effort required to detect a small population increases at an accelerating pace as population size dwindles.” If claiming this is under-appreciated, it probably pays to at least cite some of the literature that acknowledges this occurs, such as Rout et al.,2014. The references cited in McCarthy et al. (2013) also have empirical evidence of detection probability declining with abundance. Introduction, 4th paragraph: “Given limited resources, it generally will not make sense to dedicate a lot of resources to ensure a high probability of detecting....” It seems worth citing some of the literature on allocation of limited resources to surveillance. The following search on Google Scholar pulls up various papers: “Allocate limited resources to surveillance ecology”. Introduction, 4th paragraph: “Given limited resources, it generally will not make sense to dedicate a lot of resources to ensure a high probability of detecting....” The term “generally” is vague. Do you mean “universal” (i.e. as in a general theory), or “commonly”, which is perhaps the more colloquial use. It would be better to be more specific about when this objective of finding viable populations is preferred to other possibilities. I could see this objective being useful for invasive species. But for endangered species, we might sometimes be more interested in detecting non-viable populations (so they can receive conservation actions) than viable populations. Introduction, 4th paragraph: “In other words, it is inefficient to spend limited resources to detect a species...” This is a claim, that may well be true under particular (and perhaps quite common) circumstances – but those circumstances are not described, and the case is not formally established. Introduction, 4th paragraph: “It would generally be even more efficient...” That problematic word again. Introduction, 4th paragraph: “...the gain in efficiency would not be as large as...” This will all come down to the specific objective and how “efficiency” is assessed. Introduction, 5th paragraph: “Ideally a survey design avoids two sources of inefficiency: failing to detect a population that persists into the near future and detecting a population that fails to persist into the near future.” As per previous comments, this depends on the implied objective being reasonable. The scope of this objective needs to be framed more clearly. Analytical framework, 2nd paragraph: “If all individuals within the surveyed population have the same chance of being detected, trap efficiency will be related to population size as E=1-(1-e)N, where e is the probability that a particular individual is detected and N is the number of individuals in the survey area.” This statement is not correct. The derivation of this in ref. 7 is actually based on assumptions of both independence and equal detection probability. However, the modelled relationship between detection probability and population size that is used here only depends on the assumption of independence (not equality as claimed here). Details are in McCarthy et al. (2013). Analytical framework, 2nd paragraph: “The nonlinear relationship between T and N as N approaches zero...” The relationship is non-linear everywhere, not just near zero. I think you mean something else apart from ‘non-linear’. Analytical framework, 3rd paragraph: “Here we assume that there is some relationship between near-term population viability and the current populations size Z(N)...” As used in the equations, Z(N) is the probability of local extinction within some relevant time horizon as a function of N, yet here it is defined vaguely, and seems to be “viability” (the complement of extinction). It should be defined more carefully. Analytical framework, 4th paragraph: “The exact probability is equal to the integral of the product of P(N) and the probability that the population surveyed as N individuals over all possible values of N” This approach to dealing with uncertainty in N is actually a Wald’s maximin approach. You haven’t shown how well the maximum of P(N) approximates the integral. I think this would be better framed in terms of ignorance about N, so you take a maximin approach to this ignorance when finding a solution. Analytical framework, 4th paragraph: “Approximating this interval at the maximum value of P(N) results in an estimate of P(N) that is biased high, which for most applications of this analysis will be a conservative bias.” Which interval? You have not previously mentioned any intervals as far as I can tell. Do you mean integral? If so, the wording is not correct. Analytical framework, 4th paragraph: “Approximating this interval at the maximum value of P(N) results in an estimate of P(N) that is biased high, which for most applications of this analysis will be a conservative bias”. Strictly, this is not an estimate of P(N). Analytical framework, 4th paragraph: “Approximating this interval at the maximum value of P(N) results in an estimate of P(N) that is biased high, which for most applications of this analysis will be a conservative bias” Conservative in what sense? Analytical framework, 4th paragraph: “Approximating this interval at the maximum value of P(N) results in an estimate of P(N) that is biased high, which for most applications of this analysis will be a conservative bias.” This sentence needs a lot of work. What I think is meant is: “Rarely if ever will N be known. Instead, if decisions about setting survey effort are based on assuming a value of N for which the required search effort is maximized, we get a reasonable result.” What “reasonable” means is unclear to me. “Conservative” is even more vague. If framed in terms of Wald’s maximin, then I think the result will be much clearer. By assuming the worst possible value of N such that the required search effort is maximized, then the calculated value of T will be sufficient to achieve the required value of P(N) for all values of N. Analytical framework, 5th paragraph: “Because most power analyses calculate the probability of success...” Most power analyses calculate the probability of rejecting a false null hypothesis, and have nothing to do with surveillance.Analytical framework, 5th paragraph: “Because most power analyses calculate the probability of success (e.g., that a study will show a significant difference given that observed groups do differ), it is tempting to estimate survey power as 1-P(N). We caution against doing so because this quantity is difficult to interpret...” This seems like a distraction. Survey protocols have typically aimed to achieve a particular probability of detection, achieve a particular posterior probability of presence given non-detection (e.g. Wintle et al. Divs and Distributions), or tried to minimize overall monitoring and management costs. I think setting up this hypothetical scenario that no-one has proposed (as far as I am aware) is unnecessary. Analytical framework, 6th paragraph: “To determine the optimal trap effort...” Optimal implies something is being minimized or maximised. You are really finding the smallest amount of survey effort that is required to reach some target for all possible population sizes. I think the word “optimal” is not correct. Analytical framework, 6th paragraph: “If Z(N) is a differentiable function, Tmax can be calculated analytically.” I guess it depends on what is meant by “analytical”, but I’m not sure that having Z(N) differentiable is not sufficient to get an explicit expression for Tmax (the resulting expression also needs to be solvable in terms of N I think). Application to Sierra Nevada wolverines, 5th paragraph: “The average distance covered by a dispersing wolverine is 51km, so we divided the population size by 8171 km2 to calculate the corresponding density.” I don’t know how a dispersal distance of 51 km leads to this area measurement. Application to Sierra Nevada wolverines, 5th paragraph: “The average distance covered by a dispersing wolverine is 51 km, so we divided the population size by 8171 km2 to calculate the corresponding density.” How sensitive are the results to this assumed area? Discussion, 1st paragraph: “First, explicitly acknowledging that extremely small populations are very difficult to detect but unlikely to persist facilitates efficient survey designs with enough power to detect species which need to be managed without wasting effort on effectively extinct populations.” This depends on context. I agree that in some contexts, managers might not want to manage a small population that might be doomed to extinction. In other cases, managers might actually want to find these populations so that they can be managed. If a population can persist by itself, do we need to detect it? The context might vary depending on whether the possible population is the last remaining, or whether other populations exist. I think the formulation in this paper is much better suited, in general, to eradication – in this case we don’t need to find populations that are not self-sustaining. Discussion, 2nd paragraph: “Choosing a target population density based on the effects of small populations on both detection and persistence probabilities was critical to design of an efficient survey.” This is a truism based on how the problem was framed. Discussion, 3rd paragraph: “We see this as a useful uncertainty when making management decisions.” The meaning of this is unclear. What is the uncertainty? Figure 2: “Preliminary and post-hoc power analysis for wolverine survey.” Is this the correct term? Power is usually used for null hypothesis significance testing (NHST). I’m not sure this is strictly NHST. Figure 2: “The apparent dip of the second point from the left reflects a dramatic decrease in population viability but not detection probability for a population of three compared to one of four individuals.” Typo? “one of four” is unclear.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-234
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https://f1000research.com/articles/2-232/v1
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04 Nov 13
|
{
"type": "Research Article",
"title": "Prism adaptation does not alter object-based attention in healthy participants",
"authors": [
"Janet H. Bultitude",
"Alexandra List",
"Anne M. Aimola Davies",
"Alexandra List",
"Anne M. Aimola Davies"
],
"abstract": "Hemispatial neglect (‘neglect’) is a disabling condition that can follow damage to the right side of the brain, in which patients show difficulty in responding to or orienting towards objects and events that occur on the left side of space. Symptoms of neglect can manifest in both space- and object-based frames of reference. Although patients can show a combination of these two forms of neglect, they are considered separable and have distinct neurological bases. In recent years considerable evidence has emerged to demonstrate that spatial symptoms of neglect can be reduced by an intervention called prism adaptation. Patients point towards objects viewed through prismatic lenses that shift the visual image to the right. Approximately five minutes of repeated pointing results in a leftward recalibration of pointing and improved performance on standard clinical tests for neglect. The understanding of prism adaptation has also been advanced through studies of healthy participants, in whom adaptation to leftward prismatic shifts results in temporary neglect-like performance. Here we examined the effect of prism adaptation on the performance of healthy participants who completed a computerised test of space- and object-based attention. Participants underwent adaptation to leftward- or rightward-shifting prisms, or performed neutral pointing according to a between-groups design. Significant pointing after-effects were found for both prism groups, indicating successful adaptation. In addition, the results of the computerised test revealed larger reaction-time costs associated with shifts of attention between two objects compared to shifts of attention within the same object, replicating previous work. However there were no differences in the performance of the three groups, indicating that prism adaptation did not influence space- or object-based attention for this task. When combined with existing literature, the results are consistent with the proposal that prism adaptation may only perturb cognitive functions for which normal baseline performance is already biased.",
"keywords": [
"Hemispatial neglect (‘neglect’) is a disabling condition that can follow brain injury. Patients with neglect show difficulty in responding to or orienting towards objects and events that occur on the contralesional side of space1. Symptoms of neglect can be reduced following a brief period of a sensory-motor training technique called prism adaptation2. Patients who spend a few minutes pointing to visual targets while wearing prismatic glasses that shifted the visual image to the right demonstrate a leftward shift",
"or ‘adaptation’",
"in their pointing as well as improved performance on standard clinical tests such as target cancellation and line bisection. The benefits of prism adaptation have been observed in multiple sensory modalities: vision3",
"tactile detection4",
"haptic exploration5",
"pressure sensitivity and finger position sense6",
"and auditory processing7. Furthermore",
"prism adaptation results in improvements in tasks that have direct relevance for recovery of independence such as reading8",
"9",
"postural control10",
"and wheelchair navigation11",
"12. The broad generalisation of prism adaptation treatment has generated considerable interest in understanding the mechanisms by which the technique reduces symptoms."
],
"content": "Introduction\n\nHemispatial neglect (‘neglect’) is a disabling condition that can follow brain injury. Patients with neglect show difficulty in responding to or orienting towards objects and events that occur on the contralesional side of space1. Symptoms of neglect can be reduced following a brief period of a sensory-motor training technique called prism adaptation2. Patients who spend a few minutes pointing to visual targets while wearing prismatic glasses that shifted the visual image to the right demonstrate a leftward shift, or ‘adaptation’, in their pointing as well as improved performance on standard clinical tests such as target cancellation and line bisection. The benefits of prism adaptation have been observed in multiple sensory modalities: vision3, tactile detection4, haptic exploration5, pressure sensitivity and finger position sense6, and auditory processing7. Furthermore, prism adaptation results in improvements in tasks that have direct relevance for recovery of independence such as reading8,9, postural control10, and wheelchair navigation11,12. The broad generalisation of prism adaptation treatment has generated considerable interest in understanding the mechanisms by which the technique reduces symptoms.\n\nIn healthy participants, adaptation to leftward-shifting prisms (producing a rightward reaching bias) temporarily induces neglect-like performance on tests of lateralised spatial attention such as a non-manual version of the line bisection test called the landmark test13–17. Although the changes shown by healthy participants are generally smaller in magnitude than those shown by neglect patients, they too have been observed in multiple sensory modalities14,16–19. Interestingly, Michel and colleagues13 showed that the magnitude of midpoint shift increased with more leftward line placement and longer line length, replicating the so-called ‘position’ and ‘length’ effects that have been described in neglect patients. Therefore, although adaptation to rightward-shifting prisms can reduce symptoms in neglect patients by shifting the response bias towards the left side of space, adaptation to leftward-shifting prisms can induce a similar but opposite change in healthy participant, inducing a small rightward response bias. The similarity in the effects of prism adaptation on the performance of neglect patients and healthy controls makes it possible to gain insights into the potential therapeutic effects of the technique through experiments on healthy volunteers.\n\nThe primary motivation of the present study was to investigate whether prism adaptation can alter object-based attention: that is, the extent to which object boundaries influence the allocation and redirection of selective attention. Considerable research provides support for the existence of object-based attention mechanisms that are distinct from a purely spatially-based allocation of attention in which object boundaries are irrelevant20–22. A particularly compelling demonstration of object-based attention was provided by Egly, Driver and Rafal23. In their task (the ‘Egly task’), two rectangles were arranged horizontally or vertically on either side of a central fixation cross. Targets appeared at one end of one rectangle. The targets were preceded by a cue in either the same location (validly-cued) or a different location (invalidly-cued) to the target. Critically, for invalidly-cued trials the cue location was either at the same end of the adjacent rectangle as the target (requiring a between-object shift of attention), or in the opposite end of the same rectangle as the target (requiring a within-object shift of attention). By setting the length of the rectangles to be equidistant to the distance separating the rectangles, the authors ensured that any difference in reaction times (RT) for the two invalidly-cued conditions could only be attributed to object-based attention mechanisms (because the spatial separation of the cue and target locations was the same across conditions). Larger RT costs were found for the between-object condition than for the within-object condition, providing evidence for object-based attention.\n\nIn neglect patients, symptoms can manifest in both space- and object-based frames of reference. For example, a neglect patient may fail to copy all objects on the left side of a page (space-based neglect), or may fail to copy the left half of every object in the scene regardless of where they appear on the page (object-based neglect). Although patients often demonstrate a combination of space- and object-based neglect, the two are considered to be separable and have partially distinct neurological bases24,25. The broad generalisation of benefits induced by prism adaptation suggests that the therapy can influence core aspects of neglect symptoms. However, although several studies suggest that prism adaptation can alter space-based orienting in neglect patients26–29 and healthy participants14–16, there is relatively little evidence that prism adaptation would have equal efficacy in treating patients whose deficits are primarily object-based.\n\nSome insights into whether prism adaptation can influence object-based attention were provided by Schindler and colleagues30. Healthy participants, right-hemisphere lesioned patients without neglect, and right-hemisphere patients with neglect completed the Egly task before and after adaptation to rightward-shifting prisms. Their task had a similar format to the original Egly task, in that there were two rectangles presented either vertically or horizontally, and these were positioned one on each side of a central fixation cross. Compared to the two control groups, the neglect patients were significantly worse at shifting their attention from one object in the ipsilesional field to another in the contralesional visual field, and this deficit was abolished by prism adaptation. This finding suggests that prism adaptation can influence object-based attention. However there are still some outstanding questions regarding both object-based attention and how it is influenced by prism adaptation. First, for healthy participants adaptation to leftward-shifting prisms can alter cognitive performance, however adaptation to rightward-shifting prisms generally has no such effect (although see Berberovic and Mattingley19 for an exception). Because Schindler and colleagues only tested adaptation to rightward-shifting prisms, it is not yet known whether prism adaptation can induce neglect-like changes in object-based attention in healthy participants. Second, the object-based deficit reported by Schindler and colleagues is potentially confounded by the requirement to shift attention between the two visual fields. Neglect patients were slower at shifting their attention between-objects only when it required a shift of attention from the ipsilesional to contralesional field: the RTs of neglect patients were normal for between-object shifts vertically within a visual field, and for horizontal within-object shifts from the ipsilesional to contralesional field. It is therefore unclear whether the pattern shown by their neglect patients at baseline can be attributed to a general deficit in shifting attention horizontally between two objects, or to a more specific deficit in shifting attention between two objects that are presented in different visual hemifields. By extension, it is unclear whether the improvements observed following prism adaptation were driven by a change in object-based attention, or an improvement in inter-hemisphere signalling.\n\nIn the present study we examine the effect of prism adaptation on space- and object-based attention in healthy participants using an adapted version of the Egly paradigm31 in which all shifts of attention were restricted within one of the two visual fields (see Figure 2). Four rectangles (two in each visual field) were presented either horizontally or vertically in each trial. Cues and targets for each trial appeared in the same visual field according to three conditions (validly-cued, within-object shift and between-object shift). In a between-group design, participants completed blocks of the Egly task after adaptation to leftward- or rightward-shifting prisms, or neutral pointing.\n\nUsing this four-rectangle version of the Egly paradigm we examined the effect of prism adaptation on three aspects of attention that are known to be altered in hemispatial neglect. First, we examined the effect of prism adaptation on object-based attention by comparing reaction times for validly-cued trials, within-object trials and between-object trials in each visual field across the three participant groups.\n\nSecond, we examined the effect of prism adaptation on the tonic distribution of attention across the visual field. Kinsbourne32 argued that the distribution of visuo-spatial attention across space is determined by two opposing gradients controlled by the contralateral cerebral hemispheres. One possible way that prism adaptation influences attention is by altering the balance between these opposing gradients: i.e., by rebalancing the hemispheric competition in patients and unbalancing the hemispheric competition in healthy controls. If this is the case, then adaptation to leftward-shifting prisms would induce a neglect-like gradient of attention in healthy participants, with lowest attention (highest RTs) to the left-most part of the visual field, and highest attention (lowest RTs) to the right-most part of the visual field.\n\nFinally, we examined the effect of prism adaptation on horizontal shifts of attention. Patients with neglect are impaired at shifting their attention in a contralesional direction (the ‘disengage deficit’33). There is some evidence that prism adaptation can reduce the disengage deficit in neglect patients28,34 and can alter reaction times for horizontal shifts of attention in healthy participants35, although there are some inconsistencies in the effects that have been observed for different cue types across these studies. We predicted that adaptation to leftward-shifting prisms would result in a neglect-like deficit in shifting attention leftward. This neglect-like deficit may be restricted to between-object shifts of attention, in keeping with the findings of Schindler and colleagues30.\n\n\nMethods\n\nSixty healthy undergraduates (13 males) participated in the experiment in exchange for course credits (mean age=19.6 years, SEM=0.3). Each participant had normal or corrected-to-normal vision and was right-handed according to the Edinburgh Handedness Inventory (mean=-0.84, SEM=0.02, where -1 denotes exclusive right-handedness36). Informed consent was obtained in accordance with guidelines approved by the Bangor University ethics committee and the 2008 Declaration of Helsinki.\n\nThe general procedure is outlined in Figure 1. Throughout the experiment the participant was seated in a standard computer chair that could be wheeled and rotated by the experimenter between the computer and a custom-built prism adaptation box. The participant completed the Egly task in four sets of three blocks (twelve blocks in total), with prism adaptation preceding each set. Multiple sets of prism adaptation were used to ensure that the participant was fully adapted throughout all of the Egly blocks. Similar ‘top-up’ adaptation sets have been used previously30,37. The participant completed one practice Egly block before the first sham adaptation session during which the experimenter gave verbal feedback on the participant’s performance. To confirm visuo-motor adaptation, open-loop pointing tests were performed immediately before and after the first session of prism adaptation, and at the end of the experiment (the ‘pre’, ‘post’ and ‘late’ open-loop pointing sessions, respectively). Prism direction was manipulated between groups, with each participant randomly allocated to undergo adaptation to neutral, leftward-shifting or rightward-shifting lenses.\n\nPA = prism adaptation.\n\nFor prism adaptation, the participant faced a 90 cm wide × 35 cm high × 70 cm deep prism adaptation box. The box was open at two opposite ends at which the participant and experimenter were positioned, as well as at the top, allowing the participant vision of the inside of the box. Three 1.5 cm diameter targets were placed on the base of the box at arm’s length from the participant and at angles of -10°, 0° and +10° from their body midline (negative numbers indicate a leftward displacement). The participant was fitted with welding goggles containing neutral lenses, or 25-diopter (~17°) Fresnel lenses that induced a visual shift to the left or right. While resting their chin on the edge of the box, the participant reached out to touch the targets in a pre-determined sequence (left-middle-right-middle) that was repeated for 90 pointing movements, returning their hand to rest in front of their torso between each movement. The participants could see only the distal half of each reaching movement. Pointing was performed in time with a metronome set to 1 Hz to encourage a constant, ballistic pointing speed. After completing the 90 pointing movements, the participant closed their eyes and the goggles were removed. To minimise de-adaptation, the participant was asked to keep their eyes closed between the different parts of the experiment, with the exception that after each set of three Egly blocks a more extended break was offered in which the participant could keep their eyes open before undergoing another set of prism adaptation.\n\nA lid was placed on the box with lines drawn on the upper surface radiating at angles of -10°, 0° and +10° from the participant’s body midline. These served as target lines for the open-loop pointing task. The participant rested their chin on the top of the box and pointed with their right arm under each of the target lines four times in pseudorandom order as directed by the experimenter, returning their hand to rest in front of their torso between each pointing movement. The participant was instructed to point with their elbow straight and their index finger extended. Pointing error was measured by the experimenter to the nearest 0.5 degrees with the aid of markings drawn on the underside of the lid.\n\nStimuli were generated by Eprime software on a Dell PC running Windows XP. They appeared on a 17-inch monitor running at 85 Hz, positioned 60 cm from the participant’s eyes. Head movements were prevented by a chin rest that also prevented vision of the responding (right) hand.\n\nFigure 2 and Figure 3 show examples of stimuli used in the experiment. In a pilot version of this experiment we tested a simple target detection response in which participants pressed a button when a square target appeared. A third of pilot participants responded to more than 30% of the catch trials (‘false alarms’, i.e., trials in which no target appeared and the participants were required to refrain from responding). Because the high number of false alarms brings into question the reliability of the responses to true targets, we opted to use a discrimination task38 in the reported experimental study in order to encourage participants to respond appropriately to all targets.\n\nStimuli appeared as grey figures on a white background unless otherwise indicated. Four rectangles were presented, two on each side of a central 0.2° × 0.2° fixation point. The rectangles were presented in the same orientation: either all horizontal or all vertical. Each rectangle subtended visual angles of 5.0° by 1°. Adjacent rectangles' outer edges were separated by a visual angle of 5.0°. Therefore, the angular distance between the two rectangles' outer edges in each hemifield was equal to the angular length of the longest side of the individual rectangles. The distance of the closest edge of any rectangle was 1.3° from the edge of the central fixation cross. The four rectangles and the fixation cross remained on the screen for the entire trial.\n\nThe figure shows the timing and duration of each event (ms). A vertical arrangement of rectangles (as shown) was used in half of the trials, and a horizontal arrangement was used in the remaining half of the trials. A within-object condition is shown.\n\nThe three possible target locations are shown relative to the cue location given in Figure 2. For the three possible trial types the target ‘T’ is circled in red for illustrative purposes (no red circle was displayed in the experiment).\n\nAfter 1000 ms a cue was presented for 100 ms in the form of a blackening of the outline at one end of one of the rectangles (see Figure 2). After a further 100 ms delay, the cue was followed by a target appearing for 200 ms in 1 of 3 locations relative to the cue. The target was a black T or L, measuring 0.4° × 0.4°, which could appear at any orientation. In the majority of trials (71.4%) the target appeared in the same position on the same rectangle as the cue (validly-cued trials, see Figure 3). In 28.6% of trials the target appeared at a position other than that indicated by the cue (i.e., invalid cueing), requiring a shift of attention. In these trials, the target appeared either at the opposite end of the same rectangle as the cue (within-object shift) or at the same end of the rectangle adjacent to that which the cue had appeared in (between-object shift). These shifts were confined within the visual field such that the target always appeared on the same side of the fixation cross as the cue. Within-object and between-object shifts occurred equiprobably, and the angular distance of the attentional shift was equal for both of these conditions. Distractors in the form of 0.4° × 0.4° black T/L hybrids (Fs) appeared in all other locations for the same duration as the target (200 ms).\n\nThe participant was asked to indicate whether the target was a T or an L. Responses were given by pressing either of two keys of a standard keyboard with the index and middle fingers of the right hand, with response mapping counterbalanced between participants. The response keys were placed directly in front of the participant’s body midline. The trial ended when the participant gave their response, or after 3000 ms, whichever came first. There was a 500 ms inter-trial interval in which only the fixation cross remained on the screen. Each participant completed twelve experimental blocks of 112 trials each.\n\nThe participant was instructed to respond as quickly and as accurately as possible, and to maintain central fixation. Fixation was monitored by the experimenter through a closed-circuit television. For trials in which eye movements occurred the experimenter clicked a mouse button, and these trials were later removed from analysis.\n\nThe data from the open-loop pointing and Egly tasks were analysed using repeated-measures ANOVAs. Sphericity was violated in some datasets (p for Mauchley’s W > .05), in which case Greenhouse-Geisser-corrected alpha levels were used and are indicated. Follow-up analyses were performed using paired- and independent-samples t-tests with Bonferroni-corrected alpha levels.\n\nPointing errors were analysed using a Group (L, N, R) × Session (pre, post, late) ANOVA.\n\nFor the Egly task, trials in which participants made an eye movement or gave an incorrect response were excluded from the RT analyses. In addition, because there were a substantially greater number of valid than invalid trials, each participant’s data were screened for outliers in the valid, within-object and between-object conditions, with RTs more than 2SD from the mean for each condition excluded from analysis. These criteria resulted in excluding less than 1% of the data. For each participant, mean RTs and percentage accuracy were calculated for each condition and grouped for further analysis. Three sets of repeated-measures ANOVAs were performed to examine the effects of prism adaptation on different aspects of space- and object-based attention.\n\nFirst, to examine the effects of prism adaptation on object-based attention in each visual field, we performed repeated-measures ANOVAs of mean RTs and percentage accuracy with three factors: Group (L, N, R) × Visual Field [left visual field (LVF), right visual field (RVF)] × Validity (valid, within, between).\n\nSecond, to examine the effects of prism adaptation on the tonic distribution of attention across the visual field, the mean RTs for validly-cued trials were pooled according to which of the four horizontal locations they appeared in. The mean RTs and percentage accuracy were subject to Group (L, N, R) × Horizontal Location (left-LVF, right-LVF, left-RVF, right-RVF) ANOVAs with the prediction that adaptation to leftward-shifting prisms would result in slower RTs and lower accuracy in the LVF, and that this decrement in performance would be most evident in the leftmost location in the LVF.\n\nThe third set of analyses tested for any effects of prism adaptation on location-based costs of shifting attention horizontally within an object or between two objects in each visual field. The data for invalidly-cued trials involving a horizontal shift in attention were subjected to two Group (L, N, R) × Visual Field (left, right) × Shift Type (between, within) × Horizontal Shift Direction (left shift, right shift) ANOVAs. If adaptation to leftward-shifting prisms results in a neglect-like disengage deficit, participants in the leftward-shifting group would demonstrate increased RT costs and/or reduced accuracy for shifting attention leftward, which could be observed only for between-object shifts.\n\n\nResults\n\nThree participants performed no better than chance in the discrimination task for the invalidly-cued trials in the Egly task (chi-squares p>0.05), and were excluded from further analyses.\n\nThe raw pointing error data are provided in Dataset 1. The Group (L, N, R) × Session (pre, post, late) ANOVA revealed a main effect of Session [F(1.7,93.7)=7.4, p<0.005, Greenhouse-Geisser corrected] and a main effect of Group [F(1,54)=53.2, p<0.001]. These were driven by a significant Group × Session interaction [F(3.4,93.7)=61.0, p<0.001, Greenhouse-Geisser corrected], which is plotted in Figure 4.\n\nHorizontal pointing errors (°) are shown for the Group × Session interaction. L = leftward-shifting; N = neutral pointing; R = rightward-shifting. Error bars indicate ±1SEM; ** indicates p<0.001.\n\n\n\nThe mean pointing errors for the neutral group were unchanged across sessions (Pre: M=0.0, SEM=0.43; Post: M=-0.2, SEM=0.46; Late: M=-0.6, SEM=0.57; ps>0.05). For the leftward-shifting prism group there was a significant rightward shift in pointing errors between the pre-adaptation (M=0.4, SEM=0.44) and post-adaptation sessions [M=4.3, SEM=0.47; t(18)=9.6, p<0.001]. Pointing errors at the late test (M=3.0, SEM=0.58) were also significantly rightward of the pre-adaptation error level [t(18)=4.7, p<0.001].\n\nSimilarly, for the rightward-shifting prism group there was a significant leftward shift in pointing errors between pre-adaptation (M=-1.1, SEM=0.46) and post-adaptation [M=-5.3, SEM=0.48; t(19)=13.5, p<0.001]. Pointing errors at the late test were also significantly leftward of pre-test error [M=-5.7, SEM=0.60; t(19)=14.2, p<0.001].\n\nIn summary, as expected, there was no change in pointing error for the neutral group, and the leftward- and rightward-shifting prism groups showed significant changes in pointing errors that were in the predicted direction for the prismatic shift and were sustained throughout the entire post-adaptation Egly task.\n\nThe analyses of accuracy and mean RTs produced identical results with respect to the experimental hypotheses. For the sake of brevity, only the RT analyses are reported here, however both raw datasets are provided (Dataset 2–Dataset 7). Mean accuracy was 85%.\n\nThe Group (N, L, R) × Visual Field (left, right) × Validity (valid, within, between) ANOVA revealed a main effect of Visual Field [F(1,54)=20.0, p<0.001] and a main effect of Validity [F(2,108)=199.0, p<0.001]. These were driven by a significant Visual Field × Validity interaction [F(2,104.5)=3.36, p<0.05; Greenhouse-Geisser corrected], which is shown in Figure 5. In the LVF, RTs for validly-cued trials were 115 ms faster than RTs for trials requiring a within-object shift of attention [valid: M=398, SEM=10; within-object: M=513, SEM=15; t(56)=15.0, p<0.001], and trials requiring a between-object shift in attention (M=553, SEM=18) were a further 40 ms slower compared to within-object shifts [t(56)=6.2, p<0.001]. In the RVF, RTs for validly-cued trials were 106 ms faster than for the within-object shift condition [valid: M=388, SEM=10.6; within-object: M=494, SEM=14; t(56)=14.5, p<0.001], and RTs for the between-object shift condition (M=526, SEM=16) were a further 32 ms slower than for trials requiring a within-object shift of attention [t(56)=5.3, p<0.001]. Finally, for each of the three trial types, RTs were significantly faster for trials in the RVF compared to the LVF (ps<0.005). There were no further significant main effects or interactions, including no main effects or interactions involving Group (Fs<2.1, ps>0.05). The raw percentage accuracy and mean RTs for this analysis are provided in Datasets 2 and 3.\n\nMean RTs (ms) are shown for the three different target types in the left visual field (LVF) and right visual field (RVF). Error bars indicate ±1SEM; ** indicates p<0.001.\n\n\n\nThese results replicate the classic Egly effect: invalidly-cued trials are associated with slower RTs compared to validly-cued trials, and this RT cost is significantly larger for between-object shifts of attention than within-object shifts. This pattern was present in both visual fields and was unaltered by prism adaptation.\n\nThe percentage accuracy and mean RT data of each participant for the Group (L, N, R) × Horizontal Location (left-LVF, right-LVF, left-RVF, right-RVF) analyses are provided in Datasets 4 and 5. The ANOVA of mean RTs revealed a significant main effect of Horizontal Location [F(1,105)=116.0, p<0.001], and a non-significant trend for the Group × Horizontal Location interaction [F(3.1,84.7)=2.6, p=0.054; Greenhouse-Geisser corrected]. Paired-samples t-tests revealed that in each group the RTs for targets appearing closer to fixation (i.e., right-LVF and left-RVF) were significantly faster than those appearing further away to fixation (i.e., left-LVF and right-RVF, ps<0.001; Figure 6).\n\nMean RTs (ms) are shown for validly-cued trials according to horizontal location for the three different groups. L = leftward-shifting; R = rightward-shifting, N = neutral pointing; LVF = left visual field; RVF = right visual field; Lloc = left location, Rloc = right location. Error bars indicate ±1SEM.\n\n\n\nIndependent-samples t-tests comparing the mean RTs for the three groups at each location revealed that the leftward-shifting prism group responded to targets in the rightmost location of the RVF 70 ms slower than the neutral pointing group [left: M=466, SEM=80.4; neutral: M=396, SEM=79.8; t(37)=2.7, p<0.012], however there were no significant differences in the RTs of these groups in the three other locations (ps>0.012, Bonferroni corrected). There were also no significant differences between the leftward- and rightward-shifting prism groups, or between the rightward-shifting prism group and the neutral pointing group in any of the four locations (ps>0.012, Bonferroni corrected). Therefore, these results are not consistent with a systematic neglect-like change in the RTs of the leftward-shifting prism group, but instead reflect a typical eccentricity-driven distribution of attention39–41.\n\nThe Group (N, L, R) × Visual Field (LVF, RVF) × Shift Type (between, within) × Horizontal Shift Direction (left-shift, right-shift) ANOVA was performed on the mean RTs for invalidly-cued trials only. There was a significant main effect of Visual Field [F(1,54)=9.0, p<0.005], a significant main effect of Shift Type [F(1,54)=28.2, p<0.002] and a significant main effect of Horizontal Shift Direction [F(1,54)=4.7, p<0.05]. There was also a significant Visual Field × Horizontal Shift Direction interaction [F(1,54)=170.7, p<0.005]. These were driven by a significant three-way interaction of Visual Field × Shift Type × Horizontal Shift Direction [F(1, 54)=4.3, p<0.05), which is plotted in Figure 7. The percentage accuracy and mean RTs for each participant for this analysis is provided in Datasets 6 and 7.\n\nMean RTs (ms) are shown for invalidly-cued trials that involved a horizontal shift in attention. LVF = left visual field; RVF = right visual field; L shift = left shift; R shift = right shift. Error bars indicate ±1SEM; ** indicates p<0.005 for each t-test comparison of between- and within-object shifts.\n\n\n\nFor between-object shifts, mean RTs for shifting rightward in the LVF were 109 ms faster than mean RTs for shifting leftward in the LVF [leftward: M=602, SEM=21.4; rightward: M=493, SEM=20; t(56)=8.6, p<0.001]. Similarly, for between-object shifts, mean RTs for shifting leftward in the RVF were 143 ms faster than mean RTs for shifting rightward in the RVF [leftward: M=454, SEM=14; rightward: M=597, SEM=19.8; t(56)=10.9, p<0.001]. For within-object shifts, mean RTs for shifting rightward in the LVF were 99 ms faster than mean RTs for shifting leftward in the LVF [leftward: M=568, SEM=18.5; rightward: M=469, SEM=14.7; t(56)=9.2, p<0.001]. Similarly, for within-object shifts, mean RTs for shifting leftward in the RVF were 116 ms faster than mean RTs for shifting rightward in the RVF [leftward: M=440, SEM=14.0; rightward: M=556, SEM=18.8; t(56)=9.7, p<0.001]. Furthermore, t-tests comparing RTs for between- to within-object shifts for each shift direction in each visual field revealed that the RT advantage for within-object shifts was only significant for shifts away from fixation (ps<0.005). Finally, there were no main effects or interactions involving Group (Fs<2.0, ps>0.05).\n\n\nDiscussion\n\nThe results demonstrate that adaptation to leftward-shifting prisms did not alter the performance of healthy participants on the Egly task. Specifically, there were no differences between the three groups in their relative RTs for validly-cued, within-object and between-object trials (Figure 5), indicating no effect of prism adaptation on object-based attention. RTs for validly-cued trials in four locations across the visual field were numerically larger for both prism groups compared to the neutral pointing group (Figure 6). However, this difference was not significant, and the responses of the leftward-shifting prism group were not consistent with a neglect-like decrement in LVF attention relative to the neutral group. Finally, there were no differences between the three groups in the speed with which attention was shifted laterally in each visual field (Figure 7), indicating that adaptation to leftward-shifting prisms did not produce a neglect-like disengage deficit. Therefore, although significant motor after-effects were obtained for both prism groups, the data do not provide any evidence that prism adaptation alters space- or object-based attention in healthy participants.\n\nThe existing literature provides a number of examples of tasks for which prism adaptation has been shown to improve the performance of neglect patients and also produce neglect-like changes in the performance of healthy participants: for example line bisection13,19,42, mental number bisection14,43, haptic circle centring5,17 and postural control10,18. However, there are some tasks for which prism adaptation has been shown to reduce neglect symptoms, but has no impact on the performance of healthy participants: visual search27,44 and a temporal order judgement task sensitive to biases in spatial attention26. Our results are consistent with the latter category in that there is substantial evidence that prism adaptation can influence the spatial distribution of attention in neglect patients26,28,34, and specific evidence that it influences the performance of neglect patients on the Egly task30, however we find no evidence for any prism-related differences in the performance of healthy participants on our Egly task. These results are consistent with those of Schindler and colleagues30, who found that adaptation to rightward-shifting prisms did not alter the performance of healthy participants on a version of the Egly task, and we have extended their findings by showing that there are also no changes following adaptation to leftward-shifting prisms.\n\nWe have previously noted that any aspects of performance that have been altered by prism adaptation in healthy individuals are ones for which ‘normal’ behaviour is already biased45. The term ‘pseudoneglect’ refers to the commonly observed leftward bias that is shown by healthy participants on visuo-spatial tasks such as line bisection, even in the absence of motor responses46. One explanation for this bias is that engaging in visuo-spatial tasks, which favour right-hemisphere processing, results in a slight imbalance in the opponent attentional gradients of the two hemispheres such that the leftward orienting controlled by the right hemisphere is dominant. Many studies that demonstrate changes in the performance of healthy participants following prism adaptation could be interpreted as ‘treating’ or reducing pseudoneglect rather than inducing neglect-like patterns13,15,16,26. Consistent with this explanation, we demonstrated that prism adaptation altered the extent to which participants processed the local elements of a visual image compared to the global configuration45. Like pseudoneglect, global-local processing has been related to opponent processes of the left and right hemispheres47,48, and healthy participants normally show a bias in favour of right-hemisphere global processing49.\n\nIn contrast, healthy participants do not normally show a pseudoneglect-style spatial bias when performing the Egly task , visual search44 or temporal order judgement50. Although the participants in the present study did show an overall spatial bias (see Figure 4), this was present as a RVF RT advantage, rather than a LVF RT advantage as would be indicative of pseudoneglect. Because all participants gave their responses using their right hands, the RVF advantage can be attributed to the common phenomenon of speeded responses when stimuli are presented in the same side of space as the response button or responding hand (the ‘Simon effect’51). That is, the RT imbalance that we observed is probably not due to a hemispheric or visual field difference in the way that the central cognitive task is performed per se. Our results are therefore consistent with our previous proposal that prism adaptation is more likely to perturb cognitive functions for which the baseline performance is already biased (either by brain damage or due to normal cognitive phenomena such as pseudoneglect45).\n\nAssuming that the mechanisms through which prism adaptation influences the performance of healthy participants and neglect patients are similar, it is possible that the absence of any effects of left-shifting prisms in inducing a neglect-like effect on healthy participants in the current study can be attributed to our design. Schindler and colleagues30 used a simple detection task to demonstrate the benefits of rightward-shifting prisms for patients with neglect whereas the present study used a discrimination task. It is possible that prism adaptation influences only early or rapid attentional processes and changes in performance do not manifest in the longer time period that it takes to identify a target. Furthermore, we restricted the cues and targets for each trial within a visual field. Prism adaptation may only influence object-based attention in situations in which attention is shifted between objects in different visual fields (e.g., due to the demand for interhemispheric communication). In the present study, limiting the stimuli for each trial to within a visual hemifield may have meant that once cued, attention remained focussed within the visual field so that the demands of shifting attention to a different object in the same visual field were smaller than what would be required for a between-object shift across visual fields. A smaller overall effect of object-based attention would mean that any differences between the groups could be too small to detect.\n\nPrism effects aside, our study extended the findings of Egly and colleagues23 by demonstrating that the RT cost associated with shifting attention between-objects compared to within-objects are present when each visual field is probed separately. Furthermore, for horizontal shifts of attention these differences are only significant when shifting attention outward, away from fixation. This adds to an extensive literature supporting the existence of object-based attention21,22,52.\n\nTo understand the cognitive and neural mechanisms that underlie prism adaptation, it is important to examine tasks on which this technique has no impact, as well as those for which changes are observed. Our results show that prism adaptation does not alter object- or space-based attention in healthy participants performing the Egly task. Although it is possible that particular features of our design, such as the use of a discrimination task rather than a detection task, contributed to this null finding, our results are also consistent with the proposal that prism adaptation may only perturb cognitive functions for which normal baseline performance is already biased.",
"appendix": "Author contributions\n\n\n\nJB conceived of the study, contributed to the design of the study, assisted in data collection, analysed the data and prepared the draft manuscript. AL contributed to the design of the study and to interpreting the data. AAD contributed to the conception and design of the study, to interpreting the data, and secured funding for the research. 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 relevant competing interests were disclosed.\n\n\nGrant information\n\nFunding for this work was provided by the British Federation of Women Graduates (to JB), the National Institute of Health (to AL, fellowship number NS055531), and the Australian National University (to AAD).\n\n\nAcknowledgements\n\nThe authors wish to thank Julia Taylor for assistance with data collection.\n\n\nReferences\n\nBisiach E, Vallar G: Unilateral neglect in humans. In: Boller F Grafman J Rizzolatti G editors. Handbook of neuropsychology. Elsevier Science, B.V.; ST – Unilateral neglect in humans. 2000; p. 459–502.\n\nRossetti Y, Rode G, Pisella L, et al.: Prism adaptation to a rightward optical deviation rehabilitates left hemispatial neglect. Nature. 1998; 395(6698): 166–169. PubMed Abstract | Publisher Full Text\n\nPisella L, Rode G, Farne A, et al.: Dissociated long lasting improvements of straight-ahead pointing and line bisection tasks in two hemineglect patients. Neuropsychologia. 2002; 40(3): 327–334. PubMed Abstract | Publisher Full Text\n\nMaravita A, McNeil J, Malhotra P, et al.: Prism adaptation can improve contralesional tactile perception in neglect. Neurology. 2003; 60(11): 1829–1831. PubMed Abstract | Publisher Full Text\n\nMcintosh R, Rossetti Y, Milner A: Prism adaptation improves chronic visual and haptic neglect: a single case study. Cortex. 2002; 38(3): 309–320. PubMed Abstract | Publisher Full Text\n\nDijkerman HC, Webeling M, ter Wal JM, et al.: A long-lasting improvement of somatosensory function after prism adaptation, a case study. Neuropsychologia. 2004; 42(12): 1697–702. PubMed Abstract | Publisher Full Text\n\nJacquin-Courtois S, Rode G, Pavani F, et al.: Effect of prism adaptation on left dichotic listening deficit in neglect patients: glasses to hear better? Brain A J Neurol. 2010; 133(Pt 3): 895–908. PubMed Abstract | Publisher Full Text\n\nAngeli V, Meneghello F, Mattioli F, et al.: Mechanisms underlying visuo-spatial amelioration of neglect after prism adaptation. Cortex. 2004; 40(1): 155–156. PubMed Abstract | Publisher Full Text\n\nAngeli V, Benassi MG, Làdavas E: Recovery of oculo-motor bias in neglect patients after prism adaptation. Neuropsychologia. 2004; 42(9): 1223–1234. PubMed Abstract | Publisher Full Text\n\nTilikete C, Rode G, Rossetti Y, et al.: Prism adaptation to rightward optical deviation improves postural imbalance in left-hemiparetic patients. Curr Biol. 2001; 11(7): 524–528. PubMed Abstract | Publisher Full Text\n\nWatanabe S, Amimoto K: Generalization of prism adaptation for wheelchair driving task in patients with unilateral spatial neglect. Arch Phys Med Rehabil. 2010; 91(3): 443–7. PubMed Abstract | Publisher Full Text\n\nJacquin-Courtois S, Rode G, Pisella L, et al.: Wheel-chair driving improvement following visuo-manual prism adaptation. Cortex. 2008; 44(1): 90–96. PubMed Abstract | Publisher Full Text\n\nMichel C, Pisella L, Halligan PW, et al.: Simulating unilateral neglect in normals using prism adaptation: implications for theory. Neuropsychologia. 2003; 41(1): 25–39. PubMed Abstract | Publisher Full Text\n\nLoftus AM, Nicholls ME, Mattingley JB, et al.: Left to right: representational biases for numbers and the effect of visuomotor adaptation. Cognition. 2008; 107(3): 1048–58. PubMed Abstract | Publisher Full Text\n\nLoftus AM, Vijayakumar N, Nicholls ME: Prism adaptation overcomes pseudoneglect for the greyscales task. Cortex. 2009; 45(4): 537–543. PubMed Abstract | Publisher Full Text\n\nNicholls ME, Kamer A, Loftus AM: Pseudoneglect for mental alphabet lines is affected by prismatic adaptation. Exp Brain Res. 2008; 191(1): 109–115. PubMed Abstract | Publisher Full Text\n\nGirardi M, McIntosh RD, Michel C, et al.: Sensorimotor effects on central space representation: prism adaptation influences haptic and visual representations in normal subjects. Neuropsychologia. 2004; 42(11): 1477–1487. PubMed Abstract | Publisher Full Text\n\nMichel C, Rossetti Y, Rode G, et al.: After-effects of visuo-manual adaptation to prisms on body posture in normal subjects. Exp Brain Res. 2003; 148(2): 219–226. PubMed Abstract | Publisher Full Text\n\nBerberovic N, Mattingley JB: Effects of prismatic adaptation on judgements of spatial extent in peripersonal and extrapersonal space. Neuropsychologia. 2003; 41(4): 493–503. PubMed Abstract | Publisher Full Text\n\nFink GR, Dolan RJ, Halligan PW, et al.: Space-based and object-based visual attention: shared and specific neural domains. Brain. 1997; 120(Pt 1): 2013–28. PubMed Abstract | Publisher Full Text\n\nLavie N, Driver JO: On the spatial extent of attention in object-based visual selection. Percept Psychophys. 1996; 58(8): 1238–1251. PubMed Abstract | Publisher Full Text\n\nDuncan J: Selective attention and the organization of visual information. J Exp Psychol Gen. 1984; 113(4): 501–517. PubMed Abstract\n\nEgly R, Driver J, Rafal R: Shifting visual attention between objects and locations: evidence from normal and parietal lesion subjects. J Exp Psychol Gen. 1994; 123(2): 161–77. PubMed Abstract | Publisher Full Text\n\nWalker R: Spatial and object-based neglect. Neurocase. 1995; 1: 371–383. Reference Source\n\nVerdon V, Schwartz S, Lovblad KO, et al.: Neuroanatomy of hemispatial neglect and its functional components: a study using voxel-based lesion-symptom mapping. Brain. 2010; 133(pt 3): 880–94. PubMed Abstract | Publisher Full Text\n\nBerberovic N, Pisella L, Morris AP, et al.: Prismatic adaptation reduces biased temporal order judgements in spatial neglect. Neuroreport. 2004; 15(7): 1199–1204. PubMed Abstract | Publisher Full Text\n\nSaevarsson S, Kristjánsson A, Hildebrandt H, et al.: Prism adaptation improves visual search in hemispatial neglect. Neuropsychologia. 2009; 47(3): 717–25. PubMed Abstract | Publisher Full Text\n\nNijboer TC, McIntosh RD, Nys GM, et al.: Prism adaptation improves voluntary but not automatic orienting in neglect. Neuroreport. 2008; 19(3): 293–8. PubMed Abstract | Publisher Full Text\n\nNijboer T, Vree A, Dijkerman C, et al.: Prism adaptation influences perception but not attention: evidence from antisaccades. Neuroreport. 2010; 21(5): 386–389. PubMed Abstract | Publisher Full Text\n\nSchindler I, McIntosh RD, Cassidy TP, et al.: The disengage deficit in hemispatial neglect is restricted to between-object shifts and is abolished by prism adaptation. Exp Brain Res. 2009; 192(3): 499–510. PubMed Abstract | Publisher Full Text\n\nEgly R, Rafal R, Jon D, et al.: Covert orienting in the split brain reveals hemispheric specialization for object-based attention. Psychol Sci. 1994; 5(6): 380–383. Publisher Full Text\n\nKinsbourne M: Orientational bias model of unilateral neglect: Evidence from attentional gradients within hemispace. Unilateral Negl Clin Exp Stud. 1993. Reference Source\n\nPosner MI, Walker JA, Friedrich FA, et al.: How do the parietal lobes direct covert attention? Neuropsychologia. 1987; 25(1A): 135–45. PubMed Abstract | Publisher Full Text\n\nStriemer C, Danckert J: Prism adaptation reduces the disengage deficit in right brain damage patients. Neuroreport. 2007; 18(1): 99–103. PubMed Abstract | Publisher Full Text\n\nStriemer C, Sablatnig J, Danckert J: Differential influences of prism adaptation on reflexive and voluntary covert attention. J Int Neuropsychol Soc. 2006; 12(3): 337–49. PubMed Abstract\n\nOldfield RC: The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia. 1971; 9(1): 97–113. PubMed Abstract | Publisher Full Text\n\nBultitude JH, Van Der Stigchel S, Nijboer TCW: Prism adaptation alters spatial remapping in healthy individuals: Evidence from double-step saccades. Cortex. 2013; 49(3): 759–70. PubMed Abstract | Publisher Full Text\n\nMoore CM, Yantis S, Vaughan J: Object-based visual selection: Evidence from perceptual completion. Psychol Sci. 1998; 9(2): 104–110. Publisher Full Text\n\nCarrasco M, Evert DL, Chang I, et al.: The eccentricity effect: target eccentricity affects performance on conjunction searches. Percept Psychophys. 1995; 57(8): 1241–1261. PubMed Abstract | Publisher Full Text\n\nWolfe JM, O'Neill P, Bennett SC: Why are there eccentricity effects in visual search? Visual and attentional hypotheses. Percept Psychophys. 1998; 60(1): 140–156. PubMed Abstract | Publisher Full Text\n\nWoodman GF, Luck SJ: Electrophysiological measurement of rapid shifts of attention during visual search. Nature. 1999; 400(6747): 867–869. PubMed Abstract | Publisher Full Text\n\nFarnè A, Rossetti Y, Toniolo S, et al.: Ameliorating neglect with prism adaptation: visuo-manual and visuo-verbal measures. Neuropsychologia. 2002; 40(7): 718–729. PubMed Abstract | Publisher Full Text\n\nRossetti Y, Jacquin-Courtois S, Rode G, et al.: Does action make the link between number and space representation? Visuo-manual adaptation improves number bisection in unilateral neglect. Psychol Sci. 2004; 15(6): 426–430. PubMed Abstract | Publisher Full Text\n\nMorris AP, Kritikos A, Berberovic N, et al.: Prism adaptation and spatial attention: a study of visual search in normals and patients with unilateral neglect. Cortex. 2004; 40(4–5): 703–721. PubMed Abstract | Publisher Full Text\n\nBultitude J, Woods J: Adaptation to leftward-shifting prisms reduces the global processing bias of healthy individuals. Neuropsychologia. 2010; 48(6): 1750–1756. PubMed Abstract | Publisher Full Text\n\nJewell G, McCourt ME: Pseudoneglect: a review and meta-analysis of performance factors in line bisection tasks. Neuropsychologia. 2000; 38(1): 93–110. PubMed Abstract | Publisher Full Text\n\nDelis DC, Robertson LC, Efron R: Hemispheric specialization of memory for visual hierarchical stimuli. Neuropsychologia. 1986; 24(2): 205–214. PubMed Abstract | Publisher Full Text\n\nMarshall JC, Halligan PW: Seeing the forest but only half the trees? Nature. 1995; 373(6514): 521–523. PubMed Abstract | Publisher Full Text\n\nNavon D: Forest before the trees. the precedence of global features in visual perception. Cogn Psychol. 1977; 9(3): 353–383. Publisher Full Text\n\nSchneider KA, Bavelier D: Components of visual prior entry. Cogn Psychol. 2003; 47(4): 333–366. PubMed Abstract | Publisher Full Text\n\nSimon JR: Reactions toward the source of stimulation. J Exp Psychol. 1969; 81(1): 174–176. PubMed Abstract | Publisher Full Text\n\nValsangkar-Smyth MA, Donovan CL, Sinnett S, et al.: Hemispheric performance in object-based attention. Psychon Bull Rev. 2004; 11(1): 84–91. PubMed Abstract"
}
|
[
{
"id": "2659",
"date": "18 Dec 2013",
"name": "Daniel Smith",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written paper describing a rigorously conducted study. The title accurately reflects the results and the abstract does a good job of summarizing the key results. It's not clear from the introduction whether patients experience attenuation of neglect only when wearing the prisms, or whether the effects persist after removal of the prisms. Perhaps the authors could add a sentence or two to clarify? My only comment on the results is that I would like to see estimates of effect sizes reported to help interpret the data. The conclusions are justified given the data.",
"responses": [
{
"c_id": "655",
"date": "21 Dec 2013",
"name": "Janet Bultitude",
"role": "Author Response",
"response": "Many thanks for these comments and suggestions. The journal editors have suggested that we await further reviewer comments before amending the text of the article. In the mean-time I am happy to address your comments here.Patients with hemispatient neglect demonstrate reduced symptoms after undergoing prism adaptation, and this amelioration generally persists for at least two hours after a single treatment session (Rossetti et al., 1998), and in some studies has been reported to persist for as much as a day (Farné et al., 2002) or even a week (McIntosh et al., 2002) following prism adaptation.We will revise the results section to include effect sizes."
}
]
},
{
"id": "3134",
"date": "17 Jan 2014",
"name": "Radek Ptak",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper presents a methodologically sound study that examined the effects of prism adaptation on within- and between-object shifts of attention in healthy participants. The main finding is negative, namely that prism adaptation neither affects space-based nor object-based attention. This finding was observed despite robust adaptation effects found in open-loop pointing. The conclusion of the authors is that prism adaptation may ‘only perturb cognitive functions for which normal baseline performance is already biased’, which according to them is not the case for the task used in the present study. The title of the article is appropriate and the abstract adequately summarizes the main findings. I recommend this article, but have three minor comments concerning the introduction, methods and interpretation of the results. IntroductionThe authors should be cautious when discussing the beneficial effects of prism adaptation on spatial neglect. Rehabilitation of neglect certainly is a great challenge and therapeutic techniques that appear to have lasting effects following very short interventions should undergo rigorous assessment with randomized, controlled clinical trials. It should be noted that the positive results of Rossetti et al. (1998), were not replicated in a more recent study using the same methodology (Rousseaux et al., 2006). In addition, a randomized study on a much larger group of neglect patients did not find any benefits of prism adaptation, as compared to repeated pointing with sham prisms (Turton et al., 2010).I think that in the domain of rehabilitation negative results are at least as important as positive results, and should therefore be acknowledged by the authors. MethodsThe fact that all participants responded with their right hand in the RT task is problematic, as small effects due to prism adaptation might be masked by larger effects due to space-hand mapping. I appreciate that the authors are aware of this limitation. ResultsI was surprised that the analysis of results presented in Figure 6 did not yield a main group effect (the authors only describe a marginal group x horizontal location interaction). Interestingly, both adaptation groups show increased RTs when compared to the non-adapted group, an effect that is consistent across all four locations. What was the p-value of the main group effect in the ANOVA and is there a possibility to further examine this group effect (e.g., by pooling the two prism adaptation groups and comparing them to the non-adapted group)? I think this slowing of RTs following prism adaptation, irrespective of direction of the adaptation, is worth at least a comment if not further study. Suggested literatureRousseaux M., Bernati T., Saj A., Kozlowski O., 2006. Ineffectiveness of prism adaptation on spatial neglect signs. Stroke 37, 542-543.Turton A.J., O'Leary K., Gabb J., Woodward R., Gilchrist I.D., 2010. A single blinded randomised controlled pilot trial of prism adaptation for improving self-care in stroke patients with neglect. Neuropsychol Rehabil 20, 180-196.",
"responses": []
},
{
"id": "2657",
"date": "07 Mar 2014",
"name": "Igor Schindler",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneralThis is a well designed study using appropriate methods. The authors demonstrate convincingly that prism adaptation does not impact on either within or between object attention shifts. The introduction is, overall, scholarly and concise and the discussion provides a comprehensive explanation of the findings by arguing that a biased normal baseline performance is a pre-requisite for observing significant changes in attention biases following prism adaptation. I only have a few very minor suggestions/comments the authors may want to consider:IntroductionAs Radek Ptak pointed out not all studies using prism adaptation in neglect yielded positive results.I agree regarding the inclusion of conflicting results but it is clear that the methods used varied between studies such as the extent to which the hand/arm was seen by the subject during pointing (full view of the arm vs. view of the finger only during adaptation). In addition, the type of prisms used varied across studies i.e. some used Fresnel prisms that do not provide a seamless shift of the visual field whereas others used prisms that fused magnifying lenses with prism lenses providing a seamless visual percept.Finally, the nature of brain damage itself may account for some patients showing larger prismatic after-effects than others. A recent fMRI study by Luauté et al. (2009) in healthy participants indicated increased activation of inferior parietal areas during the early stage of error detection and correction and temporal areas during the later stage of sensory realignment. Thus the combination of different prisms, adaptation protocols and lesion location/extent as well as lesion age might contribute in part to these conflicting findings.ResultsOn page 7 it is stated that the mean accuracy was 85% which could be further specified by including the accuracy range across adaptation groups to provide information on whether the accuracy levels were comparable.Also one interesting finding is that the neutral group seems to show faster reaction times compared to both prism groups irrespective of target location in the visual field. It would be useful to evaluate whether the two prism groups (pooled) showed overall higher reaction times than the neutral group as suggested by Radek Ptak but also whether the increase in reaction times was paralleled by a change in accuracy. If this is the case, it would suggest a perturbing overall impact of prism adaptation in healthy participants in the Egly task. This would potentially strengthen the argument of non–beneficial effects of prism adaptation in tasks that elicit unbiased performance in healthy participants.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-232
|
https://f1000research.com/articles/2-111/v1
|
18 Apr 13
|
{
"type": "Research Article",
"title": "Association between digital dermatoglyphics and handedness among Sinhalese in Sri Lanka",
"authors": [
"Buddhika TB Wijerathne",
"Geetha K Rathnayake",
"Geetha K Rathnayake"
],
"abstract": "Background\nThe relationship between handedness and digital dermatoglyphic patterns has never been investigated in the Sinhalese population. The goal of this study is to establish the above mentioned relationship, which would positively aid personal identification. Findings One hundred Sinhalese students (50 right-handed and 50 left-handed) were studied for their digital dermatoglyphic pattern distribution. The results show that a statistically significant correlation exists for digit 3 (P=0.002, Fisher’s exact test) of the right hand and digit 1 (P=0.002, Fisher’s exact test) and digit 2 (P=0.021, Fisher’s exact test) of the left hand. Further, whorl patterns are more common in right-handed than left-handed Sinhalese students.Conclusions Statistically significant differences in handedness and digital dermatoglyphic patterns were evident among Sinhalese people. Further study with a larger sample size is recommended.",
"keywords": [
"Forensic science",
"handedness",
"digital dermatoglyphics",
"fingerprints",
"personal identification",
"Sinhalese",
"Sri Lankans"
],
"content": "Introduction\n\nFingerprints (digital dermatoglyphics) are a unique form of evidence that greatly contribute towards personal identification in forensic science1. Because they are unique for each individual and are strongly influenced by genetics, they also perform a significant role in anthropology, human genetics, ethnology and medicine. They are characterized by alternating strips of raised friction ridges and grooves present in a variety of patterns2. These patterns start to develop between the 5th and 6th week of intrauterine life, and are fully formed by the 21st week3. These patterns do not change throughout postnatal life and their development is determined by several genes4.\n\nHandedness (i.e. hand dominance) is defined as the uneven distribution of fine motor skills between the left and right hand5. Determination of the handedness of both the assailant and the victim are important in various aspects of forensic science, including personal identification6. Hence, establishing the relationship between handedness and digital dermatoglyphics will aid forensic identification.\n\nTo date, scarce amount of studies7–13 have investigated whether there is a correlation between handedness and digital dermatoglyphics. In 1940 Cummins discovered a slight association in the sex differences of asymmetrical occurrence of dermatoglyphic patterns8. Cromwell and Rife in 1942 found that left-handers are characterized by slightly less bimanual asymmetry than right-handers among on Caucasian school children in southwestern Ohio9. In 1943 Rife found associations characteristic of autosomal linkage between the whorl frequencies on the fingers and handedness among descended from northern European stock10. In 1994 Coren reported an increased number of arches, fewer whorls in left-handers as compared to the right-handers among Canadians11. Cho in 2010 found significant difference of dermatoglyphics patterns on digit 3, 4 and 5 among Koreans12. None have investigated this association in a Sinhalese population (an Indo-Aryan ethnic group who are native to the island of Sri Lanka14). The main goal of the current study is to determine the relationship between handedness and digital dermatoglyphics in a sample of Sinhalese population.\n\n\nMethods\n\nThe study was conducted at the Department of Forensic Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka. Ethical clearance for this study was obtained from the Ethical Clearance Committee of the institute. A total of one hundred Sinhalese students (46 females, 54 males), who gave informed written consent, were included in the study. Their ages ranged between 22 and 28 years (mean ± s.d. = 25 ± 2 years). Firstly, handedness was assessed using the Edinburgh Handedness Inventory15. This required participants to demonstrate 10 unimanual tasks (preferred hand for writing, drawing, throwing, striking a match, opening a box, holding scissors, holding a toothbrush, holding a spoon, holding a broom and holding a knife). These tasks are common to Sri Lankans and they were advised to state the degree of preference for the hand used in each case as either strong (two points) or weak (one point). The handedness measure was calculated by subtracting the score for the left hand from the score for the right hand, dividing by the sum of both, and multiplying it by 100, providing an absolute range from -100 (completely left-handed) to +100 (completely right-handed). We recruited 50 predominant right-handers and 50 predominant left-handers after evaluating handedness.\n\nAll eligible students were asked to wash their hands thoroughly to remove dirt and dry them before obtaining fingerprints. Rolled prints were obtained by the ink and paper method as described by Cummins and Midlo2. The subject was asked to roll their finger from the radial side to the ulnar side on an ink pad and then transfer their fingerprints in the same manner onto the allocated area of a double sheet of plain A4 paper (Figure 1). In this way, fingerprints for all the ten fingers were obtained for each individual. Digits are numbered as follows; digit 1 (thumb), digit 2 (index finger), digit 3 (middle finger), digit 4 (ring finger) and digit 5 (little finger).\n\nA and B show the rolling of the finger from the radial side to ulnar side on an ink pad. C and D show the transference of fingerprints onto the allocated area of the paper.\n\nDigital dermatoglyphic patterns (Figure 2) were classified as follows; ulnar loop, radial loop, whorl (double loop whorl, plain whorl, central pocket loop and accidental whorl were counted as whorl) and arch (plain arch and tented arch were counted as arch). In this way, fingerprints of all the ten fingers were obtained for each individual.\n\nA: Ulnar loop, B: Radial loop, C: Plain Whorl, D: Double loop whorl, E: Plain arch, F: Tented arch, G: Accidental whorl, H: Central pocket loop.\n\nAnalysis was carried out using SPSS 17. Descriptive statistics were used to express the data. Correlations between handedness and digital dermatoglyphics were evaluated by a two-sided Fisher’s exact test. P values less than 0.05 were considered statistically significant.\n\n\nResults\n\nIn this study we observed the handedness-wise digital dermatoglyphics pattern distribution of 100 individuals (50 left-handed [31 females, 19 males] and 50 right-handed [27 males, 23 females]).\n\nTable 1 shows the digital dermatoglyphic pattern distribution of the right-handed Sinhalese students. Digit 3 of the right hand of right-handed students was found to have more ulnar loops (88%) compared to left-handers (60%), followed by 26% whorl on left-handed compared to 8% whorl in right-handed. Arches and radial loop patterns occurred less frequently. The rest of the digits on the right hand were not shown to be significantly different. Table 2 shows the digital dermatoglyphics pattern distribution of the left-handed Sinhalese students.\n\nThe right hand thumb (digit 1) of right-handed individuals had a higher ulnar loop frequency (54%) than whorl (46%) while the reverse was true for the left hands of right-handed persons (ulnar loop 40%, whorl 56%). The right hand index finger (digit 2) of right-handed persons had a higher ulnar loop frequency (52%) than whorl (36%), although whorls were more frequent (38%) than ulnar loops (30%) on the left hand of right-handed people for this digit. Radial loops were more common on the left hand compared to the right hand in both handedness groups.\n\nThe percentage of digital dermatoglyphics pattern distributions for both hands in both right- and left-handed Sinhalese individuals are shown in Figure 3.\n\nIn both hands of the total population 35.6 percent of whorls were found on right-handed people while 31.2 percent on left-handed. Radial loops were more commonly found on both hands of left-handed (6.2%) than right-handed (1.6%) people.\n\nA statistically significant correlation was observed in digital dermatoglyphic patterns between right- and left-handed people for digit 3 of the right hand (P=0.002, Fisher’s exact test), as well as for digits 1 (P=0.002, Fisher’s exact test) and 2 (P=0.021, Fisher’s exact test) of the left hand.\n\n\nDiscussion\n\nIt has been affirmed that the digital dermatoglyphic pattern of the skin is unique and unchallengeable for an individual1. This is valuable as a means of identification. In this study, effort has been made to study the relationship between dermatoglyphic and handedness in 100 Sinhalese students.\n\nThe statistically significant difference between handedness and the dermatoglyphic pattern in digit 3 on the right hand and digit 1 and digit 2 on the left hand were the most important finding of our study.\n\nIn the past, few studies have been conducted on different ethnic groups with the idea of establishing a relationship between handedness and dermatoglyphic pattern. Results of some studies are in line with the present study.\n\nIn their study on Caucasian school children in southwestern Ohio, Cromwell and Rife (1942)9 observed a slightly higher frequency of whorls (1.3%) on left ring fingers (digit 4) of left-handers than of right-handers. Whorls were absent on the right ring finger of both right- and left-handers. They further observed that the incidence of arches only on digit 3 of right hands shows highly significant differences between left-handers and right-handers (P<0.001).\n\nCoren (1994)11 in his study on Canadians found that left-handers were more likely to have arches and radial loops, while fewer whorls than right-handers. The correlation of handedness and digital dermatoglyphics was most marked on the left hand, which showed significant differences on four digits except digit 1. On the right hand, handedness was associated with a digital dermatoglyphics patterns only on digit 4.\n\nCho (2010)12, in their study on Koreans, found that both hands of left handers exhibited more arch and ulnar loop types than the right-handers and less whorl and radial loop types than the right-handers. The digital dermatoglyphic pattern of digit 3, digit 4 and digit 5 of the left hand showed a statistically significant relationship between left- and right-handed people.\n\nIn Karev's study on Bulgarian individuals13, he found that whorls were significantly less frequent, and ulnar loops significantly more frequent in all digits for right-handed people when compared to left-handed people. The ulnar fluctuating asymmetries of digits 1 and 4 showed a highly significant relationship with handedness.\n\nRife (1955)16, in his study on students at Ohio State University, USA, observed that arches were more common on the left middle finger of right-handed students than left-handed students.\n\nThe major limitation of our study is the small sample size, even though it was obvious that there is a significant handedness-wise difference of dermatoglyphics. The results can be used as supporting evidence for individual identification. Additional research involving larger samples are needed to further confirm current findings.\n\n\nConclusion\n\nThe present study supports the hypothesis that handedness and digital dermatoglyphics are correlated in members of the Sinhalese population. Our results show that there is a statistically significant difference in fingerprint patterns between right- and left-handed people for digit 3 on the right hand and for digits 1 and 2 on the left hand. The results of this study support the relationship between handedness and digital dermatoglyphics in the Sinhalese population.",
"appendix": "Author contributions\n\n\n\nBTBW was involved in study conception and design. BTBW and RMGK assisted with data collection, study coordination and data analysis. Both authors were involved with drafting the manuscript. Both authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGirard J: Criminalistics: Forensic science and crime. Illustrated ed. USA: Jones & Bartlett Learning; 2007; 128–152. Reference Source\n\nCummins H, Midlo C: Finger prints, palms and soles: An introduction to dermatoglyphics. Illustrated Ed. New York: Dover Publications; 1961. Reference Source\n\nMiličić J, Pavićević R, Halbauer M, et al.: Analysis of qualitative dermatoglyphic traits of the digito-palmar complex in carcinomas. In The State of Dermatoglyphics: The Science of Finger and Palm Prints. Illustrated Edition. Edited by Norris M Durham, Kathleen M Fox, Chris C Plato: Pennsylvania State University; Edwin Mellen Press; 2000: 29(2): 637–42. Reference Source\n\nBonnevie K: Studies on papillary patterns of human fingers. J Genet. 1924; 15(1): 1–111. Publisher Full Text\n\nRaymond M, Pontier D: Is there geographical variation in human handedness? Laterality. Asymmetries of Body, Brain and Cognition 2004; 9(1): 35–51. PubMed Abstract | Publisher Full Text\n\nStark MM: Clinical forensic medicine: a physician's guide. Humana Press; 2011; 473. Reference Source\n\nCummins H, Leche S, McClure K: Bimanual variation in palmar dermatoglyphics. Am J Anat. 1931; 48(1): 199–230. Publisher Full Text\n\nCummins H: Fingerprints correlated with handedness. Am J Phys Anthropol. 1940; 26(1): 151–66. Publisher Full Text\n\nCromwell H, Rife DC: Dermatoglyphics in relation to functional handedness. Hum Biol. 1942; 14: 517–526. Reference Source\n\nRife DC: Genetic interrelationships of dermatoglyphics and functional handedness. Genetics. 1943; 28(1): 41–8. PubMed Abstract | Free Full Text\n\nCoren S: Are fingerprints a genetic marker for handedness? Behav Genet. 1994; 24(2): 141–8. PubMed Abstract | Publisher Full Text\n\nCho KJ, Kim SI: Characteristics of Fingerprints According to Type of Handedness. Korean J Phys Anthropol. 2010; 23(1): 21–31. Reference Source\n\nKarev GB: Finger dermatoglyphics and their asymmetry in Bulgarian right-, mixed- and left-handers. Anthropol Anz. 2008; 66(3): 281–93. PubMed Abstract\n\nMinahan JB: Ethnic Groups of South Asia and the Pacific: An Encyclopedia. ABC-CLIO, 2012. Reference Source\n\nOldfield RC: The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia. 1971; 9(1): 97–113. PubMed Abstract | Publisher Full Text\n\nRife DC: Hand prints and handedness. Am J Hum Genet. 1955; 7(2): 170–9. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "931",
"date": "07 May 2013",
"name": "Esperanza Gutierrez Redomero",
"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 study was based on data from 100 individuals (50 left-handed [31 females, 19 males] and 50 right-handed [27 males, 23 females]). The greatest limitation of this study is the small sample size, as authors indicate; this size would be even smaller if the sample was analyzed by sex, as should have been done. The distribution of digital patterns presents sex differences in most of the samples analyzed, and so, the results cannot be accepted because they are affected by the effect of sex differences.",
"responses": [
{
"c_id": "495",
"date": "08 Jul 2013",
"name": "Buddhika Wijerathne",
"role": "Author Response",
"response": "We would like to thank Professor Esperanza Gutierrez Redomero for the valuable time spent reviewing our manuscript and the important comments she has made. We have attempted to address and incorporate most of the concerns that were raised in version 2 of our article."
}
]
}
] | 1
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https://f1000research.com/articles/2-111
|
https://f1000research.com/articles/2-231/v1
|
01 Nov 13
|
{
"type": "Research Article",
"title": "Translate gene sequence into gene ontology terms based on statistical machine translation",
"authors": [
"Wang Liang",
"Zhao Kai Yong",
"Zhao Kai Yong"
],
"abstract": "This paper presents a novel method to predict the functions of amino acid sequences, based on statistical machine translation programs. To build the translation model, we use the “parallel corpus” concept. For instance, an English sentence “I love apples” and its corresponding French sentence “j’adore les pommes” are examples of a parallel corpus. Here we regard an amino acid sequence like “MTMDKSELVQKA” as one language, and treat its functional description as “0005737 0006605 0019904 (Gene Ontology terms)” as a sentence of another language. We select amino acid sequences and their corresponding functional descriptions in Gene Ontology terms to build the parallel corpus. Then we use a phrase-based translation model to build the “amino acid sequence” to “protein function” translation model. The Bilingual Evaluation Understudy (BLEU) score, an algorithm for measuring the quality of machine-translated text, of the proposed method reaches about 0.6 when neglecting the order of Gene Ontology words. Although its functional prediction performance is still not as accurate as search-based methods, it was able to give the function of amino acid sequences directly and was more efficient.",
"keywords": [
"GFP",
"gene function prediction",
"machine translation"
],
"content": "Introduction\n\nDetermining the functions of proteins is a central problem in biology. There are many databases like RefSeq (http://www.ncbi.nlm.nih.gov/refseq/) that store amino acid sequences and their corresponding functions. However, almost all protein functional prediction methods rely on the identification, characterization, or quantification of sequence similarities between the available proteins of interest1. Even sequences that are similar do not necessarily have identical function. A sequence may be similar to many other sequences, so it can be difficult to choose the most appropriate one. In addition, there is no way to deduce function if there are no similar sequences in any available database.\n\nAt present, many machine learning research methods are being applied to deal with the above mentioned problems. Two examples are, Support Vector Machines (SVM); a supervised learning model which is mainly used for classification and regression analysis, and network-based methods such as protein interactome networks2,3. In addition, the sequence features, structures, evolution of amino acid sequences, and other characteristics are also utilized for functional prediction4,5.\n\nThe same sequence may have different meanings in different contexts. Different sequences may also represent the same meaning. These problems are normally called “lexically/structurally ambiguous” in the language translation field. Current machine translation research could effectively avoid these problems by building a translation model between two ‘languages’.\n\nSince many amino acid sequences and their corresponding functional description are already publically available, it is possible to build their translation relationships by mature machine translation technology. We address this task in this paper. When presented with a new amino acid sequence we aimed to provide a function-based translation.\n\nTo build the model, we need to construct the “parallel corpus” of amino acid sequences and their functional descriptions. The term “parallel corpus” is typically used in linguistic areas to refer to texts that are translations of each other. A parallel corpus contains the data for two languages.\n\nFor example, a parallel corpus from English to French translation:\n\nEnglish sentence: I love apples\n\nFrench sentence: J’adore les pommes\n\nHere, we selected amino acid sequences and their functional descriptions in Gene Ontology form to build the corpus6. The reason for using the Gene Ontology is that it gives the unified descriptions for protein functions of all species. The Gene Ontology data can be found on the website www.geneontology.org (http://www.geneontology.org/GO.downloads.annotations.shtml). The corresponding amino acid sequence data can be downloaded from the website www.uniprot.org (http://www.uniprot.org/downloads), which is a central repository of amino acid sequence. We use the database ID to identify a sequence and find its correspondences in above two data sources.\n\nAn example of a parallel corpus from “amino acid sequence” to “function” translation:\n\nAmino acid sequence (amino acid):\n\nMTMDKSELVQKAKLAEQAERYDDMAAAMKAVTEQGHELSNEERNLLSVAYKNVV\n\nFunction description (Gene ontology term ID):\n\n0005737 0006605 0019904 0035308 0042470 0042826 0043085\n\nThe basic idea of machine translation is very simple. It tries to infer “rules” from the parallel corpus and build a translation model. The “rules” are mainly the words or phrase correspondences between two languages. Similar sequences will get near translation results. Although the application of translation technology in protein functional prediction seems reasonably simple, as far as we know, there is no existing program that applies machine translation to protein sequence function analysis.\n\n\nParallel corpus\n\nHere we build three groups of gene parallel corpora. Group 1 only contains human amino acid data. It has 45,538 amino acid sequences and related Gene Ontology descriptions. Group 2 is a mixed dataset of several model species. It contains about 100,000 amino acid sequences. Group 3 is the largest dataset. It contains more than 2,000,000 amino acid sequences. All of the corpora can be downloaded from www.geneontology.org (Gene Ontology data) and www.uniprot.org (amino acid sequence data). We also provide these Gene ontology and amino acid sequence data in ‘data’ directory of Supplementary material with this paper.\n\nFirst, we need to “clean” the corpus. Data cleaning is the process of detecting and correcting (or removing) corrupted or inaccurate records from a dataset. For machine translation, sequences with significant differences in length between the sources and targets should be removed. The empty and overlong sequences should also be deleted. For amino acid data, we deleted long sequences (amino acid sequence length<1000 (amino acids), Gene ontology terms number<100) and empty sequences.\n\nAfter data cleaning, we had about 14,000 pieces of data for 45,538 human amino acids. Data of groups 2 and 3 are also reduced to about 1/4 of the original data size.\n\nAmino acid sequences are not naturally segmented. So we still need to “segment” them.\n\nText segmentation means dividing a string of written language into its component words. In English and many other languages using some form of the Latin alphabet, the space is a good approximation of a word delimiter. But for some East-Asia languages like Chinese, there are no natural delimiters. To process these languages, we need to segment sentences into “words” sequences first. For example:\n\nA Chinese sentence:\n\n我愛苹果(Iloveapple)\n\nWe need its word sequence form:\n\n我 愛 苹果(I love apple)\n\nSegmenting methods on processing East-Asia languages are applied into amino acid sequence segmentation. Here we segment/tokenise amino acid sequences by an unsupervised segmentation method9. Its main idea is to evaluate the probabilities of all possible words by Expectation Maximization (EM), an iterative method for finding maximum likelihood estimates of parameters in statistical models10, or other statistical methods like the n-grams language method11. Then for a sequence, we select the segmenting form with maximum probability as the segmentation of this sequence.\n\nAn example of parallel corpus about “segmented amino acid sequence” to “function”:\n\nAmino acid sequence (amino acid, segmented):\n\nMTMDKS ELVQKA KLAEQA ERYDDM AAA MKAVTE QGH ELSNEE RNLLSV AYKNVV\n\nFunction description (Gene ontology terms ID):\n\n0005737 0006605 0019904 0035308 0042470 0042826 0043085\n\nAfter segmenting, we also delete the obviously mis-aligned sequences (‘word’ number ratio>9. For above example, there are 10 amino acid words and 7 Gene ontology word. So its word number ratio is 10/7) in corpus.\n\n\nTranslation model\n\nThere have been many open source translation systems. Normally, the Moses system is treated as the baseline system12. Moses is a statistical machine translation system that allows you to automatically train translation models for any language pair. All you need is a collection of translated texts (parallel corpus). Once you have a trained model, an efficient search algorithm quickly finds the highest probability translation among the exponential number of choices. Here we also use the Moses system to build the translation model for amino acid sequence to Gene ontology terms. Moses offers two types of translation models: phrase-based and tree-based. We selected the phrase-based model.\n\nWe used 95% of the data to train the model, and the remaining 5% of the data for testing.\n\nThe input for Moses is the parallel corpus. All parameters are set by default values (more details reference in the “data” and “code” sections of the Supplementary materials). The language model for the Gene ontology sequence is set as 2-grams (in the fields of computational linguistics and probability, an n-gram is a contiguous sequence of n items from a given sequence of text. For more information, see Xiaping Ge, Wanda Prat, and Padhratic Smyth10). Then we could get an amino acid sequence to Gene ontology translation model. The detailed process is provided in the ‘code’ directory of Supplementary materials. Its main steps are described as in Figure 1.\n\nWe used the Bilingual Evaluation Understudy (BLEU) score to judge the performance of the translation system13. BLEU is an algorithm for evaluating the quality of text which has been machine-translated from one natural language to another. The main idea of BLEU score can be described by formula 1.\n\n\n\nIn this formula m is the number of words from the candidate translation that are found in the reference translation, and wt is the total number of words in the candidate translation.\n\nFor example, a candidate translation is “I air apple”. The reference translation is “I love apple”. There are 3 words in the candidate translation. Two words, “I” and “apple”, appear in the reference translation. So the BLEU score of this translation is 2/3=0.67.\n\nThe improved version of BLEU normally considers the n-grams match. Since we don’t care about the order of Gene ontology words, we only needed to use the unigram form of BLEU.\n\nBecause different amino acid sequence segmentation methods will produce completely different “word” sequences, and the segment methods mainly rely on the selection of maximal word length, we tried different maximal word lengths. The relation of maximal word length and the BLEU score are shown in Figure 2.\n\nThe red line corresponds to group 1, human dataset. Blue line is group 2, mixed dataset and green is group 3, the big dataset.\n\nFigure 2 shows maximal word length of 7 or 8 letters is a good choice for amino acid translation. In theory, we need 20n letters corpus to train an n word length segmenting model. For n=8, we need about 26 Gigabytes amino acid sequence data. For n=9, we need 512 Gigabytes of data. Now there are only about 20 Gigabytes in annotated amino acid sequence data in Gene ontology databases (http://www.geneontology.org/GO.downloads.annotations.shtml). So a practical word length selection could be 7.\n\nIf we don’t segment the amino acid sequence and treat every amino acid letter as a “word”, 20+ amino acid letters could only express 20+ functions. But from Figure 2, we deduct that we could still get a translation model if we have sufficient corpus. This was mainly because the training system could combine some consecutive letters into “phrases” in the alignment process. So such “phrases” could represent more things.\n\nIn Figure 2, we could also find the increasing of the size of corpus could obviously improve the performance of translation. So far now, we tried a 5,000,000 amino acid sequences data set. Its BLEU score could reach 0.6. We also provide the method to build 40,000,000 amino acid sequences corpus data in ‘get_gene_data’ directory of supplied materials.\n\nFor a usable translation system, its unigram BLEU score should be more than 0.5. Our amino acid sequence to Gene ontology translation model has reached this level. So we could compare it with conventional search based method.\n\n\nComparing with search-based method\n\nThe sections above show that the “amino acid sequence” to “protein function” translation is workable. Next, we compared it with current protein functional prediction methods.\n\nWe designed the comparison as follows:\n\n(1) Dataset: we divided the parallel corpus into 95% training corpus and 5% test corpus.\n\n(2) For the translation-based method: we trained a “amino acid sequence” to “Gene ontology” translation model based on the training corpus and then predicted the functions of a test amino acid sequence based on this translation model.\n\n(3) For the search-based method: we indexed the amino acid sequences in the training corpus by BLAST to build a training sequences database. To predict the function of the test sequences, we searched the test amino acid sequence in database of training sequences respectively. The Gene ontology description of the best match sequence was regarded as the function of this test sequence.\n\n(4) Comparing method: we compared the predicted functions of two methods by the BLEU score. The results are shown in Table 1.\n\nFrom Table 1, we found that the functional prediction performance of the translation method was still not as good as BLAST, but there were about 1%–5% sequences whose functions the BLAST method could not predict. On the other hand, the translation method could predict the functions of all amino acid sequences. Here, we mainly want to show the feasibility of the new method. More datasets and translation methods should be tested in the future.\n\n\nSummary\n\nBased on statistical machine translation technology, we present a novel method to predict the function of amino acid sequences. Although its performance remains to be improved, it shows that the application of machine translation in protein functional prediction promising. For statistical translation methods, the more corpuses we generate, the better results we would obtain. Google’s translation system has proven that vast amounts of corpus with a few rules, or even without any rules, can produce excellent translation results14. Since the protein functional prediction problem is so close to ‘language translation’, translation based protein functional prediction is likely to be the most promising approach. Source codes are permanently accessible from 10.5281/zenodo.7506.",
"appendix": "Author contributions\n\n\n\nWang Liang designed the experiments, wrote the codes and trained the translation model. Zhao Kaiyong prepared the experimental data and provided the biology knowledge supports.\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 materials\n\nThere are 5 directories in Supplementary materials.\n\nDirectory list:\n\n1. “data”: parallel corpus for Gene ontology sequence and corresponding amino acid sequence.\n\n2. “code”: codes and directions for building translation model.\n\n3. “get_gene_data”: direction for downloading data from geneontology.org(Gene ontology data) and uniprot.org(amino acid sequence data)\n\n4. “pre_process_gene_data”: convert the format of original Gene ontology data and amino sequence data to the required format of Mose system.\n\n5. “build_dict”: direction for building amino acid dictionary.\n\nhuman.pr: human protein sequence (amino acid form) for training, which has been segmented (set maximal word length as 7, same for other gene corpus)\n\nhuman.go : human gene function description in Gene ontology terms, for training\n\nhuman.pr.test : human amino acid sequence (amino acid form) for test, has been segmented\n\nhuman.go.test : human gene function description in Gene ontology terms, for test\n\nmixed.pr : mixed amino acid sequence (amino acid form) for training\n\nmixed.go : mixed gene function description in Gene ontology terms, for training\n\nmixed.pr.test : mixed amino acid sequence (amino acid form) for test, has been segmented\n\nmixed.go.test : mixed gene function description in Gene ontology terms, for test\n\nbig.pr : big amino acid sequence (amino acid form) for training\n\nbig.go : big gene function description in Gene ontology terms, for training\n\nbig.pr.test : big amino acid sequence (amino acid form) for test, has been segmented\n\nbig.go.test : big gene function description in Gene ontology terms, for test\n\nIt contains the source and example to train the gene translation model, we use the mixed data set to train the translation model, include:\n\nreadme.txt: direction about how to train the model\n\nContent in readme:\n\n(1) You need install Moses system. http://www.statmt.org/moses/?n=Development.GetStarted\n\n(2) You’d better run the baseline of Moses. http://www.statmt.org/moses/?n=Moses.Baseline\n\n(3) Python 2.7.\n\nHere we use irstlm to train language model, as this baseline suggest.\n\nfor train:\n\ngene.6.pr : amino acid sequence, have been segmented, maximal word length 6\n\ngene.6.go: gene ontology sequence\n\nfor test:\n\ngene.6.pr.test: amino acid sequence\n\ngene.6.go.test: gene ontology sequence\n\nfile: train_lm.sh\n\n(attention: please revise the moses_path and irstml_path to your own path!)\n\nrun:\n\n./train_lm.sh gene.6.go\n\nYou will get gene.6.go.blm.\n\nFile: train_ts.sh\n\n(attention: please revise the moses_path and giza_path to your path!)\n\nrun:\n\n./train_ts.sh gene.6 gene.6.go.blm\n\nWait about 4 hours (1 CPU. 10+G memory requirement)\n\nyou will get a \"train\" directory.\n\nthe train/model/moses.ini is just your translation model file\n\nFile: get_bleu.sh\n\n(please revise the moses_path)\n\nrun:\n\n./get_bleu.sh gene.6.go.test gene.6.pr.test\n\nit will translate gene.6.pr.test and then compare it with gene.6.go.test. If there is no error, you will get the result:\n\nBLEU = 26.38, 47.4/30.0/21.4/15.9 (BP=1.000, ratio=1.571, hyp_len=48369, ref_len=30786)\n\nBecause we don't consider the order of go terms, 47.4 is just its BLEU score (unigram). Here we didn’t run the tuning process. You could divide the test corpuses into test part and tuning part, and then run the tuning process. It could bring some improvement for BLEU score, but it may take several days.\n\nIf you want build your own corpus from original database source (www.geneontology.org, Gene ontology data. www.uniprot.org, amino acid sequence data), see this section:\n\nHere we will show how to get corpus from original data source. You need install gunzip.\n\nUsing human data:\n\nFile: get_gene_data.sh, its operation\n\n(1). get original gene ontology data. (ftp of www.geneontology.org).\n\n(2). get amino acid sequence data. (ftp of ftp.uniprot.org).\n\n(3). gunzip\n\n(4). convert amino acid sequence format. (get_gene.py)\n\n(5). convert gene ontology format. (get_go.py)\n\n(6). Get parallel corpus according to gene database id. (get_corpus.py)\n\njust run ./get_gene_data.sh\n\nWe will get parallel corpus\n\nhuman.pr.filter gene sequence\n\nhuman.go.filter corresponding gene ontology sequence\n\nThis corpus contain about 1,8000 data.\n\nYou could also download the big corpus:\n\nGene ontology files (UniProt [multispecies]):\n\nhttp://www.geneontology.org/gene-associations/submission/gene_association.goa_uniprot.gz\n\ncontain about 40 million data.\n\nRelated sequence data could be found at:\n\nUniProtKB/Swiss-Prot,\n\nftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz\n\nUniProtKB/TrEMBL,\n\nftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_trembl.fasta.gz\n\nYou could download these two files then merge (normally Linux shell ‘cat’ command) them together. Then revise the ./get_gene_data.sh. (refer to get_big_gene_data.sh). Finally, you could get about 20 million parallel corpus.\n\nIf you want to build your own corpus, you will need segment the amino acid sequence and clean the corpus.\n\n(1) mix.go original gene ontology sequence\n\n(2) mix.pr original amino acid sequence\n\n(Here we give these data. You could also copy the human.pr.filter in previous to mix.go, and human.go.filter to mix.go to run your own experiment, also for the big data set)\n\n(3) protein.dict dict file\n\nFile : pre_process.sh\n\njust run:\n\n./pre_process.h\n\nIts operation:\n\nstep 1: segment the amino acid sequence. (segment_gene_sequence.py)\n\nstep 2: clean the corpus.( clean_corpus.py)\n\nstep 3: divide the corpus into train and test parts.( divide_corpus.py)\n\nfinally, you will get 4 files\n\ngene.pr : amino acid sequence, have been segmented, maximal word length 6\n\ngene.go: gene ontology sequence\n\ngene.pr.test: amino acid sequence for test\n\ngene.go.test: gene ontology sequence for test\n\nThese files could be used to train the translation model.\n\nMoreover, if you want to build your own “protein.dict” for different maximal word length, we also give an example:\n\nStep 1, install SRILM\n\nA simple method to build dictionary is to use language model. Here use SRILM. You should install it first. (http://www.speech.sri.com/projects/srilm/)\n\nThen cp the executable file “ngram-count” to this directory. Normally in your install dir. There have been an “ngram-count”, but it could only run in a special Linux version, so just overwrite it.\n\nStep 2, you should make 2 file\n\n(1) In ./get_gene_word: run make\n\n(2) In ./get_gene_word_prob: you should set the srilm install path and MACHINE_TYPE in Makefile, then run make.\n\nStep 3, uncompress the data file\n\nRun: tar -xzvf protein.fa.tar.gz\n\nStep 4, train n-gram language model, n=1–5\n\nRun: ./build_all_lm_model.sh\n\n(Attention, you must cp “ngram-count” to this directory. See step1)\n\nStep 5, Build dictionary with different maximal word length\n\nRun: ./build_dict_all.sh\n\nIts operations:\n\n(1) Get all possible gene word, filter them by frequency. (get_word)\n\n(2) Get probability of all gene words. (get_prob)\n\n(3) Filter the gene words in dictionary by MI method. (mi_filter.py)\n\nFinally, you will get protein.dict.*.mi, (* is 1,2,…,5) is the dictionary with maximal word length *. Then you could use these dictionary file to segment the amino acid sequence (in section 3).\n\nIf you have map/reduce cluster, you could use EM method to build the gene word. See our open source project (https://code.google.com/p/dnasearchengine/). To train an 8 maximal word length dictionary, you need at least 4G amino acid sequence data. More amino acid sequence could be found in “Get gene data” section or ftp of gene Refseq databases. ftp://ftp.ncbi.nih.gov/refseq/.\n\n\nReferences\n\nHawkins T, Chitale M, Luban S, et al.: PFP Automated prediction of gene ontology functional annotations with confidence scores using protein sequence data. Proteins. 2009; 74(3): 566–82. PubMed Abstract | Publisher Full Text\n\nGuan Y, Myers CL, Hess DC, et al.: Predicting gene function in a hierarchical context with an ensemble of classifiers. Genome Biol. 2008; 9(Suppl 1): S3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPavlidis P, Gillis J: Progress and challenges in the computational prediction of gene function using networks [v1; ref status: indexed, http://f1000r.es/SqmJUM]. F1000Res. 2012; 1: 1–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEisen JA: Phylogenomics: Improving Functional Predictions for Uncharacterized Genes by Evolutionary Analysis. Genome Res. 1998; 8(3): 163–167. PubMed Abstract | Publisher Full Text\n\nWhisstock JC, Lesk AM: Prediction of protein function from protein sequence and structure. Q Rev Biophys. 2003; 36(3): 307–40. PubMed Abstract\n\nAshburner M, Ball CA, Blake JA: 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\nBrown PF, Cocke J, Stephen A, et al.: A Statistical Approach to Machine Translation. Computational Linguistics. 1990; 16(2): 79–85. Reference Source\n\nChiang D: Hierarchical phrase-based translation. Computational linguistics. 2007; 33(2): 201–228. Publisher Full Text\n\nLiang W, Kaiyong Z: Segmenting DNA sequence into 'words'.Reference Source\n\nGe X, Prat W, Smyth P: Discovering Chinese words from unsegmented text.Proceedings on the 22 Annual International ACM SIGIR Conference On Research and Development in Information Retrieval. Berkeley CAUSA. 1999; 271–272. Publisher Full Text\n\nRosenfeld R: Two decades of statistical language modeling: where do we go from here? Proceedings of the IEEE. 2000; 88(8): 1270–1278. Publisher Full Text\n\nKoehn P, Hoang H, Birch A, et al.: Moses: Open Source Toolkit for Statistical Machine Translation.Annual Meeting of the Association for Computational Linguistics (ACL), demonstration session, Prague, Czech Republic. 2007; 177–180. Reference Source\n\nPapineni K, Roukos S, Ward T, et al.: BLEU: a method for automatic evaluation of machine translation.ACL-2002: 40th Annual meeting of the Association for Computational Linguistics. 2002; 311–318. Publisher Full Text\n\nMikolov T, Chen K, Corrado G, et al.: Efficient Estimation of Word Representations in Vector Space.In Proceedings of Workshop at ICLR.2013. Reference Source"
}
|
[
{
"id": "2296",
"date": "04 Nov 2013",
"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\nThis paper describes a method for predicting gene function based on amino acid sequence, using a method inspired by automatic translation of human language. The idea is to break the sequence into segments, and each segment is assigned a GO term. During training the association between segments and GO terms is learned, and then this learned model is applied to new sequences. In a benchmark against BLAST performance it is much worse at identifying the GO terms associated with a protein. The defence offered that the work shows the feasibility of the approach falls a bit flat for me. It is feasible in the sense that “it can be attempted”. I do not find the results encouraging. I disagree with the claim that protein function prediction is \"so close\" to language translation, except in some rarefied technical sense. Indeed, using translation seems to be obscuring the actual task, which (in this context) is to find sequences that are similar and transfer GO annotations from one to the other. At the heart of it, that is all that is happening here.I know I am not supposed to address novelty, but I have to point out that many feature extraction methods have been proposed for sequence analysis (for example, string kernels combined with SVMs1). Many methods for sequence scoring are based on methods also used for analyzing human language (e.g. HMMs2), but without the baggage of “translation”. The authors do not make these connections in the introduction. In addition:The method is not described in enough detail, especially for bioinformatics researchers who may not be familiar with the translation methods (including myself). The provision of source code, while laudable, is insufficient. For example, for the segmentation method, the reader is directed to a non-peer reviewed manuscript on DNA segmentation. Why does each segment get at most one GO term? If that is necessary, it seems a big problem, since GO does not work that way. BLAST also does not have the limitations that are exposed by the requirements for cleaning described here. What makes a protein sequence “corrupted” or “inaccurate”? I do not get what the authors mean by \"obviously misaligned\" in comparing GO terms to protein sequences; it makes little sense and the strained relationship of the task to translation is exposed. Then authors seem to be requiring exact matches; this is also not very biological. It may be that these are fundamental limitations of the approach; if not, they must be addressed. (I did not take the time to review the source code). The results are not described in enough detail and the evaluation is weak. By optimizing the word length on the same corpus used for testing, the authors have probably inflated the performance scores, not that it helps much. The authors claim that an advantage of the method is that it always gives a prediction, even when BLAST does not. Is it really reasonable to trade specificity for sensitivity in this way? Can the authors show any cases where this yields a useful answer? Showing some cases where a correct functional annotation was found by this method, but not by BLAST, and providing some insight into why that happened, is necessary. The authors mention in the introduction that sequences that are similar may not have the same function; are they claiming that this new method addresses this? I do not think so, so it seems irrelevant.Given all these issues, I nearly checked the \"not approved\" box. But on consideration I think the work could be fixed up, from a technical standpoint.There are also quite a few English language errors.",
"responses": [
{
"c_id": "604",
"date": "05 Nov 2013",
"name": "亮 王",
"role": "Author Response",
"response": "We thank the referee for their suggestions.In next version of this paper: 1:We will give a more in depth introduction to machine translation and segment technologies. We will also add detail as to why we applied translation to protein functional prediction. Although these technologies are well known in the area of natural language processing, it is completely new for most researchers in bioinformatics. I have written a short PPT for sequence segmentation:http://www.slideshare.net/maris205/segmenting-dna-sequence-into-words We will also build an online demo for this work and its codes will also be uploaded.http://www.dnasearchengine.com/transMost current machine translation researches are based on the work of IBM in 1990:Brown et al Statistical Approach to Machine Translation, Computational Linguistics.16(2),79-85(1990).This translation model (IBM model 5) mainly contains 5 steps: word alignment, phrase extraction, language model training, decoder training, and evaluation.The translation model also uses the string features but it builds the phrase corresponding model (one phrase corresponds to a phrase list with probability, not one to one), but not the classification model like SVM. Asides from language translation, translation models are also applied in query rewrite, multi-label classification and many other areas. Because protein functional prediction could be regarded as a kind of multi-label classification problem (one sequence to several GO terms), we argue that one could use translation methods in protein functional prediction. 2: The referee requests more evaluations - There still seems to be no standard data set and evaluation methods for protein functional predictions. We will find a proper data set and try different evaluation metrics. More available function prediction methods will also be tried."
}
]
},
{
"id": "6149",
"date": "20 Oct 2014",
"name": "Karin Verspoor",
"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 provides an intriguing approach to protein function prediction -- to treat the task as a word translation process. However, the results that the authors provide to not justify the statement that the approach is likely to be \"the most promising approach\" and are not appropriately compared with state of the art methods. Furthermore, no biological intuition appears to have informed the approach; this results in an approach that, while novel, doesn't capture some fundamental characteristics of the data.Protein sequence - function relationship: The fundamental assumption embodied by the method is that there is a strong relationship between protein (sub-) sequences and functions, as represented by sequences of GO terms. While this is likely true for some sub-sequences, it won't be true for all, as the authors themselves point out.Does the method allow for some sequence \"words\" to be effectively silent in the prediction phase? Figure 1 shows each word being included in some phrase which is \"translated\". Can some words remain untranslated? How often does this happen for this task?What about sequence words that should correspond to multiple GO terms?Is the sequence of words biologically important? The \"Best BLAST\" method that the authors compare with (NB: I wouldn't call that approach \"search-based\" as that name is extremely underspecified; rather I would suggest \"sequence similarity-based\") uses local sequence similarity; some regions of the sequence will not be considered.It certainly isn't obvious to me that linear order should matter at all on the output side; it appears that the GO sequences are listed in numeric order by ID and a \"2-gram language model\" over GO sequences should not carry any more information than a 1-gram model would, as the IDs themselves are arbitrary. This representation is not well-justified.Segmentation: The authors use a EM-based approach to drive segmentation, which essentially captures the statistical properties of particular subsequences. There should be some relationship to work on motif finding here; the authors should identify some relevant work on that topic (e.g., Tompa et al. 1, although there are likely many more reviews available; as this is not my area specifically I suggest the authors do their own search). The authors should also compare to a far simpler segmentation approach: just splitting the sequence into words of fixed length. This is a character-level n-gram representation. Perhaps all the work of running EM (and the associated large number of data points) is completely unnecessary. (The authors would of course need to try a few different lengths for this to be meaningful.) Essentially, the strategy needs to be justified, and the impact on the broader function prediction task must be quantified. What is the relationship between the maximal word length and the number of output terms included in the output GO sequence? Is there a length bias on the output side that is also reflected in Figure 2? Again, this hinges on the appropriateness of characterising this task in terms of the translation model.Data: The authors do not clearly state whether they consider all GO annotations available for a protein, or if non-experimental evidence codes are excluded. If \"Inferred by Electronic Annotation\" codes are included, this means that \"Best BLAST\" has likely already been used to produce the annotations, which would explain at least to some extent why the performance of that method shows such strong results.Experiment details: The authors do not specify what e-value threshold they assume for BLAST. Filtering: The authors incorporate various filters along the way, and it is unclear how strongly this biases the training/test data that they utilise. The authors delete long sequences and proteins with many associated GO terms. The cleaning reduces the data set substantially, but the rationale for doing this cleaning appears to have more to do with the limitations of the approach that the authors have adopted than because of some biologically valid reason. The exclusion of such a large number of {sequence,GO annotations} pairs will certainly undermine the findings. This also applies to the deletion of the \"obviously mis-aligned\" sequences. How many instances does this filter? Why are these obvious mis-alignments? The number of GO terms associated with a sequence has more to do with (a) annotation bias [some sequences are far more heavily studied/annotated than others] and (b) overall progress on curating protein function information (see Baumgartner Jr. et al. 2).Evaluation: Recently, the CAFA challenge explored protein function prediction from sequence 3, and in association with that there has been detailed consideration of evaluation of this task 4 5 -- the key point being that the structure of the GO should be taken into consideration in the evaluation. The authors present their results in terms of BLEU, but in their use of the unigram form, I think that BLEU simply collapses to Precision. This, then, ignores Recall completely. The evaluation presented should certainly measure Recall, and the authors should provide results that are comparable to the results of systems that participated in CAFA. They should then compare their results to the state-of-the-art methods.SummaryIn summary, the applicability of the method to this task should be more strongly justified and the experimental set-up (including the extensive filtering of data points and the evaluation metrics) should be more carefully considered. The authors' conclusions have not been adequately justified on the basis of their results.",
"responses": []
},
{
"id": "6146",
"date": "22 Oct 2014",
"name": "Juliana Bernardes",
"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 present a new method to predict protein function based on machine translation concepts. In this scenario, protein sequences and their gene ontology terms are interpreted as words in different languages. The idea is to find the best translation for a protein sequence, that is, its most likely gene ontology terms. The method has two phase: training and inference. During the training, protein sequences are broken into several segments and then these segments are related to gene ontology terms. This mapping between protein segments and gene ontology terms constitutes the parallel corpus and it is learned during the training phase. Next, the inference is carried out by an statistical machine translation system that translates a new protein sequence into gene ontology terms by using the collection of translated pairs (parallel corpus) learned previously.My main issue with this paper is the way how sequences are broken into segments. It is well known that some segments such as motifs and domains are essential to predict protein functions. The author must explore them to enrich their model. A second point concerning the results, they are disappointing. The method did not outperform BLAST that is one of the simpler existing method for protein function prediction. The comparison was not properly done, what is the false positive rates for both methods? Authors must compare the methods by using classical measures such as precision, recall and F-measure and a ROC curve could be plotted. Moreover, the authors did not compare their approach with state-of-art methods such as SVM and HMMs. The discussion of results is really poor, the authors did not defend their method.The training and testing strategy is unusual. 95% of the data to train and just 5% to test, the author have performed a cross validation? If yes how many folds? how many times it was repeated? It is not clear in the text.The paper is not well written and there are several incompressible parts. Just to cite some of them:\"Then we use a phrase-based translation model to build the “amino acid sequence” to “protein function” translation model. (I think it is \"to map\" instead of \"to build\") Here we build three groups of gene parallel corpora. Group 1 only contains human amino acid data. (the expression “human amino acid data” is so unusual in bioinformatics community).The author wrote in the conclusion \"Since the protein functional prediction problem is so close to ‘language translation’, translation based protein functional prediction is likely to be the most promising approach.\"I do not agree that protein functional prediction is close to language translation problem. There are a lot additional points, for example evolution. In my opinion, we cannot interpret proteins as words. Anyway, the results have not showed that it is the most promising approach.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-231
|
https://f1000research.com/articles/2-193/v1
|
20 Sep 13
|
{
"type": "Observation Article",
"title": "A pre-zygotic barrier to hybridization in two con-generic species of scleractinian corals",
"authors": [
"Andrew H. Baird",
"Vivian R. Cumbo",
"Joana Figueiredo",
"Saki Harii",
"Vivian R. Cumbo",
"Joana Figueiredo",
"Saki Harii"
],
"abstract": "Hybridization is often cited as a potential source of evolutionary novelty in the order Scleractinia. While hybrid embryos can be produced in vitro, it has been difficult to identify adult hybrids in the wild. Here, we tested the potential for hybridization between two closely related species in the family Fungiidae. We mixed approximately 5000 eggs of Ctenactis echinata with sperm from Ctenactis crass. No hybrid embryos were produced. This observation adds to a growing body of evidence for pre-zygotic barriers to hybridization in corals and challenges the claim that hybridization is a major source of evolutionary novelty in the order.",
"keywords": [
"Hybridization is a controversial topic in coral reef ecology1",
"2. While small numbers of hybrid embryos can be produced in a few species in vitro3",
"the evidence for hybrids in the field is often equivocal because the genetic techniques used for corals cannot distinguish between hybridization and incomplete lineage sorting4. In fact",
"only one of the over 1300 species in the order is generally accepted to be unequivocally of hybrid origin: Acropora prolifera1",
"5. Nonetheless",
"hybridization is often invoked as a source of evolutionary novelty in the order Scleractinia6",
"7."
],
"content": "Observation\n\nHybridization is a controversial topic in coral reef ecology1,2. While small numbers of hybrid embryos can be produced in a few species in vitro3, the evidence for hybrids in the field is often equivocal because the genetic techniques used for corals cannot distinguish between hybridization and incomplete lineage sorting4. In fact, only one of the over 1300 species in the order is generally accepted to be unequivocally of hybrid origin: Acropora prolifera1,5. Nonetheless, hybridization is often invoked as a source of evolutionary novelty in the order Scleractinia6,7.\n\nHere, we report an incidental observation on the potential for hybridization between two closely related scleractinian corals species in the family Fungiidae, Ctenactis echinata and Ctenactis crassa. These species are sympatric, often dominating large multi-specific assemblages of fungiid corals throughout the central Indo-Pacific8. Colonies of these species are superficially very similar (Figure 1A and B) but can readily be distinguished by the shape of the costal dentitions9. Both species are gonochoric, that is each colony is either male or female, and reproduce by broadcast spawning, releasing gametes into the water column for fertilization8 (Figure 1C and D). At our study site on Sesoko Island (26°38'13.00\"N; 127°51'56.24\"E), Okinawa, Japan, spawning occurs following the full moons from July to August8. Furthermore, both species release gametes at the same time8 and consequently there is the potential for hybridization. In the days before the predicted date of spawning in July 2013, we collected four colonies of C. echinata and six colonies of C. crassa, to produce larvae for other experiments.\n\nLive Ctenactis echinata (A) and C. crassa (B) in aquaria prior to being isolated for spawning. Each colony is approximately 20 cm in length. Coral species in the family Fungiidae, such as these colonies of Fungia repanda, are gonochoric broadcast spawners: each individual releases either eggs (C) or sperm (D) into the water column where fertilization takes place (arrows indicate gametes).\n\nWhile the species are relatively easy to identify, determining the sex of each individual prior to spawning is impossible without destructive sampling to expose the gametes. Consequently, we placed each individual in a separate 20 L bucket containing sea water in the open air at approximately 20:00 h in order to sex each individual once gametes had been released. On the night of 27 July between 22:30 and 23:30 h three C. echinata and five C. crassa spawned revealing that the three spawning C. echinata were female, while four C. crassa were females and one was a male. The size of the eggs of each species at the time of release was distinct with a range in maximum diameter of 244–266 μm in C. echinata and 133–155 μm in C. crassa. In contrast to earlier work on C. echinata10, we saw no symbiotic algae in the eggs of either species. We collected approximately 5000 eggs from the three C. echinata females and mixed them with sperm from the C. crassa male. As a positive control we mixed eggs from the four C. crassa females with the C. crassa sperm. Approximately 100 eggs were observed under a stereo-dissecting microscope for cleavage, indicating fertilization, every 2 to 6 h over the next 24 h. At no point did we observe cleavage in the cross between species indicating that no hybrid embryos were produced. In contrast, over 90% of C. crassa eggs in the positive control were fertilized within 2 h. We conclude that despite synchrony in the time of gamete release between these two closely related sympatric species there appears to be strong pre-zygotic mechanism to avoid hybridization. While our observations are preliminary and in only one direction (i.e. we did not cross C. echinata males with C. crassa females) we predict that hybridization between these species is unlikely. This observation adds to a growing body of evidence indicating strong pre-zygotic barriers to hybridization in many scleractinian corals11–13.",
"appendix": "Author contributions\n\n\n\nAHB, VRC & JF conceived the study and performed the experiment. All authors contributed to writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding was provided by the Australian Research Council Centre of Excellence for Coral Reef Studies COE561432 (AHB), a Queensland Smart Futures Fellowship (JF) and a Sesoko Tropical Biosphere Research Station Travel Award 2013 (VRC).\n\n\nAcknowledgements\n\nWe thank the staff at the Sesoko Tropical Biosphere Research Station, University of the Ryukyus, for their assistance.\n\n\nReferences\n\nVollmer SV, Palumbi SR: Hybridization and the evolution of reef coral diversity. Science. 2002; 296: 2023–2025. PubMed Abstract | Publisher Full Text\n\nMiller DJ, van Oppen MJH: A 'fair go' for coral hybridization. Mol Ecol. 2003; 12(4): 805–807. PubMed Abstract | Publisher Full Text\n\nWillis BL, Babcock RC, Harrison PL, et al.: Experimental hybridization and breeding incompatibilities within the mating systems of mass spawning reef corals. Coral Reefs. 1997; 16: S53–S65. Publisher Full Text\n\nFlot JF, Couloux A, Tillier S: Haplowebs as a graphical tool for delimiting species: a revival of Doyle's \"field for recombination\" approach and its application to the coral genus Pocillopora. in Clipperton. BMC Evol Biol. 2010; 10: 372. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFogarty ND: Caribbean acroporid coral hybrids are viable across life history stages. Marine Ecology Progress Series. 2012; 446: 145–159. Publisher Full Text\n\nWillis BL, van Oppen MJH, Miller DJ, et al.: The role of hybridization in the evolution of reef corals. Annual Review of Ecology Evolution and Systematics. 2006; 37: 489–517. Publisher Full Text\n\nRichards ZT, van Oppen MJH, Wallace CC, et al.: Some rare Indo-Pacific coral species are probable hybrids. Plos One. 2008; 3: e3240. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoya Y, Heyward A, Sakai K: Reproductive patterns of fungiid corals in Okinawa, Japan. Galaxea. 2009; 11: 119–129. Publisher Full Text\n\nHoeksema BW: Systematics and ecology of mushroom corals. (Scleractinia: Fungiidae) [Ph D] 1989.\n\nCombosch DJ: Preliminary investigations about the hybridization potential in Japanese Fungiidea (Scleractinia). In: Hidaka M, Sakai K, Harii S, editors; 2007; Proceedings of the 21st Century Centre of Excellence International Summer Programme, University of the Ryukyus, Okinawa, Japan. University of the Ryukyus.\n\nWolstenholme JK: Temporal reproductive isolation and gametic compatibility are evolutionary mechanisms in the Acropora humilis.species group (Cnidaria; Scleractinia). Marine Biology. 2004; 144: 567–582.Publisher Full Text\n\nMorita M, Nishikawa A, Nakajima A, et al.: Eggs regulate sperm flagellar motility initiation, chemotaxis and inhibition in the coral Acropora digitifera,. A. gemmifera. and A. tenuis. J Exp Biol. 2006; 209(Pt 22): 4574–4579. PubMed Abstract | Publisher Full Text\n\nFogarty ND, Lowenberg M, Ojima MN, et al.: Asymmetric conspecific sperm precedence in relation to spawning times in the Montastraea annularis. species complex (Cnidaria: Scleractinia). J Evol Biol. 2012; 25(12): 2481–2488. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2160",
"date": "22 Oct 2013",
"name": "Yossi Loya",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate for the content of the article. The abstract represents a suitable summary of the work. Please correct: crass to crassa, in the 3rd line of the Abstract. Article content: The design, methods and analysis of the results been clearly explained and are appropriate for the topic being studied. Figures 1C and 1D appear to be irrelevant to the article since they show different species. I suggest deleting them. A proper reference to the statement 'spawning occurs following the full moons from July to August' (reference 8 in the manuscript) is: Loya Y. & K. Sakai (2008). Bidirectional sex change in mushroom corals. Proc. Roy. Soc. Biol. B 275: 2335-2343. Data and Conclusions: The authors note that they did not cross C. echinata males with C. crassa females; however they also did not test the positive control of crossing C. echinata males with C. echinata females (all their experimental C. echinata specimen were females). Nevertheless, this does not diminish their prediction that hybridization between these species is unlikely. The paper contributes further information to the controversial topic of potential hybridization and breeding incompatibilities within the mating systems of broadcast spawning reef corals.",
"responses": [
{
"c_id": "591",
"date": "26 Oct 2013",
"name": "Andrew Baird",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Dear Yossi,Thank you for your comments. We have corrected the typos you identified, changed the reference as requested and added a sentence to clarify the controls that were used to test for gamete viability. Images 1 C & D are presented as an example of the spawning behavior of fungiids."
}
]
},
{
"id": "1878",
"date": "24 Oct 2013",
"name": "Bernie Degnan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Observation Article reports the lack of cross-fertilization between Ctenactis echinata and Ctenactis crassa, closely related fungiid corals that naturally release gametes at the same time. The authors recognise the limitations of this observation - only small numbers of eggs (100's) were observed and only eggs from C. echinata were available to be fertilized (i.e. they did not cross C. echinata males with C. crassa females) - but rightly point out that this study provides further evidence that hybridization is not as widespread amongst scleractinian corals as often portrayed in the literature. However, the analysis of reciprocal crosses and a larger numbers of eggs is necessary before it can be said with some confidence that these congeners can not hybridize.",
"responses": [
{
"c_id": "590",
"date": "26 Oct 2013",
"name": "Andrew Baird",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Dear Bernie,Thank you for your comments. We would just like to point out that while we only examined 100 eggs every few hours under the microscope, we used approximately 5000 eggs in the fertilization experiment, all of which had broken down after 24 h suggesting none had fertilized. We have added a sentence to the revised text to draw attention to this."
}
]
}
] | 1
|
https://f1000research.com/articles/2-193
|
https://f1000research.com/articles/2-230/v1
|
31 Oct 13
|
{
"type": "Opinion Article",
"title": "Progress and challenges in the computational prediction of gene function using networks: 2012-2013 update",
"authors": [
"Paul Pavlidis",
"Jesse Gillis"
],
"abstract": "In an opinion published in 2012, we reviewed and discussed our studies of how gene network-based guilt-by-association (GBA) is impacted by confounds related to gene multifunctionality. We found such confounds account for a significant part of the GBA signal, and as a result meaningfully evaluating and applying computationally-guided GBA is more challenging than generally appreciated. We proposed that effort currently spent on incrementally improving algorithms would be better spent in identifying the features of data that do yield novel functional insights. We also suggested that part of the problem is the reliance by computational biologists on gold standard annotations such as the Gene Ontology. In the year since, there has been continued heavy activity in GBA-based research, including work that contributes to our understanding of the issues we raised. Here we provide a review of some of the most relevant recent work, or which point to new areas of progress and challenges.",
"keywords": [
"One of the problems with network-based approaches we documented in our previous papers1–3 is their tendency to converge on “easy answers”",
"by which we mean picking genes as candidates for a given disease or function simply because they are involved in many diseases (multifunctional) or are prominent in the network (e.g.",
"hubs)1. A possible solution would be to tailor the network data to particular contexts. Multifunctional genes would then have less of a dominant role",
"because fewer of their functions would be relevant to the network",
"and the network might reflect this. Fortuitously",
"several studies that improve our understanding of the utility of context-specific networks recently appeared",
"though they do not address the questions of whether they reduce multifunctionality and node degree biases."
],
"content": "Building better networks\n\nOne of the problems with network-based approaches we documented in our previous papers1–3 is their tendency to converge on “easy answers”, by which we mean picking genes as candidates for a given disease or function simply because they are involved in many diseases (multifunctional) or are prominent in the network (e.g., hubs)1. A possible solution would be to tailor the network data to particular contexts. Multifunctional genes would then have less of a dominant role, because fewer of their functions would be relevant to the network, and the network might reflect this. Fortuitously, several studies that improve our understanding of the utility of context-specific networks recently appeared, though they do not address the questions of whether they reduce multifunctionality and node degree biases.\n\nGuan et al. (2012) constructed 107 tissue-specific networks for the laboratory mouse to be used in disease-gene prioritization4. They used a combination of training data from Gene Ontology (GO) and tissue-specific expression signatures to customize their networks before moving to predicting disease candidate genes. The networks are not built from tissue-specific data, but various data used in combination, with each given a weight computed using \"tissue-specific gold standards\". The cross-validation performance improvement was significant but modest across most tasks (appearing to be approximately 0.03 on top of areas under receiver operating characteristic curves (AUROCs) ranging from 0.7 to 0.8).\n\nMagger et al. (2012) took a different approach, choosing to construct tissue-specific protein interaction networks by down-weighting edges involving genes not expressed in the given tissue5. Their baseline performances are somewhat higher than Guan et al. and also show improvement with tissue-specificity (from a mean AUROC of 0.82 to ~0.88). However, the bulk of this performance improvement comes with simply removing genes not expressed in a given tissue. Magger et al. provide some evidence that edges involving such genes are the source of prediction errors, as simply down-ranking the genes after analyzing a generic (non-tissue-specific) network was not as effective. While Magger et al. primarily restricted themselves to examining disease-gene associations where the causal gene was judged to be tissue-specific, they did examine the full disease-gene data where gains from tissue-specificity were much more modest (approximately AUROC 0.83 to 0.845). In contrast to the specialized task, node removal and attenuation of unexpressed genes performed particularly badly but only at low false positive rates (FPR). At high FPRs, node removal outperformed other methods, suggesting a trade-off between high precision and low precision prediction.\n\nPiro et al. (2012) created co-expression networks from groups of genes expressed in specific mouse brain regions and used these together with other information to predict gene-disease relations from OMIM6. They report that integrating tissue-specific data substantially raises their candidate disease prioritization performance. However, the final performance does not appear to be better than reported using quite old methods (~AUROC of 0.8 overall). Because they do not present results using a comparable “generic” network, it is difficult to tell if anything was gained.\n\nDowell et al. (2013) describe the creation and analysis of a mouse embryonic stem cell (mEPSC) specific gene network, relying on extensive manual curation7. This paper caught our attention in part because Dowell et al. acknowledge the potential for node degree bias and other issues, and claim “we address many of these potential pitfalls”. However, we were unable to identify the evidence that their methods do so; indeed, the focus on network hubs combined with very high performance of negative control data (assembled from datasets excluding mESCs), suggests multifunctional biases may have had a role. They suggest the use of cell-type-specific data should “reduce the impact of multi-functional genes”, but do not report whether this was indeed the case. This might have been of value in explaining how their network results were more specific, even if performance was not higher than some of the negative controls overall. If context-specific data reduces generic effects, it is of utility even if it yields no improvements in performance as judged by the usual metrics.\n\nThese reports can be considered encouraging, but still leave open the question of whether parsing data into more specific subsets is worthwhile, despite the hopes we expressed last year on this count. The noisiness of biological data may be such that breaking data into smaller bins can cost more in terms of robustness than we gain in terms of specificity. We also note that some earlier approaches combine a wide array of expression data, and treat the data sets as features to be weighted in the prioritization method8,9. Thus information such as “gene A is expressed in tissue X” may have been implicitly used. Choosing “tissue-specific functions” to assess such approaches is another challenge, and it is unknown if multifunctionality effects are reduced. None of this eliminates the possibility that network specificity provides crucial value, but more data are required.\n\n\nUsing better controls\n\nThe GBA studies discussed in the previous section did not take the opportunity to test the effect of multifunctionality or node degree bias, despite this control being easy to perform. The clearest attempt to control for multifunctionality and node degree of which we are aware is reported by Singh-Blom and colleagues in characterizing their prediction tool, CATAPAULT. Singh-Blom et al. conducted an analysis of disease and drug-target genes with a variety of networks and algorithms10. They report that a ranking of genes by multifunctionality (which they refer to as “degree”) performs poorly but not negligibly as a predictor, outperforming some of the methods they tested in cross-validation.\n\nBefore we comment further, there are some nuances to how Singh-Blom et al. (2013) use the multifunctionality ranking, compared to how we did. First, to avoid confusion the multifunctionality ranking (or a node degree ranking) should not be treated as a “method” for prediction, as it is referred to by Singh-Blom et al. It should be considered a null. In addition, the multifunctionality ranking is expected to “perform best” when performance is measured using ROC curves; Singh-Blom et al. use something more akin to precision-recall, which tends to obscure the influence of multifunctionality (and node degree), while being more heavily influenced by critical edges. Finally, multifunctionality ranking may be a too-stringent control (when using ROC) because it is literally optimized and performs better than many real algorithms on actual data1; rather, the correlation of the “real” prediction results with multifunctionality ranking is often a more helpful measure.\n\nIn any case, the fact that the multifunctionality ranking yields even modest performance in the evaluation scheme of Singh-Blom et al. hints that this ranking is also correlated with network node degree (as explained in our work1), and furthermore that such effects will have a strong impact on their results. Accordingly, Singh-Blom et al. report that highly prioritized genes tend to have high network node degree. They also report that of the top 10 candidates for eight diseases, almost all were shared among two or more of the diseases, confirming our finding that GBA too often yields “generic” predictions, not function-specific ones. These results, using different algorithms, networks and evaluation metrics than us, provide strong independent support of our claims.\n\nWhile Singh-Blom et al. confirmed some of our key findings, we may differ with them in interpretation, as they argue that the results are unproblematic. We agree with them that non-specific predictions could be correct, but this does not absolve concern about how their methods are actually operating. For example, INSR (insulin receptor) was predicted by CATAPAULT as a leukemia candidate gene, in addition for other diseases (including other cancers and diabetes). We are led to suspect this is at least partly explained by the high node degree of INSR, and not specific “guilt by association” of INSR with known leukemia genes. Can one find literature that connects insulin receptors and leukemia? Of course, since both are highly studied (one can find papers linking insulin receptors or cancer to many things) and the metabolism of cancer cells is of interest from a therapeutic standpoint. We also note that TP53 was predicted to be a diabetes-related gene by CATAPAULT. It remains possible that INSR is a bona fide leukemia gene; regardless, we strongly believe that a biologist wanting to use the output of CATAPAULT would also want to know about the specificity of the predictions.\n\nWe hope that other researchers interested in why their methods work take the step of attempting to control for “generic” results. Otherwise methodological performance is open to profound misinterpretation as to true utility. This is true even in the case where authors are clearly aware of the potential for problems. For example, Zuberi et al. (2013) report that the GeneMANIA edge weight normalization “helps to reduce the impact that the pleiotropy of high degree nodes has on functional predictions”9. We showed previously GeneMANIA’s results (with the normalization) are strongly affected by node degree, and indeed a substantial fraction of performance as measured by ROC curves could be explained by node degree effects1. Likewise, Verbeke et al. (2013) describe a gene prioritization method based on local networks that they “assume” reduces the effect of hubs, but provide no direct test11. As we have documented1, various attempts to modify networks to reduce extremes of node degree at best hide the problems from detection. We are open to the possibility that the approach of Verbeke et al. has the desired effect.\n\n\nFinding better algorithms\n\nIn the last year, there have been several interesting evaluations of gene function prediction methods. Our interest lies less in which method does best than in what these evaluations expose about the state of the field as a whole.\n\nBörnigen et al. (2012) performed a comparison of eight disease prioritization tools on a set of 42 disease genes12. The task was to prioritize the correct candidate, given whatever input the method requires (typically involving definition of a training set of genes already associated with the target function, and often a list of ~100 candidates in a genomic interval as starting points rather than a genome-wide list). The results were evaluated with ROC curves and with true positive rates at a given threshold. The authors’ method, Endeavour13, was among the top evaluation performers. No evaluation of the impact of multifunctionality was undertaken, but our experience suggests that multifunctional genes tend to be prioritized by these types of methods14. The problem of multifunctionality biasing prioritizations may be at least partly due to the difficulty of obtaining less biased training data. The “known genes” are often going to be biased towards highly-studied genes which do not form a sufficiently specific starting point for making functionally specific predictions. Regardless, Börnigen et al. were unable to clearly distinguish a best or poorest method, and the reasons for differences were not identified; it was speculated that differences in the underlying data used were important.\n\nThe more ambitious Critical Assessment of Functional Annotation (CAFA)15 was set up in a model very similar to the (now discontinued) function prediction component of CASP6 and CASP716,17. Participating groups predicted GO annotations for poorly-annotated proteins, followed by a waiting period during which some of the targets happened to be annotated by GO curators. The submitted algorithms were then assessed for correctness, relying primarily on a novel gene-centric metric that allowed partial credit for predicting a “similar” term, based on proximity in the GO term graph. Unfortunately, it emerged that this metric led to many methods (including BLAST) being outperformed by a naïve ranking of functions by prevalence (e.g. simply predict functions which are common overall; this approach ranks third or fourth in molecular function prediction), leading the organizers to exclude some results15. Radivojac et al. (2013) concluded that simple sequence analysis methods such as BLAST perform poorly, while more sophisticated methods based on integrating diverse data types are a substantial boon. However, in our separate assessment of a substantial portion of the CAFA data, we found that by more conventional metrics BLAST was among the top performers18.\n\nOur concerns about how function prediction works are further supported by a closer inspection of the methods that did well in CAFA. The best performing of them frequently have embedded in them aspects of the naive scoring method; that is, they successfully use knowledge of GO structure and term prevalence. In combination with the gene-centric evaluation metric, this creates a misleading impression of predictive power, in much the same way that a cold-reading mentalist can exploit the prevalence of names and medical conditions to impress a gullible audience. It was also possible to benefit from existing annotations for the targets (hot reading18). While successful in the narrow confines of the assessment, if put to use such approaches would only serve to increase the already strong biases in GO. In other words, in a sense CAFA turned out to be less about predicting gene function than about predicting which proteins would be annotated by GO curators and with which terms.\n\nOne of the major issues faced by CAFA is how to operationalize “function”. They (understandably) pass the buck on this issue and take the GO as an appropriate way to define function, with a number of consequent problems. In contrast, DREAM19 is a set of critical assessments motivated, like CAFA, as a means of understanding and improving inference methods (particularly network based ones), but DREAM largely focuses on more specific problems with associated datasets. One DREAM assessment we found interesting (even though it is not strictly about function prediction) focused on breast cancer survival analysis, with the goal of using molecular data (expression and genomic copy number) to improve prediction beyond that provided by clinical features. While a number of performance comparisons were made, one result was that the baseline method - simple Cox regression on clinical features – outperforms most methods across most conditions, even when they include the use of the molecular data. In only 10 out of 28 submissions were models incorporating molecular feature data with clinical able to outperform the baseline clinical predictor. On the one hand, this is encouraging: molecular data may be able to contribute something. On the other hand, the very best method using clinical data only was very close in performance to the best performing method using combined data. As is typically the case in machine learning, ensemble methods performed well (this would also have been true in CAFA), although investigator-based choices also appear to have been critical, since the class of purely automated methods performed particularly badly. In addition, the control of incorporating random gene signatures (or generic/multifunctional ones) with clinical data appears not to have been attempted (as might be suggested by previous research20), with permuted case labelling serving as the negative control instead. This leaves open the possibility that to the extent molecular data is of any predictive value at all, it does not provide us with any guidance as to molecular mechanism (by singling out relevant subsets of genes).\n\nThe last assessment we consider is the Critical Assessment of Genome Interpretation (CAGI), which focuses on using sequence data to predict elements of clinical or molecular phenotypes. While the results have not been published formally, the data presented on the CAGI web site are informative (https://genomeinterpretation.org/). The issue of appropriate controls again rears its head. For example, in the 2011 “personal genome project” assessment of phenotype prediction, the top performing submission appears to have primarily obtained performance by predicting that rare phenotypes would not occur (“due to predicting absence of rare characteristics”). In another competition, ROC curves for predicting Crohn’s disease from exome data appear to show close to half of teams performing below random (although not significantly so, apparently due to low sample size).\n\nWhy are critical assessments done? An admirably thoughtful discussion of algorithm comparisons noted that most scientists read new papers thinking “well, of course they say their method is better, but…”21. In part, critical assessments were intended to solve this problem: to help us move forward by making truly representative comparisons. It is not clear this is what is happening for function prediction assessments. Instead, we now have a system where researchers agree to participate and organizers have an obligation not to embarrass them. Thus, often only the top-performing methods are discussed, and the organizers have the same capacity to tweak as the original algorithm developers would have, and many of the same incentives. Discovering that most methods perform quite badly should be headline news, but could reduce enthusiasm for participation to the point of killing off future assessments. This is the usual problem of negative results, but scaled up to apply to the whole field through “consensus”. Perhaps publications of critical assessment should devote equal space to characterizing why methods failed; DREAM’s characterization of the poor performance of molecular data offers a toehold on this issue.\n\nTo summarize this section, because there are decreasing returns in tweaking methods, in our view comparisons of algorithms are less important than asking if they work at all and if so, how. Unfortunately this is often very difficult to discern from most of the work that has emerged in the last year, which often vary data as well as algorithms, and do not provide enough information to judge potential drivers of performance such as multifunctionality effects.\n\n\nUsing prior knowledge\n\nAs we noted last year, gene function prediction shouldn’t simply reduce to information retrieval, at least not unwittingly. Organizing existing knowledge and finding overlaps is useful, but is not the principal motivation of network-based methods, which are intended to find novel features in rich data. One way of drawing a distinction is that information retrieval GBA does not as readily suggest novel experiments. Normally, using some experimental feature to draw a functional conclusion suggests that one should try perturbing that experimental feature and observing the result; this will seem redundant if the feature is purely a property of the way the data was explicitly organized. However, the influence of prior knowledge is often hard to discern in the output of prediction methods, so information retrieval can masquerade as de novo function prediction.\n\nIt is important to realize that methods motivated by information retrieval are still forms of GBA, and are subject to the same potential problems. For example, Hoehndorf et al. (2013) created a network of genes based on semantic similarity of phenotypes of genetic diseases and animal models of diseases (PhenomeNet)22. They then use sets of genetic disease genes from various human databases, and their orthologs in mouse, to evaluate the relevance of this network for identifying gene-disease associations. For example, they rank mouse genes by the similarity of their mutant phenotypes to a target human disease’s phenotypes. They claim their approach is not GBA because “it does not require prior knowledge of the genetic basis of diseases for its predictions”. This is incorrect: because their method uses associations (semantic similarity) and infers “guilt” (involvement of a gene in a disease) based on this, it is obviously GBA, albeit a simple one where the prediction algorithm is a simple ranking of nodes by similarity. The authors may have been hoping that they don’t need to worry about node degree effects and multifunctionality, but we disagree. Using semantic similarity to identify diseases that resemble mouse models seems reasonable; using this to predict disease genes is most definitely GBA and suffers all the same potential pitfalls (and then some).\n\nTo see why, we note that the nodes in the network used by Hoehndorf et al. can be regarded as the set of both genes and diseases/phenotypes with edges indicating high semantic similarity across phenotypes. A disease node then provides the training data (a set of associated genes), and nearby gene nodes are the predicted relevant genes. By taking the gene-centred data (mutants, etc.) and treating it as equivalent to disease the authors are incorporating a hypothesis in addition to GBA, not instead of it. That is, a disease is treated conceptually as if it was gene-like. Consider that the same model should work if we were trying to predict effects of mutations from other known mutation effects through cross-validation, which would then be GBA (but also including an information retrieval component).\n\nThe use of various flavors of annotation similarity to build or influence networks is already endemic in function prediction, as we noted previously. A recent example is the work of Youngs et al. (2013), who use information on GO annotations to compute priors for function prediction23. In this manner, the likelihood that a gene is predicted to be annotated with a certain GO term is influenced by whether its other annotated GO terms tend to co-occur with desired GO term. This method, which is influenced by early work24 performs strongly in cross-validation, but we see two issues. The first is that, once again, the authors claim their evaluation approach addresses biases we have reported, without providing evidence. Second, it treats annotation biases as something to exploit (somewhat like Singh-Blom et al.), which we regard as a shaky proposition when it comes to predicting gene function, as opposed to performing information retrieval.\n\nOne interesting feature of the attempts to improve networks, methods, and priors in GBA is that researchers in each area can take the other area to be a gold standard. Thus, researchers focusing on protein interaction networks may use GO to obtain “better” interaction data25. Conversely, researchers wish to treat the network as a gold standard to improve GO26,27. In the meantime, algorithm developers treat both networks and annotations as a gold standard when comparing methods. In all these cases, researchers are performing what we would call “GBA” and working with the alignment between how genes form groups (using some method) as characterized in data and how those genes are grouped by prior annotations. The fact that there is some form of alignment is repeatedly rediscovered. A problem we perceive is that the duality of the gold-standards is increasingly blurring the lines between predictions and data. For example, Dutkowski et al. (2013), when benchmarking their method against GO, initially used as input some networks that were influenced by data from GO (e.g., YeastNet25), and so had to perform separate experiments to remove this confound. Similarly, the work of Magger et al. discussed above used data on disease gene expression patterns28 that were derived in part from the same protein interaction data that Magger et al. then use to perform tissue-specific predictions, though the implications of this are unclear. Recently we documented how protein interactions and gene ontology annotations are in many cases derived from the same publications29. Data resources used in genomics are becoming more intertwined, so ever greater care is required to avoid contaminating computational experiments with unwanted biases.\n\n\nGBA success stories?\n\nGuilt by association is widely agreed to be a valid method for investigating gene function. As mentioned, our concerns largely have to do with how GBA is performed and evaluated computationally (though the biases in existing knowledge could have impact on GBA even when it is conducted by hand). We also want to know, when GBA does work, is it because of “generic features” such as node degree, or are the GBA methods working the way most computational biologists hope they are working, which is inferring specific things about a gene based on specific features of its network neighbors. It is therefore of great interest to us and the rest of the computational GBA field to see use of GBA “in the wild”.\n\nOur review of the literature reveals different stories for disease gene prioritization and for other function prediction tasks. It is uncommon to see papers that report using computational GBA as an important means of identifying genes with a desired function (ignoring the role of sequence similarity, which is no doubt the most-used GBA method; methods that use more complex network-based approaches are our focus here). In contrast, genetics researchers faced with a genomic interval or a set of candidates seem to more readily turn to prioritization tools for assistance. This may be because the task of prioritizing a few genes (often 10–20) is simpler than prioritizing the entire genome, or that the task of identifying a disease gene is more clearly defined.\n\nTo take a well-known method as example of how algorithms are used in practice, GeneMANIA is implemented in a web-based tool described by its developers as a gene recommender system – essentially, using GBA9. A survey of recent citations suggests that the function prediction aspect is not the focus of most users of GeneMANIA30–34; in some cases users express an interest in predicting interactions, but not functions35. This may be because GeneMANIA’s tools do not operate on functions as usually defined in GBA settings; they take as an input a set of genes chosen by the user, and show a gene network with nodes selected using the GeneMANIA algorithm. In this way it is very similar to STRING36 and HumanNet37, which generate gene networks, which are adjusted by their agreement with inputs such as GO. We suspect some users of GeneMANIA, STRING and HumanNet do not realize that they are looking at the output of a guilt-by-association-influenced approach.\n\nThis is not to say that computational methods are not directly used successfully for function prediction. But even in such cases, it is often very difficult to determine how exactly GBA worked. Users of GBA are justifiably relatively uninterested in how they get to an answer, only that it is correct and leads to a new set of testable hypotheses or insights. Thus GBA success stories tend to be somewhat light on details and heavy on ad hoc aspects. We briefly mentioned several success stories in our original commentary. Some additional examples have since come to light and bear discussion.\n\nTacutu et al. (2012) provide a valuable rare large-scale assessment of a computational GBA task38. They wished to predict genes involved in regulating the longevity of Caenorhabditis elegans. Their input is a set of 205 known longevity-associated genes (LAGs) in a worm protein interaction network of 871 genes. A similar network was constructed for human orthologs. They then made predictions in a simple way, considering any gene that was a network neighbor of known LAGs (or orthologs), limited to the subset of candidates that are required for development (essential genes). This yielded 500 candidates of which 374 were tested. They report 19 of these validated, a success rate of 5%, compared to a rate of 2.4% based only on genes critical for development. It is notable that most of the predictive information came from exploiting the prior knowledge that essential genes were good candidates: the success rate went up five-fold from ~0.5% (for genome-wide screens) to 2.4% for essential genes, whereas adding the protein interaction data increased success by another two-fold. Tacutu et al. do not report any information on potential node degree effects, but obviously the largest number of candidates would have to come from the highest node-degree LAGs given the simplicity of the method. We suspect that many GBA experts would want to know how it would have worked with “better data” and a “more sophisticated algorithm”.\n\nPutnam et al. (2012) sought to identify yeast genes suppressing genomic instability39. Their initial input was 75 genes already known to be involved in genomic instability, and 928 genes in which mutations cause sensitivity to DNA damage. This set was expanded by choosing genes which have similar profiles of genetic interactions – this appears to be their key GBA step. The final set of genes numbered 1041, which were further prioritized in a second GBA-like step, based on their genetic interaction profiles with known DNA damage response genes, and by apparently manual selection, leading to selection of 87 genes for experimental follow-up. Of these, 40% had a detectable effect on genomic stability when mutated. This is impressive, but it is difficult to quantify the contribution of computation versus manual selection, and relatively few candidates were entirely novel. The authors speculate that their success rates might have been even higher if they had genetic interaction from a more relevant phenotype. For example, many genes that clustered with known DNA damage-related genes – and which thus looked “guilty” - failed to validate. As in the case with Tacutu et al., we note the relatively simple data used and the simple approach, combined with hand-tuning.\n\nWe have also reviewed some recent applications of disease gene prioritization tools, which as we comment above are seemingly used more commonly than function prediction tools (even though they are conceptually similar). We are struck by two trends. First, many (perhaps most) papers that apply such methods make no strong conclusion as to whether they have found the right gene40–48. That is, the results are treated a bit like GO enrichment analyses: as suggestive or exploratory. Second, some papers that report prioritization tool results supplant them with more precise or manually-identified information, such as the existence of an orthologous mouse mutant that has similar phenotypic features49–52.\n\nWe note a few trends from these reports. Black-box application of existing prioritization methods played at best a supporting role. The use of custom methods for creating initial target sets were important, sometimes based on experiments under the investigator’s control, rather than existing annotations from public databases. Data that was specific to the biology was deemed important: using generic data is a fallback. Not surprisingly, even with these ingredients, success in converting computationally-prioritized genes into documented hits is far from guaranteed. And while these examples bolster the claim that GBA can work, exactly how they are working with regards to multifunctionality bias is still left unclear.\n\n\nConclusions\n\nA theme that emerges from our review is brought out by the difference between the practices of computational biologists and those who actually use function prediction tools (loosely defined). These differences should come as no real surprise, but it has important implications that we feel are not being attended to sufficiently. It may be that biologists are happy with high-quality information retrieval tools, and are not actually very interested in function prediction at all. That creates a difficulty for those who are interested in predicting function, who feel compelled to develop new methods to do so, and who want their tools to be used by others. Such practitioners are left to test their methods on the GO, which we are increasingly certain is a waste of time, in the sense that it isn’t realistic, it isn’t what interests biologists, and it is easily confounded with the data used for prediction. It remains difficult to tell when methods are actually doing something useful, because evaluations have been weak, and the “in the wild” uses are obfuscated by various hand-tunings, publication bias, or inadvertent cherry-picking.\n\nWe regard these as major issues, but this is a far cry from disagreeing with functional inference overall. Some authors appear to have interpreted our papers as concluding that GBA is useless53,54, but this is too broad a brush. We fully believe in the GBA principle, and computational methods can be useful. The difficulty is in telling when and how they are working. The concern about “when” is summarized by our findings that cross-validation analysis can be very misleading – to the point of being potentially irrelevant - for predicting future performance2. The concern about “how” is reflected in our demonstrations that gene multifunctionality and node degree effects are often more important in determining the outcome of a GBA analysis than details about the connections in the network1. These two realizations should affect practice, but they do not mean that the predictions one makes are always incorrect.",
"appendix": "Author contributions\n\n\n\nPP and JG conceived and wrote the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nGillis J, Pavlidis P: The impact of multifunctional genes on \"guilt by association\" analysis. PLoS One. 2011; 6(2): e17258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillis J, Pavlidis P: 'Guilt by association’ is the exception rather than the rule in gene networks. PLoS Comput Biol. 2012; 8(3): e1002444. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPavlidis P, Gillis J: Progress and challenges in the computational prediction of gene function using networks. F1000 Res. 2012; 1: 1–14. 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\nMagger O, Waldman YY, Ruppin E, et al.: Enhancing the prioritization of disease-causing genes through tissue specific protein interaction networks. PLoS Comput Biol. 2012; 8(9): e1002690. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPiro RM, Molineris I, Di Cunto F, et al.: Disease-gene discovery by integration of 3D gene expression and transcription factor binding affinities. Bioinformatics. 2013; 29(4): 468–475. PubMed Abstract | Publisher Full Text\n\nDowell KG, Simons AK, Wang ZZ, et al.: Cell-type-specific predictive network yields novel insights into mouse embryonic stem cell self-renewal and cell fate. PLoS One. 2013; 8(2): e56810. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHibbs MA, Hess DC, Myers CL, et al.: Exploring the functional landscape of gene expression: directed search of large microarray compendia. Bioinformatics. 2007; 23(20): 2692–2699. PubMed Abstract | Publisher Full Text\n\nZuberi K, Franz M, Rodriguez H, et al.: GeneMANIA prediction server 2013 update. Nucleic Acids Res. 2013; 41(Web Server issue): W115–W122. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh-Blom UM, Natarajan N, Tewari A, et al.: Prediction and validation of gene-disease associations using methods inspired by social network analyses. PLoS One. 2013; 8(5): e58977. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVerbeke LP, Cloots L, Demeester P, et al.: EPSILON: an eQTL prioritization framework using similarity measures derived from local networks. Bioinformatics. 2013; 29(10): 1308–1316. PubMed Abstract | Publisher Full Text\n\nBörnigen D, Tranchevent LC, Bonachela-Capdevila F, et al.: An unbiased evaluation of gene prioritization tools. Bioinformatics. 2012; 28(23): 3081–8. PubMed Abstract | Publisher Full Text\n\nTranchevent LC, Barriot R, Yu S, et al.: ENDEAVOUR update: a web resource for gene prioritization in multiple species. Nucleic Acids Res. 2008; 36(Web Server issue): W377–W384. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQiao Y, Harvard C, Tyson C, et al.: Outcome of array CGH analysis for 255 subjects with intellectual disability and search for candidate genes using bioinformatics. Hum Genet. 2010; 128(2): 179–194. PubMed Abstract | Publisher Full Text\n\nRadivojac P, Clark WT, Oron TR, et al.: A large-scale evaluation of computational protein function prediction. Nat Methods. 2013; 10(3): 221–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLópez G, Rojas A, Tress M, et al.: Assessment of predictions submitted for the CASP7 function prediction category. Proteins. 2007; 69(Suppl 8): 165–174. PubMed Abstract | Publisher Full Text\n\nPellegrini-Calace M, Soro S, Tramontano A: Revisiting the prediction of protein function at CASP6. FEBS J. 2006; 273(13): 2977–2983. PubMed Abstract | Publisher Full Text\n\nGillis J, Pavlidis P: Characterizing the state of the art in the computational assignment of gene function: lessons from the first critical assessment of functional annotation (CAFA). BMC Bioinformatics. 2013; 14(Suppl 3): S15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStolovitzky G, Monroe D, Califano A: Dialogue on reverse-engineering assessment and methods: the DREAM of high-throughput pathway inference. Ann N Y Acad Sci. 2007; 1115: 1–22. PubMed Abstract | Publisher Full Text\n\nVenet D, Dumont JE, Detours V: Most random gene expression signatures are significantly associated with breast cancer outcome. PLoS Comput Biol. 2011; 7(10): e1002240. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoulesteix AL: On representative and illustrative comparisons with real data in bioinformatics: response to the letter to the editor by Smith et al. Bioinformatics. 2013; 29(20): 2664–2666. PubMed Abstract | Publisher Full Text\n\nHoehndorf R, Schofield PN, Gkoutos GV: An integrative, translational approach to understanding rare and orphan genetically based diseases. Interface Focus. 2013; 3(2): 20120055. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoungs N, Penfold-Brown D, Drew K, et al.: Parametric Bayesian priors and better choice of negative examples improve protein function prediction. Bioinformatics. 2013; 29(9): 1190–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKing OD, Lee JC, Dudley AM, et al.: Predicting phenotype from patterns of annotation. Bioinformatics. 2003; 19(Suppl 1): i183–189. PubMed Abstract | Publisher Full Text\n\nLee I, Li Z, Marcotte EM: An improved, bias-reduced probabilistic functional gene network of baker’s yeast, Saccharomyces cerevisiae. PLoS One. 2007; 2(10): e988. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDolinski K, Botstein D: Automating the construction of gene ontologies. Nat Biotechnol. 2013; 31(1): 34–35. PubMed Abstract | Publisher Full Text\n\nDutkowski J, Kramer M, Surma MA, et al.: A gene ontology inferred from molecular networks. Nat Biotechnol. 2013; 31(1): 38–45. 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): 20870–20875. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillis J, Pavlidis P: Assessing identity, redundancy and confounds in Gene Ontology annotations over time. Bioinformatics. 2013; 29(4): 476–482. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLipchina I, Elkabetz Y, Hafner M, et al.: Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response. Genes Dev. 2011; 25(20): 2173–2186. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMulvey CM, Tudzarova S, Crawford M, et al.: Subcellular proteomics reveals a role for nucleo-cytoplasmic trafficking at the DNA replication origin activation checkpoint. J Proteome Res. 2013; 12(3): 1436–1453. PubMed Abstract | Publisher Full Text\n\nO’Roak BJ, Deriziotis P, Lee C, et al.: Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet. 2011; 43(6): 585–589. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSookoian S, Pirola CJ: Metabolic syndrome: from the genetics to the pathophysiology. Curr Hypertens Rep. 2011; 13(2): 149–157. PubMed Abstract | Publisher Full Text\n\nVeerappa AM, Vishweswaraiah S, Lingaiah K, et al.: Unravelling the complexity of human olfactory receptor repertoire by copy number analysis across population using high resolution arrays. PLoS One. 2013; 8(7): e66843. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumimoto RW, Siriwardana CL, Gayler KK, et al.: NUCLEAR FACTORY transcription factors have both opposing and additive roles in ABA-mediated seed germination. PLoS One. 2013; 8(3): e59481. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranceschini A, Szklarczyk D, Frankild S, et al.: STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013; 41(Database issue): D808–D815. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee I, Blom UM, Wang PI, et al.: Prioritizing candidate disease genes by network-based boosting of genome-wide association data. Genome Res. 2011; 21(7): 1109–1121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTacutu R, Shore DE, Budovsky A, et al.: Prediction of C. elegans longevity genes by human and worm longevity networks. PLoS One. 2012; 7(10): e48282. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPutnam CD, Allen-Soltero SR, Martinez SL, et al.: Bioinformatic identification of genes suppressing genome instability. Proc Natl Acad Sci U S A. 2012; 109(47): E3251–E3259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorra VM, Waterval JJ, Stokroos RJ, et al.: Localization of the gene for hyperostosis cranialis interna to chromosome 8p21 with analysis of three candidate genes. Calcif Tissue Int. 2013; 93(1): 93–100. PubMed Abstract | Publisher Full Text\n\nBreckpot J, Thienpont B, Bauters M, et al.: Congenital heart defects in a novel recurrent 22q11.2 deletion harboring the genes CRKL and MAPK1. Am J Med Genet A. 2012; 158A(3): 574–580. PubMed Abstract | Publisher Full Text\n\nChabchoub E, Cogulu O, Durmaz B, et al.: Oculocerebral hypopigmentation syndrome maps to chromosome 3q27.1q29. Dermatology. 2011; 223(4): 306–310. PubMed Abstract | Publisher Full Text\n\nChang S, Zhang W, Gao L, et al.: Prioritization of candidate genes for attention deficit hyperactivity disorder by computational analysis of multiple data sources. Protein Cell. 2012; 3(7): 526–534. PubMed Abstract | Publisher Full Text\n\nHitz MP, Lemieux-Perreault LP, Marshall C, et al.: Rare copy number variants contribute to congenital left-sided heart disease. PLoS Genet. 2012; 8(9): e1002903. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLopezJimenez N, Gerber S, Popovici V, et al.: Examination of FGFRL1 as a candidate gene for diaphragmatic defects at chromosome 4p16.3 shows that Fgfrl1 null mice have reduced expression of Tpm3, sarcomere genes and Lrtm1 in the diaphragm. Hum Genet. 2010; 127(3): 325–336. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMelchionda L, Fang M, Wang H, et al.: Adult-onset alexander disease, associated with a mutation in an alternative GFAP transcript, may be phenotypically modulated by a non-neutral HDAC6 variant. Orphanet J Rare Dis. 2013; 8: 66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang J, Qian J, Hoeksema MD, et al.: Integrative genomics analysis identifies candidate drivers at 3q26-29 amplicon in squamous cell carcinoma of the lung. Clin Cancer Res. 2013; 19(20): 5580–5590. PubMed Abstract | Publisher Full Text\n\nZhu J, Cui L, Wang W, et al.: Whole exome sequencing identifies mutation of EDNRA involved in ACTH-independent macronodular adrenal hyperplasia. Fam Cancer. 2013. PubMed Abstract | Publisher Full Text\n\nHo DW, Yap MK, Ng PW, et al.: Association of high myopia with crystallin beta A4 (CRYBA4) gene polymorphisms in the linkage-identified MYP6 locus. PLoS One. 2012; 7(6): e40238. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHussain MS, Baig SM, Neumann S, et al.: A truncating mutation of CEP135 causes primary microcephaly and disturbed centrosomal function. Am J Hum Genet. 2012; 90(5): 871–878. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThiel C, Kessler K, Giessl A, et al.: NEK1 mutations cause short-rib polydactyly syndrome type majewski. Am J Hum Genet. 2011; 88(1): 106–114. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu L, Wynn J, Cheung YH, et al.: Variants in GATA4 are a rare cause of familial and sporadic congenital diaphragmatic hernia. Hum Genet. 2013; 132(3): 285–292. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMichailidis G: Statistical challenges in biological networks. J Comput Graph Stat. 2012; 21(4): 840–855. Publisher Full Text\n\nVey G: Metagenomic guilt by association: an operonic perspective. PLoS One. 2013; 8(8): e71484. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2276",
"date": "08 Nov 2013",
"name": "Stefano Toppo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 opinion article deals with the long standing issue of protein function prediction in its broader sense. The authors express an interesting and most of the time shareable point of view about the negative impact of gene multifunctionality that influences gene network-based guilt-by-association studies.The paper is really well written and organized in sections that focus on different aspects of function prediction and its pitfalls. Nonetheless, there are some minor points that I would suggest mitigating, as they sound too harsh and are, as far as I’m concerned, partly incorrect.In the “Finding better algorithms” section, CAFA is mentioned and commented on but I would like to pinpoint some aspects about how this is done and some related issues (below). Even the glorious series of CASP experiments, that the authors have mentioned, suffered a lot in their first editions but what is more important is that both assessors and participants are aware of this and that improvements are planned, as far as I know.The prediction results from the CAFA experiment could perhaps be framed around some different points of view:1:Looking at the F-measure results for top performing methods there is little to be happy about. There are the following additional issues coming out from CAFA: are we really sure that some of the predicted functions are not correct? Is this rather an effect caused by the possible incompleteness of some experimental data? In other words, can anyone assert firmly that there is nothing else to discover about the function of a protein? I would definitively say NO. There is more than meets the eye and besides, the benchmark is incomplete by definition as it will never complete in the future either, no matter what information is added. Not only that, but even novel experimental evidence can turn false positive predictions of protein function into a true positive. On the flip side, true positive predictions can equally be refuted by fresh experimental data and consequently turn into a false positive. 2:Some functions of the CAFA experiment were extremely difficult to predict and hard to “guess” in any way, both from a simple sequence similarity approach or other more sophisticated techniques based on machine learning. In summary, some CAFA targets were not so easy to predict. 3:Additionally with CAFA, much marginally informative experimental data was collected for many targets that mainly derived from PPI experiments. The term under indictment is “protein binding” which is heavily present, for example, in the GOA database of annotated proteins. The assessors’ decision to discard “protein binding” in the final evaluation was, consequently, correct. The good performance of naïve and BLAST methods (when “protein binding” is considered in the assessment) depends on the high occurrence (multifunctional?) of one function in the database and its prevalence over the others so that it is very easy “to predict”. In this sense, it is not exactly correct to assert that BLAST and naïve are (almost) the best performing tools because many tools participating to CAFA, whenever possible, tried to provide more informative annotations in place of the less informative “protein binding”. Taking this into account, “protein binding” would have been inappropriate to use in the final evaluation because it would have rewarded BLAST and naïve methods artificially but penalized others.In contrast, the main issue is that databases contain biased annotations and a few scarcely informative terms dominate the scene. In this respect, I totally agree with the authors that multifunctionality poses serious problems to function prediction algorithms. So how might one mitigate the effect of multifunctional and scarcely informative annotations? Perhaps CAFA will need to settle in the next editions and the contribution to this process of renewal should be constructive and proactive rather than purely critical. 4:The authors recognize that successful stories may be limited, simple and hand-tuned. Reverse engineering results is demanding and I agree with the authors, but I would note that the excess in the analysis, as suggested in the when/how methods perform section, could lead to an overestimate/underestimate of the behavior of the tools and miss their general action. As a matter of fact, biology is made of more exceptions than rules and tools are designed to follow only the rules. Can the authors suggest some possible ways in which a tentative solution can be set up that could be discussed and adopted in critical assessments of function prediction tools? 5:To me, the distinction in this paper between between GO and protein annotations using GO are not clear enough. I would, for instance, rephrase the following: “…. We also suggested that part of the problem is the reliance by computational biologists on gold standard annotations such as the Gene Ontology ….”To something like:“…. We also suggested that part of the problem is the reliance by computational biologists on gold standard annotations such as the Gene Ontology Annotation database (GOA) ….”I know the authors know the difference between the two and for this reason I would recommend that they clarify this aspect and do not confound what GO and its countless instances are (one of them is GOA).As an obvious reminder, GO is an abstraction of the knowledge tentatively organized in a directed acyclic graph and is a controlled vocabulary intended as the rosetta stone of different interpretations and expressions of the same concepts. I strongly believe that GO is rigorous and we can trust it. On the contrary, GOA contains GO instances used to describe proteins. Using the metaphor of programming language, the GO term is the “object” and its use in GOA, or other databases containing GO annotated proteins, is the “instance” of that “object”. This is an important difference because the “object” is abstract and may be varied in a number of ways that can be right or wrong when thinking about protein annotation. It is not the GO term definition per se in the dock but rather its utilization as a descriptor of protein function stored in public databases. The authors already published a paper on GO based annotations and their distribution in GOA over time. In other words, one can rely on the GO descriptions and their positions in the graph (though GO is continuously revisited, it is rather stable) but must pay attention to the proteins annotated with GO terms because they can be inappropriate and change over time, as already evaluated by the authors in a previous work (indeed, GO annotations in GOA change frequently).It would be very interesting to know what the authors think about the latter phenomenon, i.e. the updating of old annotations and their syncing with novel and more precise GO ontologies.Most of the issues, fully and carefully described by the authors, may be due more to this aspect than others. Generic annotations may have been used at the beginning of the story when GO was still incomplete and with poor coverage of biological knowledge. Thinking about “Inferred from Electronic Annotations” (IEA), these GO terms may have created the multifunctional phenomenon as they had time to spread and have consequently become both pervasive and difficult to eradicate or update.",
"responses": []
},
{
"id": "3158",
"date": "05 Feb 2014",
"name": "Vivek Anantharaman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 discussed the pitfalls of automated GBA and ways to improve functional prediction. Automated methods for GBA are only as good as ontologies and curated reference datasets. Ontologies like GO suffer from poor quality annotation being propagated throughout their data that result in “Garbage in – Garbage out” phenomenon. A generic functional prediction is the best one can expect from existing automated methods.From my experience, I have found that accurate functional prediction requires a mix of local sequence similarity and sequence profile searches, proper sequence analysis with study of sequence and phyletic conservation, structural analysis, network studies of various data points, and correlation with experimental data, all done with a heavy dose of manual tuning. None of the automated methods of GBA give consistent accurate prediction, without manual intervention.The review laid out by the authors is a good analysis of the challenges and limitation of gene function prediction. One area that the authors do not explicitly discuss is the great difference between eukaryotes and prokaryotes. GBA is currently far more effective in the latter due to operons, which are not available in eukaryotes.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-230
|
https://f1000research.com/articles/2-229/v1
|
29 Oct 13
|
{
"type": "Research Article",
"title": "Fra-1 regulation of Matrix Metallopeptidase-1 (MMP-1) in metastatic variants of MDA-MB-231 breast cancer cells",
"authors": [
"Eric Henckels",
"Ron Prywes",
"Eric Henckels"
],
"abstract": "Matrix Metallopeptidase 1 (MMP-1) expression has repeatedly been correlated to tumorigenesis and metastasis. Yet, MMP-1 regulation in a metastatic context remains largely unknown. Here we confirm differential MMP-1 expression in mammary carcinoma cells with varied metastatic potentials. We show that MMP-1 expression is regulated by an AP-1 element in its promoter in highly metastatic MDA-MB-231 mammary carcinoma cell derivatives. Fra-1, an AP-1 family transcription factor, differentially binds this element in highly metastatic cells compared to low metastatic cells and is required for MMP-1 expression. Overexpression of Fra-1 also caused increased MMP-1 expression. Fra-1 mRNA levels are unchanged in the cell variants, however its protein levels are higher in the metastatic cells. While there was no change in Fra-1 protein degradation rates, protein synthesis of Fra-1 was increased in the metastatic cell variant. These results demonstrate that Fra-1 and MMP-1 levels are differentially regulated in metastatic cell variants at the level of Fra-1 protein translation. Consistent with the importance of Fra-1 for tumor growth, we found that Fra-1 overexpression was sufficient to increase cell motility and anchorage independent growth. These results suggest that increased Fra-1 translation is critical for regulation of MMP-1 and tumor cell metastasis.",
"keywords": [
"Matrix metallopeptidase-1 (MMP-1) expression is highly correlated to several forms of cancer1. In breast cancer patients",
"MMP-1 expression has been correlated to primary tumor progression",
"metastatic potential",
"and survival2–6. Further",
"in glioblastoma",
"melanoma and breast cancer",
"higher incidence has been associated with a single nucleotide polymorphism in an Ets-binding site which increases MMP-1 expression7",
"8."
],
"content": "Introduction\n\nMatrix metallopeptidase-1 (MMP-1) expression is highly correlated to several forms of cancer1. In breast cancer patients, MMP-1 expression has been correlated to primary tumor progression, metastatic potential, and survival2–6. Further, in glioblastoma, melanoma and breast cancer, higher incidence has been associated with a single nucleotide polymorphism in an Ets-binding site which increases MMP-1 expression7,8.\n\nOutside the clinic, MMP-1 expression has been measured in a variety of breast cancer cell lines. In general, its expression is greater in cells with higher metastatic potential (e.g. MDA-MB-231) when compared to cells of low metastatic potential (e.g. MCF-7)9–11. Similarly, MDA-MB-231 cell variants with different metastatic potentials demonstrate the correlation between MMP-1 expression and metastasis12–14.\n\nMMP-1 regulation has been well studied in HeLa and other cell culture systems15. However, less is known about how MMP-1 is regulated in metastasis. Recent studies have identified several promoter regions and factors that may play a role in MMP-1 regulation. For example, in melanoma cells, Twist binding to the MMP-1 promoter was found to increase expression of MMP-116. In MCF-7 cells, HER2, which is upregulated in 15% – 25% of breast tumors and associated with poor prognosis, was found to upregulate MMP-1 through the ERK1/2 pathway17.\n\nThe AP-1 consensus site is the archetype for tumor associated gene expression. It was discovered in the MMP-1 promoter as being activated by tumor promoting phorbol esters18,19. Since its discovery, the role of AP-1 in tumorigenesis has been further substantiated20. Tissue immunohistochemistry revealed that expression of Fra-1, an AP-1 family member, correlates with breast cancer malignancy21,22.\n\nTo study the altered regulation of gene expression in metastatic breast cancer cells, we have utilized a series of MDA-MB-231 breast adenocarcinoma cell variants developed by the Massague lab12,14. MDA-MB-231 cells, which were derived from a pleural effusion of a breast cancer patient with relapsed disease, cause a low level of metastasis when injected into immunocompromised mice by various routes. Metastatic cells from these xenografts had greater metastatic potential when subsequently cultured and reinjected into mice. These cells derived from metastatic tumors in secondary organs (e.g. lung and bone) also showed greater organ-specificity. Alternatively, MDA-MB-231 cells were single cell cloned. These propagated single cell populations (Scp cell lines) had varied metastatic potential. Analysis of genomic expression using microarrays on these cell lines of varying metastatic potential provided us with an opportunity to identify genes correlated with metastatic potential12–14. In addition, these cell lines provided us with a well controlled system to understand the mechanism of how gene expression is altered in highly metastatic cells.\n\nThe gene whose expression was most strongly increased in the highly metastatic cell variants was MMP-1. In this study we have compared the expression of MMP-1 in the high and low metastatic MDA-MB-231 variants and present evidence for the role of an AP-1 site in the MMP-1 promoter and the translational regulation of the AP-1 family member Fra-1.\n\n\nMaterials and methods\n\nMicroarray gene expression data was available as supplemental data in several publications12–14 (http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S1535610803001326/1-s2.0-S1535610803001326-mmc1.xls/272618/FULL/S1535610803001326/8e4e6bf4cf8c1acc68ed4588b192b303/mmc1.xls, http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S1535610803001326/1-s2.0-S1535610803001326-mmc2.xls/272618/FULL/S1535610803001326/2c83b94783f578fb7aeff9fb3c2b6c0d/mmc2.xls, http://www.nature.com/nature/journal/v436/n7050/extref/nature03799-s10.xls). To parse the data, Affymetrix comparison sheets were used with Microsoft Excel Vlookup functions to match primer coding with gene name, symbol and reference sequence ID. Expression values from cell lines with high metastatic potential to the bone (1833, Scp-2, Scp-25 and Scp-46), to the lung (1834, 3481, 4142, 4173, 4175, 4180, Scp-3 and Scp-28), or with low metastatic potential (MDA-MB-231, Scp-6, Scp-21 and Scp-26) were averaged for each gene. The ratio of high to low metastatic potential expression levels was calculated and ordered by highest ratio. A T-test (two tailed distribution, equal variance) using Microsoft Excel was used to calculate the p value for the significance of the differences between each group.\n\nScp-2, Scp-3, Scp-21, Scp-26, Scp-28, and MDA-MB-231 cell lines were a generous gift from Joan Massague (Memorial Sloan Kettering Research Institute)14. Cells were grown in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (Gemini Bio-Products). Phoenix amphotropic helper cells from Gary Nolan (Stanford University)23 were grown in DMEM supplemented with 10% Fetal Bovine Serum.\n\nCell lines stably expressing Fra-1 or a control vector were made in Scp-21 cells. The Fra-1 retroviral expression vector, p6599 MSCV-IP N-HAonly FosL124, and pBabe-Puro vector25 plasmids were independently transfected into Phoenix amphotropic helper cells23 using Lipfectamine LTX (Life Technologies) per the manufacturer’s instructions to generate defective retroviruses. The media was changed after 16 hours to DMEM/10% fetal bovine serum. After 24 hours the media containing the virus was removed and polybrene was added to 4 μg/mL. This viral media was filtered with 0.45 μm polyethersulfone filters (Thermo Scientific) and added to Scp-21 cells. This infection media was removed after 24 hours and selection in puromycin (10 μg/mL; Sigma Aldrich) was started 24 hours later. These Scp-21 cells expressing Fra-1 or control vector were maintained in DMEM supplemented with 10% fetal bovine serum and 5 μg/mL puromycin.\n\nScp-2 and Scp-21 cells used to measure protein degradation with cycloheximide were plated at 2×106 cells in a 6 cm plate overnight. Plates were then treated with cycloheximide (10 μg/mL) for the indicated times.\n\nRNA was purified from adherent cells with Trizol Reagent (Life Technologies) per the manufacturer’s instructions. RNA was reverse transcribed with the ImProm-II Reverse Transcriptase (Promega) according to the manufacturer’s instructions with random hexamer primers (Integrated DNA Technologies).\n\nIn Figure 1B where pre-mRNA was measured with intronic primers and signal from contaminating genomic DNA can be problematic, samples measured by quantitative RT-PCR were treated with DNase I (Sigma) per the manufacturer’s instructions.\n\nA) qPCR of MMP-1 mRNA expression in Scp-2 (high metastatic potential), Scp-21 (non-metastatic) and MDA-MB-231 (low metastatic) cells. B) qPCR of MMP-1 mRNA and pre-mRNA in Scp-2 and Scp-21 cells. Mean relative values +/- standard deviation from three independent experiments are shown. C) Immunoblot with anti-MMP-1 antibody of whole cell lysates from Scp-2, Scp-21, and MDA-MB-231 cells. Anti-actin antibody served as a loading control. D) Mean MMP-1 protein band intensity from immunoblots +/- standard deviation from three independent experiments. **, p < 0.005 for two-tailed t-tests.\n\nqPCR was performed with standard protocols with the StepOne Plus System (Life Technologies) with Power SYBR master mix (Life Technologies) per the manufacturer’s instructions. Briefly, cDNA samples were combined with master mix and primers (final concentration 0.5 μM; shown in Table 1). Expression was normalized to 18S rRNA expression. Standard deviations were calculated from three independent experiments. p-values were determined by Student’s two-tailed t-tests with significance thresholds as labeled.\n\nPrimers (Integrated DNA Technologies) were designed using Primer Express (Life Technologies) with standard parameters. Primer sequences for the human genes are shown in Table 1.\n\nThe matrix metallopeptidase-1 (MMP-1) promoter was analyzed with the UCSC Genome Browser26. Analysis was performed on the following tracks: 1) Base Position, 2) Human mRNAs, 3) Placental Mammal Conservation by PhastCon with all 23 species, and 4) Vertebrate Conservation by PhastCon with all 46 species.\n\nAll constructs were made using the pGL3-Basic promoter backbone with inserts at the BglII and HindIII sites of the multiple cloning sequence. The MMP-1 promoter regions were amplified from human genomic DNA (Bioline). Amplified inserts spanned -819/+71, -514/+71, -174/+71, -810/-174, -172/-27, -115/-27, and -94/-27 bases from the transcription start site. Shorter promoter inserts were annealed from oligonucleotide sequences as shown in Table 2.\n\nThe indicated forward and reverse oligonucleotides for MMP-1 promoter fragments were annealed and cloned upstream of the c-Fos minimal promoter in a pGL3 Basic backbone. The AP1, PEA3 and HoxA5 point mutants were made in the -107/-57 fragment. The 3xAP1 and 3xPEA3 sites were cloned upstream of the c-Fos minimal promoter.\n\nThe -819/-174, -172/-27, -115/-27, -94/-27, -74/-27, and -59/-27 inserts and synthetic promoters were added upstream of a c-Fos minimal promoter insert27 in the pGL3-Basic backbone. -819/+71 AP-1 point mutations were made by PCR driven overlap extension28. pRL-SV40P with the SV40 promoter driving Renilla luciferase29 served as an internal control. pCMV-Luciferase30 served as a control.\n\nThe luciferase plasmids were transfected into cells using Lipofectamine 2000 (Life Technologies) per the manufacturer’s instructions. Cells were lysed in passive lysis buffer (Promega) 16 hours post transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega) per the manufacturer’s instructions with a 20/20 luminometer (Turner Biosystems) for a 10-second interval measurement. Mean and standard deviation for the ratio of firefly-luciferase to renilla-luciferase signals were calculated from three independent experiments. p-values were determine by Student’s two-tailed t-tests, with significance thresholds as indicated.\n\nWhole cell lysates were prepared with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-100, 1 mM DTT, 1 mM PMSF, Protease Inhibitor Cocktail III [1:200; Calbiochem], pH 7.6). After 10 minutes at 4°C, the lysates were centrifuged at 20,000 g for 15 minutes at 4°C, and lysate supernatants were normalized for protein levels with BCA Assays (Pierce) per the manufacturer’s instructions. Normalized lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto Trans-Blot transfer medium (Bio-Rad), and immunoblotted with primary antibody at 4°C for 16 hours. Antibodies used were against Fra-1 (rabbit polyclonal; sc-605X), MMP-1 (goat polyclonal; sc-12348), JunD (rabbit polyclonal; sc-74X), c-Jun (rabbit polyclonal; sc-1694X), HSP-90 (mouse monoclonal; sc-101494) and Actin (goat polyclonal; sc-1616) from Santa Cruz Biotechnology. Dilutions of 1:1000 of these antibodies were used in immunoblots. Membranes were then washed three times with Tris-buffered saline (TBS) and incubated with secondary antibody at a 1:10,000 dilution for one hour. Secondary antibodies used were: Goat anti-Rabbit IRDye 800CW, Goat anti-Rabbit IRDye 680LT, and Donkey anti-Goat IRDye 800CW from LiCor. Membranes were then washed three times with TBS. Membranes were measured for fluorescence with an Odyssey infrared imager (LiCor). Means and standard deviations were calculated from Odyssey quantitation of specific band intensities in three independent experiments. p-values were determined by Student’s two-tailed t-tests with significance thresholds as indicated.\n\nDouble stranded siRNA duplexes (Integrated DNA Technologies, Sigma-Aldrich), as indicated in Table 3, were transfected with RNAiMax Lipofectamine transfection reagent (Life Technologies) per the manufacturers instructions. Duplexes were designed as shown in Table 3.\n\nThe two oligonucleotides shown for each gene were annealed for use as siRNA. The Manufacture and catalog number for each are indicated.\n\nNuclear extracts were made from 4×107 cells grown on four 15 cm plates. Cells were washed with PBS at 4°C, and scraped into 3 mL of PBS. Samples were centrifuged at 400 g for 1 minute at 4°C in a J6B centrifuge (Beckman). The cell pellets were resuspended in 4 mL of Buffer A (10 mM Tris, 1.5 mM MgCl2, 10 mM KCl, 0.4 mM DTT, 0.04 mM PMSF, pH 7.9) and incubated for 10 minutes at 4°C. Samples were dounced 50 times with a type B 15 mL glass douncer (Kontes Glassware Co.). Dounced samples were centrifuged at 400 g for 10 minutes at 4°C in the J6B centrifuge. The nuclear pellets were resuspended in 300 μl Buffer C (20 mM Tris, 0.3 M KCl, 1.5 mM MgCl2, 25% Glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, pH 7.9) and rotated at 4°C for 30 minutes. Samples were centrifuged at 20,000 g for 15 minutes at 4°C. Nuclear extract supernatants were then removed, normalized for total protein levels by BCA Assays (Pierce) and used for DNA binding reactions.\n\nProbes and competitors for DNA binding assays were made with annealed complementary oligonucleotides (Integrated DNA Technologies), as shown in Table 4.\n\nThe Forward and Reverse oligonucleotides were annealed for each probe.\n\nThe annealed probes were end-labeled with γ-32P-ATP (Perkin Elmer) and poly nucleotide kinase (New England Biolabs), per the manufacturer’s instructions, to a final concentration of 1 ng/µl. DNA binding reactions contained 5 μl nuclear extract (approximately 10 μg), 1 ng of 32P-labeled probe, 50 ng poly dI-dC, 250 ng of competitor (as indicated), and 2 μg antibody (in supershift experiments) for 30 minutes at room temperature with binding buffer (final concentration: 10 mM Tris HCl (pH 8.0), 50 mM KCl, 0.5 mM EDTA, 0.1% Triton-X 100, 12.5% Glycerol, 0.2 mM DTT). Samples were then loaded on a 5% polyacrylamide gel in 1/4× TBE, and run for 2.5 hours at 100 V with 1× TBE running buffer. The gel was then dried and exposed to x-ray film (Kodak) for 16 hours.\n\nEMSAs for SRF were performed as above except with a high affinity SRF binding site, XGL, derived from the c-fos Serum Response Element31.\n\nChIP was performed as described32, with minor modifications. Briefly, 4×107 cells were crosslinked with 1% formaldehyde for 15 minutes at 25°C and quenched with 125 mM glycine. Crosslinked plates were lysed in RIPA buffer (as described in immunoblot methods above), sonicated with a Sonicator 3000 (Misonix) for 1 minute total, in 5 seconds on – 15 seconds off intervals, and centrifuged at 20,000 g for 15 minutes at 4°C. Lysates were normalized by BCA Assay (Pierce), per the manufacturer’s instructions, and immunoprecipitated with 2 μg of anti-Fra-1 antibody (Santa Cruz Biotechnology; Catalog #: sc-605) overnight rotating at 4°C. Protein-A agarose beads (7.5 μL) (Santa Cruz Biotechnology) diluted with 22.5 μL RIPA were added to purify immunoprecipitated protein for 90 minutes rotating at 4°C. The beads were washed three times in RIPA buffer and reconstituted in 200 μL elution buffer (70 mM Tris HCl pH 8.0, 1 mM EDTA, 1.5% SDS) for 10 minutes at 65°C. Beads were centrifuged at 1700 g for 1 minute at room temperature. The salt of the transferred supernatant was adjusted to a concentration of 200 mM NaCl and incubated for 5 hours at 65°C to reverse the crosslinks. DNA from ChIP samples was then purified with Qiaquik PCR Purification Kits (Qiagen) per the manufacturer’s instructions.\n\nPurified DNA was measured by qPCR (as previously described in the qRT-PCR method) with primers shown in Table 5.\n\nSamples were normalized to input DNA purified from reversed cross-linked input samples and measured through qPCR. Mean and standard deviations were calculated from three independent experiments. p-values were determine by Student’s two-tailed t-tests, with significance thresholds as indicated.\n\nCells (1×105) were plated in 6 cm plates for labeling. After 16 hours, cells were washed twice with warm PBS and starved for 30 minutes at 37°C with 4 mL methionine and cysteine free DMEM (Life Technologies). Media was changed to 2 mL 35S-Translabel metabolic labeling reagent (100 μCi/mL; MP Biomedicals) in methionine and cysteine free DMEM and incubated at 37°C for the indicated times. Plates were washed twice with cold PBS, lysed in ice cold RIPA buffer, centrifuged at 20,000 g for 15 minutes at 4°C, and immunoprecipitated with 2 μg of anti-Fra-1 antibody (Santa Cruz Biotechnology; #sc-605) overnight rotating at 4°C. Protein-A agarose beads (Santa Cruz Biotechnology) were used to purify immunoprecipitated protein by incubation for 90 minutes at 4°C. Washed beads were reconstituted in SDS-PAGE sample buffer (described above) and boiled for 5 minutes. Boiled samples were centrifuged at 1700 g for 1 minute at 4°C, and resolved on 12% SDS-PAGE for 2.5 hours at 150 V. The gel was placed in fixative (50% methanol/10% acetic acid) for 30 minutes rocking at 25°C. The gel was then enhanced with Amplify Fluorographic Reagent (GE) for 30 minutes rocking at 25°C. After enhancement, the gel was dried and exposed to film (Kodak) for 5 days. Autoradiographs were quantitated by ImageJ software analysis. Means and standard deviations were calculated from three independent experiments. p-values were determine by Student’s two-tailed t-tests with significance thresholds as indicated.\n\nSoft agar plating of the cell lines was performed as described33, with minor modifications. Briefly, 35-mm plates were coated with 1.5 mL 0.6% agar in DMEM. Cells (5×103) were reconstituted in 1.5 mL 0.3% agar in DMEM, and plated on top of the 0.6% agar layer. Agar layers were then covered with 1.5 mL DMEM/10% Fetal Bovine Serum. Cells were grown for 21 days with the media being changed every 5 days. Colonies were stained with 0.005% Crystal Violet in water for one hour and counted. Mean colony number and standard deviation were calculated from three independent experiments. p-values were determined by Student’s two tailed t-tests with significance thresholds as indicated.\n\nCells were grown to confluency and the monolayer was scratched and monitored by phase contrast microscopy. Cells were allowed to grow to 95% confluency and scratched with a pipette tip. Pictures of the cells were taken at 0 and 18 hours after the scratch. Images were taken at 100X magnification on a Nikon Diaphot 300 microscope. Triplicate images at each time point were used to count the number of cells that passed the scratch threshold. Mean and standard deviation were calculated from three independent experiments. p-values were determined by Student’s two-tailed t-tests, with significance thresholds as indicated.\n\n\nResults\n\nWe analyzed microarray gene expression data from a set of 16 breast carcinoma cell lines with well-characterized metastatic potential14 for a correlation between gene expression and metastasis. To identify genes that were specifically upregulated in cells with high bone or lung metastatic potential, we grouped cell lines as either highly metastatic to the bone, to the lung, or neither (i.e. with low metastatic potential)14. We determined the ratio of average expression in highly metastatic cell lines (bone or lung) to the low metastatic cells (Table 6; Supplementary Table 1). Microarray data from four highly metastatic bone cell lines were used, eight lung metastatic lines and four low or non-metastatic lines (described in Materials and methods). The highest differential expression was found for the MMP-1 gene. MMP-1 was expressed nearly an average of 100 fold more in bone metastatic cells than non-metastatic cells. Expression was also strongly higher in lung metastatic cells (27 fold), albeit with a weaker p value (0.056).\n\nHigh bone metastatic cell lines are: 1833, Scp-2, Scp-25 and Scp-46. High lung metastatic cell lines are: 1834, 3481, 4142, 4173, 4175, 4180, Scp-3 and Scp-28. Low metastatic cells lines are: MDA-MB-231, Scp-6, Scp-21 and Scp-26. The gene expression values for these cell lines were used in a two-tailed t-test to calculate relative p-values. Genes with p-values over 0.06 were not included.\n\nWe confirmed the microarray data for MMP-1 by measuring expression by quantitative RT-PCR (qPCR) in three cell lines with varying metastatic potential: MDA-MB-231 (low metastatic), and two MDA-MB-231 derived sub-lines: Scp-2 (highly metastatic) and Scp-21 (non-metastatic). Similar to what was found by microarray data, MMP-1 mRNA expression was 90 fold higher in Scp-2 cells than in Scp-21 cells and 17 fold higher than in MDA-MB-231 cells (Figure 1A, and Data File 1). Other MDA-MB-231 derived cell lines tested with high metastatic potential (Scp-28) and non-metastatic (Scp-3), similarly had high and low MMP1 expression, respectively, when measured by qPCR (data not shown and Data File 1). Immunoblot analysis confirmed that MMP-1 protein levels were commensurate with mRNA expression (Figure 1C and D, and Data File 1). These data indicate that MMP-1 is differentially regulated in cells with different metastatic potentials.\n\nIn order to test whether differential mRNA expression of MMP-1 is transcriptionally regulated, we used qPCR to measure the relative amounts of MMP-1 pre-mRNA. Pre-mRNA levels preceding splicing is a more direct indicator of transcription. Pre-mRNA levels of MMP-1 were also greatly elevated in Scp-2 metastatic cells compared to the non-metastatic Scp-21 cells, suggesting that this difference is due to changes in transcription (Figure 1B, and Data File 1).\n\nWe examined the human MMP-1 promoter for sequence conservation, and found blocks of conserved elements in the proximal promoter region (Figure 2A). These conserved regions overlap consensus transcription factor binding sites that have previously been identified for the MMP-1 promoter34–36.\n\nA) Genome Browser analysis of placental mammalian and vertebrate conservation by PhastCon26. Regions of conservation were compared to known transcription factor consensus sequences (shown in gray, with unmatched bases in red); numbers represent base position in reference to MMP-1’s transcription start site. B, D) Schematic of MMP-1 reporter constructs. C, E) Luciferase signal from Scp-2 and MDA-MB-231 cells transfected with the indicated reporter constructs. The signal was normalized to the Renilla luciferase levels from the co-transfected pRL-SV40P plasmid. The pCMV-luciferase construct was included as a control and its values were normalized to 1.0 for the Scp-2 cells. Its expression was approximately 10 times stronger than the MMP-1 -819/+71 luciferase reporter. The data shown represent the mean +/- standard deviation from three independent experiments. *, p < 0.05 for two-tailed t-tests.\n\nTo determine if the MMP-1 promoter is sufficient to reproduce differential transcription in reporter assays, we inserted sections of the MMP-1 promoter in luciferase reporter constructs (Figure 2B) and measured luciferase expression in Scp-2 and MDA-MB-231 cells (high and low metastatic cells, respectively). The MMP-1 promoter region from -819 to +71 was sufficient for five fold greater expression in the highly metastatic Scp-2 cells (Figure 2C, and Data File 2).\n\nIn order to determine which region of the MMP-1 promoter was required for differential transcription of MMP-1 in Scp-2, Scp-21 and MDA-MB-231 cells, 5′ and 3′ deletions were made (Figure 2B). Both -514/+71 and -174/+71 constructs were sufficient to drive significant differential expression, similar to -819/+71 (Figure 2C). As a control, we used a CMV promoter-luciferase construct that gave similar expression in the two cell lines. These results suggest that key regulatory elements for differential expression are in the -174/+71 promoter region.\n\nTo further delineate the region required for differential expression, we designed a 3′ deletion -819/-174 construct. The -819/-174 MMP-1 region was inserted into a luciferase plasmid upstream of the c-Fos minimal promoter (Figure 2B). The c-Fos minimal promoter includes the TATA box and transcription start site to give baseline expression. The -819/-174 construct was not able to drive significant expression (Figure 2C). Together, the 5′ and 3′ deletion constructs identified the -174/+70 MMP-1 promoter region as necessary and sufficient for MMP-1 transcriptional regulation.\n\nWe also used the c-Fos minimal promoter with a series of 5′ MMP-1 promoter deletions to -27, to further isolate the region required for expression in -174/+70 (Figure 2D). We found that the -94/-27 region was the minimal region required for differential expression between Scp-2 and MDA-MB-231 cells, with little differential expression seen with the -74 construct (Figure 2E, and Data File 2). However, while the ratio of expression between Scp-2 and MDA-MB-231 was consistent among -172/-27, -115/-27 and -94/-27, overall expression was significantly lower in -94/-27 and -115/-27 compared to -172/-27, suggesting that there are positively acting regulatory elements between -74 and -172. These constructs showed that the -94 to -27 region was sufficient for differential expression by the MMP1 promoter.\n\nHaving isolated a small regulatory region of the MMP-1 promoter, we sought to determine the specific transcription factor binding sites involved. Previous findings and conservation mapping pointed to several potential regulators in the -94/-27 region of the MMP-1 promoter: HoxA5, PEA3, and AP-1 (Figure 2A)37,38. To determine which, if any, of these sites are required for regulation, synthetic promoters were made with the region that contains the three consensus sites, -107 to -57, upstream of the c-Fos minimal promoter (Figure 3A). The -107/-57 region drove significantly higher expression in Scp-2 than in MDA-MB-231 cells (Figure 3B, and Data File 3). This differential expression was lower than with the constructs used in Figure 2E suggesting that sequences flanking the -107 to -57 region can modulate the induction. Nevertheless, these reporters allowed us to check this minimal region for the role of regulatory elements. Point mutations were made to each of the three conserved consensus regions (Figure 3A). Among them, only the AP-1 site mutation significantly decreased expression and decreased differential expression. While Scp-2 cells did have greater luciferase expression than MDA-MB-231 cells for the AP1 mutant, this low level was variable and the difference was not statistically significant (Figure 3B).\n\nA) Sequence of MMP-1 promoter regions inserted in reporter constructs. B) Luciferase signal from Scp-2 and MDA-MB-231 cells transfected with the indicated reporter constructs. The signal was normalized to the Renilla luciferase levels from the co-transfected pRL-SV40P plasmid. C) Sequences of the AP1 mutation in the -819/+71 reporter and of the 3× PEA3 and AP1 inserts. D) Luciferase signal from Scp-2 and MDA-MB-231 cells transfected with the indicated reporter constructs. The signal was normalized to the Renilla luciferase levels from the co-transfected pRL-SV40P plasmid. The 3x AP-1 construct values were normalized to 1.0 for the Scp-2 cells. Its expression was approximately three times stronger than the -819/+71 MMP-1 luciferase reporter. The data shown represent mean +/- standard deviation from three independent experiments. *, p < 0.05 ; **, p < 0.005 for two-tailed t-tests.\n\nTo confirm that the AP-1 site is required for expression in the context of the fuller promoter, we created a -819/+71 MMP-1 promoter construct with point mutations in the AP-1 consensus site (Figure 3C). These point mutations were sufficient to completely abrogate luciferase expression in both Scp-2 and MDA-MB-231 cells (Figure 3D, and Data File 3). The AP1 site alone was not sufficient to drive expression in Scp-2 cells as the site is present in the -74/-27 construct that was not expressed (Figure 2E). To determine whether multiple copies of the AP-1 site were sufficient, we made a synthetic promoter construct with a triple MMP-1 AP-1 consensus site and found that it gave a robust signal with significant differences between Scp-2 and MDA-MB-231 (Figure 3C and D). The ratios of luciferase expression in Scp-2 versus MDA-MB-231 cells were similar with the triple AP-1 synthetic promoter and the -819/+71 region of MMP-1 (Figure 3D). In contrast, a triple PEA3 site did not drive luciferase expression, suggesting that it is not sufficient for differential expression (Figure 3D). Together these experiments demonstrated that the AP-1 region of the promoter is both necessary and sufficient for differential transcriptional regulation of MMP-1 in Scp-2 and MDA-231 cell lines.\n\nThe AP-1 consensus site is bound by a dimer of AP-1 family members reviewed in39. There are seven AP-1 family genes: three Jun genes (c-Jun, JunB and JunD) and four Fos related genes (c-Fos, Fra-1, Fra-2, and FosB). Dimers are comprised of at least one Jun family member, but can be homo- or hetero-dimers40,41. To determine which AP-1 family members were expressed in Scp-2, Scp-21 and MDA-MB-231 cells, and would therefore be candidates for MMP-1 regulation, we performed qPCR in each of the cell lines. Fra-1, Fra-2 and JunD had the highest expression levels, with lower levels of c-Jun and nearly undetectable JunB, FosB and c-Fos (Figure 4A, and Data File 4). However, unlike the differential mRNA expression seen for MMP-1 (Figure 1A), all the detectable AP-1 family members had comparable mRNA expression among the different cell lines (Figure 4A). While there was significant variability in expression levels in experimental repeats, and hence the relatively large error bars, there was no consistent difference in expression among the cell lines.\n\nA) qPCR of AP-1 family member mRNA expression in Scp-2, Scp-21 and MDA-MB-231 cells. Mean relative values are shown +/- standard deviation from three independent experiments. B) Immunoblots with anti-AP-1 family antibodies of whole cell lysates from Scp-2, Scp-21, and MDA-MB-231 cells. Anti-MMP-1 is included for comparison and anti-actin antibody served as a loading control. C) Mean Fra-1 protein band intensity from immunoblots as in (B) +/- standard deviation from three independent experiments. **, p < 0.005 for two-tailed t-tests.\n\nTo explore whether AP-1 family member protein expression is consistent with their mRNA expression, we performed immunoblots. Specifically, we looked at Fra-1, Fra-2, c-Jun and JunD in Scp-2, Scp-21 and MDA-MB-231 cell lines. The remaining AP-1 family members, c-Fos, FosB and JunB, that were not expressed at the mRNA level were not considered further. Interestingly, contrary to Fra-1 mRNA expression levels, Fra-1 protein levels were significantly higher in Scp-2 cells than Scp-21 and MDA-MB-231 cells (Figure 4B and C, and Data File 4). However, there was no significant difference in protein expression levels of Fra-2, JunD or c-Jun. These results suggest the possibility that differences in Fra-1 protein expression in Scp-2, Scp-21 and MDA-MB-231 cell lines are responsible for regulation of MMP-1 transcription.\n\nTo test the hypothesis that Fra-1 regulates MMP-1, we inhibited expression of Fra-1 in Scp-2 cells with short interfering RNAs (siRNA). Two siRNA duplexes decreased Fra-1 mRNA expression by over 80% (Figure 5A) and Fra-1 protein levels by about 70% (Figure 5B and C, and Data File 5). This inhibition greatly reduced MMP-1 mRNA expression (Figure 5D, and Data File 5), supporting Fra-1’s role in MMP-1 regulation.\n\nA) Scp-2 cells were transfected with control or two independent siRNA duplexes for Fra-1. Fra-1 mRNA levels were measured by qPCR. B) Scp-2 cells transfected with control or Fra-1 siRNA duplexes were immunoblotted with anti-Fra-1 antibodies. Anti-actin antibody served as a loading control. C) Mean Fra-1 band intensity from immunoblots as in (B) from three independent experiments. D) qPCR of MMP-1 and GAPDH control mRNA expression of Scp-2 cells transfected with control or Fra-1 siRNA duplexes. In A, C and D, mean relative values are shown +/- standard deviation from three independent experiments. *, p < 0.05; **, p < 0.005 for two-tailed t-tests.\n\nThough other AP-1 family members were not differentially expressed in the Scp-2, Scp-21 and MDA-MB-231 cell lines, we sought to determine which other AP-1 family members were required for MMP-1 expression. As JunD is the most strongly expressed Jun family member in these cells, we first inhibited its mRNA expression with siRNA duplexes (Supplementary Figure 1). However, despite efficient reduction in JunD levels, this inhibition did not have an effect on MMP-1 mRNA expression (Supplementary Figure 1).\n\nWe had difficulty efficiently depleting Fra-2 and c-Jun with siRNAs, perhaps because mRNA expression of these genes was relatively low. As such, partial inhibition of c-Jun and Fra-2 had no statistically significant impact on MMP-1 (data not shown). Therefore, it was not possible for us to assess whether c-Jun, or c-Jun acting redundantly with JunD, were required for MMP-1 expression. Nevertheless, the requirement of the AP-1 site in the MMP1 promoter and depletion of Fra-1 clearly show that this factor is required for expression of MMP1 in the metastatic MDA-MB-231 derived cells. The differential expression of Fra-1 protein levels suggests that this mechanism may at least partially account for differences in MMP1 expression.\n\nAs previous experiments showed that Fra-1 was required for MMP-1 expression, we confirmed protein binding in vitro to the MMP-1 AP-1 site in the highly metastatic Scp-2 and non-metastatic Scp-21 cells using electrophoretic mobility shift assays (EMSA). The -107 to -57 region of the MMP-1 promoter, containing the AP-1 consensus sequence, was used as a probe for binding with nuclear extracts from Scp-2 and Scp-21 cells. Specific binding was observed (Figure 6A lanes 1 and 2) which was competed by excess non-labeled competitor (lanes 3 and 4). Mutations in the AP-1 binding site abolished this competition, suggesting that the band is indeed AP-1 (lanes 5 and 6).\n\nA) In vitro binding. Electrophoretic mobility shift assay (EMSA) with the MMP-1 AP-1 site. Scp-2 and Scp-21 nuclear extracts were incubated with a 32P-end labeled MMP-1 promoter double-stranded oligonucleotide probe spanning the AP-1 consensus site (-107 to -57 bases relative to the transcription start site). Nonspecific competitor, unlabeled, and point mutant AP-1 site oligonucleotides were added in 250 fold excess of the probe. The final three lanes included anti-AP-1 family member antibodies. B) Control EMSAs were preformed as in A except with a Serum Response Element probe to detect SRF binding. C) In vivo binding. Chromatin immunoprecipitation with Scp-2 and Scp-21 cells immunoprecipated with anti-Fra-1 antibody or mock antibody control. The immunoprecipitated DNA from the samples was measured by qPCR for binding of Fra-1 to the MMP-1 AP-1 promoter site, an upstream non-AP-1 control MMP-1 site, the IL-6 gene AP-1 site, or an upstream non-AP-1 control IL-6 site. The data shown represent the mean fold over control DNA values +/- standard deviation for three indendent experiments. **, p < 0.005 for two-tailed t-tests.\n\nInterestingly, a stronger AP-1 complex was detected in the highly metastatic Scp-2 cells compared with the low metastatic Scp-21 cells (Figure 6A, compare lanes 1 and 2). This is consistent with higher Fra-1 protein expression in Scp-2 cells and higher expression of MMP1 (Figure 4B and C). As a control for the similarity of the nuclear extracts of the two cell lines, we examined binding of the transcription factor SRF to a Serum Response Element (SRE) probe and found no significant difference (Figure 6B).\n\nTo determine which proteins in the nuclear extracts were present in the bound band, we used antibodies specific for AP-1 family members. Anti-Fra-1 antibody supershifted the band (Figure 6A, lane 7), indicating that Fra-1 is a major component of the bound complex. In contrast, Fra-2 antibodies had little effect (lane 8). Antibodies to c-Jun strongly shifted the complex into multiple bands, suggesting that it too is in the complex (lane 9). We did not observe a shift with antibodies to JunD, however the antibodies may be ineffective for supershifts (data not shown). These EMSA experiments support the conclusion that Fra-1 and c-Jun are the predominant members of the AP-1 complex bound to the MMP-1 site.\n\nTo show Fra-1 binding and regulation of the MMP-1 promoter in vivo, we performed chromatin immunoprecipitation (ChIP) experiments in Scp-2 and Scp-21 cells. Antibodies to Fra-1 demonstrated higher binding to the MMP-1 promoter in Scp-2 cells than Scp-21, consistent with the relative MMP-1 expression in these cells (Figure 6C, and Data File 6). A similar, though slightly weaker, difference was seen at a known AP-1 binding site in the IL-6 gene (Figure 6C)42,43. Background signal was seen at distal control sites in the MMP-1 and IL-6 genes or with a non-specific control antibody.\n\nSince Fra-1 is required for MMP-1 expression and binds preferentially to the MMP-1 promoter in Scp-2 cells, we analyzed Fra-1 regulation. As shown Figure 4, Fra-1 mRNA levels did not vary significantly among the metastatic variant cell lines, while Fra-1 protein levels were higher in Scp-2 cells. To better understand the post-transcriptional regulation of Fra-1, we analyzed Fra-1 protein degradation and translation.\n\nWe first measured the degradation rate by blocking new protein translation using the protein synthesis inhibitor cycloheximide. By measuring protein levels over time, without de-novo translation, we could compare degradation rates of Fra-1 in Scp-2 and Scp-21 cells. We compared Fra-1 levels at either 0 to 4 or 0 to 24 hour intervals (Figure 7A). While levels were somewhat variable in specific experiments (as seen in Figure 7A) we quantified the results from three repetitions. We found that Fra-1 protein was more abundant in Scp-2 than Scp-21 cells, as previously seen, and when we normalized to the starting relative levels in each cell line, we found that there was no significant difference in the stability of Fra-1 in these two cell lines (Figure 7B, and Data File 7).\n\nA) Fra-1 protein stability. Scp-2 and Scp-21 cells were treated with cycloheximide and whole cell lysates collected at the indicated times post-treatment. Immunoblots are shown with anti-Fra-1 antibody of representative experiments. B) The mean relative Fra-1 band intensities from immunoblots as in (A) from three independent experiments +/- the standard deviation are shown. C) 35S-metabolic labeling of Fra-1. Cells were depleted of cysteine and methionine for 30 minutes and labeled with 35S-cysteine and -methionine for the indicated times and immunoprecipitated. D) Fra-1 protein levels as in (C) were quantified and normalized to total protein labeling. The mean band intensity +/- standard deviation from three independent experiments. E) Control for general protein synthesis. Autoradiograph of total protein on an SDS-polyacrylamide gel from cells depleted of cysteine and methionine for 30 minutes and labeled with 35S-cysteine and -methionine for the indicated times.\n\nWith no difference in Fra-1 protein degradation or mRNA levels, we measured Fra-1 translation rates. Scp-2 and Scp-21 cells were labeled with 35S-labeled amino acids to measure amino acid incorporation into proteins over a one-hour interval. Immunoprecipitation of Fra-1 showed that its translation was significantly higher in Scp-2 cells than Scp-21 cells (Figure 7C and D, and Data File 7). There was a slightly higher general protein synthesis in the Scp-21 cells as seen by running the total 35S-labeled cell lysates on an SDS-polyacrylamide gel (Figure 7E). This is the opposite direction as seen for Fra-1 protein synthesis. Together with the lack of change in Fra-1 protein stability and the approximate half-life of 5 hours (Figure 7B), these results suggest that Fra-1 is regulated at the level of protein translation.\n\nTo determine the effect of higher Fra-1 expression in non-metastatic cells, we created Scp-21 cells that stably express Fra-1. Control Scp-21 cells that stably express a control vector, have low Fra-1 protein expression, while the cells infected with a Fra-1 retrovirus expressed high levels, several fold higher than that in Scp-2 cells (Figure 8B). We found that higher Fra-1 expression resulted in higher MMP1 mRNA and protein expression, suggesting that higher levels of Fra-1 are sufficient for increased MMP1 expression (Figure 8A and B, and Data File 8).\n\nA) qPCR of MMP-1 in Scp-2, Scp-21, Scp-21 control vector (Puro), and Scp-21 cells stably expressing HA-tagged Fra-1. Mean relative values are +/- standard deviation from three independent experiments. B) Immunoblots of the indicated cell lines with anti-MMP-1, anti-Fra-1 and anti-HA antibodies. Anti-HSP-90 served as a loading control. C) Effect on cell motility. Scratch-wound assays of the indicated cell lines were performed with cell motility measured 0 and 18 hours after the scratch. Cells were grown to 95% confluency and scratched with a pipette tip. Images were taken at 100X magnification. D) Quantitation of the number of cells crossing the initial scratch threshold at 18 hours. The means of three fields in three independent experiments +/- standard deviation are shown. E) Anchorage independent growth. The indicated cell lines were grown in soft agar for 21 days. The means +/- standard deviation of the relative number of colonies formed in three independent experiments are shown. *, p < 0.05. **, p < 0.005 for two-tailed t-tests.\n\nSince increased Fra-1 protein expression correlated with increased MMP-1 expression and metastasis, we tested whether it is sufficient to drive properties of metastatic cells, in particular cell motility and anchorage independent growth. A scratch assay, or wound healing assay, was used to measure cell motility. We found that Scp-2 had greater motility than Scp-21 cells, and that Scp-21 cells expressing Fra-1 had significantly greater motility than vector control Scp-21 cells (Figure 8C and D, and Data File 8). Surprisingly, Scp-21 cells expressing Fra-1 had even greater motility than highly metastatic Scp-2 cells. Therefore, Fra-1 expression increases motility.\n\nNon-metastatic cells often grow poorly in soft agar, while metastatic cells can display increased anchorage independent growth44,45. Similar to the pattern seen in motility assays, Scp-2 showed significantly greater growth in soft agar than Scp-21 cells (Figure 8E, and Data File 8). Interestingly, Scp-21 cells expressing Fra-1 greatly increased the growth of the cells in soft agar, similar to the Scp-2 levels. These results indicate that increased Fra-1 expression in Scp-21 cells was sufficient for increased anchorage independent growth.\n\n\n\n\nDiscussion\n\nWe have used a well defined system of metastatic cell variants to limit the heterogeneity of the samples and to provide a large number of closely related cell lines with variable metastatic potential14. The cell lines are all derived from MDA-MB-231 breast carcinoma cells, either by selection of metastatic clones in mouse xenografts or by analysis of single cell clones (the Scp lines). The result that the Scp cell clones have vastly different, but reproducible, metastatic potentials suggests that the cells with these properties were pre-existing in the MDA-MB-231 cultures12,14. The analysis of gene expression in these cell lines yielded a list of genes correlated to metastatic potential. We have found that MMP-1 is among the most strongly elevated genes in cells with high metastatic potential and that this expression is transcriptionally regulated by Fra-1 interaction with the AP-1 site of the MMP-1 promoter. Fra-1 expression was also regulated, but at the level of protein translation.\n\nThe mapping of sequence elements required for expression of MMP-1 in high vs. low metastatic cells did not reveal an element that was required in only one of the cell types. However, the AP-1 site was strongly required for expression in both cell lines. Furthermore, the synthetic triplicate AP-1 site promoter construct showed higher expression in the highly metastatic cells, demonstrating that it is sufficient to mediate higher expression. The single AP1 site in the MMP-1 promoter was not sufficient, suggesting that it normally requires the binding of other factors to function fully. Initial studies of phorbol ester induction of the MMP-1 promoter in fibroblasts had a similar result38. Therefore, it is likely that additional factor binding to the -96/-74 region of the MMP-1 promoter is also required. However, transcription factors with known binding sites in that region, HoxA5 and PEA3, were not required. In addition, expression of the MMP-1 reporter gene was significantly greater with the -172/-27 region compared to the -115/-27 region. While this region was not required for differential expression, it is likely that additional factor binding in this region increases expression.\n\nIn line with our findings, AP-1 regulation of MMP-1 has been well studied in several systems18,46,47 and AP-1 expression has been implicated in tumorigenesis22,48,49. In particular, expression of Fra-1 has been shown to be correlated to plastic proliferative breast disorders21 and aggressive breast cancer cells50.\n\nWe have shown that Fra-1 is required for MMP-1 expression in the MDA-MB-231 derivatives. Fra-1 binds to the AP-1 consensus sequence as a heterodimer with a Jun protein51. Therefore, a Jun protein should also be required for MMP-1 expression. However, depletion of the most highly expressed Jun protein, JunD, had no effect on MMP-1 expression. JunB could not be detected by immunoblotting and showed very low expression by qPCR. The final Jun protein, c-Jun, was detected by immunoblot and qPCR. However, five siRNA duplexes were unable to significantly reduce c-Jun expression (data not shown). Challenges inhibiting c-Jun expression may be due to its low levels or, alternatively, to a cell requirement for c-Jun expression—making c-Jun inhibition toxic to the cell. Due to the inability to strongly deplete c-Jun levels, we cannot determine whether it is required for MMP-1 expression or whether it fulfills a redundant requirement with JunD. It is also possible that there is a novel partner for Fra-1 in these cells.\n\nWe did not detect altered Fra-1 mRNA expression in the MDA-MB-231 cell variants, however higher mRNA expression has been observed in more metastatic ER negative cell lines when compared to less metastatic cells52,53. Differences in Fra-1 mRNA expression were also observed in breast cancer patients, where expression was higher in carcinomas compared with benign tumors21. Thus, besides regulation of translation, as we have found here, alternative mechanisms to regulate Fra-1 mRNA expression may be important in some breast tumors.\n\nWhile Fra-1 mRNA levels were not significantly regulated in the MDA-MB-231 cell variants, immunoblots, EMSAs and chromatin immunoprecipitations showed that the metastatic variant Scp-2 cells have higher Fra-1 protein expression and higher DNA binding in vitro and in vivo to the MMP-1 AP-1 site. As Fra-1 was the only detectable AP-1 family factor that varied in the metastatic variants, this suggests that Fra-1 is responsible for the difference in MMP-1 expression. In addition, overexpression of Fra-1 in the low metastatic Scp21 cells increased MMP-1 expression, showing that higher Fra-1 expression is sufficient, as well as necessary, for elevated MMP-1 expression.\n\nWe found that Fra-1 protein levels were regulated by altered translation rates. There were little differences in the rates of protein degradation. However, short metabolic labeling showed increased synthesis of Fra-1 in the metastatic cell variant. Several studies have demonstrated that phosphorylation of Fra-1 by ERK1/2 increases its protein stability54–57. However, this mechanism does not appear to be functioning in the MDA-MB-231 cells, since we did not detect a change in degradation rates.\n\nRecently, data from human cancer cell lines pointed to evidence of miRNA-34a regulation of Fra-1 and MMP-158,59. In breast cancers, miRNA-34a was inversely correlated to the metastatic potential of cell lines and tumor samples, but was not found to be different in paired tumor and normal breast tissue samples59. Strikingly, expression of miRNA-34a in MDA-MB-231 cells reduced Fra-1 expression, matrigel invasion, and tumors in mouse xenografts. In addition, overexpression of Fra-1 rescued the suppressive effects of miRNA-34a on migration and invasion of MDA-MB-231 cells59. While miRNA-34a regulation of Fra-1 is a strong hypothesis for MMP-1 regulation in MDA-MB-231 variants, a major difference is that we did not observe changes in Fra-1 mRNA expression as reported with miRNA-34a58,59. Separately, miRNA-143 was also found to target Fra-1 mRNA, suggesting that this and other miRNAs are also candidates for Fra-1 regulation60. It will be interesting to determine which, if any, miRNA regulates Fra-1 translation in metastatic MDA-MB-231 variants.\n\nDespite initial work supporting miRNA translational regulation without impact on mRNA levels61, more recent evidence supports miRNA regulation of both protein and mRNA expression62,63. There are several examples of other genes being regulated by miRNA without discernable differences in mRNA levels64–67. As our experiments only show translational regulation, it is possible that Fra-1 is a case where miRNA regulation is entirely translational. Alternatively, it is possible that Fra-1 translation is regulated by a mechanism other than miRNA.\n\nStable expression of Fra-1 in non-metastatic MDA-MB-231 derivative cells led to greater MMP-1 expression, motility and anchorage-independent growth. This supports Fra-1 as an upstream regulator of MMP-1 and potentially of other genes required for increased metastatic properties. These results are in line with previous colon cancer data, correlating Fra-1 expression with escape from anoikis56, and increased motility68. In spontaneous murine mammary adenocarcinoma variants with different metastatic potential, Fra-1 expression also correlated to invasiveness69. Transient transfections of Fra-1 in MDA-MB-231 and MCF-7 similarly increased matrigel cell invasion48. Contrary to our findings, this overexpression had no impact on MMP-1 expression. In other experiments, however, overexpression of Fra-1 in MCF-7 cells increased cell invasion and MMP-1 expression52. Recently, Fra-1 was also shown to be required for high metastasis in xenografts of a highly metastatic MDA-MB-231 derivative cell line70.\n\nFra-1 has many direct and indirect targets71. Fra-1 depletion in a highly metastatic MDA-MB-231 variant line altered the expression of 1,234 genes70. Among these, E-cadherin has an inverse correlation with Fra-1, confirming previous results50. Fra-1 expression has also previously been shown to alter morphology and invasiveness69 in a manner similar to the epithelial to mesenchymal transition (EMT). As such, Fra-1 regulation may function as a keystone regulator, impacting several aspects of tumorigenesis and metastasis72.\n\nHow MMP-1 function is coopted by tumor cells is an open question. MMP-1 is critical in degrading interstitial collagen, and tumor cells may require that function to invade15,73. However, MMP-1 has also been shown to be required for migration and xenograft tumor formation by MDA-MB-231 cells through cleavage and activation of protein activated receptor-1 (PAR-1), such that autocrine activation of specific cellular proteins is an alternative mechanism for MMP-1 function74.\n\n\nConclusion\n\nOur work and the work of others have clearly demonstrated effects of Fra-1 and MMP-1 in multiple cancer systems. We find that in the highly metastatic MDA-MB-231 cells, Fra-1 is activated at the level of protein translation, perhaps through the loss or inhibition of an miRNA. Increased Fra-1 protein then binds the AP-1 site of the MMP-1 promoter, increasing MMP-1 transcription and translation. It is likely that other targets of Fra-1 also contribute to increased metastasis. Further research will be necessary to determine the mechanism of Fra-1 translational regulation, which target genes are involved that can lead to increased metastasis, and how these steps might be blocked to prevent metastatic progression of breast cancer.",
"appendix": "Author contributions\n\n\n\nE.H. carried out all the experiments. E.H. and R.P. designed the experiments. The manuscript was jointly written.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has been supported by funding from the Guzik Foundation to E.H. and R.P.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis research was supported by generous gifts of cells and supplies by Joan Massague, Carol Prives, Will Freed-Pastor, and Anthony Barsotti.\n\n\nSupplementary figure\n\nA) Immunoblot with anti-JunD antibodies of Scp-2 cells transfected with control or siRNA duplexes for JunD. B) Mean JunD band intensity +/- standard deviation for immunoblots from three independent experiments as in (A). C) qPCR of JunD, MMP-1 and Fra-1 in Scp-2 cells transfected with control or siRNA duplexes for JunD. Mean relative values are +/- standard deviation from three independent experiments are shown. **, p < 0.005 for two-tailed t-tests.\n\n\nReferences\n\nBrinckerhoff CE, Rutter JL, Benbow U: Interstitial collagenases as markers of tumor progression. Clin Cancer Res. 2000; 6(12): 4823–4830. PubMed Abstract\n\nMcGowan PM, Duffy MJ: Matrix metalloproteinase expression and outcome in patients with breast cancer: analysis of a published database. Ann Oncol. 2008; 19(9): 1566–1572. PubMed Abstract | Publisher Full Text\n\nCheng S, Tada M, Hida Y, et al.: High MMP-1 mRNA expression is a risk factor for disease-free and overall survivals in patients with invasive breast carcinoma. J Surg Res. 2008; 146(1): 104–109. PubMed Abstract | Publisher Full Text\n\nPoola I, DeWitty RL, Marshalleck JJ, et al.: Identification of MMP-1 as a putative breast cancer predictive marker by global gene expression analysis. Nat Med. 2005; 11(5): 481–483. PubMed Abstract | Publisher Full Text\n\nNakopoulou L, Giannopoulou I, Gakiopoulou H, et al.: Matrix metalloproteinase-1 and -3 in breast cancer: correlation with progesterone receptors and other clinicopathologic features. Hum Pathol. 1999; 30(4): 436–442. PubMed Abstract\n\nKohrmann A, Kammerer U, Kapp M, et al.: Expression of matrix metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New findings and review of the literature. BMC cancer. 2009; 9: 188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRutter JL, Mitchell TI, Buttice G, et al.: A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter creates an Ets binding site and augments transcription. Cancer Res. 1998; 58(23): 5321–5325. PubMed Abstract\n\nMcCready J, Broaddus WC, Sykes V, et al.: Association of a single nucleotide polymorphism in the matrix metalloproteinase-1 promoter with glioblastoma. Int J Cancer. 2005; 117(5): 781–785. PubMed Abstract | Publisher Full Text\n\nGiambernardi TA, Grant GM, Taylor GP, et al.: Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol. 1998; 16(8): 483–496. PubMed Abstract | Publisher Full Text\n\nKousidou OC, Roussidis AE, Theocharis AD, et al.: Expression of MMPs and TIMPs genes in human breast cancer epithelial cells depends on cell culture conditions and is associated with their invasive potential. Anticancer Res. 2004; 24(6): 4025–4030. PubMed Abstract\n\nBalduyck M, Zerimech F, Gouyer V, et al.: Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro. Clin Exp Metastasis. 2000; 18(2): 171–178. PubMed Abstract | Publisher Full Text\n\nKang Y, Siegel PM, Shu W, et al.: A multigenic program mediating breast cancer metastasis to bone. Cancer cell. 2003; 3(6): 537–549. PubMed Abstract | Publisher Full Text\n\nBos PD, Zhang XH, Nadal C, et al.: Genes that mediate breast cancer metastasis to the brain. Nature. 2009; 459(7249): 1005–1009. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinn AJ, Gupta GP, Siegel PM, et al.: Genes that mediate breast cancer metastasis to lung. Nature. 2005; 436(7050): 518–524. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrinckerhoff CE, Matrisian LM: Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol. 2002; 3(3): 207–214. PubMed Abstract | Publisher Full Text\n\nWeiss MB, Abel EV, Mayberry MM, et al.: TWIST1 is an ERK1/2 effector that promotes invasion and regulates MMP-1 expression in human melanoma cells. Cancer Res. 2012; 72(24): 6382–6392. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim S, Han J, Shin I, et al.: A functional comparison between the HER2 (high)/HER3 and the HER2(low)/HER3 dimers on heregulin-beta1-induced MMP-1 and MMP-9 expression in breast cancer cells. Exp Mol Med. 2012; 44(8): 473–482. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAngel P, Baumann I, Stein B, et al.: 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region. Mol Cell Biol. 1987; 7(6): 2256–2266. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAngel P, Imagawa M, Chiu R, et al.: Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987; 49(6): 729–739. PubMed Abstract | Publisher Full Text\n\nYoung MR, Colburn NH: Fra-1 a target for cancer prevention or intervention. Gene. 2006; 379: 1–11. PubMed Abstract | Publisher Full Text\n\nChiappetta G, Ferraro A, Botti G, et al.: FRA-1 protein overexpression is a feature of hyperplastic and neoplastic breast disorders. BMC cancer. 2007; 7: 17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSong Y, Song S, Zhang D, et al.: An association of a simultaneous nuclear and cytoplasmic localization of Fra-1 with breast malignancy. BMC cancer. 2006; 6: 298. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPear WS, Nolan GP, Scott ML, et al.: Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993; 90(18): 8392–8396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhite EA, Sowa ME, Tan MJ, et al.: Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses. Proc Natl Acad Sci U S A. 2012; 109(5): E260–267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgenstern JP, Land H: Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990; 18(12): 3587–3596. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKent WJ, Sugnet CW, Furey TS, et al.: The human genome browser at UCSC. Genome Res. 2002; 12(6): 996–1006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y, Prywes R: Activation of the c-fos enhancer by the erk MAP kinase pathway through two sequence elements: the c-fos AP-1 and p62TCF sites. Oncogene. 2000; 19(11): 1379–1385. PubMed Abstract | Publisher Full Text\n\nHeckman KL, Pease LR: Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc. 2007; 2(4): 924–932. PubMed Abstract | Publisher Full Text\n\nChen X, Prywes R: Serum-induced expression of the cdc25A gene by relief of E2F-mediated repression. Mol Cell Biol. 1999; 19(7): 4695–4702. PubMed Abstract | Free Full Text\n\nSelvaraj A, Prywes R: Megakaryoblastic leukemia-1/2, a transcriptional co-activator of serum response factor, is required for skeletal myogenic differentiation. J Biol Chem. 2003; 278(43): 41977–41987. PubMed Abstract | Publisher Full Text\n\nPrywes R, Dutta A, Cromlish JA, et al.: Phosphorylation of serum response factor, a factor that binds to the serum response element of the c-FOS enhancer. Proc Natl Acad Sci U S A. 1988; 85(19): 7206–7210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeckerman R, Donner AJ, Mattia M, et al.: A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint. Genes Dev 2009; 23(11): 1364–1377. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreed-Pastor WA, Mizuno H, Zhao X, et al.: Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway. Cell. 2012; 148(1–2): 244–258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOverall CM: Matrix metalloproteinase substrate binding domains, modules and exosites. Overview and experimental strategies. Methods Mol Biol. 2001; 151: 79–120. PubMed Abstract | Publisher Full Text\n\nPardo A, Selman M: MMP-1: the elder of the family. Int J Biochem Cell Biol. 2005; 37(2): 283–288. PubMed Abstract | Publisher Full Text\n\nRa HJ, Parks WC: Control of matrix metalloproteinase catalytic activity. Matrix Biol. 2007; 26(8): 587–596. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGutman A, Wasylyk B: The collagenase gene promoter contains a TPA and oncogene-responsive unit encompassing the PEA3 and AP-1 binding sites. EMBO J. 1990; 9(7): 2241–2246. PubMed Abstract | Free Full Text\n\nAuble DT, Brinckerhoff CE: The AP-1 sequence is necessary but not sufficient for phorbol induction of collagenase in fibroblasts. Biochemistry. 1991; 30(18): 4629–4635. PubMed Abstract | Publisher Full Text\n\nOzanne BW, Spence HJ, McGarry LC, et al.: Transcription factors control invasion: AP-1 the first among equals. Oncogene. 2007; 26(1): 1–10. PubMed Abstract | Publisher Full Text\n\nVerde P, Casalino L, Talotta F, et al.: Deciphering AP-1 function in tumorigenesis: fra-ternizing on target promoters. Cell Cycle. 2007; 6(21): 2633–2639. PubMed Abstract | Publisher Full Text\n\nHalazonetis TD, Georgopoulos K, Greenberg ME, et al.: c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell. 1988; 55(5): 917–924. PubMed Abstract | Publisher Full Text\n\nDendorfer U, Oettgen P, Libermann TA: Multiple regulatory elements in the interleukin-6 gene mediate induction by prostaglandins, cyclic AMP and lipopolysaccharide. Mol Cell Biol. 1994; 14(7): 4443–4454. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSehgal PB, Wang L, Rayanade R, et al.: Interleukin-6-type cytokines. Ann N Y Acad Sci. 1995; 762: 1–14. PubMed Abstract | Publisher Full Text\n\nHamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science. 1977; 197(4302): 461–463. PubMed Abstract | Publisher Full Text\n\nYoneda T, Williams PJ, Hiraga T, et al.: A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res. 2001; 16(8): 1486–1495. PubMed Abstract | Publisher Full Text\n\nLee W, Mitchell P, Tjian R: Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell. 1987; 49(6): 741–752. PubMed Abstract | Publisher Full Text\n\nVincenti MP, White LA, Schroen DJ, et al.: Regulating expression of the gene for matrix metalloproteinase-1 (collagenase): mechanisms that control enzyme activity, transcription, and mRNA stability. Crit Rev Eukaryot Gene Expr. 1996; 6(4): 391–411. PubMed Abstract\n\nMilde-Langosch K: The Fos family of transcription factors and their role in tumourigenesis. Eur J Cancer. 2005; 41(16): 2449–2461. PubMed Abstract | Publisher Full Text\n\nBamberger AM, Methner C, Lisboa BW, et al.: Expression pattern of the AP-1 family in breast cancer: association of fosB expression with a well-differentiated, receptor-positive tumor phenotype. Int J Cancer. 1999; 84(5): 533–538. PubMed Abstract | Publisher Full Text\n\nZajchowski DA, Bartholdi MF, Gong Y, et al.: Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells. Cancer Res. 2001; 61(13): 5168–5178. PubMed Abstract\n\nCohen DR, Ferreira PC, Gentz R, et al.: The product of a fos-related gene, fra-1, binds cooperatively to the AP-1 site with Jun transcription factor AP-1 is comprised of multiple protein complexes. Genes Dev. 1989; 3(2): 173–184. PubMed Abstract | Publisher Full Text\n\nBelguise K, Kersual N, Galtier F, et al.: FRA-1 expression level regulates proliferation and invasiveness of breast cancer cells. Oncogene. 2005; 24(8): 1434–1444. PubMed Abstract | Publisher Full Text\n\nPhilips A, Teyssier C, Galtier F, et al.: FRA-1 expression level modulates regulation of activator protein-1 activity by estradiol in breast cancer cells. Mol Endocrinol. 1998; 12(7): 973–985. PubMed Abstract | Publisher Full Text\n\nBasbous J, Chalbos D, Hipskind R, et al.: Ubiquitin-independent proteasomal degradation of Fra-1 is antagonized by Erk1/2 pathway-mediated phosphorylation of a unique C-terminal destabilizer. Mol Cell Biol. 2007; 27(11): 3936–3950. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCasalino L, De Cesare D, Verde P: Accumulation of Fra-1 in ras-transformed cells depends on both transcriptional autoregulation and MEK-dependent posttranslational stabilization. Mol Cell Biol. 2003; 23(12): 4401–4415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVial E, Marshall CJ: Elevated ERK-MAP kinase activity protects the FOS family member FRA-1 against proteasomal degradation in colon carcinoma cells. J Cell Sci. 2003; 116(Pt 24): 4957–4963. PubMed Abstract | Publisher Full Text\n\nGruda MC, Kovary K, Metz R, et al.: Regulation of Fra-1 and Fra-2 phosphorylation differs during the cell cycle of fibroblasts and phosphorylation in vitro by MAP kinase affects DNA binding activity. Oncogene. 1994; 9(9): 2537–2547. PubMed Abstract\n\nWu J, Wu G, Lv L, et al.: MicroRNA-34a inhibits migration and invasion of colon cancer cells via targeting to Fra-1. Carcinogenesis. 2012; 33(3): 519–528. PubMed Abstract | Publisher Full Text\n\nYang S, Li Y, Gao J, et al.: MicroRNA-34 suppresses breast cancer invasion and metastasis by directly targeting Fra-1. Oncogene. 2013; 32(36): 4294–303. PubMed Abstract | Publisher Full Text\n\nHorita HN, Simpson PA, Ostriker A, et al.: Serum response factor regulates expression of phosphatase and tensin homolog through a microRNA network in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2011; 31(12): 2909–2919. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOlsen PH, Ambros V: The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol. 1999; 216(2): 671–680. PubMed Abstract | Publisher Full Text\n\nEulalio A, Huntzinger E, Izaurralde E: Getting to the root of miRNA-mediated gene silencing. Cell. 2008; 132(1): 9–14. PubMed Abstract | Publisher Full Text\n\nFilipowicz W, Bhattacharyya SN, Sonenberg N: Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008; 9(2): 102–114. PubMed Abstract | Publisher Full Text\n\nPoy MN, Eliasson L, Krutzfeldt J, et al.: A pancreatic islet-specific microRNA regulates insulin secretion. Nature. 2004; 432(7014): 226–230. PubMed Abstract | Publisher Full Text\n\nCimmino A, Calin GA, Fabbri M, et al.: miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A. 2005; 102(39): 13944–13949. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhattacharyya SN, Habermacher R, Martine U, et al.: Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell. 2006; 125(6): 1111–1124. PubMed Abstract | Publisher Full Text\n\nFazi F, Rosa A, Fatica A, et al.: A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell. 2005; 123(5): 819–831. PubMed Abstract | Publisher Full Text\n\nVial E, Sahai E, Marshall CJ: ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumor cell motility. Cancer cell. 2003; 4(1): 67–79. PubMed Abstract | Publisher Full Text\n\nKustikova O, Kramerov D, Grigorian M, et al.: Fra-1 induces morphological transformation and increases in vitro invasiveness and motility of epithelioid adenocarcinoma cells. Mol Cell Biol. 1998; 18(12): 7095–7105. PubMed Abstract | Free Full Text\n\nDesmet CJ, Gallenne T, Prieur A, et al.: Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting breast cancer metastasis. Proc Natl Acad Sci U S A. 2013; 110(13): 5139–5144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChinenov Y, Kerppola TK: Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene. 2001; 20(19): 2438–2452. PubMed Abstract | Publisher Full Text\n\nFleischmann A, Hafezi F, Elliott C, et al.: Fra-1 replaces c-Fos-dependent functions in mice. Genes Dev. 2000; 14(21): 2695–2700. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPage-McCaw A, Ewald AJ, Werb Z: Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol. 2007; 8(3): 221–233. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoire A, Covic L, Agarwal A, et al.: PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell. 2005; 120(3): 303–313. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2520",
"date": "26 Nov 2013",
"name": "Sekhar Reddy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIncreased expression levels of matrix metallopeptidase 1 (MMP-1) and the FRA-1/AP-1 transcription factor have been implicated in breast cancer cell progression. This manuscript by Henckels and Prywes addresses the mechanisms underlying the transcriptional activation of MMP-1 by FRA-1 in metastatic variants of breast adenocarcinoma cells. They have performed comprehensive experiments to delineate the upregulation of MMP-1 in breast cancer cells and found that FRA-1 is required for the transcriptional induction of MMP-1 and metastasis of the highly invasive cancer cell line, Scp-2. While these studies confirm previous findings that FRA-1 regulates MMP-1 expression and breast cancer cell metastasis and that FRA-1 mRNA expression is greater in breast cancer cells; the discovery that FRA-1 translation is an additional regulatory mechanism that contributes to the increase in MMP-1 expression in breast cancer cells is novel and significant. They have performed elegant studies and demonstrated that neither the mRNA expression and degradation nor protein stability of FRA-1 are altered, but its protein synthesis rate is increased, leading to increased FRA-1 expression in these highly metastatic cells. Furthermore, they have performed depletion studies with RNAi to address the role of Jun family members and have shown that c-Jun (but not Jun-B or Jun-D) is a potential dimeric partner of FRA-1 in the regulation of MMP-1 expression. While their studies clearly support the authors’ overall conclusions, additional experiments involving ChIP and re-ChIP assays with c-Jun antibody could have further strengthened the EMSA results and indicated whether the “FRA-1/c-Jun” or “FRA-1/X” complex regulates the MMP-1 transcription. It is also unclear whether depletion of MMP-1 phenocopies the effects of FRA-1-siRNA knockdown in Scp-2 cells. Nonetheless, the study raises an important issue: that increased FRA-1 expression is differentially regulated in breast cancer cells. Whether such (transcriptional and translational) regulation of FRA-1 is indicative of different grades of breast metastasis is a question worth pursuing. Further defining the mechanisms that underlie increased FRA-1 expression levels is critical to devising novel strategies for breast cancer treatment.",
"responses": []
},
{
"id": "4351",
"date": "22 Apr 2014",
"name": "Danny N Dhanasekaran",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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-designed study with solid data to support the claim that Fra-1 regulated MMP-1 is involved in the metastatic phenotype of a variant cell line derived from MDA-MB-231 cells. The study is carried out with well-controlled experiments. The authors have nicely presented their voluminous data with a very succinct discussion.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-229
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https://f1000research.com/articles/2-228/v1
|
29 Oct 13
|
{
"type": "Case Report",
"title": "Pseudoaneurysmatic complication of an arteriovenous graft",
"authors": [
"Konstantina Triga"
],
"abstract": "Pseudoaneurysm is a rare complication of arteriovenous-grafts (AVGs) used in hemodialysis patients. Rupture and bleeding are the most common complications. In this case report, I present the case of a successful repair of an infected pseudoaneurysm that occurred at the AVG, its surgical ligation and the insertion of a new graft segment.",
"keywords": [
"Hemodialysis",
"arteriovenous graft",
"pseudoaneurysm"
],
"content": "Introduction\n\nImprovements in hemodialysis techniques have led to an extended life expectancy such that the number of patients with end-stage renal disease is increasing1. Complications regarding vascular access are the main causes of hospitalization in dialysis patients2,3.\n\nNowadays, it is recognised that autologous arteriovenous fistulas (AVF) lead to better long-term results when compared to other possible vascular accesses, including polytetrafluoroethylene (PTFE) grafts4–6. Although the creation of an AVF is a common practice, many patients require secondary or tertiary access procedures. Indeed, an important complication of AVFs is aneurismal dilatation of the venous end which can rupture and cause hemorrhages that can even be fatal7.\n\nPseudoaneurysm is also a relatively rare1 (2% to 10%) complication of dialysis access graft1 and comes from repeated needle punctures. An arteriovenous bridge graft, usually with PTFE, continues to be a reasonable alternative form of hemodialysis access1,8. The creation and maintenance of hemodialysis access occupies a significant portion of most vascular and general surgery practices9.\n\n\nPresentation of the case\n\nA 78-year-old Caucasian Greek man presented with a well defined round region of 2–3 cm diameter, with discoloration, swelling and pain in the arteriovenous graft anastomosis in his left upper extremity. He had been on hemodialysis for 6 years as a result of end-stage renal disease. Hemodialysis was originally initiated with an arteriovenous cimino fistula in his left arm, which provided low blood supply and was thus considered non-functional. The access was switched to a new arterio-venous fistula in his right upper arm, which thrombosed after 3 years. Eventually an arteriovenous graft was placed between the brachial artery and axillary vein in his left upper arm. After two years, upon physical examination, a pulsatile mass was found in the middle of the arteriovenous graft anastomosis. A murmur was detected on auscultation. The size of the pseudoaneurysm was approximately 2 × 3 cm. Within a month’s time, he presented with swelling and discolouration of the aneurismatic mass which had developed a purulent outflow. The patient was referred to surgeons who performed a longitudinal incision and explored the pseudoaneurysm sac.\n\n\nDiagnosis and therapeutic intervention\n\nAn infection was confirmed after taking cultures when the aneurismal sac was opened. There was a destructed segment of about 6 cm in length at the initial AVG. The defect in the graft was repaired with the interposition of a second PTFE graft that was placed next to the initial graft and was anastomosed in an end-to-side fashion by performing a bypass procedure in the same section. The surgical procedure was successful and the patient was discharged without any complications on the same day. The patient continued hemodialysis sessions after this for three years without any complications.\n\n\nDiscussion\n\nThe progressive increase in the number of hemodialysis patients makes vascular access creation a common procedure. There are three different choices for hemodialysis access, an AVF, AVG and a central catheter. The brachiocephalic AVF in the forearm should be the first choice1. However, some patients lack available veins for AVF creation or have unsuitable superficial veins due to their use for repeated arteriovenous fistula procedures. In these cases, upon the exhaustion of all autologous AVF possibilities, a PTFE prosthetic graft becomes an alternative necessity in order to bridge arteries and veins. It is usually placed as an arm or forearm loop graft or even as an axillary-brachial bypass graft. A successful procedure requires adequate blood flow, and, therefore, the rotation or angulation of the prosthesis needs to be avoided.\n\nCommon complications of a vascular access include thrombosis, infection, venous hypertension, and aneurysmal degeneration10,11. Thrombosis is the most common complication in 90% of AVFs12. The use of antiplatelet agents, marcumar or other agents may reduce the risk of thrombosis13, but there is a risk of bleeding and further studies need to prove this hypothesis.\n\nTrue aneurysms are most likely to occur in native AVFs, compared with AVGs where a pseudoaneurysm may occur. The indications for surgical intervention of venous aneurysms are progressively increasing size, thrombosis or an open skin lesion14.\n\nFocal aneurysms can be managed either with manual ligation, embolization and thrombin injection under ultrasonographic guidance15, interposition, replacement with vein or a prosthesis, or resection and imbrication.\n\nPTFE grafts can also lead to bleeding, dilatation, infection, pseudoaneurysm, seroma, steal, swelling, stenosis or thrombosis1,8. Duplex scans can define the exact nature and extension of the aneurysm1.\n\nThe development of a graft–related pseudoaneurysm is multifactorial16. Repeated canulations may cause disruption and fragmentation of the PTFE graft material16,17. Further, progressive enlargement of a pseudoaneurysm can lead to the breakdown of the overlying skin, spontaneous bleeding, and rupture16. Surgical repair is recommended16,18. The main treatment includes ligation and resection of the graft followed by insertion of a new interposition graft segment16,19.\n\nIn this case study, the aneurysm was infected to such an extent that it caused the patient’s arteriovenous graft to rupture. The surgeons repaired the graft with the interposition of a second PTFE AVG placed right next to the initial one. They performed a bypass procedure in the same section by anastomosing the second graft in an end-to-side fashion. After completing the resection procedure of the AVF aneurysm, the patient’s new AVG was canulated without any complications. Our patient was therefore satisfied with the outcome of the surgery.\n\nConcluding, AV dialysis accesses are associated with multiple complications20. The traditional repair of hemodialysis graft pseudoaneurysms requires the surgical replacement of the segment that involves the PTFE graft material or autogenous vein16,19,21. Careful attention to technical detail is required, such as focusing on patients with development of arm swelling or discomfort after surgical access placement or hemodialysis sessions; decreased flow rates during dialysis, suspected of having a pseudoaneurysm; AVF/graft stenosis; or adjacent fluid collection and prolonged immaturity of a surgically created AVF, in order to avoid several diagnostic pitfalls22.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nYasim A, Kabalci M, Eroglu E, et al.: Complication of hemodialysis graft: anastomotic pseudoaneurysm: a case report. Transplant Proc. 2006; 38(9): 2816–2818. PubMed Abstract | Publisher Full Text\n\nMarques AB, Pereira D, Ribeiro RCHM: Motivos e frequência de internação dos pacientes com IRC em tratamento hemodialítico. Arq Cienc Saude. 2005; 12(2): 67–72. Reference Source\n\nCoronel F, Herrero JA, Mateos P, et al.: Long-term experience with the Thomas shunt, the forgotten permanent vascular access for haemodialysis. Nephrol Dial Transplant. 2001; 16(9): 1845–1849. PubMed Abstract | Publisher Full Text\n\nAguiló JM, Galleguillos IO, Rodríguez OO: Accesos vasculares para hemodiálisis: experiencia con PTFE. Rev Chil Cir. 1992; 44(4): 451–454. Reference Source\n\nHerskovic J, Kappes J, Ramírez M, et al.: Accesos vasculares: consejos para su construcción y manejo. Rev Hosp Clin Univ Chile. 1993; 4(1/2): 54–56. Reference Source\n\nSánchez AH: Accesos vasculares para hemodiálisis. Cuad Cir. 1994; 8(1): 31–37. Reference Source\n\nWinsett OE, Wolma FJ: Complications of vascular access for hemodialysis. South Med J. 1985; 78(5): 513–517. PubMed Abstract\n\nBaşaran O, Karakayali H, Emiroğlu R, et al.: Complications and long-term follow-up of 4416 vascular access procedures. Transplant Proc. 2003; 35(7): 2578–2579. PubMed Abstract | Publisher Full Text\n\nRodriguez HE, Leon L, Schalch P, et al.: Arteriovenous access: managing common problems. Perspect Vasc Surg Endovasc Ther. 2005; 17(2): 155–166. PubMed Abstract | Publisher Full Text\n\nChávez AA, Silva JCS, Díaz PD, et al.: Complicaciones de los accesos vasculares para hemodiálisis. Bol Hosp Vina del Mar. 2003; 59(4): 145–154. Reference Source\n\nGarcía de Cortázar L, Gutiérrez E, Delucchi MA, et al.: Vascular accesses for chronic hemodialysis in children. Rev Med Chil. 1999; 127(6): 693–697. PubMed Abstract\n\nBurkhart HM, Cikrit DF: Arteriovenous fistulae for hemodialysis. Semin Vasc Surg. 1997; 10(3): 162–165. PubMed Abstract\n\nDember LM, Beck GJ, Allon M, et al.: Effect of clopidogrel on early failure of arteriovenous fistulas for hemodialysis: a randomized controlled trial. JAMA. 2008; 299(18): 2164–2171. PubMed Abstract | Publisher Full Text\n\nMackrell PJ, Cull DL, III Carsten CG : Hemodialysis access: placement and management of complications. In: Hallett Jr JW, Mills JL, Earnshaw JJ, Reekers JA. Comprehensive vascular and endovascular surgery. 1st ed. New York: Mosby Publishing Co. 2004.\n\nKarabay O, Yetkin U, Silistreli E, et al.: Surgical management of giant aneurysms complicating arteriovenous fistulae. J Int Med Res. 2004; 32(2): 214–217. PubMed Abstract | Publisher Full Text\n\nVesely TM: Use of stent grafts to repair hemodialysis graft-related pseudoaneurysms. J Vasc Interv Radiol. 2005; 16(10): 1301–1307. PubMed Abstract | Publisher Full Text\n\nDelorme JM, Guidoin R, Canizales S, et al.: Vascular access for hemodialysis: pathologic features of surgically excised ePTFE grafts. Ann Vasc Surg. 1992; 6(6): 517–524. PubMed Abstract | Publisher Full Text\n\nBesarab A, Work J, Brouwer D, et al.: III. NKF-K/DOQI Clinical Practice Guidelines for Vascular Access. Am J Kidney Dis. 2001; 37(1 Suppl 1): S137–S181. PubMed Abstract\n\nLin PH, Johnson CK, Pullium JK, et al.: Transluminal stent graft repair with Wallgraft endoprosthesis in a porcine arteriovenous graft pseudoaneurysm model. J Vasc Surg. 2003; 37(1): 175–181. PubMed Abstract | Publisher Full Text\n\nTaylor SM, Eaves GL, Weatherford DA, et al.: Results and complications of arteriovenous access dialysis grafts in the lower extremity: a five year review. Am Surg. 1996; 62(3): 188–191. PubMed Abstract\n\nNajibi S, Bush RL, Terramani TT, et al.: Covered stent exclusion of dialysis access pseudoaneurysms. J Surg Res. 2002; 106(1): 15–19. PubMed Abstract | Publisher Full Text\n\nLockhart ME, Robbin ML: Hemodialysis access ultrasound. Ultrasound Q. 2001; 17(3): 157–167. PubMed Abstract"
}
|
[
{
"id": "2238",
"date": "04 Nov 2013",
"name": "Joyce Popoola",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a case report on the management of a pseudoaneurysm of a fistula by interposing a graft. Aneurysms are becoming increasingly common as patient timelines on dialysis are increasing. The management strategy is not novel however there is little reported in the literature on this subject. The article would benefit from pre and post procedure pictures. It would also be useful to hear about other case experiences in the unit that have long term outcomes.Overall this case report is useful for other practitioners as it promotes monitoring/referral and highlights a management strategy for pseudoaneurysms.",
"responses": []
},
{
"id": "2241",
"date": "04 Nov 2013",
"name": "Michael Mayr",
"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 is a case report describing the revision of an arterio-venous graft with infected pseudoaneurysm in a patient on chronic haemodialysis.Major comments:The current status of the manuscript provides insufficient details to be useful for other practitioners. Additionally, important details of diagnostic tests are missing. However, most importantly the discussion does not demonstrate why the finding is important and why it should be relevant for the future treatment of infected pseudoaneurysms. Thus, it is not clear what the key message of the case report is. Is it an example of a specific surgical procedure? Is it that a new prosthetic material was placed in an infectious site or is there another aspect? In any case, the author should include a key message in this case presentation which has importance for practitioners who care for patients on hemodialysis. Therefore, the manuscript needs major revision to be indexed.Minor comments:References Some statements are not sufficiently supported by the cited references. Some examples are listed:To support the statement \"Improvements in hemodialysis techniques have led to an extended life expectancy such that the number of patients with end-stage renal disease is increasing\", epidemiological and outcome data is needed. The cited reference, a case report in which an anastomotic pseudoaneurysm developed in a patient during the early period of hemodialysis treatment, is insufficient to confirm the author`s statement.Also, to support the statement \"Complications regarding vascular access are the main causes of hospitalization in dialysis patients\" the cited reference 3 is insufficient, since the paper exclusively describes the outcome of Thomas shunts. To support this statement the author should search for confirming epidemiological data.\"The progressive increase in the number of hemodialysis patients makes vascular access creation a common procedure. There are three different choices for hemodialysis access, an AVF, AVG and a central catheter. The brachiocephalic AVF in the forearm should be the first choice.\" These statements are not sufficiently supported by the cited reference. Epidemiological and outcome data are needed as reference.Introduction and DiscussionThe introduction and discussion should be more focused on the case presentation. Especially the second and third paragraph of the introduction, which could be more focused and serve as a background on \"pseudoaneurysm in arteriovenous-grafts (AVGs)\".The last two sentences of the third paragraph add nothing to the subject of the manuscript. Further, most of the first three paragraphs of the discussion can be omitted and the discussion can be substantially shortened. Some examples are listed: \"The use of antiplatelet agents, marcoumar or other agents may reduce the risk of thrombosis, but there is a risk of bleeding and further studies need to prove this hypothesis.\" \"The indications for surgical intervention of venous aneurysms are progressively increasing size, thrombosis or an open skin lesion.\"Presentation of the case\"He had been on hemodialysis for 6 years as a result of end-stage renal disease.\" The second part of this sentence is redundant; ESRD is virtually always the reason to be on chronic hemodialysis.The beginning and end of the presentation are also redundant. The author should be more stringent in the case presentation. Further, it would be of interest to know whether there was an increase in systemic inflammatory markers and whether blood cultures were taken.In the discussion, the author writes: \" In this case study, the aneurysm was infected to such an extent that it caused the patient’s arteriovenous graft to rupture.\" Could the author explain what exactly happened? Was a rupture of the graft noted? If yes, the author should add this to the case presentation.Were there risk factors for an infection? Does the author have any information about the underlying kidney disease and the metabolic situation, e.g. diabetes mellitus, cardiovascular risk factors and so on?Diagnosis and therapeutic intervention\"An infection was confirmed after taking cultures when the aneurismal sac was opened.\" It would be of interest to know the result of a tissue culture.The information about the antibiotic therapy, type of antibiotics given and duration is missing.Further remarksI suggest rephrasing the term \" alternative necessity\".How would you define \" venous hypertension\" in patients on hemodialysis?",
"responses": []
},
{
"id": "2237",
"date": "25 Nov 2013",
"name": "Rajesh Agarwala",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is well written and will be helpful to practitioners.Major concerns:The case report needs to add a few additional details, especially:A graphical representation of the blood flow over certain time periods.The gauge of the needle used for this patientAccess recirculation, if any.KT/VThe ratio of AVF fistula vs AVG vs catheter in the institute.Ultrasonography pictures of pre and post procedures.The time duration between the placement of AVG and cannulation.Details of the organism(s) detected from the culture.These are all the educational points which will help the practitioners at the outset.The case reports' stress is currently on the procedure, which is not unique. Instead, as this article is addressed to the nephrology community, I would suggest that the stress should be changed so as to lean more towards the event that lead to the pseudo-aneurysm.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-228
|
https://f1000research.com/articles/2-226/v1
|
28 Oct 13
|
{
"type": "Case Report",
"title": "Minoxidil induced hypertrichosis in a 2 year-old child",
"authors": [
"Ingrid Herskovitz",
"Joshua Freedman",
"Antonella Tosti",
"Joshua Freedman",
"Antonella Tosti"
],
"abstract": "We report a case of a 2 year-old male patient who developed generalized hypertrichosis after 2 months of treatment with 5% minoxidil foam for alopecia areata. This report highlights the danger of prescribing topical minoxidil to young children and the need to correctly instruct caretakers about its administration.",
"keywords": [
"Topical minoxidil is widely utilized as an off-label therapy for alopecia areata in adults and children. We report here a case of generalized hypertrichosis in a 2 year old child."
],
"content": "Case\n\nTopical minoxidil is widely utilized as an off-label therapy for alopecia areata in adults and children. We report here a case of generalized hypertrichosis in a 2 year old child.\n\nThe patient was a 2-year old hispanic boy with no other significant medical history, who was affected by patchy alopecia areata involving 40% of the scalp since the age of 1 year. Three months before the patient came to our clinic he was prescribed 5% minoxidil foam to be applied to affected areas of the scalp twice a day. After two months the parents noticed hair regrowth but also growth of long pigmented hairs on his face, trunk and limbs. The patient’s mother admitted that she had possibly been applying more product than originally instructed.\n\nClinical examination showed patchy alopecia areata involving 10% of the scalp and generalized hypertrichosis (Figure 1). No other side effects were observed. The patient was referred to a pediatric endocrinologist who excluded underlying endocrinological abnormalities. Minoxidil was discontinued and considerable clinical improvement of the hypertrichosis and the scalp alopecia areata was observed at two month follow up.\n\n\nDiscussion\n\nMinoxidil affects hair growth through incompletely understood mechanisms; known effects include increased duration of the anagen growth phase, agonistic affects on adenosine-triphosphate (ATP)-sensitive potassium channels, and prostaglandin stimulation in the dermal papillae. It is clinically indicated as a therapy for androgenetic alopecia, however off-label uses include topical application in alopecia areata in both adults and children.\n\nSystemic administration of minoxidil either by oral administration to the mother during pregnancy or by oral ingestion by the child, has led to rarely reported instances of diffuse hypertrichosis in children1,2 and newborns via maternal–fetal transmission3,4.\n\nHypertrichosis is a common side effect of topical minoxidil treatment in women. Although usually localized to the face, it may occasionally involve limbs and other body areas5,6. To our knowledge there are no reports of generalized hypertrichosis in a pediatric population.\n\nSystemic absorption of the drug is typically minimal with topical therapy, with 1.4% of the applied dose being absorbed7. However, hypotheses on the pathogenesis of the diffuse hypertrichosis reaction routinely include systemic absorption, as well as high sensitivity of the follicular apparatus to minoxidil5. In our patient, the excessive dose (both in terms of concentration and daily quantity) in combination with the patient’s low body weight favoured systemic adsorption. Further support for systemic effects are noted in the reported cardiovascular side affects in three patients from 10 to 14 years of age treated for alopecia areata with minoxidil 2% topically twice a day8. These effects included sinus tachycardia, sensation of palpitation and dizziness.\n\n\nConclusion\n\nThe efficacy of topical minoxidil in alopecia areata has never been definitively proven9. The possibility of systemic absorption contraindicates, in our opinion, this treatment in young children, who can develop serious cutaneous or systemic side effects. Furthermore, there are some alternative treatments of alopecia areata in children that are considered safer, for example topical immunotherapy and topical anthraline application.",
"appendix": "Author contributions\n\n\n\nIH made a substantial contribution to the conception and design of the study, acquisition and interpretation of data and the drafting of article. JF made a substantial contribution to the design of the study, interpretation of data and drafting of article. AT made a substantial contribution to the conception and interpretation of data. In addition, they were responsible for revising the article critically for important intellectual content and giving final approval of the version to be published.\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\nMiwa LJ, Shaefer MS, Stratta RJ, et al.: Drug-induced hypertrichosis: case report and review of the literature. DICP. 1990; 24(4): 365–8. PubMed Abstract\n\nLorette G, Nivet H: [Diffuse hypertrichosis caused by minoxidil in a 2-and-a-half-year-old child]. Ann Dermatol Venereol. 1985; 112(6–7): 527–8. PubMed Abstract\n\nKaler SG, Patrinos ME, Lambert GH, et al.: Hypertrichosis and congenital anomalies associated with maternal use of minoxidil. Pediatrics. 1987; 79(3): 434–6. PubMed Abstract\n\nVeyrac G, Chiffoleau A, Bailly C, et al.: [Cutaneous application of monoxidil during pregnancy: hairy infant]. Therapie. 1995; 50(5): 474–6. PubMed Abstract\n\nPeluso AM, Misciali C, Vincenzi C, et al.: Diffuse hypertrichosis during treatment with 5% topical minoxidil. Br J Dermatol. 1997; 136(1): 118–20. PubMed Abstract | Publisher Full Text\n\nGonzález M, Landa N, Gardeazabal J, et al.: Generalized hypertrichosis after treatment with topical minoxidil. Clin Exp Dermatol. 1994; 19(2): 157–8. PubMed Abstract | Publisher Full Text\n\nRoy K, Forman S: Miscellaneous topical agents (ch. 55); in Wolverton S (ed). Comprehensive dermatologic drug therapy. 3rd ed. online. Elsevier, Philadelphia 2013.\n\nGeorgala S, Befon A, Maniatopoulou E, et al.: Topical use of minoxidil in children and systemic side effects. Dermatology. 2007; 214(1): 101–2. PubMed Abstract | Publisher Full Text\n\nDelamere FM, Sladden MM, Dobbins HM, et al.: Interventions for alopecia areata. Cochrane Database Syst Rev. 2008; 16(2): CD004413. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2220",
"date": "05 Nov 2013",
"name": "Mohamed Badawy Hassan Tawfik Abdel-Naser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is well written and informative. Here are a few comments:1. \"The patient was referred to a pediatric endocrinologist who excluded underlying endocrinological abnormalities.\"- It would be very helpful if a short paragraph about the possible endocrinologic causes and the lab work done by the endocrinologist to exclude them could be added.2. \"Minoxidil was discontinued and considerable clinical improvement of the hypertrichosis and the scalp alopecia areata was observed at two month follow up\"- Are there any photographs to demonstrate the improvement of hypertrichosis?",
"responses": []
},
{
"id": "2221",
"date": "11 Nov 2013",
"name": "Johannes Steffen Kern",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written and instructive case report. It emphasizes that minoxidil, which is not a standard first line therapy in alopecia areata, should probably not be used in young children because of possible side effects.Some comments:Detailed information about the endocrinological work-up performed would be of interest.Were minoxidil levels in the blood of the child assessed?Was there a family history of hypertrichosis?Is there a follow up picture that shows the improvement of the hypertrichosis after discontinuation of minoxidil?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-226
|
https://f1000research.com/articles/2-191/v1
|
18 Sep 13
|
{
"type": "Web Tool",
"title": "taxize: taxonomic search and retrieval in R",
"authors": [
"Scott A. Chamberlain",
"Eduard Szöcs",
"Eduard Szöcs"
],
"abstract": "All species are hierarchically related to one another, and we use taxonomic names to label the nodes in this hierarchy. Taxonomic data is becoming increasingly available on the web, but scientists need a way to access it in a programmatic fashion that’s easy and reproducible. We have developed taxize, an open-source software package (freely available from http://cran.r-project.org/web/packages/taxize/index.html) for the R language. taxize provides simple, programmatic access to taxonomic data for 13 data sources around the web. We discuss the need for a taxonomic toolbelt in R, and outline a suite of use cases for which taxize is ideally suited (including a full workflow as an appendix). The taxize package facilitates open and reproducible science by allowing taxonomic data collection to be done in the open-source R platform.",
"keywords": [
"Evolution by natural selection has led to a hierarchical relationship among all living organisms. Thus",
"species are categorized using a taxonomic hierarchy",
"starting with the binomial species name (e.g",
"Homo sapiens)",
"moving up to genus (Homo)",
"then family (Hominidae)",
"and on up to Domain (Eukarya). Biologists",
"whether studying organisms at the cell",
"organismal",
"or community level",
"can put their study objects into taxonomic context",
"allowing them to know close and distant relatives",
"find relevant literature",
"and more."
],
"content": "Introduction\n\nEvolution by natural selection has led to a hierarchical relationship among all living organisms. Thus, species are categorized using a taxonomic hierarchy, starting with the binomial species name (e.g, Homo sapiens), moving up to genus (Homo), then family (Hominidae), and on up to Domain (Eukarya). Biologists, whether studying organisms at the cell, organismal, or community level, can put their study objects into taxonomic context, allowing them to know close and distant relatives, find relevant literature, and more.\n\nThe use of taxonomic names is, unfortunately, not straightforward. Taxonomic names often vary due to name revisions at the generic or specific levels, lumping or splitting lower taxa (genera, species) among higher taxa (families), and name spelling changes. For example, a study found that a compilation of 308,000 plant observations from 51 digitized herbarium records had 22,100 unique taxon names, of which only 13,000 were accepted names1,2. In addition, there is no one authoritative taxonomic names source for all taxa - although, there are taxon specific sources that are used by many scientists. Different sources (e.g., uBio, Tropicos, ITIS) may use different accepted names for the same taxon. For example, while the Integrated Taxonomic Information Service (ITIS) has Helianthus x glaucus as an accepted name, The Plant List (http://www.theplantlist.org) gives that name as unresolved. But Helianthus glaucus is an accepted name in The Plant List, while ITIS does not list this name.\n\nOne attempt to help inconsistencies in taxonomy is the use of numeric codes. For example, ITIS assigns a Taxonomic Serial Number (TSN) to each taxon, while the Universal Biological Indexer and Organizer (uBio) assigns each taxon a NameBank identifier (namebankID), and Tropicos assigns their own identifier to each taxon. Codes are helpful within a database as they can easily refer to, for example, Helianthus annuus with a code like 123456 instead of its whole name. However, each database uses their own code; in this case for Helianthus annuus, ITIS uses 36616, uBio uses 2658020, and Tropicos uses 40022652. As there are no universal codes for taxa across databases, this can lead to additional confusion. Last, name comparisons across databases have to be done with the actual names, not the codes.\n\nTaxonomic data is getting easier to obtain through the web (e.g., http://eol.org/). However, there are a number of good reasons to obtain taxonomic information programatically rather than through a web interface. First, if you have more than a few names to look up on a website, it can take quite a long time to enter each name, get data, and repeat for each species. Programatically getting taxonomic names solves the problem by looping over a list of names. In addition, doing taxonomic searching, etc. becomes reproducible. With increasing reports of irreproducibility in science3,4, it is extremely important to make science workflows repeatable. Science workflows can now easily incorporate text, code, and images in a single executable document5. Reproducible documents should become mainstream in biology to avoid mistakes, and make collaboration easier.\n\nThe R language is widely used by biologists, and now has over 5,000 packages on the Comprehensive R Archive Network (CRAN) to extend R. R is great for manipulating, visualizing and fitting statistical models to data. Gentleman et al.6 give a detailed discussion of advantages of R in computational biology. Getting data from the web will be increasingly common as more and more data gets moved to the cloud. Therefore there is a need to get data from the web directly into R. Increasingly, data is available from the web via application programming interfaces (API). These allow computers to talk to one another using code that is not human readable, but is machine readable. Web APIs often define a number of methods that allow users to search for a species name, or retrieve the synonyms for a species name, for example. A further advantage of APIs is that they are language agnostic, meaning that data can be consumed in almost any computing context, allowing users to interact with the web API without having to know the details of the code. Moreover data can be accessed from every computer, whereas for example an Excel file can only be opened in a few programs.\n\nThe goal of taxize, an R package in development, is to make many use cases that involve retrieving and resolving taxonomic names easy and reproducible. In taxize, we have written a suite of R functions that interact with many taxonomic data sources via their web APIs (Table 1). The interface to each function is usually a simple list of species names, just as a user would enter when interacting with a website. Therefore, we hope that moving from a web to an R interface for taxonomic names will be relatively seamless (if one is already nominally familiar with R).\n\nHere, we justify the need for programmatic taxonomic resolution tools like taxize, discuss our data sources, and run through a suite of use cases to demonstrate the variety of ways that users can use taxize.\n\n\nWhy do we need taxize?\n\nThere is a large suite of applications developed around the problem of searching for, resolving, and getting higher taxonomy for species names. For example, Linnaeus http://linnaeus.sourceforge.net/ provides the ability to search for taxonomic names in documents and normalize those names found. In addition, there are many web interfaces to search for and normalize names such as Encyclopedia of Life’s Global Names Resolver http://resolver.globalnames.org/, uBio tools http://www.ubio.org/index.php?pagename=sample_tools, and iPlant’s Taxonomic Name Resolution Service http://tnrs.iplantcollaborative.org/.\n\nAll of these data repositories provide ways to search for taxonomic names and resolve them in some cases. However, scientists ideally need a tool that is free and can be used programmatically, thereby facilitating reproducible research. The goal of taxize is to facilitate the creation of reproducible and easy to use workflows for searching for taxonomic names, resolving them, getting higher taxonomic names, and other tasks related to research dealing with species.\n\n\nData sources and package details\n\ntaxize uses many data sources (Table 1), and more can easily be added. There are two common tasks provided by the data sources: name search and name resolution. Other functionality in taxize includes retrieving a classification tree for a species, or retrieving child taxa of a focal taxon. One of the data sources (Phylomatic) returns phylogenies, while another (NCBI) returns genetic sequence data. However, there are other R packages that are focused solely on sequence data, such as rsnps10, rentrez11, BoSSA12, and ape13, so taxize does not venture deeply into these other domains.\n\nSome of the data sources taxize interacts with require authentication. That is, in addition to the search terms the user provides (e.g., Homo sapiens), the data provider requires an alphanumeric identification key so that they can better manage their servers, collect analytics, and shut down users that abuse the API. The services that require an API key in taxize are: Encyclopedia of Life (EOL) http://eol.org/, the Universal Biological Indexer and Organizer (uBio) http://www.ubio.org/index.php?pagename=sample_tools, Tropicos http://www.tropicos.org/, and Plantminer9. One can easily obtain API keys by visiting the website of each service (see Table 1 for links to each site). There are two typical ways of using API keys. First, you can pass in your API key in a function call (e.g., ubio_namebank(srchName=’Ursus americanus’, key=’your_alphanumeric_key’)). Second, you can store your key in the .Rprofile file, which is a common place to store settings. We recommend the second option as it simplifies function calls as taxize detects the stored keys.\n\nOne available data source in taxize is The Plant List http://www.theplantlist.org. The connection in taxize is done via the taxonstand package14,15 that solely interacts with The Plant List. We provide a few convenience functions that wrap taxonstand into taxize.\n\ntaxize would not have been possible without the work of others. taxize uses httr16 and RCurl17 for performing calls to web APIs, XML18 for parsing XML, RJSONIO19 for parsing JSON, and stringr20 and plyr21 for manipulating data.\n\nNew data sources can be added; for example, we plan to add the following sources: Wikispecies and The Tree of Life. A connection to www.freshwaterecology.info (a database with autecological characteristics, ecological preferences and biological traits as well as distribution patterns of more than 12,000 European freshwater organisms belonging to fish, macro-invertebrates, macrophytes, diatoms and phytoplankton) will be finished when their new API is released. In addition, the authors welcome further suggestions of data sources to be added.\n\n\nUse cases\n\nFirst, one must install and load taxize into the R session.\n\n\n\nAdvanced users can also download and install the latest development copy from GitHub http://github. com/ropensci/taxize_, also permanently available at http://dx.doi.org/10.5281/zenodo.7097.\n\nThis is a common task in biology. We often have a list of species names and we want to know a) if we have the most up to date names, b) if our names are spelled correctly, and c) the scientific name for a common name. One way to resolve names is via the Global Names Resolver (GNR) service provided by the Encyclopedia of Life http://resolver.globalnames.org/. Here, we are searching for two misspelled names:\n\n\n\nThe correct spellings are Helianthus annuus and Homo sapiens. Another approach uses the Taxonomic Name Resolution Service via the Taxosaurus API http:// taxosaurus.org/ developed by iPLant and the Phylotastic organization. In this example, we provide a list of species names, some of which are misspelled, and we’ll call the API with the tnrs function.\n\n\n\nIt turns out there are a few corrections: e.g., Madia sateva should be Madia sativa, and Rosa california should be Rosa californica. Note that this search worked because fuzzy matching was employed to retrieve names that were close, but not exact matches. Fuzzy matching is only available for plants in the TNRS service, so we advise using EOL’s Global Names Resolver if you need to resolve animal names.\n\ntaxize takes the approach that the user should be able to make decisions about what resource to trust, rather than making the decision on behalf of the user. Both the EOL GNR and the TNRS services provide data from a variety of data sources. The user may trust a specific data source, and thus may want to use the names from that data source. In the future, we may provide the ability for taxize to suggest the best match from a variety of sources.\n\nAnother common use case is when there are many synonyms for a species. In this example, we have six synonyms of the currently accepted name for a species.\n\n\n\nAnother task biologists often face is getting higher taxonomic names for a taxa list. Having the higher taxonomy allows you to put into context the relationships of your species list. For example, you may find out that species A and species B are in Family C, which may lead to some interesting insight, as opposed to not knowing that Species A and B are closely related. This also makes it easy to aggregate/standardize data to a specific taxonomic level (e.g., family level) or to match data to other databases with different taxonomic resolution (e.g., trait databases).\n\nA number of data sources in taxize provide the capability to retrieve higher taxonomic names, but we will highlight two of the more useful ones: Integrated Taxonomic Information System (ITIS) http://www.itis.gov/ and National Center for Biotechnology Information (NCBI)7. First, we’ll search for two species, Abies procera and Pinus contorta within ITIS.\n\n\n\n\n\nIt turns out both species are in the family Pinaceae. You can also get this type of information from the NCBI by excuting the following code in R: classification (specieslist, db = ’ncbi’).\n\nInstead of a full classification, you may only want a single name, say a family name for your species of interest. The function tax_name is built just for this purpose. As with the classification-function you can specify the data source with the db argument, either ITIS or NCBI.\n\n\n\nIf a data source does not provide information on the queried species, the result could be taken from another source and the results from the different sources could be pooled.\n\nAs mentioned previously most databases use a numeric code to reference a species. A general workflow in taxize is: Retrieve Code for the queried species and then use this code to query more data/information. Below are a few examples. When you run these examples in R, you are presented with a command prompt asking for the row that contains the name you would like back; that output is not printed below for brevity. In this example, the search term has many matches. The function returns a data.frame of the matches, and asks for the user to input which row number to accept.\n\n\n\nIn another example, you can pass in a long character vector of taxonomic names:\n\n\n\nIn another example, note that no match at all returns an NA:\n\n\n\nEcologists are increasingly taking a phylogenetic approach to ecology, applying phylogenies to topics such as the study of community structure22, ecological networks23, and functional trait ecology24. Yet, many biologists are not adequately trained in reconstructing phylogenies. Fortunately, there are some sources for getting a phylogeny without having to know how to build one; one of these is for angiosperms, called Phylomatic8. We have created a workflow in taxize that accepts a species list, and taxize works behind the scenes to get higher taxonomic names, which are required by Phylomatic to get a phylogeny. Here is a short example, producing the tree in Figure 1.\n\nThis phylogeny was produced using the phylomatic_tree function, which queries the Phylomatic database, and prunes a previously created phylogeny of plants.\n\n\n\nBehind the scenes the function phylomatic_tree retrieves a Taxonomic Serial Number (TSN) from ITIS for each species name, then a string is created for each species like this poaceae/oryza/oryza_sativa (with format \"family/genus/genus_epithet\"). These strings are submitted to the Phylomatic API, and if no errors occur, a phylogeny in newick format is returned. The phylomatic_tree() function also cleans up the newick string and converts it to an ape phylo object. The output from phylomatic_tree() is a phylo object, which can be used for plotting and phylogenetic analyses. Be aware that Phylomatic has certain limitations - refer to the paper describing Phylomatic8 and the website http://phylodiversity.net/phylomatic/.\n\nThere are currently no resources for getting a phylogeny of animals simply from species names. However, a few projects are working on this problem, including the Open Tree of Life http://blog.opentreeoflife.org/. We will incorporate these resources when the appropriate APIs are available.\n\nIf someone is not a taxonomic specialist on a particular taxon they probably do not know what children taxa are within a family, or within a genus. This task becomes especially unwieldy when there are a large number of taxa downstream. You can of course go to a website like Wikispecies http://species.wikimedia.org/wiki/Main_Page or Encyclopedia of Life http://eol.org/ to get downstream names. However, taxize provides an easy way to programatically search for downstream taxa, both for the Catalogue of Life (CoL) http://www.catalogueoflife.org/ and the Integrated Taxonomic Information System http://www.itis.gov/. Here is a short example using the CoL in which we want to find all the species within the genus.\n\n\n\nThe result from the above call to col_downstream() is a data.frame that gives a number of columns of different information.\n\nThere are a number of things we can do once we have the correct taxonomic names. One thing we can do is ask about the conservation status of a species (IUCN Red List of Threatened Species http://www.iucnredlist.org). We have provided a set of functions, iucn_summary and iucn_status, to search for species names, and extract the status information, respectively. Here, we search for the panther and lynx.\n\n\n\nIt turns out that the panther has a status of endangered (EN) and the lynx has a status of least concern (LC).\n\nAnother use case available in taxize deals with genetic sequences. taxize has three functions to interact with GenBank to search for available genes (get_genes_avail), download genes by GenBank ID (get_genes), and download genes via taxonomic name search, including retrieving a congeneric if the searched taxon does not exist in the database (get_seqs). In this example, we search for gene sequences for Umbra limi.\n\n\n\nThen we can ask if ’RAG1’ exists in any of the gene names.\n\n\n\nIt turns out that there are 430 different unique records found. However, this doesn’t mean that there are 430 different genes found as the API does not provide metadata to classify genes. However, you can use regular expressions (e.g., grep) to search for the gene of interest.\n\nBiologists often need to match different sets of data tied to species. For example, trait-based approaches are a promising tool in ecology25. One problem is that abundance data must be matched with trait databases such as the NCBI Taxonomy database26. These two data tables may contain species information on different taxonomic levels and data might have to be aggregated to a joint taxonomic level, so that the data can be merged. taxize can help in this data-cleaning step, providing a reproducible workflow.\n\nWe can use the mentioned classification-function to retrieve the taxonomic hierarchy and then search the hierarchies up- and downwards for matches. Here is an example to match a species with names on three different taxonomic levels.\n\n\n\nIf we find a direct match (here Gammarus roeseli), we are lucky. But we can also match Gammaridae with Gammarus roeseli, but on a lower taxonomic level. A more comprehensive and realistic example (matching a trait table with an abundance table) is given in Appendix B.\n\nIn biology, one can asks questions at varying taxonomic levels. This use case is easily handled in taxize. A function called tax_agg will aggregate community data to a specific taxonomic level. In this example, we take the data for three species and aggregate them to family level. Again we can specify if we want to use data from ITIS or NCBI. The rows in the data.frame are different communities.\n\n\n\nThe two Juncus species are aggregated to the family Juncaceae and their abundances are summed. There was only a single species in the family Asteraceae, so the data for Bellis perennis are carried over.\n\n\nConclusions\n\nTaxonomic information is increasingly sought out by biologists as we take phylogenetic and taxonomic approaches to science. Taxonomic data are becoming more widely available on the web, yet scientists require programmatic access to this data for developing reproducible workflows. taxize was created to bridge this gap - to bring taxonomic data on the web into R, where the data can be easily manipulated, visualized, and analyzed in a reproducible workflow.\n\nWe have outlined a suite of use cases in taxize that will likely fit real use cases for many biologists. Of course we have not thought of all possible use cases, so we hope that the biology community can give us feedback on what use cases they want to see available in taxize. One thing we could change in the future is to make functions that fit use cases, and then allow users to select the data source as a parameter in the function. This could possibly make the user interface easier to understand.\n\ntaxize is currently under development and will be for some time given the large number of data sources knitted together in the package, and the fact that APIs for each data source can change, requiring changes in taxize code. Contributions to taxize are strongly encouraged, and can be easily done using GitHub here http://github.com/ropensci/taxize_. We hope taxize will be taken up by the community and developed collaboratively, making it progressively better through time as new use cases arise, bug reports are squashed, and contributions are merged.",
"appendix": "Author contributions\n\n\n\nSC and ES equally contributed to the software discussed in this paper, and contributed equally to writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe taxize package is part of the rOpenSci project http://ropensci.org/. We thank Carl Boettiger, Karthik Ram, Owen Jones, Naim Matasci, and Ralf Schäfer for comments on previous versions of this manuscript. We thank all API maintainers for their work making their databases open to the public.\n\n\nReferences\n\nWeiser MD, Enquist BJ, Boyle B, et al.: Latitudinal patterns of range size and species richness of new world woody plants. Global Ecology and Biogeography. 2007; 16(5): 679–688. Publisher Full Text\n\nBoyle B, Hopkins N, Lu Z, et al.: The taxonomic name resolution service: an online tool for automated standardization of plant names. BMC Bioinformatics. 2013; 14(1): 16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStodden VC: Reproducible research: Addressing the need for data and code sharing in computational science. Comput Sci Eng. 2010; 12(5): 8–13. Publisher Full Text\n\nZimmer C: A sharp rise in retractions prompts calls for reform. New York Times. 2012. Reference Source\n\nXie Y: Dynamic Documents with R and knitr. Chapman and Hall/CRC, 2013. Reference Source\n\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFederhen S: The ncbi taxonomy database. Nucleic Acids Res. 2012; 40(Database issue): D136–D143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWebb CO, Donoghue MJ: Phylomatic: tree assembly for applied phylogenetics. Mol Ecol Notes. 2005; 5(1): 181–183. Publisher Full Text\n\nCarvalho GH, Cianciaruso MV, Batalha MA: Plantminer: a web tool for checking and gathering plant species taxonomic information. Environmental Modelling & Software. 2010; 25(6): 815–816. Publisher Full Text\n\nChamberlain S, Ushey K: rsnps: Interface to SNP data on the web. R package version 0.0.4 2013. Reference Source\n\nWinter D: rentrez: Entrez in R, R package version 0.2.1 2013. Reference Source\n\nLefeuvre P: BoSSA: a Bunch of Structure and Sequence Analysis, R package version 1.2 2010. Reference Source\n\nParadis E, Claude J, Strimmer K: APE: analyses of phylogenetics and evolution in R language. Bioinformatics. 2004; 20(2): 289–290. PubMed Abstract | Publisher Full Text\n\nCayuela L, Granzow-de la Cerda I, Al-buquerque FS, et al.: taxonstand: An r package for species names standardisation in vegetation databases. Methods Ecol Evol. 2012; 3(6): 1078–1083. Publisher Full Text\n\nCayuela L: Taxonstand: Taxonomic standardization of plant species names. R package version 1.0 2012. Reference Source\n\nWickham H: httr: Tools for working with URLs and HTTP. R package version 0.2 2012. Reference Source\n\nLang DT: RCurl: General network (HTTP/FTP/...) client interface for R. R package version 1.95–4.1 2013. Reference Source\n\nLang DT: XML: Tools for parsing and generating XML within R and S-Plus. R package version 3.95–0.2 2013. Reference Source\n\nLang DT: RJSONIO: Serialize R objects to JSON, JavaScript Object Notation. R package version 1.0–3 2013. Reference Source\n\nWickham H: stringr: Make it easier to work with strings. R package version 0.6.2 2012. Reference Source\n\nWickham H: The split-apply-combine strategy for data analysis. J Stat Softw. 2011; 40(1): 1–29. Reference Source\n\nWebb CO, Ackerly DD, McPeek MA, et al.: Phylogenies and community ecology. Annu Rev Ecol Syst. 2002; 33: 475–505. Publisher Full Text\n\nRafferty NE, Ives AR: Phylogenetic trait-based analyses of ecological networks. Ecology. 2013. Publisher Full Text\n\nPoff NL, Olden JD, Vieira NKM, et al.: Functional trait niches of north american lotic insects: traits-based ecological applications in light of phylogenetic relationships. Journal of the North American Benthological Society. 2006; 25(4): 730–755. Publisher Full Text\n\nStatzner B, Bêche LA: Can biological invertebrate traits resolve effects of multiple stressors on running water ecosystems? Freshwater Biology. 2010; 55(Supplement s1): 80–119. Publisher Full Text\n\nUsseglio-Polatera P, Bournaud M, Richoux P, et al.: Biological and ecological traits of benthic freshwater macroinvertebrates: relationships and definition of groups with similar traits. Freshw Biol. 2000; 43(2): 175–205. Publisher Full Text\n\nBaird DJ, Baker CJ, Brua RB, et al.: Toward a knowledge infrastructure for traits-based ecological risk assessment. Integr Environ Assess Manag. 2011; 7(2): 209–215. PubMed Abstract | Publisher Full Text\n\nKleyer M, Dray S, Bello F, et al.: Assessing species and community functional responses to environmental gradients: which multivariate methods? Journal of Vegetation Science. 2012; 23(5): 805–821. Publisher Full Text\n\n\n\n\n"
}
|
[
{
"id": "1855",
"date": "19 Sep 2013",
"name": "Will Pearse",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 software this article describes is well-written and of use to ecologists. The guide and appendices in this article give a good overview of the features of the package, and are well-written. The title and abstract are well-written.My only serious comment would be that the authors refer to species' taxonomy as if it perfectly reflects species' phylogeny (e.g. the end of the first paragraph in the introduction and the first paragraph in the section \"retrieve higher taxonomic names\"). More often than not, taxonomy does reflect phylogeny, but a sentence clarifying this distinction somewhere might be helpful.A few minor things:Table 1 - \"National Center for Biotechnology Information Federhen\" - the author's surname, but not the citation, is in the text; \"searchbycommonname\" and \"searchbyscientificname\" seem to be the only elements not in alphabetical order in the table.Phylomatic can now create phylogenies for mammals, not just angiosperms (e.g. \"retrieve a phylogeny\").Some sentences could perhaps be linked more neatly in the introduction (e.g. \"gives that name as unresolved. But Helianthus\", \"moved to the cloud. Therefore there is a need\", \"additional confusion. Last, name\"). Similarly, page 3 has some very short paragraphs; could the descriptions of taxonstand and The Plant List be moved to table 1? \"Science workflows\" or \"scientific workflows\" (fourth paragraph of introduction)? These are all very minor points!Perhaps an example of what to type in order to add API keys into the .Rprofile would be helpful, or maybe could just be added to the help file for \"ubio_namebank\" as this is the function named when it's mentioned.",
"responses": [
{
"c_id": "580",
"date": "14 Oct 2013",
"name": "Scott Chamberlain",
"role": "Author Response",
"response": "We appreciate Dr. Pearse's comments on our manuscript. We agree that species taxonomy does not equate to phylogenetic history, so we added the following sentence to the first paragraph: \"Although taxonomic classifications are human constructs created to understand the real phylogeny of life \\cite{benton2000}, they are nonetheless essential to organize the vast diversity of organisms.\" We fixed the citation in Table 1, and reordered the functions in the table so as to be alphabetical. Thanks for pointing out that Phylomatic now accepts mammals in addition to Angiosperm plants - we have adjusted the language accordingly.We removed the description of Taxonstand and the Plantlist.org (and associated references) that this reviewer referred to as it wasn't necessary and improves reading.This reviewer asked for a better explanation of how to use the API keys and the .Rprofile file. In response, we have added a new appendix (Appendix C) that explains using API keys and installing the development version of taxize."
}
]
},
{
"id": "1854",
"date": "23 Sep 2013",
"name": "Gavin L. Simpson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nChamberlain and Szöcs present the taxize R package, a set of functions that provides interfaces to several web tools and databases, and simplifies the process of checking, updating, correcting and manipulating taxon names for researchers working with ecological/biological data. A key feature that is repeated throughout is the need for reproducibility of science workflows and taxize provides a means to achieve this within the R software ecosystem for taxonomic search.The manuscript is well-written and nicely presented, with a good balance of descriptive text and discourse and practical illustration of package usage. A number of examples illustrate the scope of the package, something that is fully expanded upon in the two appendices, which are a welcome addition to the paper.As to the package, I am not overly fond of long function names; the authors should consider dropping the data source abbreviations from the function names in a future update/revision of the package. Likewise there is some inconsistency in the naming conventions used. For example there is the 'tpl_search()' function to search The Plant List, but the equivalent function to search uBio is 'ubio_namebank()'. Whilst this may reflect specific aspects of terminology in use at the respective data stores, it does not help the user gain familiarity with the package by having them remember inconsistent function names.One advantage of taxize is that it draws together a rich selection of data stores to query. A further suggestion for a future update would be to add generic function names, that apply to a database connection/information object. The latter would describe the resource the user wants to search and any other required information, such as the API key, etc., for example:\n\nfoo <- taxizeDB(what = \"uBio\", key = \"1646546164694\")The user function to search would then be 'search(foo, \"Abies\")'. Similar generically named functions would provide the primary user-interface, thus promoting a more consistent toolbox at the R level. This will become increasingly relevant as the scope of taxize increases through the addition of new data stores that the package can access.In terms of presentation in the paper, I really don't like the way the R code inputs merge with the R outputs. I know the author of Knitr doesn't like the demarcation of output being polluted by the R prompt, but I do find it difficult parsing the inputs/outputs you show because often there is no space between them and users not familiar with R will have greater difficulties than I. Consider adding in more conventional indications of R outputs, or physically separate input from output by breaking up the chunks of code to have whitespace between the grey-background chunks. Related, in one location I noticed something amiss with the layout; in the first code block at the top of page 5, the printed output looks wrong here. I would expect the attributes to print on their own line and the data in the attribute to also be on its own separate line.Note also, the inconsistency in the naming of the output object columns. For example, in the two code chunks shown in column 1 of page 4, the first block has an object printed with column names 'matched_name' and 'data_source_title', whilst camelCase is used in the outputs shown in the second block. As the package is revised and developed, consider this and other aspects of providing a consistent presentation to the user.I was a little confused about the example in the section Resolve Taxonomic Names on page 4. Should the taxon name be \"Helianthus annuus\" or \"Helianthus annus\"? In the 'mynames' definition you include 'Helianthus annuus' in the character vector but the output shown suggests that the submitted name was 'Helianthus annus' (1 \"u\") in rows with rownames 9 and 10 in the output shown.Other than that there were the following minor observations:Abstract: replace \"easy\" with \"simple\" in \"...fashion that's easy...\", and move the details about availability and the URI to the end of the sentence.Page 2, Column 1, Paragraph 2: You have \"In addition, there is no one authoritative taxonomic names source...\", which is a little clumsy to read. How about \"In addition, there is no one authoritative source of taxonomic names...\"?Pg 2, C1, P2-3: The abbreviated data sources are presented first (in paragraph 2) and subsequently defined (in para 3). Restructure this so that the abbreviated forms are explained upon first usage.Pg 2, C2, P2: Most R packages are \"in development\" so I would drop the qualifier and reword the opening sentence of the paragraph.Pg 2, C2, P6: Change \"and more can easily be added\" to \"and more can be easily added\" seems to flow better?Pg 5, paragraph above Figure 1: You refer to converting the object to an **ape** *phylo* object and then repeat essentially the same information in the next sentence. Remove the repetition.Pg 6, C1: The header may be better as \"Which taxa are children of the taxon of interest\".Pg 6: In the section \"IUCN status\", the term \"we\" is used to refer to both the authors and the user. This is confusing. Reserve \"we\" for reference to the authors and use something else (\"a user\" perhaps) for the other instances. Check this throughout the entire manuscript.Pg 6, C2: in the paragraph immediately below the 'grep()' for \"RAG1\", two consecutive sentences begin with \"However\".Pg 7: The first sentence of \"Aggregating data....\" reads \"In biology, one can asks questions...\". It should be \"one asks\" or \"one can ask\".Pg 7, Conclusions: The first sentence reads \"information is increasingly sought out by biologists\". I would drop \"out\" as \"sought\" is sufficient on its own.Appendices: Should the two figures in the Appendices have a different reference to differentiate them from Figure 1 in the main body of the paper? As it stands, the paper has two Figure 1s, one on page 5 and a second on page 12 in the Appendix.On Appendix Figure 2: The individual points are a little large. Consider reducing the plotting character size. I appreciate the effect you were going for with the transparency indicating density of observation through overplotting, but the effect is weakened by the size of the individual points.Should the phylogenetic trees have some scale to them? I presume the height of the stems is an indication of phylogenetic distance but the figure is hard to calibrate without an associated scale. A quick look at Paradis (2012) Analysis of Phylogenetics and Evolution with R would suggest however that a scale is not consistently applied to these trees. I am happy to be guided by the authors as they will be more familiar with the conventions than I.",
"responses": [
{
"c_id": "581",
"date": "14 Oct 2013",
"name": "Scott Chamberlain",
"role": "Author Response",
"response": "We appreciate Dr. Simpson's very thorough comments on our manuscript. The following are responses to Dr. Simpson's comments:- ...there is some inconsistency in the naming conventions used. For example there is the 'tpl_search()' function to search The Plant List, but the equivalent function to search uBio is 'ubio_namebank()'. Whilst this may reflect specific aspects of terminology in use at the respective data stores, it does not help the user gain familiarity with the package by having them remember inconsistent function names.We agree that we should definitely improve naming conventions for functions. However, we think it's better to change the function names as needed in an upcoming version of the software after we have had time work on the problem.- Consider adding in more conventional indications of R outputs, or physically separate input from output by breaking up the chunks of code to have whitespace between the grey-background chunks.We have used comments (pound signs) for the results of function calls within each code block to indicate output as separate from code input. This way users can copy/paste code directly into R to try it out.- in one location I noticed something amiss with the layout; in the first code block at the top of page 5, the printed output looks wrong here. I would expect the attributes to print on their own line and the data in the attribute to also be on its own separate line.This was a problem with the typesetting, and we have fixed it.- the inconsistency in the naming of the output object columns. For example, in the two code chunks shown in column 1 of page 4, the first block has an object printed with column names 'matched_name' and 'data_source_title', whilst camelCase is used in the outputs shown in the second block.We agree that we should definitely improve naming conventions for object columns. However, we think it's better to change the column names as needed in an upcoming version of the software after we have had time work on the problem.- I was a little confused about the example in the section Resolve Taxonomic Names on page 4. Should the taxon name be \"Helianthus annuus\" or \"Helianthus annus\"? In the 'mynames' definition you include 'Helianthus annuus' in the character vector but the output shown suggests that the submitted name was 'Helianthus annus' (1 \"u\") in rows with rownames 9 and 10 in the output shown.Fixed.- Abstract: replace \"easy\" with \"simple\" in \"...fashion that's easy...\", and move the details about availability and the URI to the end of the sentence.Fixed.- Page 2, Column 1, Paragraph 2: You have \"In addition, there is no one authoritative taxonomic names source...\", which is a little clumsy to read. How about \"In addition, there is no one authoritative source of taxonomic names...\"?Changed.- Pg 2, C1, P2-3: The abbreviated data sources are presented first (in paragraph 2) and subsequently defined (in para 3). Restructure this so that the abbreviated forms are explained upon first usage.Changed.- Pg 2, C2, P2: Most R packages are \"in development\" so I would drop the qualifier and reword the opening sentence of the paragraph.Changed.- Pg 2, C2, P6: Change \"and more can easily be added\" to \"and more can be easily added\" seems to flow better?Changed.- Pg 5, paragraph above Figure 1: You refer to converting the object to an ape phylo object and then repeat essentially the same information in the next sentence. Remove the repetition.Removed.- Pg 6, C1: The header may be better as \"Which taxa are children of the taxon of interest\".Changed.- Pg 6: In the section \"IUCN status\", the term \"we\" is used to refer to both the authors and the user. This is confusing. Reserve \"we\" for reference to the authors and use something else (\"a user\" perhaps) for the other instances. Check this throughout the entire manuscript.Fixed.- Pg 6, C2: in the paragraph immediately below the 'grep()' for \"RAG1\", two consecutive sentences begin with \"However\".Changed.- Pg 7: The first sentence of \"Aggregating data....\" reads \"In biology, one can asks questions...\". It should be \"one asks\" or \"one can ask\"Changed.- Pg 7, Conclusions: The first sentence reads \"information is increasingly sought out by biologists\". I would drop \"out\" as \"sought\" is sufficient on its own.Changed.- Appendices: Should the two figures in the Appendices have a different reference to differentiate them from Figure 1 in the main body of the paper? As it stands, the paper has two Figure 1s, one on page 5 and a second on page 12 in the Appendix.Fixed.- On Appendix Figure 2: The individual points are a little large. Consider reducing the plotting character size. I appreciate the effect you were going for with the transparency indicating density of observation through overplotting, but the effect is weakened by the size of the individual points.Although we agree that the styling of the figure could be improved, we are going to leave it as is because it's not important for understanding the material.- Should the phylogenetic trees have some scale to them? I presume the height of the stems is an indication of phylogenetic distance but the figure is hard to calibrate without an associated scale. A quick look at Paradis (2012) Analysis of Phylogenetics and Evolution with R would suggest however that a scale is not consistently applied to these trees. I am happy to be guided by the authors as they will be more familiar with the conventions than I.A scale could be used for sure. However, our focus is on showing readers that they can get data which can be used to make a phylogeny, not on how to properly create and display a phylogeny. Thus, we are leaving the phylogeny as is without a scale."
}
]
},
{
"id": "1853",
"date": "24 Sep 2013",
"name": "Ethan White",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis software paper describes an R package that provides an integrated R interface for the APIs of over a dozen taxonomically related web services. This is a valuable contribution because it will save researchers time and energy (for those capable of wrapping the APIs themselves), and will allow scientists who lack the technical knowledge to interact with web services themselves to use this data from R. In addition, some of the functions combine existing APIs in useful ways.The software was developed using version control, on a public development site (https://github.com/ropensci/taxize_), and using a bug tracker. The code is well modularized and includes an extensive test suite. This level of good software practice is notable for scientific software and is indicative of well built and maintained code. It also has a clearly declared CC0 license making it easy for others to use and build on the software.The software installed easily in R using the standard approach and generally works as expected based on the examples in the paper and on the project's website.My one major suggestion is to reinforce what the authors' have already suggested in the Conclusions, that it would be an improvement to move to a design that focuses on having a single top-level function for each type of task that the library handles with different data sources being selected using a parameter. This would allow the users to benefit maximally from one of the stronger aspects of this library, which is that it combines access to large numbers of data sources, by making it more of an integrated system and less of a collected set of API wrappers.Minor issues:The code snippets are images rather than text. This is probably a limitation of the publishing platform, but it does make it more difficult to learn about the software by executing the code snippets.The following two sentences seem rather tangential to the paper and could be removed:\"Science workflows can now easily incorporate text, code, and images in a single executable document. Reproducible documents should become mainstream in biology to avoid mistakes, and make collaboration easier.\"Given that many of the target users of this package will not be particularly familiar with web services and APIs, I would recommend adding another sentence or two to the paragraph on authentication so that readers understand why this is required (i.e., most readers won't understand \"users that abuse the API\") and what it really is (i.e., an individual login of sorts, similar to a username/password).The section on \"Aggregating data to a specific taxonomic rank\" refers to an example, but none appears to be present.",
"responses": [
{
"c_id": "579",
"date": "14 Oct 2013",
"name": "Scott Chamberlain",
"role": "Author Response",
"response": "We appreciate Dr. White's comments on our manuscript.We removed the sentences \"Science workflows can now easily incorporate text, code, and images in a single executable document. Reproducible documents should become mainstream in biology to avoid mistakes, and make collaboration easier.\"In response to this reviewer's comment about clarification on APIs and authentication, and Dr. Pearse's comments on the same issue, we have added a new appendix (Appendix C) that explains how to use API keys and install the development version of taxize.This reviewer commented that the section on Aggregating data to a specific taxonomic rank referred to an example, but none appeared to be present. The example is now in the paper."
}
]
}
] | 1
|
https://f1000research.com/articles/2-191
|
https://f1000research.com/articles/2-223/v1
|
23 Oct 13
|
{
"type": "Short Research Article",
"title": "The expression profile of miR-23b is not altered in peripheral blood mononuclear cells of patients with idiopathic inflammatory myopathies",
"authors": [
"Martina Remakova",
"Tana Svitalkova",
"Marek Skoda",
"Jiri Vencovsky",
"Peter Novota",
"Martina Remakova",
"Tana Svitalkova",
"Marek Skoda",
"Jiri Vencovsky"
],
"abstract": "Idiopathic inflammatory myopathies (IIM) belong to a group of autoimmune disorders, primarily characterized by chronic inflammation of human skeletal muscle tissue. The etiology of these diseases is unknown, however, genetic predisposition plays a significant role in disease onset. Beside the known genetic risk located in the MHC complex, the epigenetic modifications including changes in miRNAs expression profiles have been recently implicated recently in many autoimmune diseases. Micro RNA molecules are involved in many physiological processes, including the regulation of the immune response.In our study we have focused on the miR-23b, as it represents a novel promising autoimmunity regulator molecule. Downregulation of miR-23b was recently described in patients with rheumatoid arthritis and systemic lupus erythematosus. We have measured the expression miR-23b peripheral blood mononuclear cells of patients with dermatomyositis and polymyositis. No meaningful difference was found in comparison with healthy controls.",
"keywords": [
"Idiopathic inflammatory myopathies (IIM) are chronic autoimmune disorders characterized by skeletal muscle weakness",
"inflammatory immune cells in muscle tissues and frequent presence of serum autoantibodies. IIM can be divided into several groups: dermatomyositis (DM)",
"polymyositis (PM)",
"inclusion body myositis (IBM)",
"and immune mediated necrotizing myopathy (NM). The etiology of IIM remains unclear",
"however current findings indicate that epigenetic mechanisms may contribute to the disease pathogenesis1",
"2."
],
"content": "Introduction and aims\n\nIdiopathic inflammatory myopathies (IIM) are chronic autoimmune disorders characterized by skeletal muscle weakness, inflammatory immune cells in muscle tissues and frequent presence of serum autoantibodies. IIM can be divided into several groups: dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), and immune mediated necrotizing myopathy (NM). The etiology of IIM remains unclear; however current findings indicate that epigenetic mechanisms may contribute to the disease pathogenesis1,2.\n\nMicroRNA molecules are part of the epigenetic regulatory network. They act as negative regulators of gene expression and participate in the regulation of key biological processes such as cell growth, differentiation, proliferation or apoptosis3–6. Recent findings highlight the crucial importance of miRNAs in development, homeostasis and function of innate and adaptive immunity7–10. Aberrant expression patterns have been documented in a broad range of diseases including autoimmune disorders11.\n\nIn our study we have focused on the miR-23b type of microRNA, as it represents a novel promising autoimmunity regulator molecule with considerable therapeutic potential12. Recent research has revealed downregulation of this miRNA in resident cells present in inflammatory lesions of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and its role during the pathogenesis of autoimmune disease has been functionally characterized12.\n\nThe aim of our study was to analyze differential expression of miR-23b and miR-23b* in peripheral blood mononuclear cells (PBMCs) of patients with PM/DM, and to examine potential involvement of these miRNA types in the pathogenesis of IIM.\n\n\nMaterial and methods\n\nA cohort of 25 adult patients (age range 21–86 years, 7 males) suffering from idiopathic inflammatory myopathy and 22 healthy controls (age range 25–79 years, 5 males), both from the middle region of Czech Republic were analyzed. Diagnosis of PM or DM was determined according to the Bohan and Peter criteria13,14. In total 12 of patients suffered from PM, 13 patients were diagnosed as DM. All individuals involved in this study signed an informed consent. PBMCs of all individuals were obtained and purified by density gradient centrifugation on Ficoll in Leucosep tubes (Greiner Bio-One GmbH, Germany). 10 ml of peripheral blood of patients with myositis and healthy control subjects was collected into EDTA coated tubes under sterile conditions. The blood was added to a LeucoSep tube. The cell separation tubes were centrifuged (Jouan CR 3i; Jouan, St. Herblain, France) for 10 min at 2400 rpm without braking at room temperature. The cell suspension was collected, and the cells were washed three times in PBS (for 10 min at 1000 rpm), resuspended in 1 ml PBS and transferred into a 1,5 ml microcentrifuge tube. The microcentrifuge tube was centrifuged at 8000 rpm for 1 minute, supernatant was removed and the cell pellet was frozen using liquid nitrogen and stored at -80°C. The total RNA preparation from PBMCs was carried out using the conventional TRIZOL® reagent (Invitrogen, Carlsbad, USA) extraction procedure15. Afterwards, the RNA was treated with RQ1 RNase free DNase (Promega, USA) for 20 min at 37°C in order to remove genomic DNA contamination, purified with phenol-chloroform-isoamylalcohol (25:24:1, PENTA, Czech Republic) reextraction and precipitated by 1 volume 2-propanol and 1/10 volume 300 mM sodium acetate (pH 4.8). Then, the RNA was washed twice with 70% ethanol and dissolved in RNase-free water (Life Technologies, Carlsbad, CA, USA). The quantity of total RNA was measured with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, USA). A microfluidic electrophoresis 2100 Bioanalyzer (Agilent Technologies, USA) was then used to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%].\n\nTaqMan gene specific miRNA assays were used to quantify the expression levels of mature miR-23b and miR-23b* (microRNA 000400, 002126) from Life Technologies (Carlsbad, CA, USA). Total RNA was reverse-transcribed by the TaqMan microRNA reverse transcription kit (Applied Biosystems) in a reaction mixture containing a miR-specific stem-loop reverse transcription (RT) primer. The quantification of mature miRNAs was performed using the TaqMan miRNA assay kit (Applied Biosystems) containing TaqMan primers in a universal PCR master mix without AmpErase UNG. 4 small nuclear RNAs - RNU44, RNU48, U47 and RNU6B (microRNA 001094, 001006, 001223, 001093, Applied Biosystems) were amplified as an internal control. qPCR was conducted at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression level of miR-23b (miR-23b*) is shown as delta Ct. The delta Ct value was normalized to the mean value of four other miRNA molecules used in this experiment as housekeeping molecules (RNU44, RNU48, RNU6B, U47). This process was used to overcome the differences in cDNA quantity between different samples. All analyses were done in technical duplicates.\n\nStatistical significance was determined by the Student's t-test using Microsoft Excel®2007. A value of P<0.05 was considered to be statistically significant.\n\nThe whole study, including the laboratory work was performed at the Institute of Rheumatology in Prague. Collection of clinical data and blood from patients as well as the laboratory analysis of the biological material was undertaken under the regulation of the relevant local research ethics committee (Ref. Nr. 5371/2012).\n\n\nResults and discussion\n\nWe have examined the differential expression of the miR-23b and miR-23b* in PBMCs of patients with PM/DM and healthy controls. The detection of expression of miR-23b and miR-23b* was performed by quantitative real-time PCR. The median expression level (delta Ct) of miR-23b in the group of IIM patients was -3.90, the expression (delta Ct) in the control group reached -4.03. The median expression level of miR-23b* was -11.69 in IIM patients and -11.68 in the controls (Figure 1). The expression level (delta Ct) of both tested miR-23b molecules did not show any significant differences between patients suffering from myositis and control cohort (Figure 1). Furthermore, we did not find any difference in miR-23b (or miR-23b*) expression between DM and PM patients. The table with raw qPCR expression data used for statistical evaluation is provided in supplementary material.\n\nThe expression level of miR-23b was not significantly changed in patients suffering from idiopathic inflammatory myopathy when compared with healthy controls (A). No significant difference in expression of miR-23b* was found between myositis patients and controls (B).\n\n\n\nThe role of miR-23b has been studied on inflammatory lesions of humans with lupus erythematosus or rheumatoid arthritis, as well as on corresponding tissues of murine models of lupus, rheumatoid arthritis and multiple sclerosis12. The authors Zhu et al.12 demonstrated that miR-23b targets TAB2, TAB3 and IKK-α mRNA transcripts and thereby suppresses autoimmune symptomatology by limiting the actions of the key proinflammatory cytokines IL-17, IL-1β and TNF. As it was found that the in vitro treatment of resident cell lines with IL-17 led to the downregulation of miR-23b, the expression of miR-23b and IL-17 in tissue resident cells seems to be mutually antagonistic12. IL-17 is mainly produced by activated T-cells, has the ability to activate other T-cells, and is also capable of triggering the inflammatory process in many other tissues/cells16,17. Involvement of miR-23b in the pathogenesis of myositic muscle was recently reported12 and the etiopathogenetic role of IL-17 in IIM has also been previously described18,19. The aim of our research was to analyze the changes in expression of miR-23b/miR-23b* in PBMCs from patients with myositis and to evaluate the importance of the IL-17/miR-23b signaling network in the PMBCs of these patients. Our results clearly show that the expression profile of miR-23b (as well as miR-23b*) is not changed in the PBMCs of IIM patients. Therefore, the regulative role of miR-23b in the development of myositis is not taking place in PMBCs. We cannot exclude involvement of this system in the local inflammatory process within the inflamed myositis muscle tissue.",
"appendix": "Author contributions\n\n\n\nJV and PN conceived and designed the study. MR, MS and TS carried out the research. MS prepared the first draft of the manuscript. MR and TS contributed to the experimental design and preparation of the manuscript. PN provided direct supervision of the research. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Internal Grant Agency of the Ministry of Health in the Czech Republic (Nr. NT 12452) and a grant from the Charles University Grant agency Nr. 621812.\n\n\nAcknowledgements\n\nWe are grateful to Jitka Smekalova and Marketa Kubalkova for excellent technical assistance.\n\n\nReferences\n\nCeribelli A, Yao B, Dominguez-Gutierrez PR, et al.: MicroRNAs in systemic rheumatic diseases. Arthritis Res Ther. 2011; 13(4): 229. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaleazzi M, Balistreri E, Giannitti C, et al.: MicroRNAs in autoimmune rheumatic diseases. Reumatismo. 2012; 64(1): 7–17. PubMed Abstract | Publisher Full Text\n\nBasu A, Jiang X, Negrini M, et al.: MicroRNA-mediated regulation of pancreatic cancer cell proliferation. Oncol Lett. 2010; 1(3): 565–568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeng S, Cong S, Zhang X, et al.: MicroRNA-192 targeting retinoblastoma 1 inhibits cell proliferation and induces cell apoptosis in lung cancer cells. Nucleic Acids Res. 2011; 39(15): 6669–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLe MT, Teh C, Shyh-Chang N, et al.: MicroRNA-125b is a novel negative regulator of p53. Genes Dev. 2009; 23(7): 862–76. PubMed Abstract | Publisher Full Text | Free 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\nO’Connell RM, Rao DS, Baltimore D: microRNA regulation of inflammatory responses. Annu Rev Immunol. 2012; 30: 295–312. PubMed Abstract | Publisher Full Text\n\nBaltimore D, Boldin MP, O’Connell RM, et al.: MicroRNAs: new regulators of immune cell development and function. Nat Immunol. 2008; 9(8): 839–845. PubMed Abstract | Publisher Full Text\n\nO’Connell RM, Rao DS, Chaudhuri AA, et al.: Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol. 2010; 10(2): 111–122. PubMed Abstract | Publisher Full Text\n\nDai R, Ahmed SA: MicroRNA, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases. Transl Res. 2011; 157(4): 163–179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlevizos I, Illei GG: MicroRNAs as biomarkers in rheumatic diseases. Nat Rev Rheumatol. 2010; 6(7): 391–398. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu S, Pan W, Song X, et al.: The microRNA miR-23b suppresses IL-17–associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-α. Nat Med. 2012; 18(7): 1077–86. PubMed Abstract | Publisher Full Text\n\nBohan A, Peter JB: Polymyositis and dermatomyositis (first of two parts). N Engl J Med. 1975; 292(7): 344–7. PubMed Abstract | Publisher Full Text\n\nBohan A, Peter JB: Polymyositis and dermatomyositis (second of two parts). N Engl J Med. 1975; 292(8): 403–7. PubMed Abstract | Publisher Full Text\n\nChomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162(1): 156–159. PubMed Abstract | Publisher Full Text\n\nYao Z, Fanslow WC, Seldin MF, et al.: Herpesvirus Saimiri Encodes a New Cytokine , IL-17 , which Binds to a Novel Cytokine Receptor. Immunity. 1995; 3(6): 811–821. PubMed Abstract | Publisher Full Text\n\nYao Z, Painter SL, Fanslow WC, et al.: Human il-17: A novel cytokine derived from T cells. J Immunol. 1995; 155(12): 5483–6. PubMed Abstract\n\nZhu W, Streicher K, Shen N, et al.:45 Genomic signatures characterize leukocyte infiltration in myositis muscles. BMC medical genomics. 2012; 5: 53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTournadre A, Miossec P: Interleukin-17 in inflammatory myopathies. Curr Rheumatol Rep. 2012; 14(3): 252–6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2167",
"date": "11 Nov 2013",
"name": "Maria Rescigno",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 I approve of this article; I would just add in the discussion that one caveat of this work is the use of PBMC and that by taking resident inflammatory cells the authors may be able to see a difference in miR-23 expression.",
"responses": []
},
{
"id": "2169",
"date": "11 Dec 2013",
"name": "Stefano Casola",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the authors provide convincing evidence that expression of miRNas mir23 and mir23b* in peripheral blood mononuclear cells of patients affected by inflammatory myopathies is unchanged. This result warrants future investigation to elucidate the role of the same miRNAs in cells that take an active part in the inflammatory response in affected muscles.Minor comment: The authors should spend one sentence to describe the existence of two separate mir23b miRNAs. Also, in the main text the authors should correct the acronym PBMC in a few places towards the end of the discussion.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-223
|
https://f1000research.com/articles/2-75/v1
|
05 Mar 13
|
{
"type": "Research Article",
"title": "Casanovas are liars: behavioral syndromes, sperm competition risk, and the evolution of deceptive male mating behavior in live-bearing fishes",
"authors": [
"David Bierbach",
"Amber M Makowicz",
"Ingo Schlupp",
"Holger Geupel",
"Bruno Streit",
"Martin Plath",
"Amber M Makowicz",
"Ingo Schlupp",
"Holger Geupel",
"Bruno Streit",
"Martin Plath"
],
"abstract": "Mate choice in many species is sensitive to social cues from neighboring individuals; for example, animals can copy mate choice decisions. If males copy other males’ choices, sperm of two or more males can compete for fertilization of the female’s ova. In the internally fertilizing fish Poecilia mexicana, males respond to the presence of rivals with reduced expression of mating preferences (audience effect), thereby lowering the risk of by-standing rivals copying their mate choice. Also, males interact initially more with a non-preferred female when observed by a rival, which has been interpreted in previous studies as a strategy to mislead rivals, again reducing sperm competition risk (SCR). Using a comparative approach, we tested the hypothesis that SCR is indeed a driving force explaining the occurrence of audience-induced changes in poeciliid male mate choice behavior. If this were true, then males of species with higher overall sexual activity — and, thus, higher potential for multiple mating — should show stronger audience effects. We investigated ten poeciliid species (in two cases including multiple populations) and found support for our hypothesis as mean sexual activity correlated positively with the occurrence of potentially deceptive behavior. An alternative explanation for audience effects would be that males attempt to avoid aggressive encounters, which would predict stronger audience effects in more aggressive species, and so we also characterized the examined species for aggressiveness using staged contests of size-matched males. We demonstrate a positive correlation between mean aggressiveness and sexual activity (suggesting a hormonal link as a mechanistic explanation), but we detected no correlation between aggressiveness and audience effects. Suites of correlated behavioral tendencies are termed behavioral syndromes, and our present study provides correlational evidence for the evolutionary significance of SCR in shaping a behavioral syndrome at the species level across poeciliid taxa.",
"keywords": [
"behavioral syndromes",
"mate choice",
"non-independent mate choice",
"social eavesdropping",
"sperm competition"
],
"content": "Introduction\n\nFemale mate choice and male competition are widely acknowledged as the principal forces of sexual selection1,2, while male mate choice has received comparatively little attention (but see3–5). Over the past decades however, it has become apparent that males also express mating preferences3,6–12, especially if females show pronounced differences in mate quality (e.g., through size–fecundity relationships13). Nonetheless, male reproductive biology is clearly influenced by competition over mates1,14–16, and at least in species in which females tend to mate with multiple males, this competition extends well into the period after a successful copulation, as sperm of several males can compete for fertilization of the female’s ova17–19. Our present study provides novel insights into the presumed role this sperm competition risk (SCR) plays for the occurrence of so-called audience effects20 during male mate choice, where males alter their mate choice behavior in the presence of a rival, probably as a strategy to reduce SCR19,21.\n\nMost communication events in group-living animals take place in front of conspecifics22–24, and social information can be used to choose among possible mating partners (social eavesdropping;25–27). For example, quail (Coturnix japonica) and crayfish (Procambarus clarkii) females ‘eavesdrop’ on the outcome of male contests and choose their mating partners accordingly28,29. Furthermore, in various species, both females and males copy other individuals’ mate choice decisions (mate choice copying;30–33) to reduce their own mate searching effort and possibly to improve on mate quality assessment34–36. Male mate choice copying, however, brings about an increased SCR for both the copied male and the copier10,19,35.\n\nTheory predicts that males should adjust their mating behavior strategically to imminent SCR threat19,21,37, and several studies on species exhibiting frequent multiple mating confirm that perceived SCR affects male mate choice behavior10,11,18,38–40. In the livebearing Atlantic molly, Poecilia mexicana, for instance, males temporarily decrease their sexual activity and cease showing mating preferences when another male is eavesdropping9,18,41–43. It has been hypothesized that those audience-induced changes in male mating behavior prevent rivals from copying mate choice decisions19,21. Moreover, focal males initially interact more with a previously non-preferred female in the presence of a rival, which has again been interpreted in the context of mate choice copying – and ultimately, SCR – as males could thus lead the copying male away from the preferred mate (“deceptive mating behavior”;41,42,44). Using a comparative approach, our present study provides an empirical test of the hypothesis that SCR indeed drives the evolution of behavioral responses to the presence of rivals. Specifically, if SCR plays a role, then males of species with higher overall sexual activity – and thus, higher potential for multiple mating and male mate choice copying – should show stronger audience effects, including deceptive mating behavior.\n\nTheoretical considerations identify avoidance of aggressive interactions as another potential mechanism explaining audience-induced changes in male mating behavior21. Specifically, if different males share intrinsic mating preferences (e.g., for large female body size8,42), focal males could interact more equally with different females to reduce the risk of injuries resulting from aggressive interactions over commonly preferred female phenotypes21. If avoiding aggression plays a role, then the magnitude of audience-induced changes in male mating behavior (at the species level) should correlate positively with mean aggressiveness. To test this hypothesis, we examined the intensity of aggressive interactions in size-matched dyadic (paired) male combats for the set of poeciliid species included herein.\n\nIn summary, we assembled a unique data-set comprising ten different poeciliid species (in some cases, several sub-species or ecotypes, or multiple populations) and sought for variation at the taxon level in (1) audience-induced changes in male mate choice, (2) deceptive male mating behavior (previously published data re-analyzed, see Table 1), (3) mean sexual activity, and (4) mean aggressiveness (newly generated data, Table 1), and we tested for correlations of these behavioral tendencies.\n\nIn (a) SL differences between the two opponents are given along with the results from paired t-tests comparing winner and loser SL after dominance was established. In (b) Naudience indicates the number of trials with an audience presented during the second part. * indicates species imported by “Aquarium Dietzenbach GmbH”.\n\n\nMethods\n\nThe experiments reported here comply with the current laws of Germany (approved by Regierungspräsidium Darmstadt V-54-19c-20/15-F104/Anz.18) and the USA (approved by the Institutional Animal Care and Use Committee of the University of Oklahoma; AUS- IACUC approved protocols: R06-026 and R09-023).\n\nTest subjects were lab-reared descendants of wild-caught fish. We included Atlantic mollies from the coastal lagoons around the Central Mexican city of Tampico (belonging to the subspecies P. mexicana limantouri); another population was collected in the Río Oxolotan in Tabasco, South México (P. mexicana mexicana). Recent phylogenetic analyses argue in favor of full species status of the two subspecies45. We further included a locally adapted and genetically differentiated (i.e., independently evolving) ecotype from the P. mexicana mexicana clade: the hydrogen sulfide-adapted form inhabiting El Azufre, a tributary to the Río Oxolotan46,47. As another representative of short-fin mollies48,49 we included mangrove mollies (P. orri) from Roatán Island, Honduras. Two species of long-fin mollies were tested: sailfin mollies (P. latipinna) stemmed from the Comal River in Central Texas, USA, while Tamési mollies (P. latipunctata) were collected near Ciudad Mante in Tamaulipas, México. We further included guppies (P. reticulata) from Venezuela and a feral population from the San Antonio River, Texas, USA50, as well as Venezuelan swamp guppies (P. picta). As representatives of the genus Limia, we included L. tridens and sulfur limia (L. sulphurophilia), both originating from the Dominican Republic. Gambusia sexradiatafrom the Río Teapa, and Grijalva mosquitofish (Heterophallus milleri) from the Río Oxolotán (both Tabasco, México) were included as representatives of mosquito fishes.\n\nTest fish came from large, randomly outbred single-species stocks maintained at the Department of Ecology and Evolution of the University of Frankfurt (P. m. mexicana, P. m. limantouri, P. reticulata from Venezuela, P. picta, L. tridens), or at the Department of Biology at the University of Oklahoma in Norman (P. m. mexicana from El Azufre, P. latipinna, P. latipunctata, P. orri, feral P. reticulata, L. sulphurophila, G. sexradiata, H. milleri; Table 1). Fishes were reared as single-species, mixed-sex stocks in 200-l (Frankfurt) or 1,000-l (Norman) tanks at 25–27°C under an 12:12 hours light:dark cycle (Frankfurt) or under ambient light conditions in a greenhouse (Norman). At the University of Frankfurt, fishes were fed twice daily ad libitum with commercial flake food. Stock tanks in Norman contained naturally growing algae as well as a variety of naturally occurring invertebrates such as chironomid larvae, copepods and amphipods, on which the fishes could feed. In addition, fishes were supplied with flake food every two days. However, at least 1 week prior to the behavioral experiments, fishes were fed ad libitum at least once daily with flake food.\n\n\nExperimental design\n\nWe determined male aggressive behaviors during dyadic encounters by analyzing contests staged between pairs of males in a small test tank measuring 30 × 20 × 20 cm51. To avoid confounding effects of previously established dominance and/or familiarity52,53, males were taken from different stock tanks. Males in a dyad differed by less than 15% in standard length (SL), which has previously been established as the threshold below which fights typically escalate51; nevertheless, size difference was included as a covariate in the statistical analyses (see below). We separated males by an opaque filter sponge while three sides of the test tank were taped with gray paper to minimize disturbances from the outside. The bottom of the tank was filled with black gravel, and water was aerated and maintained at 27–29°C. Males could habituate to the test tank overnight, and observations took place the next day between 09:00 and 13:00. To initiate a trial, the sponge divider was gently lifted, and we noted behavioral interactions for a maximum of 10 minutes, starting with the first interaction. We focused on three frequent aggressive behaviors54: (1) S-position: this threat display usually initiates a fight. Males swim in a parallel or anti-parallel position and bend their bodies in an S-shaped manner with all unpaired fins erect; (2) tail-beats: S-positions are often followed or superimposed by tail-beats, which are fast movements of head and tail in opposing directions that either touch the opponent’s body or send shock waves to the opponent; and (3) bites: we defined all incidences of ramming and bite-like attacks52 as bites, because both these behaviors occur extremely quickly and thus are indistinguishable to the human eye. For some species examined in this study no formal description of aggressive behavior was available from the literature, and so we confirmed in pre-trials that the aforementioned behaviors are part of their behavioral repertoire.\n\nWe also recorded fight duration until dominance was established. Contest outcome could be inferred from behavioral differences between the contestants. Folded fins, head-down posture and a position at the periphery of the tank typically characterize contest losers, while winners constantly chase and further attack the loser with fins fully erect, occasionally performing S-positions or bites51. We met all requirements for animal well-being in behavioral experiments. Apart from occasional loss of single scales, no severe injuries were observed, as we separated males immediately once dominance was established. If no dominance was established within 10 minutes of the first interaction, we terminated the fight; those trials were discarded from the analysis of fighting durations, while fight durations were scored as “0” when no aggressive behavior occurred at all (those trials were terminated after a total of 15 minutes of observation). SL of both contestants was taken after a contest by laying the fish flat on plastic foil-covered millimeter paper (Table 1). Afterwards we transferred males back to their respective stock tanks. In total, we successfully completed N = 146 trials (Table 1).\n\nWe reanalyzed previously published data on audience-induced changes in male mate choice (Table 1). Focal males were isolated in 25- to 38-l tanks for two to four days prior to the tests to ensure that they were motivated to mate12. We tested each focal male only once; however, owing to the limited number of males available from our stocks, some males were also used as audience males after they had served as a focal male, but never on the same day and not in the same dyadic constellation. As familiarity among males affects the strength of audience effects in P. mexicana9, focal and audience males were taken from different stock tanks.\n\nEach focal male was tested for its mating preference in a binary choice situation and was then retested with the same stimulus females either without audience (control treatment) or with an audience male present (50% of trials each). We were thus able to examine changes in focal males’ behavior from the first to the second part of the tests and could discern between effects induced by the audience and changes that would occur over the course of the experiment even without audience. In theory, we could have used an alternative design of presenting an audience in all trials while starting the tests with or without audience in alternating order; however, in such a design, prior exposure to the audience male (when presented during the first part) could still affect the focal males’ behavior during the second part of the tests55.\n\nThe test tank (50 × 30 × 30 cm, length × width × height) was filled to 20 cm height with aged tap water. Water temperature was maintained at 27–28°C using an aquarium heater. In addition, the water was aerated between trials, but both the heater and the air-stone were removed for all trials. Black plastic covered all sides except the front. Prior to the tests, we choose two different-sized stimulus females (for SL see Table 1) from a stock tank and introduced them into the test tank. Poeciliid males prefer to mate with larger, more fecund females (e.g.8,56–58, but see Ala-Honkola et al.59). Afterwards, we introduced a focal male into a transparent Plexiglas cylinder (10 cm diameter) located in the center of the tank and left the fish undisturbed for 5 minutes. After the habituation period, we gently lifted the cylinder. During a 10-min observation period, we scored male sexual behaviors directed toward either of the two females and noted with which female the focal male interacted first. We decided a priori to terminate trials if the male did not show any sexual behavior during the first part of the test; N = 3 trials with P. orri, N = 5 (P. latipinna), N = 2 (P. latipunctata), N = 4 (P. reticulata, Venezuela), N = 1 (P. picta), N = 1 (P. reticulata, San Antonio), and N = 6 (H. milleri) were discarded from the statistical analyses based on this criterion.\n\nGenital nipping is a typical pre-copulatory behavior in poeciliids, whereby the male approaches the female from behind and touches her genital region with his snout54,60. During thrusting, males swing their gonopodium forward while attempting to introduce it into the female’s gonopore. Courtship behavior is absent in P. mexicana60, P. orri, the examined Limia species (authors, personal observation) and Gambusia spp. (61 for G. holbrooki). Poecilia reticulata males court in front of females in an S-shaped body posture (sigmoid displays62,63), while the primary courtship display of P. picta males consists of circling around the female (the so-called ‘orbit’54,63), but males also court with their fins raised in front of the female (63; D.B., personal observation). Heterophallus milleri males circle around the female and swing their gonopodium forward when in the female’s visual field58. Large P. latipinna and P. latipunctata males occasionally court in front of females with raised dorsal fins54,64. As not all species examined herein show courtship displays and courtship was by far the least frequent behavioral category, we excluded numbers of courtship displays from our main analyses.\n\nUpon completion of the first preference test, we immediately repeated measurement of male mating preferences, but in one half of the trials, an audience male was presented, while the other half of the trials was repeated without audience (control). To initiate this second part of a trial, we reintroduced the focal male into the acclimatization cylinder. An audience male was placed in another transparent cylinder in the central back of the tank, while for the control only an empty cylinder was presented. The audience male was confined in his cylinder throughout the test. After another 5 minutes of habituation (during which all four fish could interact visually), measurement of male preferences was repeated, as described above. Interactions between males were not quantified, but aggressive displays were not observed. In total, we successfully completed N = 408 trials (Table 1). Once a trial was completed, all fish were measured for SL to the closest millimeter (Table 1).\n\n\nStatistical analyses\n\nAll statistical analyses were performed in SPSS 13. While “P. mexicana” used in our study clearly represent three phylogenetically independent groups (two sub-species and one derived ecotype45) and, thus, were treated statistically as independent species, this was not the case for the two populations of the guppy (P. reticulata). We thus re-ran all analyses also without data from the feral guppy population (San Antonio), but this did not alter the direction of the results (not shown). Furthermore, in several analyses, body size measures were included as covariates and, since species differed strongly in overall size ranges (see Table 1), we nested all covariates within species (whenever absolute values were used) to account for species-specific size ranges.\n\nWe tested for variation and compared the magnitude of differences in male aggressiveness across species. First, we employed Principal Component Analysis (PCA) to reduce the number of dependent variables (numbers of S-positions, tail-beats and bites per male dyad) and extracted one independent component (PC1; eigenvalue = 2.47) that explained 82.3% of the variance. The three aggressive behaviors had axis loadings of 0.85 (S-positions), 0.93 (tail-beats) and 0.94 (bites). PC1 was checked for normal distribution using a Kolmogorov-Smirnov test and used as a dependent variable in a univariate General Linear Model (GLM) with ‘species’ as a fixed factor. We included ‘mean male SL of a dyad’ (nested within species) as a covariate because larger males tend to be more aggressive51. Moreover, the opponents’ body size difference influences fight intensity51, and so we included arcsine (square root)-transformed body size difference (SLsmall/SLlarge) as another covariate as well as its interaction with the fixed factor in our final model. If a covariate had a significant effect, we employed Pearson’s correlation on unstandardized residuals to explore the direction of the effect.\n\nFight durations were analyzed in a separate univariate GLM with the same factor and covariate structure. Both covariates and the interaction term, however, were removed from the final model since none was significant (‘mean male SL of a dyad’: F13,67 = 1.62, P = 0.24; ‘opponent body size difference’: F1,80 = 2.84, P = 0.09; ‘opponent body size difference × species’: F12,55 = 1.04, P = 0.37).\n\nAs described for the analysis of aggressive behavior, we first used PCA to condense sexual behavior (genital nipping and thrusting) to one principle component (PC1, eigenvalue = 1.79) that explained 89.7% of the variance. Both variables had equal axis loadings of 0.95. We used PC1 as dependent variable in a univariate GLM and included ‘species’ as a fixed factor. Small males show more sexual behaviors than larger ones in at least some of the species examined here as part of a ‘sneak-like’ alternative mating strategy65, and so we included focal males’ SL as a covariate (nested within species). Also, poeciliid males typically prefer to mate with large females (see above), and we thus included the mean SL of each stimulus female dyad (nested within species) as another covariate. However, both covariates had no significant effect (‘focal male SL’: F13,382 = 1.22, P = 0.26; ‘mean stimulus SL’: F13,369= 0.97, P = 0.48) and were removed from the final model.\n\nTo compare the magnitude of audience-induced changes in individual male mate choice behavior across species, we calculated a preference score41 as:\n\n(fraction of sexual behaviors with the initially preferred female during the second part of a trial) – (fraction of sexual behaviors with the same female during the first part),\n\nsuch that negative values would indicate that individual preferences decreased. Scores were included as the dependent variable in a univariate GLM with ‘species’ and ‘treatment’ (with or without audience) as fixed factors. Beside ‘focal male SL’ (nested within species), we also included ‘stimulus female SL difference’ [arcsine (square root)-transformed SLsmall/SLlarge] since one could predict that males would show stronger audience effects the larger the size difference, as large females represent high-quality mates. All possible interactions were included in the initial model. However, neither the covariates themselves (‘focal male SL’: F13,369 = 0.48, P = 0.93; ‘stimulus female size difference’: F1,380 = 0.93, P = 0.34) nor their interaction terms were significant (P > 0.22 in all cases) and were removed from the final model.\n\nQualitatively, H. milleri did not follow the pattern of reduced preference expression shown by other poeciliids (see also Bierbach et al.58), so we re-ran all analyses while excluding H. milleri, but the direction of the results was not affected (results not shown).\n\nIn the context of deceptive mating behavior, the first sexual approach of focal males is of interest, as interacting first with the previously non-preferred female has been interpreted as an attempt to mislead the rival41. Thus, we analyzed the fraction of males that first interacted with the opposite (“1”) or same female during the second part (“0”) using a binary logistic regression, with ‘species’, ‘treatment’ and their interaction term as categorical independent variables. We also included ‘focal male SL’ and ‘female SL difference’ as covariates and used a step-wise backwards elimination approach, based on likelihood ratios, to remove effects if P > 0.1.\n\nThe central question of our present paper was whether there are correlations between the four aforementioned behaviors at the species level. Owing to the limited sample size (N = 13 groups), we used non-parametric, pair-wise Spearman rank order tests to correlate species means for (1) aggressiveness (sum of all aggressive interactions per fight), (2) sexual activity (sum of nipping and thrusting behavior during the first part of the tests), (3) the strength of changes in preference expression in male mate choice (score from audience treatment minus mean score from control treatment) and (4) the occurrence of deceptive behavior (fraction of males that changed their first interaction in the audience treatment – fraction in the control treatment). To avoid error inflation due to multiple comparisons, we used Bonferroni corrections and inferred statistical significance only if P < α = 0.017.\n\n\nResults\n\nThere was pronounced variation in aggressive behavior among species (for univariate GLM, see Table 2a; Figure 1a). Both covariates (‘mean male dyad SL’ and ‘opponent body size difference’) had significant effects (Table 2a), but species-wise post hoc Pearson correlations confirmed the predicted pattern of larger males fighting more intensely only in P. m. mexicana (sulfide ecotype, see Table 3). Only in the highly aggressive Atlantic mollies (P. m. mexicana, both ecotypes, and P. m. limantouri; Figure 1) was the predicted negative relationship between opponent size difference and mean aggressiveness uncovered (Table 3), i.e., the larger the size difference the less intense fights became.\n\nMeans (+ SE) of (a) numbers of aggressive interactions per male fight and (b) fight duration in the different poeciliid species examined.\n\nSL; standard length. Significant effects are indicated by an asterisk.\n\nUnstandardized residuals were obtained from GLM (see Table 2a). Significant correlations are indicated by an asterisk.\n\nThe GLM on mean fight durations also detected significant species differences (Table 2b; Figure 1b).\n\nGLM detected a significant effect of the factor ‘species’ (Table 2c), indicating that male sexual activity also showed pronounced variation among species (Figure 2a).\n\n(a) Mean (+ SE) numbers of male sexual behaviors during a 10 min observation period. (b) Changes in individual focal males’ mate choice behavior in the presence of an audience male. Depicted are mean (+ SE) preference scores (see main text), whereby negative values indicate that male preferences decreased in strength. (c) Proportion of males that first interacted with the opposite female when released from the acclimation cylinder in the second part of the tests. Open bars in (b) and (c) represent the control treatment (no audience) while gray bars represent the audience treatment.\n\nWhen comparing the change in individual males’ mating preferences from the first to second part of the tests (preference score), we detected a significant main effect of the factor ‘treatment’, suggesting that focal males responded to an audience male with altered mate choice behavior (Table 2d). No significant effect of the interaction term ‘species by treatment’ was uncovered (Table 2c), suggesting that species did not overall differ in their response to the audience treatment (Figure 2b). A significant effect of the main factor ‘species’ (Table 2c), by contrast, can be interpreted as species differing in the consistency of their mate choice over the course of the experiment (note that this main effect considers changes in both the audience and control treatments).\n\nWhen comparing the number of trials in which the focal males first approached the same (“0”) or different (“1”) female during the second part using logistic regression, the interaction term of ‘species by treatment’ was excluded already during the first step of the step-wise elimination process (B = -0.031, SE = 0.06, Wald = 0.233, df = 1, P = 0.629), indicating that male responses to the audience treatment did not differ among species (Figure 2c). In the final model, only the factors ‘treatment’ (B = 1.39, SE = 0.27, Wald = 34.76, df = 1, P < 0.001) and ‘female body size difference’ (not significant: B = 1.49, SE = 0.83, Wald = 3.27, df = 1, P = 0.071) were retained. Thus, focal males were more likely to change their initial interaction when an audience male was presented (Figure 2c).\n\nIn line with our prediction derived from the interpretation that SCR explains the occurrence of audience-induced behavioral changes, we found a strong, positive correlation between sexual activity and the amount of deceptive behavior at the species level (Figure 3e). The alternative prediction, that avoidance of aggressive behavior drives audience effects (leading to a positive correlation between both variables), received no support, as the correlation between mean aggression and strength of audience-induced changes in preference expression even yielded a negative correlation coefficient (not statistically significant; Figure 3b). Finally, there was a significant positive correlation between aggression and sexual activity (Figure 3a).\n\nShown are results from Spearman’s rank order tests; α’ indicates the alpha error level after Bonferroni correction.\n\n\nDiscussion\n\nWe found pronounced variation among poeciliid taxa in mean aggressiveness and male sexual activity, while variation in audience effects (reduced preference expression and deceptive mating behavior in presence of an audience) was less pronounced. Subsequent correlation analyses uncovered two effects: (a) males of species with high sexual activity are more likely to show deceptive mating behavior, i.e., they initially approached more often the non-preferred female when an audience male was presented, while mean aggressiveness did not predict the occurrence of audience effects. (b) Mean aggressiveness, by contrast, correlated positively with mean sexual activity. Hence, we detected two correlations of behavioral tendencies at the species level.\n\nConsistency in the expression of a certain behavioral type across different environmental contexts at the inter-individual level has received considerable scientific interest66–68, and suites of correlated behavioral types have been termed ‘behavioral syndromes’66,69. Réale et al.70 proposed five different axes of animal personality: shyness–boldness, exploration–avoidance, general activity, aggressiveness, and sociability, and Conrad et al.69 highlighted several correlations of those behavioral axes in teleost fishes, but audience-induced changes in male mating behavior have not yet been investigated in the context of behavioral syndromes. Recent studies exemplified the importance of population differences in behavioral syndromes71–73. Also, the concept of behavioral syndromes was expanded to the comparison of groups of animals or populations; Chapman et al.73, for example, demonstrated correlations between mean colony (and caste) behavioral types in Myrmica ants. Here, we apply this concept to the comparison of different poeciliid taxa.\n\nOne of the behavioral syndromes at the species level we uncovered in our present study – the correlation between aggressiveness and sexual activity – can be partly explained mechanistically through species differences in plasma concentrations of sexual corticosteroids (testosterone and its derivates74,75). Individual androgen concentrations predict aggressiveness in male swordtails, Xiphophorus hellerii76; furthermore, plasma testosterone levels correlate positively with sexual behavior in male mosquito fish (G. holbrooki)77, so physiological pleiotropy could also explain species differences in aggression and sexual activity as detected here.\n\nThe main focus of our present study was on audience-induced changes in male mating behavior, and we asked if those behaviors can be linked to mean sexual activity and SCR. The rationale behind our prediction was that males of taxa with high overall sexual activity face a higher risk of by-standers making use of socially acquired information when eavesdropping on sexual interactions. It seems reasonable to assume the propensity for male mate choice copying to be a ubiquitous feature of poeciliid mating systems10,35, but the likelihood of mate copying in natural systems should correlate positively with mean sexual activity. We found sexual activity (but not aggressiveness – despite some degree of inter-correlation between aggressiveness and sexual activity, see above) to correlate positively with the level of presumed deceptive mating behavior. This finding lends support to our hypothesis that SCR is a driving force behind the evolution of this behavior and is in line with our interpretation that focal males thus attempt to lead the rival away from their preferred mate, exploiting male mate choice copying to reduce SCR19,21,42.\n\nA general objection to our interpretation of deceptive mating behavior could be that leading the audience away from a preferred mating partner to deceive the rival may increase the risk of losing the preferred female, as poeciliid females tend to flee from male sexual harassment60,78,79. We argue that this male behavior still offers advantages even if the preferred female flees: on the one hand, a pattern of last male sperm precedence was uncovered in guppies80,81, which renders mate choice copying a profitable option for the eavesdropping (copying) male10. However, the longer the time between copulations by the first and second male in the mating trials conducted by Evans and Magurran81, the higher the proportion of offspring fathered by the first male was. This implies that leading the by-standing rival away from (or at least delaying its approaches toward) a recently inseminated female would indeed be beneficial for the deceiving male even though it risks losing contact with the initially preferred (but already inseminated) female.\n\nSince our analyses were based on population differences in aggressiveness, sexual activity and audience induced changes in male mate choice behavior, we strongly recommend future experiments concentrating on within-population variation (e.g., individual “behavioral types”70) that define a male’s response to a by-standing rival. For example, males are very sensitive to the perceived sexual activity of a rival when exhibiting audience effects9, and future studies should elaborate on the question of whether also perceived aggressiveness – a correlate of sexual activity – might influence the expression of audience effects.\n\nIn summary, using a comparative approach, we found correlational support for the hypothesis that SCR arising from male mate choice copying drives the evolution of audience-induced changes in male mate choice behavior. We argue that taxa with elevated sexual activity face a higher risk of males making use of socially acquired information (i.e., copying mate choice decisions), and so focal males in those species are more likely to respond to the presence of an audience with altered mate choice behavior.",
"appendix": "Author contributions\n\n\n\nDB, IS, BS and MP designed the study. DB, AMM and HG conducted the experiments. DB and MP analyzed the data. DB prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe present study was financially supported by the research funding program “LOEWE – Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz” of Hesse’s Ministry of Higher Education, Research, and the Arts (to MP) and the DFG (Pl 470/1-3; to MP).\n\n\nAcknowledgements\n\nWe thank Y Konstantin and D Schenkel for help with data collection.\n\n\nReferences\n\nAndersson M: Sexual selection. (Princeton University Press, Princeton). 1994; 624. Reference Source\n\nKokko H, Johnstone RA: Why is mutual mate choice not the norm? Operational sex ratios, sex roles and the evolution of sexually dimorphic and monomorphic signalling. Philos Trans R Soc Lond B Biol Sci. 2002; 357(1419): 319–330. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonduriansky R: The evolution of male mate choice in insects: a synthesis of ideas and evidence. Biol Rev Camb Philos Soc. 2001; 76(3): 305–339. PubMed Abstract | Publisher Full Text\n\nSargent RC, Gross MR, Van Den Berghe EP: Male mate choice in fishes. Anim Behav. 1986; 34(2): 545–550. Publisher Full Text\n\nEdward DA, Chapman T: The evolution and significance of male mate choice. Trends Ecol Evol. 2011; 26(12): 647–654. PubMed Abstract | Publisher Full Text\n\nSaether SA, Fiske P, Kålås JA: Male mate choice, sexual conflict and strategic allocation of copulations in a lekking bird. Proc Biol Sci. 2001; 268(1481): 2097–2102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong BBM, Jennions MD, Keogh JS: Sequential male mate choice in a fish, the Pacific blue-eye Pseudomugil signifer. Behav Ecol Sociobiol. 2004; 56(3): 253–256. Publisher Full Text\n\nPlath M, Seggel U, Burmeister H, et al.: Choosy males from the underground: male mating preferences in surface- and cave-dwelling Atlantic mollies (Poecilia mexicana). Naturwissenschaften. 2006; 93(3): 103–109. PubMed Abstract | Publisher Full Text\n\nBierbach D, Girndt A, Hamfler S, et al.: Male fish use prior knowledge about rivals to adjust their mate choice. Biol Lett. 2011; 7(3): 349–351. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBierbach D, Kronmarck C, Hennige-Schulz C, et al.: Sperm competition risk affects male mate choice copying. Behav Ecol Sociobiol. 2011; 65(9): 1699–1707. Publisher Full Text\n\nWronski T, Bierbach D, Czupalla LM, et al.: Rival presence leads to reversible changes in male mate choice of a desert dwelling ungulate. Behav Ecol. 2012; 23(3): 551–558. Publisher Full Text\n\nSchlupp I, Plath M: Male mate choice and sperm allocation in a sexual/asexual mating complex of Poecilia. (Poeciliidae, Teleostei). Biol Lett. 2005; 1(2): 169–171. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiesch R, Plath M, Garcia de Leon FJ, et al.: Convergent life-history shifts: toxic environments result in big babies in two clades of poeciliids. Naturwissenschaften. 2010; 97(2): 133–141. PubMed Abstract | Publisher Full Text\n\nHoward RD, Martens RS, Innis SA, et al.: Mate choice and mate competition influence male body size in Japanese medaka. Anim Behav. 1998; 55(5): 1151–1163. PubMed Abstract | Publisher Full Text\n\nHunt J, Breuker CJ, Sadowski JA, et al.: Male-male competition, female mate choice and their interaction: determining total sexual selection. J Evol Biol. 2009; 22(1): 13–26. PubMed Abstract | Publisher Full Text\n\nParker GA: Assessment strategy and the evolution of fighting behaviour. J Theor Biol. 1974; 47(1): 223–243. PubMed Abstract | Publisher Full Text\n\nParker GA: Sperm competition and its evolutionary consequences in the insects. Biol Rev. 1970; 45(4): 525–567. Publisher Full Text\n\nZiege M, Mahlow K, Hennige-Schulz C, et al.: Audience effects in the Atlantic molly (Poecilia mexicana)-prudent male mate choice in response to perceived sperm competition risk? Front Zool. 2009; 6: 17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPlath M, Bierbach D: Sex and the public: Social eavesdropping, sperm competition risk and male mate choice. Commun Integr Biol. 2011; 4(3): 276–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarler P, Dufty A, Pickert R: Vocal communication in the domestic chicken: II. Is a sender sensitive to the presence and nature of a receiver? Anim Behav. 1986; 34(Part 1): 194–198. Publisher Full Text\n\nPlath M, Schlupp I: Misleading mollies: The effect of an audience on the expression of mating preferences. Commun Integr Biol. 2008; 1(2): 199–203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcGregor PK, Peake TM: Communication networks: social environments for receiving and signalling behaviour. Acta Ethol. 2000; 2(2): 71–81. Publisher Full Text\n\nPeake TM: Eavesdropping in communication networks. In Animal Communication Networks, P.K. McGregor, ed. (Cambridge: Cambridge University Press). 2005; 13–37. Publisher Full Text\n\nEarley RL, Dugatkin LA: Fighting, mating and networking: pillars of poeciliid sociality. In Animal Communication Networks, P.K. McGregor, ed. (Cambridge: Cambridge University Press). 2005; 84–113. Publisher Full Text\n\nDabelsteen T: Public, private or anonymous? Facilitating and countering eavesdropping. In Animal Communication Networks, P.K. McGregor, ed. (Cambridge: Cambridge University Press). 2005; 38–62. Publisher Full Text\n\nJohnston RE: Eavesdropping and scent over-marking. In Animal Communication Networks, P.K. McGregor, ed. (Cambridge: Cambridge University Press). 2005; 344–371. Publisher Full Text\n\nEarley RL: Social eavesdropping and the evolution of conditional cooperation and cheating strategies. Philos Trans R Soc Lond B Biol Sci. 2010; 365(1553): 2675–2686. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOphir AG, Galef BG Jr: Female Japanese quail that ‘eavesdrop’ on fighting males prefer losers to winners. Anim Behav. 2003; 66(2): 399–407.Publisher Full Text\n\nAquiloni L, Buric M, Gherardi F: Crayfish females eavesdrop on fighting males before choosing the dominant mate. Curr Biol. 2008; 18(11): R462–R463. PubMed Abstract | Publisher Full Text\n\nDugatkin LA: Copying and Mate Choice. In Social Learning in Animals. M.H. Cecilia, G.G. Bennett, A.C.M.H. Jr and Bennett G. Galef, Jr., eds. (San Diego: Academic Press), 1996; 85–105. Reference Source\n\nWestneat DF, Walters A, McCarthy TM, et al.: Alternative mechanisms of nonindependent mate choice. Anim Behav. 2000; 59(3): 467–476. PubMed Abstract | Publisher Full Text\n\nValone TJ: From eavesdropping on performance to copying the behavior of others: a review of public information use. Behav Ecol Sociobiol. 2007; 62(1): 1–14. Publisher Full Text\n\nBonnie KE, Earley RL: Expanding the scope for social information use. Anim Behav. 2007; 74(2): 171–181. Publisher Full Text\n\nGibson RM, Höglund J: Copying and sexual selection. Trends Ecol Evol. 1992; 7(7): 229–232. PubMed Abstract | Publisher Full Text\n\nSchlupp I, Ryan MJ: Male Sailfin mollies (Poecilia latipinna) copy the mate choice of other males. Behav Ecol. 1997; 8(1): 104–107. Publisher Full Text\n\nWitte K: Learning and mate choice. In Fish cognition and behavior, LK. Brown C, Krause J, ed. (Oxford: Blackwell Publishing Ltd), 2006; 70–95. Publisher Full Text\n\nWedell N, Gage MJG, Parker GA: Sperm competition, male prudence and sperm-limited females. Trends Ecol Evol. 2002; 17(7): 313–320.Publisher Full Text\n\nWong BBM, McCarthy M: Prudent male mate choice under perceived sperm competition risk in the eastern mosquito fish. Behav Ecol. 2009; 20(2): 278–282. Publisher Full Text\n\nJeswiet SB, Lee-Jenkins SSY, Godin JGJ: Concurrent effects of sperm competition and female quality on male mate choice in the Trinidadian guppy (Poecilia reticulata). Behav Ecol. 2012; 23(1): 195–200. Publisher Full Text\n\nDubois F, Belzile A: Audience effect alters male mating preferences in zebra finches (Taeniopygia guttata). PLoS One. 2012; 7(8): e43697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPlath M, Richter S, Tiedemann R, et al.: Male fish deceive competitors about mating preferences. Curr Biol. 2008; 18(15): 1138–1141. PubMed Abstract | Publisher Full Text\n\nBierbach D, Konstantin Y, Stadler S, et al.: Presence of an audience alters mate choice of poeciliid males. In Social Learning Theory: Phylogenetic Considerations across Animal, Plant, and Microbial Taxa., K. Clark, ed. (Hauppauge NY: Nova Science Publishers) (In press) 2013. 95–112. Reference Source\n\nPlath M, Blum D, Schlupp I, et al.: Audience effect alters mating preferences in a livebearing fish, the Atlantic molly, Poecilia mexicana. Anim Behav. 2008; 75: 21–29. Publisher Full Text\n\nPlath M, Richter S, Schlupp I, et al.: Misleading mollies: surface- but not cave-dwelling Poecilia mexicana males deceive competitors about mating preferences. Acta Ethol. 2010; 13(1): 49–56. Publisher Full Text\n\nTobler M, Palacios M, Chapman LJ, et al.: Evolution in extreme environments: replicated phenotypic differentiation in livebearing fish inhabiting sulfidic springs. Evolution. 2011; 65(8): 2213–2228. PubMed Abstract | Publisher Full Text\n\nPlath M, Hauswaldt JS, Moll K, et al.: Local adaptation and pronounced genetic differentiation in an extremophile fish, Poecilia mexicana, inhabiting a Mexican cave with toxic hydrogen sulphide. Mol Ecol. 2007; 16(5): 967–76. PubMed Abstract | Publisher Full Text\n\nTobler M, Riesch R, Tobler CM, et al.: Natural and sexual selection against immigrants maintains differentiation among micro-allopatric populations. J Evol Biol. 2009; 22(11): 2298–304. PubMed Abstract | Publisher Full Text\n\nHrbek T, Seckinger J, Meyer A: A phylogenetic and biogeographic perspective on the evolution of poeciliid fishes. Mol Phylogenet Evol. 2007; 43(3): 986–998. PubMed Abstract | Publisher Full Text\n\nPtacek MB, Breden F: Phylogenetic relationships among the mollies (Poeciliidae: Poecilia: Mollienesia group) based on mitochondrial DNA sequences. J Fish Biol. 1998; 53: 64–81. Publisher Full Text\n\nHubbs C, Lucier T, Garrett GP, et al.: Survival and abundance of introduced fishes near San Antonio, Texas. Tex J Sci. 1978; 30(4): 369–376. Reference Source\n\nBierbach D, Klein M, Saßmannshausen V, et al.: Divergent evolution of male aggressive behaviour: another reproductive isolation barrier in extremophile poeciliid fishes? Int J Evol Biol. 2012; 2012: 148745PubMed Abstract | Publisher Full Text | Free Full Text\n\nHsu Y, Earley RL, Wolf LL: Modulation of aggressive behaviour by fighting experience: mechanisms and contest outcomes. Biol Rev Camb Philos Soc. 2006; 81(1): 33–74. PubMed Abstract | Publisher Full Text\n\nBeaugrand J, Goulet C, Payette D: Outcome of dyadic conflict in male green swordtail fish, Xiphophorus helleri: Effects of body size and prior dominance. Anim Behav. 1991; 41(3): 417–424. Publisher Full Text\n\nParzefall J: Zur vergleichenden Ethologie verschiedener Mollienesia-Arten einschließlich einer Höhlenform von Mollienesia sphenops. Behaviour. 1969; 33: 1–37. Publisher Full Text\n\nMakowicz AM, Plath M, Schlupp I: Male guppies (Poecilia reticulata) adjust their mate choice behaviour to the presence of an audience. Behaviour. 2010; 147: 1657–1674. Publisher Full Text\n\nBisazza A, Marconato A, Marin G: Male mate preferences in the mosquitofish Gambusia holbrooki. Ethology. 1989; 83(4): 335–343. Publisher Full Text\n\nHerdman EJE, Kelly CD, Godin JGJ: Male mate choice in the guppy (Poecilia reticulata): Do males prefer larger females as mates? Ethology. 2004; 110(2): 97–111. Publisher Full Text\n\nBierbach D, Ziege M, Zimmer C, et al.: Male Grijalva mosquitofish (Heterophallus milleri Radda, 1987) increase individual mating preferences in front of an audience. Aqua Int J Ichthyol. 2012; 18(4): 199–208. Reference Source\n\nAla-Honkola O, Säilä L, Lindström K: Males prefer small females in a dichotomous choice test in the poeciliid fish Heterandria formosa. Ethology. 2010; 116(8): 736–743. Publisher Full Text\n\nPlath M, Makowicz AM, Schlupp I, et al.: Sexual harassment in live-bearing fishes (Poeciliidae): comparing courting and noncourting species. Behav Ecol. 2007; 18(4): 680–688. Publisher Full Text\n\nEvans JP, Pierotti M, Pilastro A: Male mating behavior and ejaculate expenditure under sperm competition risk in the eastern mosquitofish. Behav Ecol. 2003; 14(2): 268–273. Publisher Full Text\n\nBaerends GP, Brouwer R, Waterbolk HT: Ethological studies on Lebistes reticulatus (Peters): I. An analysis of the male courtship pattern. Behaviour. 1955; 8(4): 249–334. Publisher Full Text\n\nLiley NR: Ethological isolating mechanisms in four sympatric species of Poeciliid fishes. Behaviour. 1966; 13(suppl 1): 197. Reference Source\n\nNiemeitz A, Kreutzfeldt R, Schartl M, et al.: Male mating behaviour of a molly, Poecilia latipunctata: a third host for the sperm-dependent Amazon molly, Poecilia formosa. Acta Ethol. 2002; 5(1): 45–49. Publisher Full Text\n\nPlath M: Male mating behavior and costs of sexual harassment for females in cavernicolous and extremophile populations of Atlantic mollies (Poecilia mexicana). Behaviour. 2008; 145(1): 73–98. Publisher Full Text\n\nSih A, Bell A, Johnson JC: Behavioral syndromes: an ecological and evolutionary overview. Trends Ecol Evol. 2004; 19(7): 372–378. PubMed Abstract | Publisher Full Text\n\nBell AM: Future directions in behavioural syndromes research. Proc Biol Sci. 2007; 274(1611): 755–761. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSih A: Behavioral syndromes: A behavioral ecologist’s view on the evolutionary and ecological implications of animal personalities. In Personality and Temperament in Nonhuman Primates, A.Weiss, J.E. King and L. Murray, eds. (New York: Springer), 2011; 313–336. Publisher Full Text\n\nConrad JL, Weinersmith KL, Brodin T, et al.: Behavioural syndromes in fishes: a review with implications for ecology and fisheries management. J Fish Biol. 2011; 78(2): 395–435. PubMed Abstract | Publisher Full Text\n\nRéale D, Dingemanse NJ, Kazem AJ, et al.: Evolutionary and ecological approaches to the study of personality. Philos Trans R Soc Lond B Biol Sci. 2010; 365(1560): 3937–3946. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBókony V, Kulcsár A, Tóth Z, et al.: Personality traits and behavioral syndromes in differently urbanized populations of house sparrows (Passer domesticus). PLoS One. 2012; 7(5): e36639. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDingemanse NJ, Wright J, Kazem AJN, et al.: Behavioural syndromes differ predictably between 12 populations of three-spined stickleback. J Anim Ecol. 2007; 76(6): 1128–1138. PubMed Abstract | Publisher Full Text\n\nChapman BB, Thain H, Coughlin J, et al.: Behavioural syndromes at multiple scales in Myrmica ants. Anim Behav. 2011; 82(2): 391–397. Publisher Full Text\n\nBook AS, Starzyk KB, Quinsey VL: The relationship between testosterone and aggression: a meta-analysis. Aggress Violent Behav. 2001; 6(6): 579–599. Publisher Full Text\n\nBreedlove SM, Becker JB, Crews D, et al.: Behavioral endocrinology, 2nd Edition. (Bradford Books) 2002. Reference Source\n\nHannes RP: Blood and whole-body androgen levels of male swordtails correlated with aggression measures in a standard-opponent test. Aggress Behav. 1986; 12(4): 249–254. Publisher Full Text\n\nToft G, Edwards TM, Baatrup E, et al.: Disturbed sexual characteristics in male mosquitofish (Gambusia holbrooki) from a lake contaminated with endocrine disruptors. Environ Health Perspect. 2003; 111(5): 695–701. PubMed Abstract | Free Full Text\n\nKöhler A, Hildenbrand P, Schleucher E, et al.: Effects of male sexual harassment on female time budgets, feeding behavior, and metabolic rates in a tropical livebearing fish (Poecilia mexicana). Behav Ecol Sociobiol. 2011; 65(8): 1513–1523. Publisher Full Text\n\nMagurran AE: Sexual coersion. In Ecology and Evolution of Poeciliid Fishes, J. Evans, A. Pilastro and I. Schlupp, eds. (Chicago: University of Chicago Press), 2011; 209–217. Reference Source\n\nEvans J, Pilastro A: Postcopulatory sexual selection. In Ecology and Evolution of Poeciliid Fishes. J. Evans, A. Pilastro and I. Schlupp, eds. (Chicago: Chicago University Press), 2011. Reference Source\n\nEvans JP, Magurran AE: Patterns of sperm precedence and predictors of paternity in the Trinidadian guppy. Proc Biol Sci. 2001; 268(1468): 719–724. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1005",
"date": "17 Jun 2013",
"name": "Katja Heubel",
"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 very much appreciate the effort of putting results together to compile a dataset that makes comparison across taxa possible. The authors could take more advantage of that. See more detailed comments below. Unfortunately, the manuscript does not have line numbers.Title: Your title does not really suit your story. In particular, I am not in favour of your Casanova analogy. It is not clear to me how and in which part of your paper you may have tested the males properties of being “Casanovas” – I would have expected you to test a males multiple mating success – which you did not. Furthermore, the “liar” is not clear to me either. You did not test how male’s fitness correlated traits are dishonest or something like this. Anyways, analogies from the liberal arts always come with the problem of being a bit inaccurate since the definition of such characters are always somewhat blurred (is a “Casanova” a male with many matings? Many mates? High reproductive success? Is also male-male competitive ability part of the syndrome? What about the male aggression level?). I suggest refraining from using such non-scientific terms in your title and rather find a suitable title. Reading the abstract and introduction would suggest a title on sperm competition risk and audience effect.Abstract: your start is a bit odd. You start with what seems to me to be the most exceptional example for the cause of sperm competition. Mate copying and even more specific male mate copying is not required for sperm completion. A simple scenario of multiple mating in a promiscuous mating system is a much more straightforward explanation.Article content: the methods section is well explained. Looking at Table 1, I started wondering how many cases were excluded because a clear winner-loser situation could not be established. Comparing N dyads and the df’s, it seems that some species (H. milleri, P. reticulata feral, L. tridens, P. latipunctata) have more difficulties in establishing hierarchies under the given size differences. In the mentioned species, no more than 50% of trials ended with a winner-loser situation (H. milleri 4 out of 14 dyads). Thus, the n of trials with established hierarchy is 103, not 146.Your experimental procedure on male mating behaviour comes with the side-effect that some of the males (and females) got to mate successfully in the first round, but others maybe not. Does this potentially affect their behaviour in front of an audience?I was wondering whether there would be an alternative and more efficient approach to test species level regressions/correlations? Maybe you can compare regression slopes. Figure 2c may suggest that real deception only occurs in L. tridens, P. mex. lim, P. mex sulf, and P. mex mex which have an average likelihood to swap preferences and approach the other female of more than 50%.Introduction: the introduction is a bit weak in underlining the novelty and achievements of the current study compared to the already existing body of articles on SCR and audience effects in poeciliids. A reader skimming through the paper may wonder why another study on SCR and audience in poecillids would be an interesting read.I very much appreciate the effort of putting results together to compile a dataset that makes comparison across taxa possible. Take more advantage of that.In particular, I was wondering how encounter rates and population density may affect SCR and related coping styles. A life-history approach by looking at shifts in the reproductive periods may be interesting. Depending on whether first or last males sperm precedence is applicable, one may predict an advancement of the mating activity on the population level. Nevertheless, I am aware that this is beyond the scope of this paper.Discussion: I am not convinced that your first sentence is supported by your data: where do you show that variation in audience effects is less pronounced among taxa? Why is personality and behavioural syndromes not touched upon in your introduction? Not sure it really belongs to your story. There is no real data on SCR in your paper. Your introduction deals with SCR in great detail, but it is not really in your data. Is there any solid data that supports your proposed link between sexual activity and SCR?Data: having 13 taxa at hand, it would be interesting to see which and how some species cluster together. Could you include multiple contrasts or a factor analysis to illustrate similarity vs dissimilarity among species?As it is, Table 2 with “species” being significant, only reports that at least one species is different from the others. Would be useful to add more information. What you really want to show is how the species are clustering and how this relates to their mating system and sperm competition risk.",
"responses": [
{
"c_id": "522",
"date": "12 Aug 2013",
"name": "David Bierbach",
"role": "Author Response",
"response": "Thank you for your positive view of our comparative approach! We tried to outline this cross-taxa character in more detail in the new version of the manuscript. I am afraid the lack of line numbers is a feature of the journal’s publishing and editing concept, sorry. You are right to be wary of analogies from the liberal arts. However, the Oxford dictionary defines a “Casanova” as a man notorious for seducing women. As the most sexually active species in our study readily switch their preferred females, we believe that referring to those males as “Casanovas” is not far from reality. The second part of our title adequately describes our current study from a scientific point of view, also incorporating sperm competition as one of your suggested key words. Abstract: Thank you for this point! We changed the beginning of the abstract, several parts of the introduction and discussion, as well as the statistical analysis to underpin the comparative approach more precisely. Article content: The reviewer is right about the number of dyads in which a clear dominance hierarchy was established. Nevertheless, even when no dominance was established we analyzed the number of aggressive behaviors that occurred and counted those trials as successful. We now precisely state how many trials dominance was established in. The reviewer is right, successful and unsuccessful matings could have influenced the behavior in front of an audience. However, in most Poeciliids it is not possible to discriminate with certainty between a successful mating (defined as a mating with sperm being transferred) and the pure mating attempt. We followed our analysis with a protocol from Hammond-Tooke et al. (2012) where Spearman’s rank order tests were used to test for behavioral syndromes. We also depict results from PCA to cluster species based on their behaviors (see below). The new analysis found significant between-species variation in the level of deceptive behavior while the general tendency to swap more often between females when confronted with an audience male was found in all but one species. Our current analysis is focused on general behaviors across Poeciliids, more species-based analysis can be found in Bierbach et al. (2013) (in press). Discussion: Our new analysis shows that between-species variation is much smaller in the audience behavior. We have switched the paragraph dealing with personality and behavioral syndromes from the discussion to the introduction so that the reader will be introduced to these considerations at the beginning. Several studies used the presence of a by-standing rival during mate choice to simulate SCR (see introduction) and sexual activity as a proxy for sperm competition in Poeciliids with high rates of multiple matings (which in turn leads to broods normally sired by multiple males). However, quantifying direct sperm competition (e.g., through offspring genotyping) was beyond the scope of our study. Data: To provide an overview about similarity vs. dissimilarity among species we now provide results from PCA."
}
]
},
{
"id": "1007",
"date": "17 Jun 2013",
"name": "Lisa Locatello",
"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\nBierbach and co-authors investigated the topic of the evolution of the audience effect in live bearing fishes, by applying a comparative method. They specifically focused on the hypothesis that sperm competition risk, arising from male mate choice copying, and avoidance of aggressive interactions play a key role in driving the evolution of audience-induced changes in male mate choice behavior. The authors found support to their hypothesis of an influence of SCR on the evolution of deceptive behavior as their findings at species level showed a positive correlation between mean sexual activity and the occurrence of deceptive behavior. Moreover, they found a positive correlation between mean aggressiveness and sexual activity but they did not detect a relationship between aggressiveness and audience effects. The manuscript is certainly well written and attractive, but I have some major concerns on the data analyses that prevent me to endorse its acceptance at the present stage.I see three main problems with the statistics that could have led to potentially wrong results and, thus, to completely misleading conclusions. •\n\nFirst of all the Authors cannot run an ANCOVA in which there is a significant interaction between factor and covariate Tab. 2 (a). Indeed, when the assumption of common slopes is violated (as in their case), all other significant terms are meaningless. They might want to consider alternative statistical procedures, e.g. Johnson—Neyman method. •\n\nSecond, the Authors cannot retain into the model a non significant interaction term, as this may affect estimations for the factors Tab. 2 (d). They need to remove the species x treatment interaction (as they did for other non significant terms, see top left of the same page 7). •\n\nThe third problem I see regards all the GLMs in which species are compared. Authors entered the 'species' level as fixed factor when species are clearly a random factor. Entering species as fixed factors has the effect of badly inflating the denominator degrees of freedom, making authors’ conclusions far too permissive. They should, instead, use mixed LMs, in which species are the random factor. They should also take care that the degrees of freedom are approximately equal to the number of species (not the number of trials). To do so, they can enter as random factor the interaction between treatment and species. Data need to be re-analyzed relying on the proper statistical procedures to confirm results and conclusions.A more theoretical objection to the authors’ interpretation of results (supposing that results will be confirmed by the new analyses) could emerge from the idea that male success in mating with the preferred female may reduce the probability of immediate female’s re-mating, and thus reduce the risk of sperm competition on the short term. As a consequence, it may be not beneficial to significantly increase the risk of losing a high quality and inseminated female for a cost that will not be paid with certainty. The authors might want to consider also this for discussion. Lastly, I think that the scenario generated from comparative studies at species level may be explained by phylogenetic factors other than sexual selection. Only the inclusion of phylogeny, that allow to account for the shared history among species, into data analyses can lead to unequivocal adaptive explanations for the observed patterns. I see the difficulty in doing this with few species, as it is the case of the present study, but I would suggest the Authors to consider also this future perspective. Moreover, a phylogenetic comparative study would be aided by the recent development of a well-resolved phylogenetic tree for the genus Poecilia (Meredith 2011).Minor comments:Page 3: the authors should specify that also part of data on male aggressiveness (3 species from Table 1) come from previous studies, as they do for data on deceptive male mating behavior.Page 5: since data on mate choice come from other studies is it so necessary to report a detailed description of methods for this section? Maybe the authors could refer to the already published methods and only give a brief additional description.Page 6: how do the authors explain the complete absence of aggressive displays between the focal male and the audience male during the mate choice experiments? This sounds curious if considering that in all the examined species aggressive behaviors and dominance establishment are always observed during dyadic encounters.",
"responses": [
{
"c_id": "523",
"date": "12 Aug 2013",
"name": "David Bierbach",
"role": "Author Response",
"response": "Thank you very much for your overall positive view! In the revised version of our manuscript we rigorously tried to answer all your questions and clear up all points of critique raised. We analyzed our data with the recommended mixed model approach and added a PCA that depicts the species’ behavioral characteristics. Statistics: We re-analyzed all data using mixed models with “species ID” included as a subject grouping factor and random intercepts for each species. We then established whether there was significant between-species variation through likelihood ratio tests (model with random intercepts for species vs. reduced model). In the new analysis, random slopes for ‘opponent size difference’ were included for each species in our model so that the final analysis appropriately accounts for species-specific reactions towards this covariate. In the new analysis only significant interaction terms and covariates were retained. For the analysis of the changes in mating preferences (linear mixed model) as well as changed first interactions (generalized linear mixed model) we included ‘treatment’ as a random factor as suggested. “male success in mating with the preferred female may reduce the probability of immediate female’s re-mating”: This idea opposes our assumption of general “male mate choice copying” which renders recently mated females more attractive to rivals. If we understand correctly, you suggest some kind of “mate guarding” that would delay re-mating. This is however not a feature of any Poeciliidae mating system known so far. In this context, we would like to refer to our paragraph in the discussion dealing with patterns of sperm precedence in Poeciliids. Up to now, last male sperm precedence is at least verified in one of the species investigated here (for guppies) but it was not the focus of investigations into the other species. Thus, as audience-induced changes in preferences are found in all but one species (namely H. milleri), we assume the occurrence of last male sperm precedence is one cause that renders the “risk of losing a high quality and inseminated female” beneficial. Phylogenetics: Phylogenetic analysis may be useful, and we re-ran our analysis while excluding the population of feral guppies that were most closely related to the Venezuelan guppies. However, the results remained unchanged. Furthermore, the new PCA that includes all behaviors investigated in the current study does not show any phylogenetic grouping. Minor comments: The reviewer is right, the protocol for the mate choice tests as well as the aggression tests are already published but we would like to keep it in the current manuscript for reasons of clarity (also taking advantage of the less restrictive word limits of an online-only journal). In our mate choice tests, focal and audience males were separated as the audience males were fixed in a Plexiglas cylinder. Thus, direct aggression was not observable. Furthermore, a recent study showed that Atlantic molly females did not prefer males after they had won a fight which could have resulted in focal males showing low aggressiveness in front of the two female stimulus fish (Bierbach et al. 2013)"
}
]
},
{
"id": "1068",
"date": "16 Jul 2013",
"name": "Clelia Gasparini",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a study in which audience effects on male mating behaviour was analysed in several species of poeciliids (a family of livebearing freshwater fish) and related to mean sexual activity (used as a proxy for sperm competition risk) and aggressiveness. This is an attempt to study if sperm competition risk (SCR) can explain the occurrence of audience effects on male choice in this family. The rationale behind this is that males should adjust their mating behaviour by modulating, or even reversing, their initial mate choice in the presence of a rival. The change in male mate choice in the presence of another male has been mainly interpreted as a deceptive signal to lead competitors away from the preferred females, therefore lowering sperm competition risk. Given the complexity of factors (abiotic or biotic) that can contribute simultaneously to shape male mating decisions, explanations other than SCR (though not necessarily mutually exclusive) are also possible, although SCR is certainly likely to be important. Indeed, sperm competition is pervasive in poeciliids, and it is therefore likely that sperm competition is a major force in shaping the evolution of male mating strategies in this family. The hypothesis tested in this paper is that a higher sperm competition risk (SCR) should positively correlate with stronger audience effects across different species. Aggressiveness was also considered, as males could adjust their mate choice to avoid aggressive rivals. This is a well written paper, addressing an interesting topic in evolutionary biology. Unfortunately, as the study is only correlative and phylogeny was not accounted for, results can only suggest a general trend, but this can certainly set the stage for future work in this area.No data was collected or analysed to directly quantify SCR in the different species, but total sexual activity (measured in the initial test) was used as a proxy.Aggressiveness tests: The authors performed aggressiveness tests, controlling for a number of factors that can possibly confound interpretation of results, for example, choosing males from different tanks to prevent previously established dominance. However, would aggressiveness scores differ when males are tested in the presence of a female during these encounters? Indeed, two males may have a lot more reasons to exhibit aggressive behaviour when a potential partner is present.Male mate choice tests: In these tests the focal male and two females were free to interact. Methods are described in detail, but I wonder if this is the exact protocol used in all experiments. I am guessing that the method used is probably similar across experiments, but it seems unlikely to me that it is exactly as described here for all of them. Authors also exclude courtship from the sexual activity variable because this behaviour is not present in all species. However, courtship is an important component of sexual behaviour in some of the species considered and including this aspect of male behaviour may therefore change results. Main conclusions: The main finding that lead the authors to support the hypothesis “SCR is a driving force behind the evolution of this behaviour” is the positive correlation (depicted in fig 3e) between the intensity of sexual behaviour (proxy for SCR) recorded in the first test and the level (occurrence) of deceptive behaviour (the fraction of males that reverse their first choice, based on the first interaction with female, page 7). I would like to know how well the first sexual interaction reflects a male’s sexual choice in these species; is there any direct evidence? In guppies, for example, researchers have tested whether the time spent in front of a female during a binary dichotomous test is a good predictor of actual mating preference (Jeswiet & Godin 2011). Are there any studies that show that first sexual interaction is a reliable sign of male sexual interest in most of the species considered here?",
"responses": [
{
"c_id": "524",
"date": "12 Aug 2013",
"name": "David Bierbach",
"role": "Author Response",
"response": "Thank you very much for the positive view of our paper! The reviewer is right, we did not account for phylogeny in our current paper and the main focus of our study was to provide a general comparison of Poeciliid male reproductive behavior given the strong SCR assumed in this family. Aggressiveness tests: Your assumptions might be right; in Siamese fighting fish effects of by-standing females on male aggressive behavior has been found (see work by McGregor). However, a recent study showed that Atlantic molly females did not prefer males after they had won a fight (Bierbach et al. 2013) which could lead to reduced aggressive behavior between males when being observed by a female in Poeciliids. Male mate choice tests: The described experimental setup was exactly the same in all studies from which we extracted the mate choice data. The reviewer is right, courtship is an important aspect of some of the investigated species’ sexual behavior. Nevertheless, in order to draw general conclusions across a wide range of Poeciliid species that differ in several behavioral and ecological aspects, we focused on sexual behaviors that are directly linked to copulations (thus sperm transfer). Surely, courtship is an aspect that should be investigated in future studies.Main conclusions: Thank you for this comment! To show that first sexual interactions and mating preferences are congruent, we added another paragraph to the methods section where we explain that in 76%-100% of the trials the first approached females were also subject to the majority of males’ sexual behaviors makes the first sexual approach a good proxy for male preference in all species examined."
}
]
}
] | 1
|
https://f1000research.com/articles/2-75
|
https://f1000research.com/articles/2-189/v1
|
16 Sep 13
|
{
"type": "Case Report",
"title": "Case Report: Delayed intra-abdominal bleeding following trans-vaginal ultrasonography guided oocyte retrieval for in vitro fertilization in patients at risk for thrombo-embolic events under anticoagulant therapy",
"authors": [
"Roy Mashiach",
"David Stockheim",
"Mati Zolti",
"Raoul Orvieto",
"Roy Mashiach",
"David Stockheim",
"Mati Zolti"
],
"abstract": "We report herein, two cases of massive delayed (2 and 4 days) intra abdominal hemorrhage following ovum pick-up (OPU), in patients at risk for thrombo-embolic events, who concomitantly used therapeutic doses of low molecular weight heparin (LMWH). We discuss the possible mechanisms involved in causing the aforementioned delayed bleeding, and call for re-evaluation of the presently accepted anticoagulant co-treatment regimen. These case reports should direct physicians' attention and keep them alert, while conducting IVF treatment to this subgroup of high risk patients.",
"keywords": [
"IVF",
"ovum pick-up",
"bleeding",
"anticoagulant"
],
"content": "Introduction\n\nNowadays, ultrasonographically guided trans-vaginal ovum pick-up (OPU) is considered an accepted and safe method for oocyte retrieval worldwide. Nevertheless, the multiple punctures of the vaginal vault can injure or tear the vaginal mucosa, ovaries, intra-abdominal organs or blood vessels, resulting in mild to severe internal and external bleeding and other complications1,2. Clinically significant blood loss after OPU is actually uncommon3, with a reported incidence of severe intra- or retroperitoneal bleeding varying from 0 to 1.3%;2.\n\nIntraperitoneal bleeding tends to be severe with acute hemodynamic deterioration, whereas retroperitoneal bleeding usually has a later and more indolent presentation. Intra-abdominal bleeding should be suspected immediately after OPU with the development of signs and symptoms of anemia. While a drop in hemoglobin concentration is an indication for prompt blood transfusion, hemodynamic deterioration dictates surgical intervention with subsequent hemostatis. Most of cases are diagnosed several hours following OPU, with a reported interval between OPU and surgical intervention, ranging from 3 to 18 hours4,5.\n\nIn patients with an underlying coagulopathy, the interval between OPU and the appearance of clinical signs and symptoms of severe intra-abdominal bleeding or surgical intervention, was reported to be longer. While, Battaglia et al.6 reported a 3 hour interval between OPU and surgical intervention in a patient with coagulation factor XI deficiency6, intervals of 2 and 10 days, were reported in patients suffering from essential thrombocythemia7 and factor VIII deficiency8, respectively. Notably, none of the aforementioned patients were treated with anticoagulant medications.\n\nThe situation is even more complicated for women with known thrombophilia and/or history of a thrombo-embolic event, who use anticoagulant drugs. The hitherto published research regarding this issue is scarce, with only one study reporting on 24 patients treated concomitantly with anticoagulation therapy, who underwent 68 oocyte retrieval procedures9. Since IVF treatment was not associated with any medical complication, such as bleeding or thromboembolic complications, Yinon et al. have concluded that among patients considered as high risk for a thromboembolic event, the introduction of low molecular weight heparin (LMWH), as a cycle protective treatment, is safe9.\n\nHere, we report two cases of massive delayed intra-abdominalhemorrhage following OPU, in patients at risk for thromboembolic events, who concomitantly used LMWH. These cases aim to direct physicians' attention and keep them alert, while treating this subgroup of at risk patients.\n\n\nCase report\n\nTwo patients were referred to our IVF clinic for egg/embryos collection for surrogacy because of the risk associated with pregnancy. Both were under therapeutic doses of LMWH, which according to a senior haematologist consultant, were co-administered during the controlled ovarian hyperstimulation (COH) for IVF, until 16 h before the oocyte retrieval and resumed 12 h after the procedure. This study was approved by the institutional review board of Sheba Medical Centre.\n\nA 37-year-old patient with essential thrombocytosis, was diagnosed incidentally following routine examination 7 years ago. One year ago, she developed sinus vein thrombosis, necessitating therapeutic doses of anticoagulant therapy. Controlled ovarian hyperstimulation included the multidose GnRH antagonist protocol, with follicle stimulating hormone (FSH) daily dose of 112.5 to 150 IU. She achieved a peak E2 level of 1538 pmol/l with one leading follicle at each ovary. OPU was reported as uneventful with a retrieval of 3 oocytes. A routine complete blood count (CBC) 2 h following OPU revealed a stable hemoglobin (Hb) concentration, as compared to her baseline level (9.92 g/dL vs 10.52 g/dL, respectively).\n\nTwo days following OPU she was re-admitted to our ward, because of severe abdominal and shoulder pain, abdominal bloating and tenesmus. On physical examination her abdomen was swollen and tender, with floating blood as evidenced by transabdominal ultrasonography. She was hospitalized and closely observed hemodynamically. During the next 4 h, her Hb concentration dropped to 7.2 g/dL with clinical deterioration, mandating resuscitation, and urgent exploration.\n\nDuring exploratory laparoscopy, a massive hemoperitoneum was found (approximately 2.5 L of blood), and a profuse bleeding was observed from a large vein in the posterior aspect of the left infundibulo-pelvic ligament. The vessel was successfully coagulated. The patient was transfused with 4 units of blood, 3 unit of cryoprecipitate, and 2 units of fresh frozen plasma. Her postoperative course was uneventful and she was discharged 4 days later with a hemoglobin level of 9.4 g/dL.\n\nA 32-year-old patient, suffering from Budd–Chiari syndrome as a consequence of a myeloprolifertive disorder, necessitating therapeutic doses of anticoagulant therapy. Controlled ovarian hyperstimulation included the long GnRH agonist protocol, with HMG daily dose of 375 to 450 IU. She achieved a peak E2 level of 2349 pmol/l with four leading follicles at both ovaries. OPU was reported as technically uneventful, however no oocytes were retrieved.\n\nThree days following OPU, the patient started to feel unwell and complained of lower abdominal pain and dyspnea with stable Hb concentration (10.43–10.95 g/dL). A day later, she gradually became pale, tachycardiac, with a drop in Hb level (8.84 g/dL) that continued (8.66 g/dL) despite a blood transfusion, mandating urgent exploratory laparoscopy. During laparoscopy, a massive hemoperitoneum was observed (approximately 2.5 L of blood), with an active bleeding from a tear of the right ovarian capsule. The tear was successfully coagulated with an accurate hemostasis. The patient was transfused with 5 units of blood, and 2 units of fresh frozen plasma. Her postoperative course was uneventful and she was discharged a week later with a hemoglobin level of 11.68 g/dL.\n\n\nDiscussion\n\nWe report herein, to our knowledge, the first 2 cases of massive delayed intra-abdominal hemorrhage following OPU, in patients at risk for thromboembolic event, who concomitantly used therapeutic doses of LMWH. We are not aware of any guidelines that have been published by any professional society, regarding OPU under LMWH. Moreover, since most guidelines relate to patients undergoing surgeries that may apply strict hemostatic measures, which are not available during US guided OPU, our report challenges and calls for re-evaluation of the anticoagulation regimen, provided to this subgroup of at risk patients, during an IVF treatment cycle.\n\nFollowing OPU, the cessation of bleeding requires the interaction of the injured blood vessel with the circulating platelets and clotting proteins in order to form a stable platelet-fibrin clot. Any abnormalities of these factors may result in clinically significant bleeding. In the present cases, both patients received therapeutic doses of LMWH (enoxaparin, 60mg bid), which were discontinued 16 h prior to OPU and resumed 12 h after the procedure. Moreover, intra abdominal bleeding occurred 2 and 4 days following OPU.\n\nSeveral plausible mechanisms might be responsible for the aforementioned delayed bleeding: (a) early postoperative use of enoxaparin and unfractionated heparin was reported to be associated with a significant increase in re-exploration for postoperative bleeding, often at a significantly delayed time period after the initial surgery10. Moreover, this delay was especially common with enoxaparin; (b) while the effect of LMWH and unfractionated heparin on the coagulation cascade is well established, we may wonder whether these substances also affect the fibrin nanostructure, which plays a major role in the mechanical strength and lysis of clots. Yeromonahos et al.11 provided quantitative evidence showing that both LMWH and unfractionated heparin, independent of their other actions on the coagulation cascade, directly alter the nanostructure of fibrin fibers, contributing to improved fibrinolysis.\n\nWe therefore believe that the presently accepted anticoagulant co-treatment regimen, including the dose and timing of administration, should be challenged and undergo a critical re-evaluation. For example, delaying and reducing the first dose of LMWH post OPU, with gradual increments might offer some advantages. However, whether this modification will increase the tendency toward thrombosis, should be further investigated. Moreover, since intra abdominal bleeding obviously occurs, and may occur several days following OPU, we recommend that the patient be kept in the ward for observation for at least 2–4 days following OPU.\n\nIn the presented cases we also observed a tear of the ovarian capsule and an inadvertent puncture of a blood vessel within the infudibullo-pelvic ligament. Both complications might have been prevented by meticulous follicular aspiration using a small diameter aspiration needle (19G), and preferably under the guide of color Doppler ultrasonography12, respectively.\n\nIn conclusion, massive delayed intra-abdominal hemorrhage may occur following OPU, in patients at risk for thromboembolic event, who concomitantly used LMWH. These case-reports should direct physicians' attention and keep them alert, while conducting an IVF treatment to this subgroup of high risk patients.",
"appendix": "Author contributions\n\n\n\nRoy Mashiach: coordinated and participated in the patients' treatment, contributed to the discussion, assisted in writing the paper and edited it in all its revisions.\n\nDavid Stockheim: participated in the patients' treatment, contributed to the discussion, assisted in writing the paper and edited it in all its revisions.\n\nMati Zolti: participated in the patients' treatment, contributed to the discussion, assisted in writing the paper and edited it in all its revisions.\n\nRaoul Orvieto: the principal investigator, designed the study, participated in the patients' treatment, contributed to the discussion, assisted in writing the paper and edited it in all its revisions.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nBennett SJ, Waterstone JJ, Cheng WC, et al.: Complications of transvaginal ultrasound-directed follicle aspiration: a review of 2670 consecutive procedures. J Assist Reprod Genet. 1993; 10(1): 72–7. PubMed Abstract | Publisher Full Text\n\nOrvieto R, Ben Rafael Z: Bleeding, severe pelvic infection and ectopic pregnancy following ART. In: Textbook of Assisted Reproductive Techniques: Volume 2: Clinical Perspectives. 4th Edition. D.K. Gardner, A. Weissman, C. Howles, Z. Shoam, Eds. Informa Healthcare, London, UK Chapt. 2012; 62: 374–81. Reference Source\n\nYih MC, Goldschlag D, Davis OK, et al.: Complete blood counts (CBC) after oocyte retrieval: what is normal? Fertil Steril. 2001; 76(2): S115–6. Publisher Full Text\n\nDicker D, Ashkenazi J, Feldberg D, et al.: Severe abdominal complications after transvaginal ultrasonographically guided retrieval of oocytes for in vitro fertilization and embryo transfer. Fertil Steril. 1993; 59(6): 1313–15. PubMed Abstract\n\nLiberty G, Hyman JH, Eldar-Geva T, et al.: Ovarian hemorrhage after transvaginal ultrasonographically guided oocyte aspiration: a potentially catastrophic and not so rare complication among lean patients with polycystic ovary syndrome. Fertil Steril. 2010; 93(3): 874–9. PubMed Abstract | Publisher Full Text\n\nBattaglia C, Regnani G, Giulini S, et al.: Severe intraabdominal bleeding after transvaginal oocyte retrieval for IVF ET and Coagulation factor XI deficiency: a case report. J Assist Reprod Genet. 2001; 18(3): 178–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEl-Shawarby SA, Margara RA, Trew GH, et al.: Thrombocythemia and hemoperitoneum after transvaginal oocyte retrieval for in vitro fertilization. Fertil Steril. 2004; 82(3): 735–7. PubMed Abstract | Publisher Full Text\n\nKart C, Guven S, Aran T, et al.: Life threatening intraabdominal bleeding after oocyte retrieval successfully managed with angiographicembolization. Fertil Steril. 2011; 96(2): e99–e102. PubMed Abstract | Publisher Full Text\n\nYinon Y, Pauzner R, Dulitzky M, et al.: Safety of IVF under anticoagulant therapy in patients at risk for thrombo-embolic events. Reprod Biomed Online. 2006; 12(3): 354–358. PubMed Abstract | Publisher Full Text\n\nJones HU, Muhlestein JB, Jones KW, et al.: Early postoperative use of unfractionated heparin or enoxaparin is associated with increased surgical re-exploration for bleeding. Ann Thorac Surg. 2005; 80(2): 518–22. PubMed Abstract | Publisher Full Text\n\nYeromonahos C, Marlu R, Polack B, et al.: Antithrombin independent effects of heparins on fibrin clot nanostructure. Arterioscler Thromb Vasc Biol. 2012; 32(5): 1320–4. PubMed Abstract | Publisher Full Text\n\nShalev J, Orvieto R, Meizner I: Use of color Doppler sonography during follicular aspiration in patients undergoing in vitro fertilization may reduce the risk of blood vessel injury. Fertil Steril. 2004; 81(5): 1408–10. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1843",
"date": "02 Oct 2013",
"name": "Fatma Ferda Verit",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this article can be accepted without any revisions. The paper quality is good and it adds new knowledge to the literature.",
"responses": []
},
{
"id": "1844",
"date": "09 Oct 2013",
"name": "Pedro Barri",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 observational case-report on two cases of delayed intra-abdominal bleeding following OPU for IVF in patients under anticoagulant therapy.This article is acceptable, but some changes should be included: There is no information either on the type of needles used for aspiration, or on the aspiration pressure. Why does a young patient (32 yrs) have a negative OPU? How many needle punctures have been performed? There is no information on the outcomes of the oocytes retrieved in case 1. The discussion section is well structured, but authors should present a final recommendation on daily doses and the duration of LMWH.",
"responses": [
{
"c_id": "573",
"date": "10 Oct 2013",
"name": "Raoul Orvieto",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We would like to thank Prof. Barri for his valuable comments.1. The follicles were aspirated by experienced physicians using a fine (19 G) needle (William A. Cook Australia PTY LTD, Australia) and 140 mmHg suction pressure.2. The negative OPU was unexplained (empty follicle?).3. Punctures were started in the ovary with the follicles closest to the vagina, and each follicle was sequentially punctured without reinserting the needle through the vaginal wall.4. In case 1, OPU was reported as uneventful with a retrieval of 3 oocytes, of which 2 were fertilized and cryopreserved.5. As we already mentioned and further clarify: \"We therefore believe that the presently accepted anticoagulant co-treatment regimen, including the dose and timing of administration, should be challenged and undergo a critical re-evaluation. For example, delaying [24 h after the procedure] and reducing the first dose [half] of LMWH post OPU, with gradual increments [after 2 days of half a daily dose] might offer some advantages. However, whether this modification will increase the tendency toward thrombosis, should be further investigated [by multidisciplinary experts]. Moreover, since intra abdominal bleeding obviously occurs, and may occur several days following OPU, we recommend that the patient be kept in the ward for observation for at least 2–4 days following OPU.\""
}
]
},
{
"id": "1846",
"date": "30 Oct 2013",
"name": "William Ledger",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 built around 2 case reports of clinically significant intra-abdominal bleeding in the days following egg collection for the purposes of IVF, in patients receiving therapeutic doses of Heparin. The authors use the case reports as a focus for discussion of the management of patients prescribed this medication prior to IVF.The reports are provisionally novel to warrant publication. The use of English is somewhat idiosyncratic and requires minor review. The text preceding the first case report includes the suggestion that both eggs and embryos may be collected for surrogacy. Only eggs can be collected, and this should be amended.The most important part of the article is in the discussion. The authors suggest a review of anticoagulant co-treatment in the light of their cases. I do not think that their experience would be sufficient risk of thrombotic complications. This problem is mentioned but should be further highlighted by the authors. I would also suggest they further emphasize the use of a smaller gage aspiration needle for egg collection in these higher risk cases.",
"responses": [
{
"c_id": "601",
"date": "03 Nov 2013",
"name": "Raoul Orvieto",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We would like to thank Prof. Ledger for his valuable comments.Eggs were collected and fertilized, and the embryos were frozen for future FET cycles to the surrogate. The issue of the thrombotic risk and the suggestion of a smaller gage needle both appear in the 2nd version."
}
]
}
] | 1
|
https://f1000research.com/articles/2-189
|
https://f1000research.com/articles/2-216/v1
|
15 Oct 13
|
{
"type": "Research Article",
"title": "Buffering the pH of the culture medium does not extend yeast replicative lifespan",
"authors": [
"Brian M Wasko",
"Daniel T Carr",
"Herman Tung",
"Ha Doan",
"Nathan Schurman",
"Jillian R Neault",
"Joey Feng",
"Janet Lee",
"Ben Zipkin",
"Jacob Mouser",
"Edward Oudanonh",
"Tina Nguyen",
"Torin Stetina",
"Anna Shemorry",
"Mekedes Lemma",
"Matt Kaeberlein",
"Brian M Wasko",
"Daniel T Carr",
"Herman Tung",
"Ha Doan",
"Nathan Schurman",
"Jillian R Neault",
"Joey Feng",
"Janet Lee",
"Ben Zipkin",
"Jacob Mouser",
"Edward Oudanonh",
"Tina Nguyen",
"Torin Stetina",
"Anna Shemorry",
"Mekedes Lemma"
],
"abstract": "During chronological aging of budding yeast cells, the culture medium can become acidified, and this acidification limits cell survival. As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium. In this study, we assessed whether a similar process occurs during replicative aging of yeast cells. We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the initial pH of the medium is able to significantly extend replicative lifespan. Thus, we conclude that, unlike chronological life span, replicative life span is not limited by acidification of the culture medium or by changes in the pH of the environment.",
"keywords": [
"Aging has been studied extensively in the budding yeast Saccharomyces cerevisiae using two fundamentally different systems: the replicative lifespan assay and the chronological lifespan assay1. Replicative life span is defined as the number of daughter cells that a mother cell can produce prior to entering an irreversible cell cycle arrest",
"while chronological lifespan is defined as the length of time that a yeast cell can maintain viability in a non-dividing state2",
"3. Numerous genetic and environmental factors have been identified that can modulate either replicative aging",
"or chronological aging",
"or both."
],
"content": "Introduction\n\nAging has been studied extensively in the budding yeast Saccharomyces cerevisiae using two fundamentally different systems: the replicative lifespan assay and the chronological lifespan assay1. Replicative life span is defined as the number of daughter cells that a mother cell can produce prior to entering an irreversible cell cycle arrest, while chronological lifespan is defined as the length of time that a yeast cell can maintain viability in a non-dividing state2,3. Numerous genetic and environmental factors have been identified that can modulate either replicative aging, or chronological aging, or both.\n\nReplicative aging has been studied almost exclusively by maintaining individual cells on the surface of a nutrient agar plate, microdissecting daughter cells away from the mother cells, and counting the number of daughter cells that the mother cell produces prior to senescence4. Generally, rich YPD medium (2% glucose) is used for replicative lifespan assays. Calorie restriction by reducing the glucose concentration of the medium to 0.5% or lower has been shown in numerous studies to extend lifespan in different wild type strain backgrounds between 10–40%5,6.\n\nSeveral methods have been described for studying chronological aging. The most widely utilized protocol involves culturing yeast cells in synthetic complete liquid medium with 2% glucose as the carbon source, either under shaking or static conditions, in culture tubes or 96-well plates7,8. Alternative, but less frequently used, liquid culture methods for chronological aging involve culturing cells in rich YPD medium, using a respiratory carbon source such as glycerol, or transferring cells to water once they have reached stationary phase growth arrest9,10. A plate-based assay for chronological life span analysis has also been described in which cells are growth arrested through limitation for tryptophan11. In all of these assays, viability over time is determined by restoring a small subset of the population to nutrient rich growth conditions and assaying their ability to re-enter the cell cycle, either through quantification of colony forming units on solid-agar plates or through outgrowth kinetics in liquid culture3,12. Similar to the case for replicative lifespan, calorie restriction by reducing the initial glucose concentration of the culture medium can extend chronological lifespan, generally by more than 100%13,14.\n\nOne important feature of the standard method for determining chronological aging is that the culture medium becomes acidified over the first few days of the experiment, with pH dropping from an initial value of around 4.0 to 2.5–2.9 within 96 hours15. This acidification of the external environment results from the production of organic acids, including acetic acid, following fermentation of glucose to ethanol and subsequent utilization of ethanol as a carbon source once the glucose is depleted. Preventing medium acidification by buffering the culture to a pH of 6.0 with either citrate phosphate buffer or low salt MES buffer results in a more than doubling of chronological lifespan15. Calorie restriction, or switching the yeast culture to a non-fermentable carbon source, such as glycerol or ethanol, also prevents acidification and results in a similar magnitude of chronological lifespan extension as buffering15,16.\n\nAlthough the two yeast aging assays are nearly always studied independently, it is clear that they share at least some overlap. As mentioned above, calorie restriction extends both replicative and chronological lifespan, as do a few genetic interventions, such as deletion of either TOR1 or SCH9, both of which are nutrient-responsive kinases7,17–19. In addition, it has been shown that chronologically aged cells have reduced replicative lifespan when returned to rich growth conditions9. This reduction in replicative lifespan following chronological aging appears to be mediated through changes in mitochondrial function, since the chronologically old cells that retain the lowest mitochondrial membrane potential also have the longest replicative lifespan following resumption of cell division20. Calorie restriction or buffering the culture medium of the cells during chronological aging also protects against subsequent replicative lifespan reduction21, raising the possibility that medium acidification directly influences both types of yeast aging. To assess this possibility, we performed replicative lifespan analysis on wild type BY4742 mother cells under either standard conditions or on rich media buffered to different pH values. We were unable to detect a significant replicative lifespan extension from buffering the culture medium under any of the conditions examined, including those conditions that robustly extend chronological lifespan.\n\n\nMethods\n\nAll lifespan experiments were performed in the BY4742 strain background (Thermo Scientific, Waltham, MA) as previously described4,22. Virgin daughter cells were isolated and allowed to grow into mother cells while their corresponding daughters were microdissected using Zeiss Axioskop 40 dissection microscopes and manually counted until the mother cell could no longer divide4. YEP agar plates (1% yeast extract, 2% bacto-peptone, 2% agar) containing 2% glucose (YPD) were utilized and strains were grown at 30°C during the day, dissected at room temperature, and placed in a refrigerator at 4°C over night. Daughter cells were removed from each mother cell roughly every 2 hours by micromanipulation4. Cells were scored as senescent when they had failed to divide for at least eight hours of incubation at 30°C. Terminal morphology was defined as the budded state of the mother cells upon senescence23. All experiments were performed by a team of dissectors who were blinded to the identity of the strains under examination in any given experiment. Prism Graphpad 5.0 was used for data analysis. Statistical significance for differences in median lifespan was determined using the Wilcoxon Rank-Sum test. Budded and unbudded states were determined visually for each mother cell assayed and statistical comparisons of budding rates utilized Fischer’s Exact two-tailed test. Multiple comparison corrections were performed using the Bonferroni correction.\n\nStock buffers were prepared at 1 M in deionized water and pH was adjusted by addition of appropriate molar ratios of conjugate acid and conjugate base, or by empirical adjustment with HCl or NaOH. Buffer reagents were obtained from Sigma-Aldrich (St Louis, MO). The following buffers were used: Tris(hydroxymethyl)aminomethane (Tris) (; 3-(N-morpholino)propanesulfonic acid (MOPS); 2-(N-morpholino)ethanesulfonic acid (MES); citrate buffer (sodium citrate and citric acid); acetate buffer (sodium acetate and acetic acid). Stock buffers were sterilized by filtration using a VWR International syringe driven 0.2 micron cellulose acetate membrane filter. Buffers were diluted to 100 mM final concentration in YPD agar after autoclaving and cooling to ~55°C. Appropriate stock buffer pH was empirically determined, as necessary to adjust the pH of YPD liquid media at room temperature to the indicated final pH. Measurement of pH was performed using an Accumet Excel XL15 pH meter. The pH of agar media was further verified by use of EMD colorpHast pH-indicator strips. For adjustment of media pH without buffer addition, the indicated acid or base was added to achieve the desired pH prior to autoclaving.\n\n\nResults\n\nLowering the pH of YPD agar plates to 5.0 by HCl (p = 0.0218) and to 6.0 using MES buffer (p = 0.0165) trended toward a decrease in lifespan that was statistically significant without adjusting for multiple comparisons (α = 0.05), but which did not reach significance after adjusting for multiple comparisons using the Bonferroni correction (α = 0.0056) (Figure 1A, Table 1). Lowering pH to 5.0 by acetic acid (p = 0.8869) or buffering to pH 5.6 using acetate buffer (p = 0.0896) had no detectable effect on lifespan. Further reduction of pH to 3.0 using a citrate buffer (p = 0.3412) also had no effect on lifespan.\n\nReplicative lifespans with pH adjusted to 7–8 by buffering or NaOH (A), or pH 6.0 or below by acids or buffers (B) and to pH 9.0 by NaOH (C).\n\nRLS, median replicative lifespan; U, unbudded; B, budded.\n\nBuffering YPD media to pH 7 by MOPS (p = 0.4161) or Tris (p = 0.3653) had no detectable effect on yeast lifespan (Figure 1B). Increasing the pH of YPD to 8.0 by NaOH (p = 0.7658) or buffering at pH 7.8 with 100 mM Tris (p = 0.1754) also has no detectable effect on lifespan. Raising pH further to 9.0 by sodium hydroxide (p < 0.0001) resulted in a significant reduction of lifespan (Figure 1C), and cells on YPD buffered to pH 9.0 with 100 mM Tris buffer did not divide and, thus, replicative lifespan could not be determined for this condition.\n\nTerminal morphology is defined as the budded state of the mother cells upon senescence23. Terminal morphology frequency was not significantly altered when pH was buffered at 7.0 by MOPS (p = 0.8224) or 7.0 (p = 0.8224) or 7.8 by Tris (p = 1) or by adjustment of pH to 8.0 by sodium hydroxide (p = 0.3711). Manipulations that lowered the pH displayed a trend toward a higher percentage of unbudded cells upon arrest (Table 1), but this did not reach statistical significance after correcting for multiple testing (α = 0.0056).\n\n\nDiscussion\n\nThe results presented here demonstrate that, unlike chronological lifespan, acidification of the culture medium does not limit replicative lifespan under standard conditions. This is relevant information, because it rules out the possibility that interventions shown to extend replicative lifespan are acting by either reducing the production and secretion of organic acids into the environment or by increasing resistance to acid stress.\n\nThe biological relevance of acidification limiting chronological lifespan has been an area of contention within the field, due in part to concerns that cell death due to acidification may be a yeast specific phenomenon1. Evidence supporting this concern has been provided by parallel analyses of replicative and chronological lifespan for yeast deletion mutants corresponding to Caenorhabditis elegans genes that increase lifespan when their expression is reduced. A significant enrichment for long replicative lifespan was found among this set of yeast deletions24, but no enrichment for increased chronological lifespan under standard conditions was observed25. On the other hand, there is evidence that a similar acid-induced mechanism of senescence occurs in mammalian cells, at least in culture, suggesting the possibility that the intracellular response to external pH may be conserved26,27.\n\nThe trend toward reduced lifespan noted under some of the conditions tested is of interest and may warrant further study. The significant reduction in lifespan associated with adjusting to pH 9.0 by NaOH may reflect a reduced ability of yeast to proliferate under basic conditions, which is consistent with the inability of yeast cells to grow in the replicative lifespan assay when the YPD was buffered to pH 9.0 by Tris buffer. Among the acidic conditions tested, any effects on lifespan are likely to be due to the composition of the buffer rather than a direct result of the lower pH. As evidence for this, we note that YPD buffered to pH 3.0, the most acidic condition tested, had no effect on lifespan.\n\nIn summary, we find no evidence that acidification of the culture medium, or pH changes in general, limit replicative lifespan in the BY4742 laboratory yeast strain under standard conditions. Buffer conditions that dramatically extend chronological lifespan of this strain do not similarly extend replicative lifespan. These data demonstrate that effects of acidification on aging in yeast are likely to be restricted to non-dividing cells.",
"appendix": "Author contributions\n\n\n\nMK and BMW conceived the study and designed the experiments. BMW, DC, HT, HD, NCS, JRN, JF, JL, BZ, JM, EO, TN, TS, AS and MT carried out the experiments. BMW performed the analyses of results. BMW and MK wrote the manuscript. All authors have critically reviewed and agreed to the 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 NIH Grant R01AG039390 to MK. BMW was supported by NIH Training Grant T32ES007032.\n\n\nReferences\n\nLongo VD, Shadel GS, Kaeberlein M, et al.: Replicative and chronological aging in Saccharomyces cerevisiae. Cell Metab. 2012; 16(1): 18–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteinkraus KA, Kaeberlein M, Kennedy BK: Replicative aging in yeast: the means to the end. Annu Rev Cell Dev Biol. 2008; 24(1): 29–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFabrizio P, Longo VD: The chronological life span of Saccharomyces cerevisiae. Aging Cell. 2003; 2(2): 73–81. PubMed Abstract | Publisher Full Text\n\nSteffen KK, Kennedy BK, Kaeberlein M: Measuring replicative life span in the budding yeast. J Vis Exp. 2009; (28). PubMed Abstract | Publisher Full Text | Free Full Text\n\nLin SJ, Defossez PA, Guarente L: Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science. 2000; 289(5487): 2126–2128. PubMed Abstract | Publisher Full Text\n\nSchleit J, Johnson SC, Bennett CF, et al.: Molecular mechanisms underlying genotype-dependent responses to dietary restriction. Aging Cell. 2013. PubMed Abstract | Publisher Full Text\n\nPowers RW III, Kaeberlein M, Caldwell SD, et al.: Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 2006; 20(2): 174–184. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLongo VD, Gralla EB, Valentine JS: Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. Mitochondrial production of toxic oxygen species in vivo. J Biol Chem. 1996; 271(21): 12275–12280. PubMed Abstract | Publisher Full Text\n\nAshrafi K, Sinclair D, Gordon JI, et al.: Passage through stationary phase advances replicative aging in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 1999; 96(16): 9100–9105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacLean M, Harris N, Piper PW: Chronological lifespan of stationary phase yeast cells; a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms. Yeast. 2001; 18(6): 499–509. PubMed Abstract | Publisher Full Text\n\nWei M, Fabrizio P, Madia F, et al.: Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension. PLoS Genet. 2009; 5(5): e1000467. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurakami C, Kaeberlein M: Quantifying yeast chronological life span by outgrowth of aged cells. J Vis Exp. 2009; (27). PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith DL Jr, McClure JM, Matecic M, et al.: Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins. Aging Cell. 2007; 6(5): 649–662. PubMed Abstract | Publisher Full Text\n\nMurakami CJ, Burtner CR, Kennedy BK, et al.: A method for high-throughput quantitative analysis of yeast chronological life span. J Gerontol A Biol Sci Med Sci. 2008; 63(2): 113–121. PubMed Abstract | Publisher Full Text\n\nBurtner CR, Murakami CJ, Kennedy BK, et al.: A molecular mechanism of chronological aging in yeast. Cell Cycle. 2009; 8(8): 1256–1270. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurakami CJ, Wall V, Basisty N, et al.: Composition and acidification of the culture medium influences chronological aging similarly in vineyard and laboratory yeast. PLoS ONE. 2011; 6(9): e24530. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaeberlein M, Powers RW III, Steffen KK, et al.: Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science. 2005; 310(5751): 1193–1196. PubMed Abstract | Publisher Full Text\n\nFabrizio P, Pozza F, Pletcher SD, et al.: Regulation of longevity and stress resistance by Sch9 in yeast. Science. 2001; 292(5515): 288–290. PubMed Abstract | Publisher Full Text\n\nFabrizio P, Pletcher SD, Minois N, et al.: Chronological aging-independent replicative life span regulation by Msn2/Msn4 and Sod2 in Saccharomyces cerevisiae. FEBS Lett. 2004; 557(1–3): 136–142. PubMed Abstract | Publisher Full Text\n\nDelaney JR, Murakami C, Chou A, et al.: Dietary restriction and mitochondrial function link replicative and chronological aging in Saccharomyces cerevisiae. Exp Gerontol. 2013; 48(10): 1006–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurakami C, Delaney JR, Chou A, et al.: pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast. Cell Cycle. 2012; 11(16): 3087–3096. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaeberlein M, Kirkland KT, Fields S, et al.: Sir2-independent life span extension by calorie restriction in yeast. PLoS Biol. 2004; 2(9): E296. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDelaney JR, Chou A, Olsen B, et al.: End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging. FEMS Yeast Res. 2013; 13(3): 267–276. PubMed Abstract | Publisher Full Text\n\nSmith ED, Kennedy BK, Kaeberlein M: Genome-wide identification of conserved longevity genes in yeast and worms. Mech Ageing Dev. 2007; 128(1): 106–111. PubMed Abstract | Publisher Full Text\n\nBurtner CR, Murakami CJ, Olsen B, et al.: A genomic analysis of chronological longevity factors in budding yeast. Cell Cycle. 2011; 10(9): 1385–1396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeontieva OV, Blagosklonny MV: Yeast-like chronological senescence in mammalian cells: phenomenon, mechanism and pharmacological suppression. Aging (Albany, NY Online). 2011; 3(11): 1078–1091. PubMed Abstract | Free Full Text\n\nKaeberlein M, Kennedy BK: A new chronological survival assay in mammalian cell culture. Cell Cycle. 2012; 11(2): 201–202. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2096",
"date": "22 Oct 2013",
"name": "Karim Mekhail",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nYeast cells have been successfully used for decades to identify conserved genetic and environmental factors that control cellular lifespan, which often underlies organismal lifespan. In yeast, the lifespan of dividing and non-dividing cells can be assessed via measurement of replicative and chronological lifespan, respectively. It was previously shown that chronological lifespan can be optimized through appropriate buffering of the cell culture media. In this brief F1000Research Research Article, Wasko et al., investigate whether replicative lifespan is also affected by environmental pH levels. The authors conduct a series of technically sound and straightforward experiments to address this question. Micromanipulation of BY4742 cells cultured under various pH conditions did not reveal any significant relationship between extracellular pH levels and replicative lifespan. The authors were quite careful in their interpretation of the findings and appropriately acknowledged limitations. For example, possible effects related to the different types of buffering agents used were noted. In addition, clear statements highlighting that the results of this study are obtained via reliance on a single yeast strain (BY4742) were included throughout the results and discussion sections. Moreover, statistical analyses were generally appropriate as presented. Overall, as suggested by the authors, the presented data suggest that the effects of acidification on aging in yeast are likely to be restricted to non-dividing cells. I only make the minor suggestion to consider adding the word “BY4742” before the word “yeast” in the title.",
"responses": []
},
{
"id": "2625",
"date": "03 Dec 2013",
"name": "Cory Dunn",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 work, Wasko et al. investigate whether altering the pH of rich medium can result in changes to the replicative lifespan of Saccharomyces cerevisiae strain BY4742. They find that replicative lifespan, except in the most extreme case of pH 9.0, is not affected by extracellular pH. Their results indicate that mutations and conditions increasing replicative lifespan are unlikely to do so through altering acidity/alkalinity of rich medium.Recently, it was demonstrated by Hughes et al. Nature 492:261-265 that deletion of the vacuolar V1VO-ATPase subunit Vma2p, which results in an increased vacuolar pH, causes a drastic shortening of replicative lifespan. Also, Hughes et al. found, by a qualitative microscopy assay, that over expression of VMA1 or VPH2 decreased vacuolar pH and that replicative lifespan was coincidentally increased.Extracellular pH has been demonstrated to impinge on vacuolar pH, more prominently in the case of increased medium pH. This has been examined most comprehensively by Brett et al. PLoS ONE 6(3):e17619, but has also been demonstrated by others using genetic, fluorophore-based, and even 31P-NMR-based assays. If extracellular pH impinges upon vacuolar pH, and if replicative lifespan depends upon vacuolar pH, then why is replicative lifespan not changed upon altering extracellular pH? A discussion of this point is definitely warranted in a revised version of this manuscript.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-216
|
https://f1000research.com/articles/2-215/v1
|
14 Oct 13
|
{
"type": "Research Article",
"title": "Prism adaptation does not alter configural processing of faces",
"authors": [
"Janet H. Bultitude",
"Paul E. Downing",
"Robert D. Rafal",
"Paul E. Downing",
"Robert D. Rafal"
],
"abstract": "Patients with hemispatial neglect (‘neglect’) following a brain lesion show difficulty responding or orienting to objects and events on the left side of space. Substantial evidence supports the use of a sensorimotor training technique called prism adaptation as a treatment for neglect. Reaching for visual targets viewed through prismatic lenses that induce a rightward shift in the visual image results in a leftward recalibration of reaching movements that is accompanied by a reduction of symptoms in patients with neglect. The understanding of prism adaptation has also been advanced through studies of healthy participants, in whom adaptation to leftward prismatic shifts results in temporary neglect-like performance. Interestingly, prism adaptation can also alter aspects of non-lateralised spatial attention. We previously demonstrated that prism adaptation alters the extent to which neglect patients and healthy participants process local features versus global configurations of visual stimuli. Since deficits in non-lateralised spatial attention are thought to contribute to the severity of neglect symptoms, it is possible that the effect of prism adaptation on these deficits contributes to its efficacy. This study examines the pervasiveness of the effects of prism adaptation on perception by examining the effect of prism adaptation on configural face processing using a composite face task. The composite face task is a persuasive demonstration of the automatic global-level processing of faces: the top and bottom halves of two familiar faces form a seemingly new, unknown face when viewed together. Participants identified the top or bottom halves of composite faces before and after prism adaptation. Sensorimotor adaptation was confirmed by significant pointing aftereffect, however there was no significant change in the extent to which the irrelevant face half interfered with processing. The results support the proposal that the therapeutic effects of prism adaptation are limited to dorsal stream processing.",
"keywords": [
"Prism adaptation",
"sensory-motor coordination",
"face perception",
"global/local processing"
],
"content": "Introduction\n\nPatients with hemispatial neglect (‘neglect’) following a brain lesion show difficulty responding or orienting to objects and events that appear on the left side of space1. A diagnosis of neglect is a strong predictor of poor functional outcome and low independence following stroke2. This may be partly because the disorder impairs perception in a broad range of sensory modalities ranging from vision, touch, proprioception, and motor control to more abstract aspects of cognition such as a patient’s awareness of their own body3 and their imagined images of familiar locations4. Furthermore, although the rightward spatial bias is the defining symptom of neglect, several other processing disturbances are associated with the disorder. These include low general arousal5, poor sustained attention6, and difficulties in keeping track of spatial locations as they move about their environment7. These non-lateralised spatial biases are thought to increase neglect severity and reduce the potential for recovery8.\n\nOver the last fifteen years a promising behavioural intervention for neglect has emerged in the form of a sensorimotor training technique called prism adaptation9. During prism adaptation, patients reach for objects viewed through rightward-deflecting prisms, leading to a leftward recalibration of reaching movements that can be measured as leftward errors once the prisms are removed. In patients with neglect this leftward recalibration of reaching is accompanied by a reduction in their symptoms. A single five-minute session of prism adaptation is sufficient to improve the performance of neglect patients on tests of visuo-motor function such as copying, cancellation and reading9,10. These effects extend to non-visual spatial processing, such as tactile perception11 and manual exploration of space while blindfolded12, and to complex mental operations such as the exploration of an internally generated map of France13; and 'bisection' of numbers14. Evidence amassed over a number of studies suggests that this simple behavioural intervention can have broadly generalised effects, and prism adaptation is considered to be a highly promising potential treatment for neglect15.\n\nWhereas adaptation to rightward-shifting prisms can reduce neglect symptoms in brain-lesioned patients, adaptation to leftward-shifting prisms, involving a rightward recalibration of reaching, leads to neglect-like changes in the spatial performance of healthy participants. These perceptual changes have been demonstrated on a similar range of visual, non-visual and mental tasks (albeit to a lesser extent than those changes observed in patients)17–19. Since prism adaptation can be used to induce similar, but opposite, changes in the performance of healthy participants as in neglect patients, it is possible to gain insights into the potential therapeutic effects of the technique by testing healthy volunteers.\n\nOne example of research from healthy participants that has complemented the understanding gained from studies in patients is in research examining the effects of prism adaptation on non-lateralised deficits. There are now several pieces of evidence from brain-lesioned patients that prism adaptation alters spatial processing deficits that cannot be described in terms of orienting to the left versus the right, including reductions in spatial dysgraphia20 and shifts21 and reductions in perseveration22. We previously demonstrated that adaptation to rightward-shifting prisms reverses the tendency of patients with right hemisphere lesions to become fixated on local details of a scene in preference to the global configuration (the ‘local processing bias’)23. Patients identified the local or global level of large letters that were built from smaller letters (‘Navon’ figures). Reaction times to the local level increased after prism adaptation, demonstrating that there was a reduction in patients’ ability to identify the local level without interference from conflicting information at the global level. Conversely, RTs to the global level decreased following prism adaptation, demonstrating that patients were better able to ignore irrelevant conflicting information from the local level. In a similar experiment with healthy participants we demonstrated that adaptation to leftward-shifting prisms temporarily increased local processing24, and led to neglect-like errors in the way in which a spatial representation or ‘map’ of the environment is updated as we move our gaze around it25. Together these results demonstrate that prism adaptation has a more pervasive influence on visual perception than merely shifting attention to one side.\n\nTo further test the extent of this influence, the present study examines the effect of prism adaptation on the perception of composite faces in healthy participants. Faces, perhaps more than any other object, undergo automatic global-level processing in which individual components are highly integrated and less available to independent evaluation. This is powerfully illustrated in the composite face illusion (Figure 1): when the upper and lower halves of two faces are recombined, the virtually unavoidable illusion is that one is viewing the face of a third, different person. When participants are asked to identify the top or bottom halves of composites that are formed from faces of well-known celebrities, they are slower compared to when performing the same task when the two face halves are offset26. This reaction time cost demonstrates that even when processing a face as an integrated Gestalt would impair our ability to perform the task at hand, we are unable to suppress such configural processing.\n\nStimuli for the task used in the present study took the same form as these examples, but were created from black-and-white publicity photographs of two well-known movie stars (Brad Pitt and George Clooney).\n\nWe had two main reasons for testing the influence of prism adaptation on configural face perception. First, by using a stimulus type for which normal processing is known to be strongly biased towards global processing, we reasoned that we could gain insight into the pervasiveness of the influence of prism adaptation on perceptual processes. Second, this experiment explores the possibility that prism adaptation could be used to improve face processing in individuals with prosopagnosia and autism, who have been shown to have reduced or absent configural face processing27,28. We predicted that adaptation to leftward-shifting prisms, which induced neglect-like processing in healthy participants, would reduce the RT cost associated with identifying composite faces. We further predicted that there would be no change in composite face processing following adaptation to rightward-shifting prisms, which does not induce perceptual changes in healthy participants.\n\n\nMaterial and methods\n\nSixty-four right-handed undergraduate women (mean age=19.8 years, SEM=0.32; mean handedness=-0.83, SEM=0.026 where a score of -1 denotes complete right-handedness;29) completed a composite face task before and after a brief (five-minute) session of prism adaptation (see below for a full description of the task). Only female participants were selected for the study as it was felt that the stimuli - images of Brad Pitt and George Clooney - might have, on average, higher saliency for women than men. To be included in the study participants were also required to have normal or corrected-to-normal vision, and full use of their right arm. Informed consent was obtained in accordance with guidelines approved by the Bangor University ethics committee and the 2008 Declaration of Helsinki. Participants received course credits for the 45-minute session.\n\nIn a repeated-measures design, participants completed one set of configural face processing tasks before prism adaptation, and one set of configural face processing tasks after prism adaptation.\n\nPrism adaptation and confirmation of sensorimotor realignment were performed using a similar procedure as that used for prism adaptation treatment of hemispatial neglect24. For prism adaptation, participants made 150 visually-guided pointing movements while wearing goggles fitted with prismatic lenses that shifted the visual field 15° to the left or right. In order to confirm adaptation, a participant pointed under target lines while vision of their pointing arm was occluded by a panel ('open-loop pointing'). Twelve open-loop pointing trials were performed immediately before and after prism adaptation (‘pre-' and ‘post-test’). In order to confirm that the sensorimotor realignment was retained throughout the entire post-adaptation configural face processing task, a third set of open-loop pointing errors were recorded at the end of the experiment (‘late-test’). Open-loop pointing error was measured by the experimenter to the nearest 0.5°, with negative numbers indicating leftward errors and positive numbers indicating rightward errors.\n\nParticipants performed a composite face task using stimuli similar to those used by Weston and Perfect30. Figure 1 (adapted from Weston and Perfect30) provides examples of the four stimulus types used in the present experiment. Stimuli for the composite faces task had the same form as these examples, but were created from black-and-white publicity photographs of two well-known movie stars (Brad Pitt and George Clooney). All stimuli were constructed from the same two images and were presented on a black background. All participants correctly named the celebrities when shown these photographs at the beginning of the experimental session. Congruent stimuli were the unaltered pictures: that is the top and bottom face-halves were from the same celebrity. Incongruent stimuli were constructed by combining top and bottom face-halves from the two different celebrities. Faces were presented with the top and bottom halves aligned, or with the top half of the face offset to the left or right with reference to the bottom half, by approximately one-third of the face half. Participants identified the top or bottom half of each face in separate, identical blocks. For each trial, a fixation cross appeared for 500 ms, followed by the face stimulus for 200 ms, then a blank screen. Responses were made by pressing one of two buttons on a keyboard with the index or middle finger of their right hand. The participant’s response ended the trial. Each block consisted of 32 repetitions of each of the four stimulus types (congruent-aligned, congruent-misaligned, incongruent-aligned and incongruent-misaligned) in pseudorandom order, resulting in a total of 128 trials per block. Block order (top first or bottom first) and key allocation (Brad-left-George-right or George-left-Brad-right) were counterbalanced between participants.\n\nStatistical analyses were performed using SPSS software31. Pointing errors and reaction time (RT) data were subjected to repeated-measures ANOVAs. Follow-up paired-t-tests were performed using Bonferroni correction for multiple comparisons.\n\n\nResults\n\nData File 1 contains the full pointing data for each participant. A mixed ANOVA of pointing errors with the factors Prism Group (leftward, rightward) and Session (pre, post, late) revealed a significant two-way interaction [F(2,124)=213.2, p<0.001]. This reflected a significant rightward shift in pointing error for the leftward-shifting prism group between the pre-test (M=-0.2, SEM=0.33) and the post-test [M=4.9, SEM=0.28; t(31)=15.0, p<0.001], which was still significant in the late-test [M=3.6, SEM=0.27; t(31)=10.4, p<0.001]. Similarly, there was a significant leftward shift in for the rightward-shifting prism group between the pre-test (M=0.7, SEM=0.35) and the post-test [M=-3.3, SEM=0.40; t(31)=12.1, p<0.001], and this was sustained to the late-test [M=-2.7, SEM=0.39; t(32)=8.7, p<0.001]. Comparison of 95% Confidence Intervals around the pre- to post-test pointing shifts indicated that there were no significant differences in the absolute magnitude of the after-effect for the two groups. Similarly, there were no significant differences in the pre- to late-test pointing shifts. Therefore prism adaptation resulted in significant shifts in open-loop pointing error in both groups, which were maintained for the entire duration of the post-adaptation composite face task.\n\n\n\nMean accuracy was at ceiling (93%), precluding meaningful analysis. For each participant, responses that were faster than 200 ms or more than 3 SD above their mean RTs were excluded from analysis. Four participants demonstrated low accuracy for incongruent trials (>3 SD from mean error rate) during one or more block of the experiment, suggesting a failure to comprehend or comply with task instructions (i.e., their responses suggested that these participants were identifying, for example, the top half of the faces in a block in which they had been instructed to identify the bottom half of the faces). These participants were excluded from the analyses. Data for one of the experimental blocks was missing for two participants due to an error made by the experimenter. Since the responses of these individuals were otherwise similar to the remaining participants (suggesting that they were able to understand the instructions) these participants were retained and their missing data was replaced by the mean for that group.\n\nFor each prism group (leftward- or rightward-prisms), repeated-measures ANOVAs were conducted on the RT cost of alignment; that is, the difference between RTs for aligned and misaligned faces. By this index, a larger RT cost indicates greater interference due to configural processing, and a small RT cost indicates that participants were able to focus on the face halves with little or no interference from configural processing. The key factors of interest for the analyses were Prism (pre, post) and Congruency (congruent, incongruent). Previous studies have demonstrated temporal limitations to the effects of Navon figure processing on changes in the recognition of pre-learned faces32 and composite halves30, with the effects decaying by the second half of the post-induction test phase. In order to test for such changes over time, we therefore included two further time-based factors in our analyses: Block Number (first, second) and Block Half (first, second). Finally, since any time-based effects may also be influenced by which half of the face participants identified immediately after prism adaptation, a between-subjects factor of Block Order (top-half-first, bottom-half-first) was also included. Therefore, the ANOVAs included five factors: Prism, Congruency, Block Order, Block Number and Block Half.\n\nThe full data for the analysis are presented in Data File 2. The analyses revealed significant main effects of Congruency for both leftward-shifting [M=-6.5 vs M=16.1; F(1,28)=28.6, p<0.001] and rightward-shifting [M=0.15 vs M=14.1; F(1,29)=913.0, p<0.005] prism groups, reflecting lower RT costs of alignment for the incongruent faces than for the congruent faces. A significant main effect of Block Half for the rightward-shifting prism group indicated higher RT costs for trials in the first half of the block compared to the second half [M=12.7 vs M=-1.6; F(1,29)=5.5, p<0.05]. A significant Block Order x Block Number interaction for the leftward-shifting prism group [F(1,27)=4.78, p<0.05] reflected trends in the RT cost of alignment depending on which face half the participants identified. That is, there was a non-significant tendency towards a higher RT cost of alignment for block two for participants in the ‘bottom half first’ group, and for block one for the participants in the ‘top half first’ group (ps>0.05).\n\n\n\nThere was no significant interaction of Prism and Congruency for the leftward-shifting prism group (ps>0.05), although a trend for a Prism x Congruency interaction arose for the rightward-shifting prism group [F(1,29)=3.4, p=0.074]. The RT costs for this interaction are plotted in Figure 2 for both groups, and follow-up t-tests were performed on an a priori basis. In contradiction of the experimental hypothesis, there was no significant change in RT cost of alignment for congruent or incongruent faces following adaptation to leftward-shifting prisms. There was, however, a trend for a reduction in RT cost for incongruent faces for participants in the rightward-shifting prism (control) group [t(30)=2.2, p=0.04, assessed to a Bonferroni-corrected alpha-level of p=0.0125].\n\nRT costs of alignment for congruent and incongruent trials before and after adaptation to leftward- and rightward-shifting prisms. Error bars represent ± SEM, *=significant to a Bonferroni-corrected alpha level of p=0.0125.\n\nThere were no significant interactions of Prism and Congruency with any other factor with Block Number or Block Half to suggest any short-lived effect of prism adaptation on composite face processing. This is apparent in Figure 3, which shows incongruent trial RT costs of the two Prism groups averaged across eight time points (2 Prism x 2 Block Number x 2 Block Halves).\n\nThe RT costs for the leftward- and rightward-shifting prism groups are shown broken into eight different time periods across the experiment, and were no different between the two groups.\n\nOverall, the results demonstrate that the RT cost of alignment became numerically smaller with time for both groups, consistent with a practice effect. Importantly, there was no significant reduction in RT cost following adaptation to leftward-shifting prisms.\n\n\nDiscussion\n\nOur results indicate that adaptation to leftward-shifting prisms did not reduce the RT cost associated with identifying individual halves of composite faces. Our data did reflect trends for reduced RT costs of alignment for incongruent faces for both the leftward- and rightward-shifting prism groups. However, this was not significant, and was in fact numerically larger for the rightward-shifting prism (control) group. With our large sample size (N=32 per group), it is unlikely that the lack of significant change in RT costs for incongruent trials can be attributed to type II error. We conclude instead that prism adaptation does not reduce configural processing of face stimuli.\n\nOur research is particularly comparable to studies examining the effects of prism adaptation on the processing of chimeric faces and objects (stimuli that are formed by joining together the left and right halves of different faces or objects). Ferber and colleagues demonstrated that prism adaptation shifted the extent to which a neglect patient33 and healthy participants34 passed their gaze over different halves of chimeric faces. However, these changes in the visual exploration were not accompanied by any alteration in perceptual judgements of the faces. Sarri and colleagues35 extended on this to demonstrate that although prism adaptation did not alter patients’ perception of chimeric faces it did dramatically improve their awareness of the identity of the left side of non-face objects. Our findings that prism adaptation alters the global versus local processing of Navon figures23,24 but not composite faces is consistent with this distinction between significant effects of prism adaptation on object but not face processing.\n\nThese results have bearing on an existing debate about whether the beneficial effects of prism adaptation on hemispatial neglect are restricted to tasks that have a direct motor or attentional component, or whether the technique also directly alters perceptual awareness per se33,35–39. Striemer and Danckert36 proposed that the beneficial effects of prism adaptation are limited to dorsal stream attentional and visuomotor behaviours, whereas ventral stream perceptual processes are relatively unaffected. Many of the tasks on which neglect patients have shown improvement following neglect, such as pen-and-paper tasks9,40, reading41, haptic exploration12, postural imbalance42 and wheel-chair navigation43, can be explained by a leftward shift in motor behaviour (including eye movements). In contrast, several studies have shown that prism adaptation does not alter the performance of neglect patients on tasks that require direct perceptual comparison of the left and right side of the stimuli33,44,45, or stimuli on the left or right sides of space46. Strikingly, the same patients showed leftward shifts in their ocular exploration of the stimuli33,46, or in similar tasks that had an overt motor component45. Overall, Striemer and Danckert argued that prism adaptation alters performance on perceptual tasks only under specific circumstances (see Nijboer and colleagues47 for data that directly contradicts this conclusion, and papers by Saevarsson and Streimer and their colleagues38,39 for further discussions of this model).\n\nWe previously attributed the effects of prism adaptation on the processing of Navon figures to changes in the relative activity of left and right temporo-parietal areas23,24. While object recognition per se is strongly attributed to dorsal stream processing, sensitivity to global versus local features of an object has been linked to differential specialisation of the left and right temporo-parietal cortices to these two levels of processing48–53. A further model of visual processing suggests that fast global processing of visual objects dominates in the dorsal stream providing rapid activation of frontoparietal attention mechanisms, whereas more detailed local processing occurs mainly through slower ventral stream mechanisms54–56. Thus, the effects of prism adaptation on the processing of Navon figures could be attributed to changes in dorsal stream mechanisms, either by altering relative processing weights of left and right temporo-parietal areas, or by a global enhancement or suppression of dorsal stream mechanisms.\n\nSimilar to other objects, it has been suggested that there is left hemisphere specialisation for processing face features and a right hemisphere specialisation for processing the face as a whole57. However these have been localised to face-selective areas of the fusiform gyrus (i.e., the dorsal stream). A mechanism of prism adaptation that operates mainly through the ventral stream would therefore explain the absence of any effect of prism adaptation on face processing.\n\nPrism adaptation is a promising treatment for hemispatial neglect. In order to understand the cognitive and neural mechanisms that underlie this intervention, it is important to examine tasks on which this technique has no impact, as well as those for which improvements are observed. Our finding that prism adaptation does not alter configural processing of faces is consistent with the dorsal versus ventral stream processing model proposed by Striemer and Danckert36. Studies that directly compare the effects of prism adaptation on classic dorsal and ventral stream tasks would further illuminate the mechanisms of the beneficial effects of this intervention on hemispatial neglect.",
"appendix": "Author contributions\n\n\n\nJB conceived of the study, collected data for the study, analysed the data and prepared the draft manuscript. PD provided expertise on face processing and aided in interpreting the data. RR aided in the design of the study and aided in interpreting the data. 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 relevant competing interests disclosed.\n\n\nGrant information\n\nFunding for this work was provided by the British Federation of Women Graduates (to JB).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors are grateful to Prof. Elinor McKone for her advice on the experimental design and Ms. Jo Mason for assistance with data collection.\n\n\nReferences\n\nBisiach E, Vallar G: Unilateral neglect in humans. In: Boller F Grafman J Rizzolatti G editors. Handbook of neuropsychology. Elsevier Science, B.V.; ST – Unilateral neglect in humans 2000; p. 459–502.\n\nJehkonen M, Ahonen JP, Dastidar P, et al.: Visual neglect as a predictor of functional outcome one year after stroke. Acta Neurol Scand. 2000; 101(3): 195–201. PubMed Abstract | Publisher Full Text\n\nBisiach E, Perani D, Vallar G, et al.: Unilateral neglect: personal and extra-personal space. Neuropsychologia. 1986; 24(6): 759–767. PubMed Abstract\n\nBisiach E, Luzzatti C: Unilateral neglect of representational space. Cortex. 1978; 14(1): 129–133. PubMed Abstract\n\nHeilman KM, Watson RT, Valenstein E: Neglect and related disorders. In: Heilman, K.M. and Valenstein, W., Eds. Clinical Neuropsychology 2. New York: Oxford University Press. 1985; pp 243–293.\n\nRoberson IH, Manly T, Beschin N, et al.: Auditory sustained attention is a marker of unilateral spatial neglect. Neuropsychologia. 1997; 35(12): 1527–1532. PubMed Abstract | Publisher Full Text\n\nHusain MN, Mannan S, Hodgson T, et al.: Impaired spatial working memory across saccades contributes to abnormal search in parietal neglect. Brain. 2001; 124(Pt 5): 941–952. PubMed Abstract | Publisher Full Text\n\nHusain M, Rorden C: Non-spatially lateralized mechanisms in hemispatial neglect. Nat Rev Neurosci. 2003; 4(1): 26–36. PubMed Abstract | Publisher Full Text\n\nRossetti Y, Rode G, Pisella L, et al.: Prism adaptation to a rightward optical deviation rehabilitates left hemispatial neglect. Nature. 1998; 395(6698): 166–9. PubMed Abstract | Publisher Full Text\n\nLuauté J, Halligan P, Rode G, et al.: Prism adaptation first among equals in alleviating left neglect: A review. Restor Neurol Neurosci. 2006; 23(2005): 1–10. Reference Source\n\nMaravita A, McNeil J, Malhotra P, et al.: Prism adaptation can improve contralesional tactile perception in neglect. Neurology. 2003; 60(11): 1829–31. PubMed Abstract | Publisher Full Text\n\nMcintosh R, Rossetti Y, Milner A: Prism adaptation improves chronic visual and haptic neglect: a single case study. Cortex. 2002; 38(3): 309–20. PubMed Abstract | Publisher Full Text\n\nRode G, Rossetti Y, Boisson D: Prism adaptation improves representational neglect. Neuropsychologia. 2001; 39(11): 1250–4. PubMed Abstract | Publisher Full Text\n\nRossetti Y, Jacquin-Courtois S, Rode G, et al.: Does action make the link between number and space representation? Visuo-manual adaptation improves number bisection in unilateral neglect. Psychol Sci. 2004; 15(6): 426–30. PubMed Abstract | Publisher Full Text\n\nLuauté J, Halligan P, Rode G, et al.: Prism adaptation first among equals in alleviating left neglect: a review. Restor Neurol Neurosci. 2006; 24(4–6): 409–18. PubMed Abstract\n\nBerberovic N, Mattingley JB: Effects of prismatic adaptation on judgements of spatial extent in peripersonal and extrapersonal space. Neuropsychologia. 2003; 41(4): 493–503. PubMed Abstract | Publisher Full Text\n\nLoftus A, Nicholls M, Mattingley J, et al.: Left to right: representational biases for numbers and the effect of visuomotor adaptation. Cognition. 2008; 107(3): 1048–58. PubMed Abstract | Publisher Full Text\n\nMichel C, Pisella L, Halligan P, et al.: Simulating unilateral neglect in normals using prism adaptation: implications for theory. Neuropsychologia. 2003; 41(1): 25–39. PubMed Abstract | Publisher Full Text\n\nNicholls M, Kamer A, Loftus A: Pseudoneglect for mental alphabet lines is affected by prismatic adaptation. Exp Brain Res. 2008; 191(1): 109–15. PubMed Abstract | Publisher Full Text\n\nRode G, Pisella L, Marsal L, et al.: Prism adaptation improves spatial dysgraphia following right brain damage. Neuropsychologia. 2006; 44(12): 2487–93. PubMed Abstract | Publisher Full Text\n\nNys GM, Seurinck R, Dijkerman HC: Prism adaptation moves neglect-related perseveration to contralesional space. Cogn Behav Neurol. 2008; 21(4): 249–53. PubMed Abstract | Publisher Full Text\n\nNijboer TC, Nys GM, van der Smagt MJ, et al.: Repetitive long-term prism adaptation permanently improves the detection of contralesional visual stimuli in a patient with chronic neglect. Cortex. 2011; 47(6): 734–40. PubMed Abstract | Publisher Full Text\n\nBultitude JH, Rafal RD, List A: Prism adaptation reverses the local processing bias in patients with right temporo-parietalction lesions. Brain. 2009; 132(Pt 6): 1669–77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBultitude JH, Woods JM: Adaptation to leftward-shifting prisms reduces the global processing bias of healthy individuals. Neuropsychologia. 2010; 48(6): 1750–6. PubMed Abstract | Publisher Full Text\n\nBultitude JH, Van der Stigchel S, Nijboer T: Prism adaptation alters spatial remapping in healthy individuals: Evidence from double-step saccades. Cortex. 2013; 49(3): 459–770. PubMed Abstract | Publisher Full Text\n\nYoung A, Hellawell D, Hay DC: Configurational information in face perception. Perception. 1987; 16(6): 747–59. PubMed Abstract | Publisher Full Text\n\nPalermo R, Willis ML, Rivolta D, et al.: Impaired holistic coding of facial expression and facial identity in congenital prosopagnosia. Neuropsychologia. 2011; 49(5): 1226–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAvidan G, Tanzer M, Behrmann M: Impaired holistic processing in congenital prosopagnosia. Neuropsychologia. 2011; 49(9): 2541–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOldfield RC: The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia. 1971; 9(1): 97–113. PubMed Abstract | Publisher Full Text\n\nWeston NJ, Perfect TJ: Effects of processing bias on the recognition of composite face halves. Psychon Bull Rev. 2005; 12(6): 1038–42. PubMed Abstract | Publisher Full Text\n\nCorp. I. IBM SPSS Statistics for Windows. Armonk, NY IBM; 2012.\n\nHills PJ, Lewis MB: Temporal limitation of navon effect on face recognition. Percept Mot Skills. 2007; 104: 501–9. PubMed Abstract | Publisher Full Text\n\nFerber S, Danckert J, Joanisse M, et al.: Eye movements tell only half the story. Neurology. 2003; 60(11): 1826–9. PubMed Abstract | Publisher Full Text\n\nFerber S, Murray LJ: Are perceptual judgments dissociated from motor processes?--A prism adaptation study. Brain Res Cogn Brain Res. 2005; 23(2–3): 453–6. PubMed Abstract | Publisher Full Text\n\nSarri M, Kalra L, Greenwood R, et al.: Prism adaptation changes perceptual awareness for chimeric visual objects but not for chimeric faces in spatial neglect after right-hemisphere stroke. Neurocase. 2006; 12(3): 127–35. PubMed Abstract | Publisher Full Text\n\nStriemer CL, Danckert JA: Through a prism darkly: re-evaluating prisms and neglect. Trends Cogn Sci. 2010; 14(7): 308–16. PubMed Abstract | Publisher Full Text\n\nFerber S, Murray LJ: Are perceptual judgments dissociated from motor processes?-A prism adaptation study. Brain Res Cogn Brain Res. 2005; 23(2–3): 453–6. PubMed Abstract | Publisher Full Text\n\nSaevarsson S, Kristjánsson Á: A note on Striemer and Danckert’s theory of prism adaptation in unilateral neglect. Front Hum Neurosci. 2013; 7: 44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStriemer CL, Danckert J: The influence of prism adaptation on perceptual and motor components of neglect: a reply to Saevarsson and Kristjansson. Front Hum Neurosci. 2013; 7: 255. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPisella L, Rode G, Farne A, et al.: Dissociated long lasting improvements of straight-ahead pointing and line bisection tasks in two hemineglect patients. Neuropsychologia. 2002; 40(3): 327–34. PubMed Abstract | Publisher Full Text\n\nAngeli V, Benassi MG, Làdavas E: Recovery of oculo-motor bias in neglect patients after prism adaptation. Neuropsychologia. 2004; 42(9): 1223–34. PubMed Abstract | Publisher Full Text\n\nTilikete C, Rode G, Rossetti Y, et al.: Prism adaptation to rightward optical deviation improves postural imbalance in left-hemiparetic patients. Curr Biol. 2001; 11(7): 524–8. PubMed Abstract | Publisher Full Text\n\nJacquin-Courtois S, Rode G, Pisella L, et al.: Wheel-chair driving improvement following visuo-manual prism adaptation. Cortex. 2008; 44(1): 90–6. PubMed Abstract | Publisher Full Text\n\nSarri M, Kalra L, Greenwood R, et al.: Prism adaptation changes perceptual awareness for chimeric visual objects but not for chimeric faces in spatial neglect after right-hemisphere stroke. Neurocase. 2006; 12(3): 127–35. PubMed Abstract | Publisher Full Text\n\nStriemer CL, Danckert J: Dissociating perceptual and motor effects of prism adaptation in neglect. Neuroreport. 2010; 21(6): 436–41. PubMed Abstract | Publisher Full Text\n\nDijkerman HC, McIntosh RD, Milner AD, et al.: Ocular scanning and perceptual size distortion in hemispatial neglect: effects of prism adaptation and sequential stimulus presentation. Exp Brain Res. 2003; 153(2): 220–30. PubMed Abstract | Publisher Full Text\n\nNijboer T, Vree A, Dijkerman C, et al.: Prism adaptation influences perception but not attention: evidence from antisaccades. Neuroreport. 2010; 21(5): 386–9. PubMed Abstract | Publisher Full Text\n\nMarshall JC, Halligan PW: Visuo-spatial neglect: a new copying test to assess perceptual parsing. J Neurol. 1993; 240(1): 37–40. PubMed Abstract | Publisher Full Text\n\nRobertson LC, Delis DC: 'Part-whole' processing in unilateral brain-damaged patients: dysfunction of hierarchical organization. Neuropsychologia. 1986; 24(3): 363–70. PubMed Abstract | Publisher Full Text\n\nHubner R: Hemispheric Differences in Global/Local Processing Revealed by Same-Different Judgements. Visual Cognition. 1998; 5(4): 457–78. Publisher Full Text\n\nMartin M: Hemispheric specialization for local and global processing. Neuropsychologia. 1979; 17: 33–40. Publisher Full Text\n\nSergent J: The cerebral balance of power: confrontation or cooperation? J Exp Psychol Hum Percept Perform. 1982; 8(2): 253–72. PubMed Abstract | Publisher Full Text\n\nVan Kleeck MH: Hemispheric differences in global versus local processing of hierarchical visual stimuli by normal subjects: new data and a meta-analysis of previous studies. Neuropsychologia. 1989; 27(9): 1165–78. PubMed Abstract | Publisher Full Text\n\nLaycock R, Cross AJ, Lourenco T, et al.: Dorsal stream involvement in recognition of objects with transient onset but not with ramped onset. Behav Brain Funct. 2011; 7(1): 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBullier J: Integrated model of visual processing. Brain Res Rev. 2001; 36(2–3): 96–107. PubMed Abstract | Publisher Full Text\n\nThoma V, Henson RN: Object representations in ventral and dorsal visual streams: fMRI repetition effects depend on attention and part-whole configuration. Neuroimage. 2011; 57(2): 513–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRossion B, Dricot L, Devolder A, et al.: Hemispheric Asymmetries for Whole-Based and Part-Based Face Processing in the Human Fusiform Gyrus. J Cogn Neurosci. 2000; 12(5): 793–802. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2070",
"date": "13 Nov 2013",
"name": "Christopher Striemer",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper the authors have examined the after-effects of directional prism adaptation on global and local visual processing using the composite face effect. Previous work by Bultitude and colleagues has demonstrated that rightward prism adaptation in patients with right brain damage helps reduce the local processing bias. Furthermore, leftward prism adaptation in healthy individuals actually increases the local processing bias. Based on these findings, the authors predicted that, in healthy individuals, leftward prism adaptation should increase local processing, thereby reducing the composite face effect (i.e., the increase in reaction time observed when processing aligned vs. misaligned composite faces).The results of the experiment indicated that there were no significant changes in the composite face effect following leftward prism adaptation. However, there was a trend towards a reduction in the composite face effect following rightward prism adaptation. Critically, the absence of any effect of leftward shifting prisms on the composite face effect cannot be attributed to de-adaptation, as participants remained significantly adapted at the conclusion of the experiment. Based on these results the authors argued that their data are consistent with the notion that prism adaptation primarily influences processing in the dorsal visual stream, and the dorsal attention network. Overall I found the study to be very interesting and well motivated. Although I found study to be quite interesting, I do have some queries regarding the methods used, as well as the interpretation of the data. In the Methods section it is not clear whether concurrent or terminal feedback was used during the prism adaptation session. Please clarify in the revised manuscript. In the Results section, when discussing the results of the composite face task (page 5, 2nd paragraph, right column) you note that, “the analyses revealed significant main effects of congruency for both leftward and rightward shifting prisms groups, reflecting lower RT costs of alignment for the incongruent faces than for the congruent faces.” Perhaps I have misinterpreted the composite face effect, but isn’t the prediction that participants should be slower (i.e., an increased RT cost) when processing aligned (compared misaligned) incongruent compared to congruent faces? The data from Figure 2 seem to support this interpretation in that participants are slower to respond for incongruent compared to congruent faces. Please clarify this in the revised manuscript. It is interesting to note that the authors observed a trend towards a reduction in the composite face effect following rightward prism adaptation. However, the possible reasons for this are not addressed in the discussion. Is it possible that rightward prism adaptation may have increased activity in left temporal-parietal cortex thereby increasing attention to local features, and, by extension, decreasing configural face processing? In the Discussion section I believe there may be a typo (or perhaps some confusion) regarding your characterization of the dorsal and ventral streams. Specifically, on the bottom of the left column on page 7 you mention that “While object recognition per se is strongly attributed to dorsal stream processing ....” I believe what you mean to say is that object recognition is strongly tied to the ventral visual stream. While it is true that some imaging studies have observed activation in dorsal stream areas during object processing tasks, it is as of yet unclear what visual information these signals are conveying. However, it is well known that damage to the ventral stream has devastating consequences for object and face recognition. Likewise, when you are describing hemispheric specialization for face processing (page 7, middle paragraph, right column) you refer to the processing of face features and configural processing of faces as being “localized to face-selective areas in the fusiform gyrus (i.e., the dorsal stream).” Again, what I believe you meant to say was face selective areas in the ventral stream. Finally, your interpretation of the results is somewhat difficult to reconcile with findings from Sarri and colleagues (Sarri, Greenwood, Kalra, & Driver, 2011; Sarri, Kalra, Greenwood, & Driver, 2006) suggesting that patients with neglect can detect chimeric non-face objects following rightward prism adaptation, as both types of patterns (i.e., chimeric objects and composite faces) require ventral stream processing. One way to interpret this is that perhaps prisms have differential effects on face vs. non-face objects. However, perhaps a simpler way of interpreting this is one of task difficulty. That is, discriminating between halves of a chimeric face, or the top and bottom halves of a composite face, require a detailed within-category discrimination. In contrast, chimeric objects typically involve a much simpler between-category discrimination.",
"responses": []
},
{
"id": "3373",
"date": "18 Feb 2014",
"name": "Anna Barrett",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this interesting study, Bultitude and colleagues demonstrate that prism adaptation does not alter the interference effect produced by irrelevant facial feature information on facial identification performance. This is consistent with the findings of Sarri and colleagues (2006, 2011). The study results are straightforward and the discussion appropriate. With respect to theoretical implications of the results, there remains a question in my mind about whether this study supports a selective effect of prism adaptation on dorsal processing, versus a selective effect on spatial action-intentional “Aiming”, because previous studies demonstrated that face recognition with affectively-loaded, dynamic properties may be strongly linked to the dorsal visual stream (Schwartz et al., 1998; Adolphs et al., 2003). However, the current study did not use dynamic or (explicitly) emotional stimuli, so this distinction of dorsal visual processing versus spatial action processing is academic to the results at hand and the link of these results to dorsal versus spatial ‘Aiming’ function can be explored in future experiments.",
"responses": []
},
{
"id": "4653",
"date": "06 May 2014",
"name": "Wolfgang Heide",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBultitude and colleagues performed a psychophysical study on the perception/recognition time of composite faces before and after prism adaptation, in 64 healthy young women. Faces were composed of top and bottom halves from either the same or 2 different familiar persons. The authors demonstrate that adaptation to leftward-shifting prisms does not significantly change change the extent to which the irrelevant face half interfered with processing. Consequently, they conclude that prism adaptation does not have a significant effect on ventral stream processing of perceptual features, but appears to affect exclusively dorsal stream processing. The study results are straightforward and the discussion is appropriate. Only I propose that the authors might add some comments and discussion on their finding that rightward-shifting prisms did lead to some significant reduction of reaction time costs.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-215
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https://f1000research.com/articles/2-214/v1
|
14 Oct 13
|
{
"type": "Opinion Article",
"title": "Ultimate drivers of native biodiversity change in agricultural systems",
"authors": [
"David A Norton",
"Nick Reid",
"Laura Young",
"Nick Reid",
"Laura Young"
],
"abstract": "The ability to address land degradation and biodiversity loss while maintaining the production of plant and animal products is a key global challenge. Biodiversity decline as a result of vegetation clearance, cultivation, grazing, pesticide and herbicide application, and plantation establishment, amongst other factors, has been widely documented in agricultural ecosystems. In this paper we identify six ultimate drivers that underlie these proximate factors and hence determine what native biodiversity occurs in modern agricultural landscapes; (1) historical legacies; (2) environmental change; (3) economy; (4) social values and awareness; (5) technology and knowledge; and (6) policy and regulation. While historical legacies and environmental change affect native biodiversity directly, all six indirectly affect biodiversity by influencing the decisions that land managers make about the way they use their land and water resources. Understanding these drivers is essential in developing strategies for sustaining native biodiversity in agricultural landscapes into the future.",
"keywords": [
"Global land use and land cover have changed markedly over the past two centuries as a result of rapid population growth and the increasing demand for food and fibre products. As much as 75% of the Earth’s ice-free surface has been directly modified by human activities",
"mainly through urbanisation",
"timber harvesting",
"cultivation",
"and livestock grazing1. These changes have resulted in land and water degradation and consequential species extinctions. Ongoing human population growth (perhaps to 9.3 billion by 20502)",
"including a massive increase in the size of the world’s middle class3 and the concomitant increase in the consumption of meat",
"dairy products and luxury goods derived from animals (e.g. leather and wool)",
"will see the demand for agricultural products increase further. This increase in demand is likely to be met through further losses of natural areas and the transformation of low-production systems to high-input farming and grazing systems. The ongoing expansion and intensification of agriculture will put more pressure on native biodiversity and the ecosystem services that biodiversity provides to humanity4",
"5. Sound environmental management of agriculture has never been more important for human well-being",
"the maintenance of global life-support systems",
"and the survival of planetary biodiversity."
],
"content": "Introduction\n\nGlobal land use and land cover have changed markedly over the past two centuries as a result of rapid population growth and the increasing demand for food and fibre products. As much as 75% of the Earth’s ice-free surface has been directly modified by human activities, mainly through urbanisation, timber harvesting, cultivation, and livestock grazing1. These changes have resulted in land and water degradation and consequential species extinctions. Ongoing human population growth (perhaps to 9.3 billion by 20502), including a massive increase in the size of the world’s middle class3 and the concomitant increase in the consumption of meat, dairy products and luxury goods derived from animals (e.g. leather and wool), will see the demand for agricultural products increase further. This increase in demand is likely to be met through further losses of natural areas and the transformation of low-production systems to high-input farming and grazing systems. The ongoing expansion and intensification of agriculture will put more pressure on native biodiversity and the ecosystem services that biodiversity provides to humanity4,5. Sound environmental management of agriculture has never been more important for human well-being, the maintenance of global life-support systems, and the survival of planetary biodiversity.\n\nOur concern in this paper is for the native biodiversity that evolved in an area rather than the exotic diversity that is associated with agriculture and came to that place from elsewhere6. Exotic biodiversity forms the basis of most agricultural systems worldwide, including crop and livestock species, as well as the many other species that have benefitted from human activities. Maintaining the genetic diversity of exotic species used in agriculture is critical, but is not our focus. Not all changes due to agricultural management will be negative for native biodiversity: some species will prosper from changes, and remnants of some native communities will persist in a matrix of agricultural development7. As a general rule however, remnant native taxa tend to be generalists and agricultural development usually results in the homogenisation of native biodiversity8.\n\nDegradation of agricultural lands and the inevitable loss of associated native biodiversity within these landscapes is one of the main consequences of the expansion in the extent and intensity of agricultural production at all scales, from local to global9. Addressing land degradation and biodiversity loss while maintaining the production of plant and animal products is a key global challenge. Understanding the pressures or drivers that lead to environmental degradation and biodiversity loss is important in meeting this challenge10. Indeed, it is not possible to restore degraded systems without understanding these drivers and addressing them11. The importance of anthropogenic drivers of change for predicting current and future ecosystem condition was strongly emphasised in the Millennium Ecosystem Assessment9,12. Drivers are any natural or human-induced factors that directly or indirectly cause ecosystem change and biodiversity loss and include both the proximate drivers that result in actual change on the ground and the ultimate drivers that underlie these. Our focus in this article on the ultimate drivers of native biodiversity loss is motivated not by any intent to undermine the obvious importance of proximate causes of change, such as vegetation clearance, cultivation, grazing, pesticide and fertiliser application or plantation establishment, but by a desire to better understand the underlying or ultimate influences leading to day-to-day management decisions by land managers that directly affect native biodiversity.\n\nIn this article we outline a framework for considering ultimate drivers of biodiversity change in agricultural landscapes under six headings (Figure 1): (1) historical legacies; (2) environmental change; (3) economy; (4) social values and awareness; (5) technology and knowledge; and (6) policy and regulation. While environmental change and historical legacies directly affect native biodiversity, all six indirectly affect biodiversity by influencing the decisions that land managers make about the way they use land and water resources. These land management decisions are critical and have a range of flow-on effects for biodiversity. We use examples from Australia and New Zealand to illustrate these underlying drivers of biodiversity change, but similar examples can be found in and applied to any agricultural system worldwide.\n\nWhile all drivers indirectly affect biodiversity through their effect on land-use practices (thin black lines), only environmental change and historical legacies directly affect biodiversity (thick black lines). Interactions (dotted lines) also occur amongst the different drivers.\n\n\nUltimate drivers\n\nThe current distribution of biodiversity in agricultural ecosystems reflects the interactions between the environment (climate, landform, soils, etc.), and the effects of past human activities (habitat creation, modification and loss, introduction of agricultural species, commensals, pesticide and fertiliser application, etc.7). Legacies from these historical influences on biodiversity, together with a range of new, primarily anthropogenic, drivers will shape future biodiversity patterns directly through their influence on biodiversity (e.g. land clearance or invasive species), but most often indirectly through their influence on land and water management practices (e.g. the intensity of management inputs), which in turn have direct effects on biodiversity9. The various drivers interact across multiple spatial and temporal scales12 and can work synergistically. It is often synergistic effects that are of most concern for biodiversity conservation13–15. Without a clear understanding of ultimate drivers of land-use (and hence biodiversity) change, it is not possible to identify and implement appropriate strategies for biodiversity conservation, strategies that are urgently required if biodiversity decline is to be reversed over the majority of the Earth’s surface16–18.\n\nThe way that land managers respond to these drivers in terms of their farm management practices will be critical for native biodiversity, and the outcomes could either be positive or negative depending on the particular response taken. Unfortunately to date, most of the outcomes for native biodiversity have been negative. Developing a good understanding of these drivers and the ways they influence biodiversity is essential if we are to have the ability to influence the way that agricultural landscapes are managed in order to obtain better outcomes for native biodiversity. We now review each of these drivers.\n\n\nHistorical legacies\n\nHistorical legacies include the many events that have occurred in the past but have an ongoing influence on both land management practices and biodiversity today. Two types of historical legacy are particularly important: effects of past land management activities on soils (e.g. erosion, salinization, compaction, pesticide application, acidification, nutrient inputs) and ongoing adjustments of remnant biotas to historical habitat destruction and fragmentation. Both of these directly affect biodiversity, while changes in soil biogeochemistry also influence farm management practices and hence biodiversity indirectly.\n\nIn some agricultural landscapes, periods of intensive land use (e.g. irrigation or fertiliser application) have resulted in fundamental changes in ecosystem attributes, especially in relation to soil properties (e.g. through salinization19). These modifications have pushed ecosystems across thresholds of change that may take decades, centuries or millennia to reverse20, with their effects influencing both land management practices and biodiversity for the foreseeable future. A good example of an historical legacy having a long-term effect on biodiversity is the influence of phosphorus fertiliser and exotic legume seed inputs in tableland pastures in south-eastern Australia21. Elevated soil P and N levels, coupled with clearance of native timber, resulted in epidemic numbers of native scarabs, particularly Christmas beetles (Anoplognathus spp., Scarabeidae). These beetles, in concert with other defoliating insects, have caused widespread dieback of pasture eucalypts, and continue to hamper establishment of eucalypts in intensively developed pasture land21,22. Elevated soil P and N are good for grass growth and the loss of tree cover has resulted in increased stocking rates because of the greater area of pasture. However, the loss of shade and shelter increases sheep mortality in extreme weather and the loss of woodland and scattered trees has caused reductions in native birds, mammals (microbats and arboreal marsupials) and lizards23,24.\n\nRemnant biotas are still adjusting to the effects of fragmentation and the extinction debt associated with past and current habitat clearance25,26. For many species, particularly those within forested habitats, the altered environmental conditions that occur as a result of fragmentation reduce the quality of the habitat as edge effects alter forest remnant interior microclimates (e.g. making them warmer, windier and dryer27). Both fragmentation itself and management practices on adjacent agricultural land can alter the disturbances that naturally occurred in these systems (e.g. fire or flooding regimes) or introduce novel disturbances such as grazing, chemical drift and weed invasion, which affect the remnant biota28. Habitat loss also reduces the number of remaining individuals of a species, increasing their vulnerability to disturbance and the likelihood of local extinction, especially as isolation reduces the chance of recolonisation29. At the same time, altering species distributions, both of natives and exotics, means that new species are becoming established in agricultural landscapes causing further changes to remnant biotas, the full impacts of which are yet to be felt30 (see next section).\n\n\nEnvironmental change\n\nWhile climate change and species invasion are only two of a range of environmental changes occurring globally (others include increased nitrogen deposition, land use change and intensification, and CO2 enrichment), they exert perhaps the strongest influence on both agriculture and biodiversity (we see land-use change as one of the proximate causes of biodiversity change). Climate change and species invasion can both directly affect biodiversity, but also have strong effects on land management practices with flow-on effects on biodiversity.\n\nWhile the long-term consequences of climate change will be shifts in average rainfall and temperature patterns, it is the changes in the frequency and intensity of extreme events such as droughts, floods, wind and snow storms or frosts that are likely to be most significant for both farm production and biodiversity in the short to medium term31,32. Native biodiversity will be affected directly through changes in species distributions33, including invasive species, and altering interactions between species34. Biodiversity will also be affected indirectly through changing land-use patterns as farmers change their management practices to cope with climate change35. While the potential direct effects of climate change on biodiversity are generally well appreciated, the indirect effects are less well understood.\n\nOne example of a possible indirect effect of climate change will be through intensification of farm management practices as farmers seek to buffer themselves against the vagaries of unpredictable weather. Irrigation can be used to guarantee grass and crop growth as summer droughts become more frequent, while exotic grasses and legumes can be used to increase productivity of native dryland grasslands to better buffer these systems36. For example, a succession of below-average annual rainfall years in the first decade of the 21st century in the Mackenzie Basin in New Zealand’s eastern South Island (Figure 2) has increased pressure on farmers to establish irrigation or plant legume crops such as lucerne as a buffer against the effects of drought. Intensification such as this has a number of flow-on effects for biodiversity, both as a result of habitat loss as remnant vegetation is cleared to accommodate more intensive farming systems37,38 and through flow-on effects to other parts of the ecosystem (e.g. eutrophication of waterways as a result of increased fertiliser inputs and irrigation, or altered river flows as a result of abstraction and regulation39).\n\nData (downloaded from www.cliflo.niwa.co.nz) are expressed as a proportional deviance from the 1927–2009 average value.\n\nInvasive species are another important part of environmental change and have the potential to be a key driver of the future condition of biodiversity. In particular, a vast pool of introduced species, especially plants, occur in gardens or as individuals in the wild but do not yet have an obvious impact on either agriculture or native biodiversity40. These species may never become a problem, but equally, should environmental conditions or land management practices change, or if they simply cross an abundance threshold, then they could have major impacts in the future. It is difficult to predict when or why a species will start to rapidly expand, but it is clear that for many species this might not occur for years or even decades after the species is first naturalised41. An example of this is the European daisy, mouse-ear hawkweed (Hieracium pilosella, Asteraceae), which was present in New Zealand as early as 1878 (it most likely arrived as a contaminant in grass seed) but wasn’t recognised as a problem until the 1970s when it started to rapidly expand across a wide range of sites in the eastern South Island (Figure 3). This species has become a major pest in the rain-shadow mountains of this region where it has invaded grasslands, significantly reducing both livestock feed and biodiversity values through outcompeting other plant species for water and nutrients42.\n\n(Peter Espie, unpublished data with permission).\n\n\nEconomy\n\nThe economy (from local to global) is perhaps the biggest driver of agricultural decision making, and hence of change in native biodiversity. Except in subsistence systems, farmers are exposed to the vagaries of international markets including the price of farm inputs (especially fuel and fertiliser), changing consumer preferences and demands, the availability of substitutes (e.g. synthetics for natural fibres) and cheap imports, and the effects of national and global economies (e.g. cycles of recessions and booms) on key farm costs such as mortgage interest rates43. Reduced farm incomes also flow on through taxation to the funding available for conservation initiatives at a regional and national government level. While the state of the global economy does not have a direct effect on biodiversity, it has two main consequences that can have marked flow-on effects for biodiversity through effects on farm management: (1) changes in the type of farming operation that is undertaken in response to economic conditions (e.g. the shift towards dairy farming in New Zealand in response to high global dairy prices), and (2) changes in the profitability of the farming operation itself, thus affecting both what the farmer can afford to do (e.g. weed or pest control) and indirectly what government can afford to do in terms of biodiversity conservation (e.g. through financial incentives to farmers to undertake conservation work) through lower taxes.\n\nIf prices squeeze profit margins44,45, farmers might seek to increase economies of scale in their operations, which might affect biodiversity, for example, through an increase in the size of individual paddocks with a resultant loss of remnant areas of biodiversity (as already occurs when large pivot irrigators are installed37). Alternatively farmers might be forced to scale back their operations and especially the use of inputs such as fertiliser, which could be good for biodiversity; this occurred in New Zealand when widespread farm subsidies were removed in the 1980s46. Another conundrum that can occur is that declines in farm returns can be good for biodiversity as farmers reduce their investment in on-farm improvements enabling native plants and animals to reoccupy areas they had been excluded from by agricultural practices. However, good financial returns could be either positive or negative for biodiversity: positive in that farmers have more disposable income to spend on non-essential activities such as biodiversity conservation, but negative in that in other situations farmers might choose to intensify their management and increase productivity to take greater advantage of higher returns.\n\nOne of the key causes of increased agricultural production over the last 50 years has been the increased use of fertiliser, especially nitrogen-based fertilisers4. Fertiliser production is dependent on both supply (especially for fertilisers based on mining naturally occurring deposits, such as phosphorus) and the energy costs of fertiliser production. Spikes in global fertiliser prices in the mid-1970s and late-2000s led to reductions in fertiliser application in New Zealand as farmers had to trade off productivity gains against the increased cost of fertiliser. Cheap fertiliser enables farmers to intensify their management with obvious impacts on biodiversity, while high fertiliser prices generally mean that fertiliser use declines. It is clear that the supply of both fertiliser and fuel is not finite47, but the consequences for biodiversity of rapidly increasing prices of both are unclear. Technology may well provide alternatives to traditional fertilisers or alternative ways to retain key nutrients within agricultural systems might be developed. For example, various types of holistic management aim to reduce or even eliminate the use of inputs such as fertilisers by adopting alternative grazing systems and through the increased use of other nutrient sources such as N-fixing trees. Holistic management (e.g. http://holisticmanagement.org/) and regenerative agriculture (e.g. http://regenag.com/web/) are two examples of these types of approaches. Both are likely to be positive for biodiversity as they encourage a more environmentally sympathetic approach to land management and are attractive to farmers who already have a personal empathy for the environment. High fertiliser prices might force farmers to effectively ‘abandon’ some parts of their properties or alternatively farmers might choose to increase their stocking rates across their whole property to effectively ‘mine’ the resources present in order to maintain a certain level of profit in the face of rising prices.\n\n\nSocial values and awareness\n\nThe way we value biodiversity and agriculture has a fundamentally important influence on what happens to native biodiversity in agricultural landscapes. This is particularly evident with respect to the way in which society’s valuation of agricultural production versus biodiversity conservation has changed through time. As our values and awareness has changed, so too have the ways that we approach land management and hence its impacts on native biodiversity.\n\nWhen European settlers first colonised Australia and New Zealand, elements of the native biodiversity were seen as a hindrance to ‘good’ land management48. The new settlers struggled to understand the local environment while land management approaches were biased by the European agricultural tradition and ‘what worked best’. Few people at that time believed that indigenous ecosystems, especially timbered areas, held any value beyond that of the timber and grass they supported, or the land on which they grew. In fact woody vegetation and wetlands were widely seen as a limitation to ‘progress’. Settlers actively sought to impose a European mantle across the land by clearing timber, draining wetlands, planting Northern Hemisphere species, and introducing livestock48. Government policy at the time in both Australia and New Zealand actively promoted this through the setting of land clearance targets that needed to be met before land ownership could be gained (e.g. the Robertson Land Acts in the 1870s in New South Wales). Links between forest cover and soil and water values were not appreciated, while stock numbers in rangelands reached densities never matched subsequently as flocks of sheep in particular exploited the available forage49.\n\nValues and awareness have changed over time, both in Australia where aridity and frequent droughts made it difficult to impose the traditional European agricultural model over most of the continent, and in New Zealand where it was realised that a lack of trees resulted in severe soil erosion and loss of the productive potential of the land that farmers were seeking to utilise50. More recently the importance of wetlands as filters to reduce the impacts of agricultural pollutants has also been recognised51 (Figure 4). Soil and water conservation became necessary and new land management practices were developed to cope with the profound changes that resulted from European-style farming. Contour banking of sloping croplands in Australia52 and tree planting with exotic species in New Zealand were undertaken to reduce soil erosion50. While the importance of setting aside natural areas for biodiversity conservation has been long recognised (e.g. the national park and reserve systems in Australia and New Zealand date back to the late nineteenth century), the intrinsic value of native biodiversity in agricultural landscapes above and beyond its utilitarian value for grazing and timber, and especially its role in providing key ecosystem services (e.g. pollination) has only been recognised in recent decades. Policy and regulation in this area still lags behind the preservation policies that underpin public conservation lands and a shift of emphasis is urgently needed within land management, research and policy to address this imbalance53.\n\n(Photo: David Norton with permission).\n\n\nTechnology and knowledge\n\nTechnology and knowledge are also important drivers of change in agricultural systems. The availability and affordability of new technology has revolutionised agriculture over the last 50 years. Technological advances in plant breeding and genetic engineering, the use of new herbicides and pesticides, the development of larger, more powerful machinery (tractors, irrigators, etc.), and the application of precision agriculture have all enabled farmers to produce more food and fibre at lower cost, and to move into environments that previously were not able to be farmed (e.g. the heavy soils on alluvial floodplains that early post-war machinery could not cultivate; Figure 5). The development of new genotypes through plant breeding programmes and the incorporation of genetic engineering technology to increase disease resistance and productivity and facilitate weed control has also allowed farmers to increase output of food and fibre production4,54.\n\n(Photo: Leah MacKinnon with permission).\n\nTechnology is both positive and negative for biodiversity: positive in that many of the new technological advances have enabled farmers to better target their management interventions, thus sparing areas with higher biodiversity values (e.g. through the use of GPS to guide application of fertiliser or herbicide) and in being able to increase productivity in one part of the farm and spare or rest other areas and avoiding adjacent natural areas such as wetlands55. Technological advances have also enabled the application of more environmentally sustainable management practices such as direct drilling as opposed to ploughing56, especially where native pastures can be direct drilled with production species without affecting the native biodiversity present (called pasture-cropping57). However, the advent of new technology has also enabled farmers to farm new areas, especially areas that might otherwise have been marginal or too difficult to crop, and to increase the size of individual management units (e.g. paddocks) and farms in order to capitalise on economies of scale, further increasing impacts on native biodiversity through homogenising farmscapes8.\n\nSome technological advances have been both positive and negative. For example, direct drilling enhances soil condition by removing the need for cultivation, but the increased use of herbicides and spray drift may affect adjacent native ecosystems. Genetic engineering has allowed farmers to grow crops that are resistant to glyphosate and certain insect herbivores. Genetically modified glyphosate-resistant crops allow over-spraying with herbicides to control weeds in the crop, both with adverse effects due to herbicide drift into adjacent native vegetation and concomitant loss of habitat of native fauna58. In addition, increasing use of glyphosate has fostered selection of glyphosate-resistant weeds, which are now widespread across Australian irrigated farming districts59. However, the development through genetic engineering of Bacillus thuringiensis (Bacillaceae) insect resistance in commercial crops such as rice and cotton has been positive for native biodiversity, as it requires less pesticide application, which may well have benefits for native invertebrates60,61. However, genetic engineering has not yet been able to address abiotic stressors of plant productivity such as drought62, although Monsanto have recently developed a genetically modified form of maize including a gene that enables plants to decrease their soil water absorption rate under dry conditions63. Such developments could potentially provide farmers with another tool for improving resilience in the face of climate change, although traditional plant breeding at this stage appears just as effective as genetic modification63.\n\nKnowledge is a key driver of change in agricultural systems, both in relation to farming practices and biodiversity. Knowledge feeds into social values and awareness as well as having a direct impact on the way land is managed and hence on biodiversity. Much of the impact of past agricultural practices on biodiversity has occurred because farmers (and land managers more generally) have not had the relevant information to enable them to realise the adverse consequences of their activities. The recent recognition of the importance of biodiversity conservation in agricultural landscapes, especially in ‘new’ countries like Australia and New Zealand, has limited the attention that biologists have given to these areas. Several studies have highlighted the lack of ecological research undertaken in agricultural landscapes64,65. For example, a survey of New Zealand ecological literature published over the period 1968–1997, found that 65% of articles focused exclusively on protected areas while 18% focused exclusively on non-protected areas (mainly agricultural and plantation forest landscapes), despite non-protected areas accounting for nearly 70% of the total land area66. While native biodiversity in agricultural landscapes has received more attention in recent years7, there are still substantial gaps in our knowledge in terms of just what native biodiversity is present there, how this biodiversity is impacted by agricultural practices, and what ecosystem services this biodiversity provides to agricultural production specifically and society more generally.\n\n\nPolicy and regulation\n\nPolicy and regulation develop in part from social value systems, but in many ways are not in tune with current societal values or awareness of issues. This occurs because the politicians and bureaucrats who develop policy are often not willing to tackle current societal issues (as is clearly evidenced by the gun debate in the USA), perhaps because of the pressures associated with short election cycles, and because the vast majority of society simply does not have any idea of the diversity of issues that are regulated and hence is not involved in debates about them. This leaves policy making and regulation in the hands of a very small group of politicians, bureaucrats and lobby groups. In addition, historical legacies of past policy, including the case law associated with legislation, drive ongoing policy development. Notwithstanding this, policy and regulation exert a very powerful influence on land management decision making67, with both positive and negative outcomes for biodiversity. Policy influences decision making at every level from international to national and regional levels. Protectionist trade tariffs are one example where international policy can directly influence the types of decisions made by farmers30, for example on the type of farming that is undertaken. The removal of trade barriers is also likely to further facilitate the flow of invasive species as trade increases facilitating these species in reaching new areas through increased dispersal opportunities68.\n\nDomestic policy and associated regulation also have a marked impact on a farmer’s decision making. Regulation can be particularly poor in mitigating the many small incremental actions of multiple actors. This is illustrated by vegetation clearance rules that are widely used to restrict the amount of native vegetation cleared in Australia and New Zealand (Supplementary material). However, if every farmer in a region cleared what was legally possible under exemptions associated with such rules, biodiversity would continue to decline. Furthermore, some farmers look at vegetation clearance rules as a cost of business, with the fine or penalty associated with breaking the rule being trivial compared to the potential profits that would result from utilising newly cleared areas. Blunt regulatory instruments are weak at controlling gradual land-use intensification. (e.g. the loss of palatable native plants from fertilised and over-sown pastures subjected to increased stocking rates), nor do they deal with the habitat loss resulting from species invasions. Some policy can have unintended positive outcomes for biodiversity. In New Zealand, a change in government in the early 1980s resulted in the removal of a range of farm subsidies. As a result of this, fertiliser price increased locally, even though global fertiliser prices were low, resulting in farmers using less fertiliser and in some cases abandoning less productive land with many of these areas now regenerating back into native forest69,70.\n\n\nConclusions\n\nThe effects of different drivers on farmers’ decision making and hence on land management activities and native biodiversity are complex and often interrelated. The following example illustrates how two of these drivers can affect native biodiversity in a livestock production system through their influence on farm management practice. Both environmental change and the international market place can place substantial pressure on the profitability of a farm business, especially where financial commitments such as mortgage payments are an issue. In the case of environmental change, this can be through changes in the frequency and intensity of extreme climatic events such as droughts, heatwaves, floods, windstorms or frosts that are significant for farm production31,32. In the case of international markets, factors such as increasing costs of external inputs such as fuel and fertiliser coupled with fluctuating returns for farm products are critical. One response to these types of pressure is to intensify farm management practices in order to buffer the farm business against the vagaries of unpredictable weather or markets. Intensification can involve one or more management actions (the proximate drivers of change), including increasing the carrying capacity of existing pastures (e.g. through cultivation or topdressing with seed and fertiliser), bringing new land into production through native vegetation clearance, or by overgrazing the existing forage base (as a short-term strategy to cope with immediate financial challenge). All of these can result in land degradation and loss of native biodiversity.\n\nAgricultural systems are of course very diverse and each driver will have a different effect depending on the nature of the particular system including the type of farming system (grazing, cropping, irrigated, rain-fed, with or without woody vegetation, etc.), the productivity of the system (annual yield of grain, milk solids, meat, etc.), and the amount and distribution of native biodiversity present. The potential to intensify farm management for whatever reason is the primary cause of native biodiversity vulnerability30, as intensification almost always results in the loss of native species, both where this involves clearing areas of remnant native vegetation or through transformation of current farmed areas from low-producing to high-producing states. In contrast, native biodiversity in systems that have relatively little potential for intensification, either because they have already been intensively developed (e.g. irrigated arable or dairy farming systems) or because productivity is limiting and cannot be readily enhanced (e.g. rangeland systems lacking water), is likely to be less vulnerable.\n\nAn appreciation of the drivers of biodiversity change within agricultural ecosystems is important as it enables us to better plan management activities in order to achieve desired outcomes for native biodiversity. Distinguishing between direct and indirect drivers is particularly important, as it is the indirect or ultimate drivers that are likely to have the greatest influence on future biodiversity given that the primary use of these ecosystems is for the production of food and fibre. The drivers discussed here are ultimately responsible for most impacts on native biodiversity in agricultural systems, both directly but especially through their strong influence on farm management practice. Management in turn dictates the nature and habitat qualities of the production matrix and shifts remnants of native ecosystems into structural and compositional states further from their historical condition71. These changes are likely to require novel approaches for the long-term management of native biodiversity72 as well as an explicit evaluation of the value of this biodiversity, both for itself (intrinsic value) and for the ecosystem services that it provides to farm production and to society more generally.\n\nIn order to address the effects of the different drivers of native biodiversity in agricultural landscapes, mechanisms are required to encourage farmers to incorporate management of native species into overall farm management practices. In some cases, all that is required is to facilitate the farmer to continue with their existing management approach. However, more often mechanisms are required to assist farmers to change their management or adopt new practices that are more sympathetic to native biodiversity7. In selecting the most appropriate approach for doing this, it is necessary to consider the relative levels of public (external) and private (internal) benefits of a given land management practice73 and to ensure that these are appropriately accounted for. Simply putting in place regulations that limit carrying capacity or vegetation clearance do not necessarily address the underlying (ultimate) reasons driving management decisions. In fact, poorly thought through regulations can actually result in unintended or perverse outcomes (e.g. proposed vegetation clearance rules resulting in a flurry of clearance before regulations are enacted). Therefore, in order to address the indirect effects of the ultimate drivers of biodiversity change considered here, other approaches need to be considered. Such approaches could better align on-farm decision making with positive biodiversity outcomes, for example, by offering financial incentives to farmers to retain native vegetation and provide ecosystem services such as clean water and carbon sequestration74,75.",
"appendix": "Author contributions\n\n\n\nDN and NR conceived the article, LY researched examples to include, DN prepared the first draft, and DN, NR and LY substantially revised the article for publication and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by a grant provided to DN by Landcare Research (1213-27-006B).\n\n\nAcknowledgements\n\nWe thank Peter Bellingham and Jason Tylianakis for constructive comments on earlier versions of this article.\n\n\nSupplementary material\n\nExamples of a vegetation clearance rules in three New Zealand district plans (local government land planning statutes).\n\n(www.waikatodistrict.govt.nz/CMSFiles/ca/cab18ac7-cba6-49e1-888b-4c9d5efdc1cb.pdf)\n\nVegetation clearance of indigenous vegetation or habitat of indigenous fauna in a Landscape Policy Area or Conservation Policy Area is a permitted activity if:\n\n(a) it does not exceed 1000m2 or 1% of contiguous indigenous vegetation or habitat of indigenous fauna, whichever is the lesser, per contiguous area per site in any 3-year period, and is limited to:\n\n(i) a building platform for a permitted or approved building, or structure(s) or access, or\n\n(ii) gathering of plants in accordance with Maori custom and values, or\n\n(b) it does not exceed 3000m2 or 3% of contiguous indigenous vegetation or habitat of indigenous fauna, whichever is the lesser, per contiguous area per site in any 3-year period, and is limited to:\n\n(i) maintaining or reinstating productive pasture and productive forests and maintenance of tracks and fences through the removal of manuka and/or kanuka and/or treeferns that are more than 10m from a water body and less than 15 years old or less than 5m in height and any under-storey under such manuka or kanuka or treeferns growing on land that was previously in productive use.\n\n(www.hurunui.govt.nz/assets/Documents/District%20Plan/2-A-07%20Natural%20Environment.pdf)\n\n(d) Wetlands\n\nThere shall be no clearance or cultivation or damage by deposition of material or infilling of indigenous vegetation in any wetland exceeding 1000 square metres in area.\n\n(e) Clearance of indigenous vegetation\n\n(i) No clearance of indigenous vegetation not already significantly modified by any farming practice, other than as provided for in\n\n- Section A2 – Landscape\n\n- Section A7 – Natural Environment\n\n- Section B2 – Coastal Environment\n\n- Section B3 – Hurunui Lakes Area\n\nshall be permitted of greater than 1 hectare over a 5-year period on any separate certificate of title.\n\n(www.mackenzie.govt.nz/Site/Documents_and_policy/key_documents/district_plan.aspx)\n\nShort Tussock Grasslands\n\nOn each of the individual farm properties existing in the Mackenzie Basin Map as at 1 January 2002 in any continuous period of five years there shall be no clearance including cultivation above the following thresholds of short tussock grasslands, consisting of silver or blue (Poa species), or Elymus solandri, or fescue tussock where tussocks exceed 15% canopy cover:\n\n(i) 40 hectares or less – Permitted Activity\n\n(ii) Greater than 40 hectares – Discretionary Activity.\n\n\nReferences\n\nEllis EC, Ramankutty N: Putting people in the map: anthropogenic biomes of the world. Front Ecol Environ. 2008; 6: 439–447. Publisher Full Text\n\nAnon World Population Prospects: The 2010 Revision, Highlights and Advance Tables. Department of Economic and Social Affairs, Population Division Working Paper No. ESA/P/WP.220. United Nations, New York. 2011.Reference Source\n\nUnited Nations Development Programme. The Rise of the South: Human Progress in a Diverse World. Human Development Report 2013. United Nations Development Programme, New York. 2013. Reference Source\n\nTilman D, Cassman KG, Matson PA, et al.: Agricultural sustainability and intensive production practices. Nature. 2002; 418(6898): 671–677. PubMed Abstract | Publisher Full Text\n\nLindenmayer D, Cunningham S, Young A: Land Use Intensification: Effects on Agriculture, Biodiversity and Ecological Processes. CSIRO Publishing, Australia. 2012. Reference Source\n\nCrosby AW: Ecological Imperialism: The Biological Expansion of Europe, 900–1900. Revised edition. Cambridge University Press, Cambridge. 2004. Reference Source\n\nNorton D, Reid N: Nature and Farming: Sustaining Native Biodiversity in Agricultural Landscapes. CSIRO Publishing, Melbourne. 2013. Reference Source\n\nLockwood JL, McKinney ML: Biotic Homogenisation. Kluwer Academic, New York. 2001. Publisher Full Text\n\nMillennium Ecosystem Assessment Ecosystems and Human Well-being. Synthesis. Island Press, Washington D.C. 2005. Reference Source\n\nMattison EH, Norris K: Bridging the gaps between agricultural policy, land-use and biodiversity. Trends Ecol Evol. 2005; 20(11): 610–616. PubMed Abstract | Publisher Full Text\n\nHobbs RJ, Norton DA: Towards a conceptual framework for restoration ecology. Restoration Ecology. 1996; 4(2): 93–110. Publisher Full Text\n\nNelson GC, Bennett E, Berhe AA, et al.: Anthropogenic drivers of ecosystem change: an overview. Ecology and Society. 2006; 11(2). 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\nDidham RK, Tylianakis JM, Gemmell NJ, et al.: Interactive effects of habitat modification and species invasion on native species decline. Trends Ecol Evol. 2007; 22(9): 489–496. PubMed Abstract | Publisher Full Text\n\nSala OE, Chapin FS 3rd, Armesto JJ, et al.: Global biodiversity scenarios for the year 2100. Science. 2000; 287(5459): 1770–1774. PubMed Abstract | Publisher Full Text\n\nNorris K: Agriculture and biodiversity conservation: opportunity knocks. Conservation Letters. 2008; 1(1): 2–11. Publisher Full Text\n\nScherr SJ, McNeely JA: Biodiversity conservation and agricultural sustainability: towards a new paradigm of 'ecoagriculture' landscapes. Philos Trans R Soc Lond B Biol Sci. 2008; 363(1491): 477–494. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalmford A, Green R, Phalan B: What conservationists need to know about farming. Proc Biol Sci. 2012; 279(1739): 2714–2724. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCramer VA, Hobbs RJ: Ecological consequences of altered hydrological regimes in fragmented ecosystems in southern Australia: impacts and possible management responses. Austral Ecol. 2002; 27(5): 546–564. Publisher Full Text\n\nSuding KN, Hobbs RJ: Threshold models in restoration and conservation: a developing framework. Trends Ecol Evol. 2009; 24(5): 271–279. PubMed Abstract | Publisher Full Text\n\nReid N, Landsberg J: Tree decline in agricultural landscapes: what we stand to lose. In, Hobbs RJ,Yates CJ, eds.Temperate Eucalypt Woodlands in Australia: Biology, Conservation, Management and Restoration. Surrey Beatty & Sons, Chipping Norton, Victoria, 2000; pp 127–166. Reference Source\n\nReid N: Maintaining tree cover health in temperate pastoral landscapes. In, Barlow T, Thorburn R, eds. Balancing Conservation and Production in Grassy Landscapes. Environment Australia, Biodiversity Group, Canberra, 2000; pp 177–187. Reference Source\n\nLumsden LF, Bennett AF: Bats in rural landscapes: a significant but largely unknown faunal component. In, Barlow T, Thorburn R, eds.Balancing Conservation and Production in Grassy Landscapes. Environment Australia, Biodiversity Group, Canberra, 2000; pp 42–50. Reference Source\n\nMaron M: Agricultural change and paddock tree loss: implications for an endangered subspecies of red-tailed black-cockatoo. Ecological Management and Restoration. 2005; 6(3): 206–211. Publisher Full Text\n\nSchrott GR, With KA, King AW: On the importance of landscape history for assessing extinction risk. Ecological Applications. 2005; 15: 493–506. Publisher Full Text\n\nTilman D, May RM, Lehman CL, et al.: Habitat destruction and the extinction debt. Nature. 1994; 371: 65–66. Publisher Full Text\n\nMurcia C: Edge effects in fragmented forests: implications for conservation. Trends Ecol Evol. 1995; 10(2): 58–62. PubMed Abstract | Publisher Full Text\n\nHobbs RJ, Huenneke LF: Disturbance, diversity, and invasion: implications for conservation. Conserv Biol. 1992; 6(3): 324–337. Publisher Full Text\n\nDiamond JM: The island dilemma: lessons of modern biogeographic studies for the design of natural reserves. Biol Conserv. 1975; 7(2): 129–146. Publisher Full Text\n\nDidham RK, Denmead LH, Deakin EL: Riches to rags: the ecological consequences of land use intensification in New Zealand. In, Lindenmayer D, Cunningham S, Young A, eds. Land Use Intensification: Effects on Agriculture, Biodiversity and Ecological Processes. CSIRO Publishing, Australia, 2012; pp 73–83. Reference Source\n\nSillmann J, Roeckner E: Indices for extreme events in projections of anthropogenic climate change. Climatic Change. 2008; 86(1–2): 83–104. Publisher Full Text\n\nSmith MD: The ecological role of climate extremes: current understanding and future prospects. Journal of Ecology. 2011; 99(3): 651–655. Publisher Full Text\n\nWalther GR, Post E, Convey P, et al.: Ecological responses to recent climate change. Nature. 2002; 416(6879): 389–395. PubMed Abstract | Publisher Full Text\n\nTylianakis JM, Didham RK, Bascompte J, et al.: Global change and species interactions in terrestrial ecosystems. Ecol Lett. 2008; 11(12): 1351–1363. PubMed Abstract | Publisher Full Text\n\nHowden SM, Soussana JF, Tubiello FN, et al.: Adapting agriculture to climate change. Proc Natl Acad Sci U S A. 2007; 104(50): 19691–19696. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWoodman RF: Trifolium ambiguum. (Caucasian clover) in montane tussock grasslands, South Island, New Zealand. New Zealand Journal of Agricultural Research. 1999; 42(3): 207–222. Publisher Full Text\n\nMaron M, Fitzsimons JA: Agricultural intensification and loss of matrix habitat over 23 years in the west Wimmera, south-eastern Australia. Biological Conservation. 2007; 135(4): 587–593. Publisher Full Text\n\nWeeks ES, Walker S, Dymond JR, et al.: Patterns of past and recent conversion of indigenous grasslands in the South Island, New Zealand. New Zealand Journal of Ecology. 2013; 37(1): 127–138. Reference Source\n\nQuinn JM, Stroud MJ: Water quality and sediment and nutrient export from New Zealand hill-land catchments of contrasting land use. New Zealand Journal of Marine and Freshwater Research. 2002; 36(2): 409–429. Publisher Full Text\n\nLee WG, Williams P, Cameron E: Plant invasions in urban environments: the key to limiting new weeds in New Zealand. In, Suckling DM, Stevens PS, eds. Managing Urban Weeds and Pests. Proceedings of a New Zealand Plant Protection Society Symposium, 9 August, 1999. The New Zealand Plant Protection Society, Lincoln, 2000; pp 43–58.\n\nMack RN, Simberloff D, Lonsdale WM, et al.: Biotic invasions: causes, epidemiology, global consequences, and control. Ecological Applications. 2000; 10(3): 689–710. Publisher Full Text\n\nEspie PR: Hieracium in New Zealand: Ecology and Management. AgResearch, Mosgiel, New Zealand. 2001; 66. Reference Source\n\nLambin EF, Geist HJ, Lepers E: Dynamics of land-use and land-cover change in tropical regions. Annual Review of Environment and Resources. 2003; 28: 205–241. Publisher Full Text\n\nGardiner B, Reid N: Sustainable livestock production. In, Cottle D, ed. International Sheep and Wool Handbook. Nottingham University Press, Nottingham, 2010; pp 445–471. Reference Source\n\nReid N, Norton D, Stafford Smith M: Environmental management. In, Cottle D, Kahn L, eds. Beef Cattle Production and Trade. CSIRO Publishing, Victoria, in press. 2013.\n\nMacLeod CJ, Moller H: Intensification and diversification of New Zealand agriculture since 1960: an evaluation of current indicators of land use change. Agriculture, Ecosystems and Environment. 2006; 115(1–4): 201–218. Publisher Full Text\n\nCzúcz B, Gathman JP, McPherson GR: The impending peak and decline of petroleum production: an underestimated challenge for conservation of ecological integrity. Conserv Biol. 2010; 24(4): 948–956. PubMed Abstract | Publisher Full Text\n\nPark G: Nga Uruora/the Groves of Life: Ecology and History in a New Zealand Landscape. Victoria University Press, Wellington. 1995. Reference Source\n\nO’Connor KF: The implications of past exploitation and current developments to the conservation of South Island tussock grasslands. New Zealand Journal of Ecology. 1982; 5: 97–107. Reference Source\n\nMaclaren JP: Environmental Effects of Planted Forests in New Zealand. New Zealand Forest Research Institute, Rotorua. 1996. Reference Source\n\nKadlec RH, Wallace SD: Treatment Wetlands, second edition. CRC Press. Boca Raton, FL 2009. Reference Source\n\nFreebairn DM, Wockner GH: A study of soil erosion on vertisols of the eastern Darling Downs, Queensland. I. Effects of surface conditions on soil movement within contour bay catchments. Australian Journal of Soil Research. 1986; 24(2): 135–158. Publisher Full Text\n\nMoller H, MacLeod CJ, Rosin C, et al.: Intensification of New Zealand agriculture: implications for biodiversity. New Zealand Journal of Agricultural Research. 2008; 51(3): 253–263. Publisher Full Text\n\nHuffman WE, Evenson RE: Science for Agriculture: A Long Term Perspective. Iowa State University Press, Ames IA. 1993. Reference Source\n\nTscharntke T, Batary P, Clough Y, et al.: Combining biodiversity conservation with agricultural intensification. In, Lindenmayer D, Cunningham S, Young A, eds. Land Use Intensification: Effects on Agriculture, Biodiversity and Ecological Processes, CSIRO Publishing, Australia, 2012; pp 7–15. Reference Source\n\nPeigné J, Ball BC, Roger-Estrade J, et al.: Is conservation tillage suitable for organic farming? A review. Soil Use and Management. 2007; 23(2): 129–144. Publisher Full Text\n\nMillar GD, Badgery WB: Pasture cropping: a new approach to integrate crop and livestock farming systems. Animal Production Science. 2009; 49(10): 777–787. Publisher Full Text\n\nCerdeira AL, Duke SO: The Current Status and Environmental Impacts of Glyphosate-Resistant Crops: A Review. J Environ Qual. 2006; 35(5): 1633–1658. PubMed Abstract | Publisher Full Text\n\nOwen MJ, Powles SB: Glyphosate-resistant rigid ryegrass (Lolium rigidum) populations in the Western Australian grain belt. Weed Technology. 2010; 24(1): 44–49. Publisher Full Text\n\nWhitehouse MEA, Wilson LJ, Fitt GP: A comparison of arthropod communities in transgenic Bt and conventional cotton in Australia. Environmental Entomology. 2005; 34(5): 1224–1241. Publisher Full Text\n\nCattaneo MG, Yafuso C, Schmidt C, et al.: Farm-scale evaluation of the impacts of transgenic cotton on biodiversity, pesticide use and yield. Proc Natl Acad Sci U S A. 2006; 103(20): 7571–7576. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSinclair TR: Challenges in breeding for yield increase for drought. Trends Plant Sci. 2011; 16(6): 289–293. PubMed Abstract | Publisher Full Text\n\nEisenstein M: Plant breeding: Discovery in a dry spell. Nature. 2013; 501(7468): S7–S9. PubMed Abstract | Publisher Full Text\n\nRobertson AI: The gaps between ecosystem ecology and industrial agriculture. Ecosystems. 2000; 3(5): 413–418. Publisher Full Text\n\nStiling P: What do ecologists do? Bulletin of the Ecological Society of America. 1994; 75(2): 116–121. Reference Source\n\nNorton DA: Is the amount and focus of ecological research in New Zealand sufficient to sustain indigenous biodiversity on private land? New Zealand Journal of Ecology. 2001; 25: 77–82. Reference Source\n\nHaggerty J, Campbell H, Morris C: Keeping the stress off the sheep? Agricultural intensification, neoliberalism, and ‘good’ farming in New Zealand. Geoforum. 2009; 40(5): 767–777. Publisher Full Text\n\nHulme PE: Trade, transport and trouble: managing invasive species pathways in an era of globalization. J Appl Ecol. 2009; 46(1): 10–18. Publisher Full Text\n\nWilson H: Living in Raoul country: the changing flora and vegetation of Banks Peninsula. In, Burrows CJ, ed. Etienne Raoul and Canterbury Botany 1840-1996. Canterbury Botanical Society, Christchurch, 1998; pp 101–121. Reference Source\n\nStandish RJ, Sparrow AD, Williams PA, et al.: A state-and –transition model for the recovery of abandoned farmland in New Zealand. In, Hobbs RJ, Suding KN, eds.New Models for Ecosystem Dynamics and Restoration. Island Press, Washington, 2009; pp 189–205. Reference Source\n\nHobbs RJ, Arico S, Aronson J, et al.: Novel ecosystems: theoretical and management aspects of the new ecological world order. Global Ecology and Biogeography. 2006; 15(1): 1–7. Publisher Full Text\n\nSeastedt TR, Hobbs RJ, Suding KN: Management of novel ecosystems: are novel approaches required? Front Ecol Environ. 2008; 6(10): 547–553. Publisher Full Text\n\nPannell DJ: Public benefits, private benefits, and policy intervention for land-use change for environmental benefits. Land Economics. 2008; 84: 225–240. Reference Source\n\nMorton SR, Stafford Smith DM, Friedel MH, et al.: The stewardship of arid Australia: ecology and landscape management. J Environ Manage. 1995; 43(3): 195–217. Publisher Full Text\n\nSalzman J: Creating markets for ecosystem services: notes from the field. New York University Law Review. 2005; 80(6): 870–961. Reference Source"
}
|
[
{
"id": "2330",
"date": "20 Dec 2013",
"name": "Bruce Clarkson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 insightful opinion piece and deserving of publication. The introduction in particular is succinct and to the point. The key scene setting statement for me is: “As a general rule however, remnant taxa tend to be generalists and agricultural development usually results in homogenisation of native biodiversity”. I have twice in my career said as much in \"Conservation at the end of the century: challenges, crises and contradictions\"1 and Lee, Meurk & Clarkson, 2008 - “only a small generalist subset of the indigenous flora and fauna can persist in, or recolonise, intensified agricultural landscapes”2.The statement is critical because it begs the question is it even realistic to think that indigenous biodiversity can ever be adequately represented within essentially intensive productive agricultural lands?My only criticisms (minor) of the paper are around areas where I would argue for a different or slightly different emphasis. We all have different backgrounds and so depending on our experiences there are likely to be questions of emphasis. I deal with these in turn below:Page 5: left hand column last sentence regarding good financial returns; the non-essential activities are perceived non-essential activities, which if an ecosystems services or natural capital accounting model was in place may not be viewed as non-essential.Page 5: right hand column last paragraph; the European settler approach was also a pragmatic one, driven (economic driver) by the products required for export to the home country. As the historian WH Oliver has succinctly noted, this agricultural land use was driven by the imperative to survive in a new land by producing trade goods demanded by colonial masters across the sea, and the land has disproportionately suffered as a result. The settlers were just responding to a market signal, and there was no other market?Page 6: left hand column last sentence; seems that this is one place that the lack of comment policy and regulatory incentives, and the development of self-organised responses to the problem e.g. QE II Trust and Landcare groups, could have been addressed? Additionally the Lee et al., 2008 paper2, in response to the Moller et al., 2008 article3, took a view that regulation needed more emphasis than incentive, but even that may still be insufficient to stop an extinction pathway for indigenous biodiversity under agricultural intensification?Page 7: left column top para; it's not just new technology that enables development of previously marginal lands, but ready access and/or cheaper availability of existing technology e.g. the swamp forests on the Egmont ringplain habitat reduction by modern drain digger vehicles.Page 7: left column last para; the claim that farmers lacked the relevant information led to an inability to realise adverse consequences of their activities seems too simplistic. What about barriers to adoption, the need for first adopters, lack of awareness, failure to observe nature, ignorance, bloody mindedness, deliberate ignoring of warnings. There are multiple reasons for a lack of realisation?Page 7: Policy and regulation section takes a very narrow view focusing on vegetation clearance rules by themselves, but without any quantification of how clearance rules may have slowed clearance or how when coupled with incentives e.g. covenanting incentives linked to SNA scheduling to retain has had a positive impact on the Egmont ringplain.Page 8 Conclusions: I reiterate here the lack of mention of policy and regulatory incentives and the development of self-organised responses to the problem e.g. QE II Trust and Landcare groups. Making statements like \"..mechanisms are required to encourage farmers…\" or \"simply putting in place regulations… do not necessarily address…\"or \"…other approaches need to be considered\" just begs a lot a questions and a single answer by offering financial incentives is not nuanced enough: what types of incentives? Environmental, Biodiversity or Carbon Credits, tax relief? Rates relief? These are positive financial incentives but what about negative financial incentives, such as trading barriers or forms of environmental accreditation etc. Is it other approaches or just a wider diversity of approaches including regulation an positive and negative financial incentives?And who is responsible for doing this - Central Government and Local Government?And if all of this new approach is put in place, is the prognosis for native biodiversity in agricultural systems: very optimistic, reservedly optimistic or not optimistic?A valuable contribution; thanks for the opportunity to comment.",
"responses": []
},
{
"id": "3305",
"date": "10 Feb 2014",
"name": "Rachel Standish",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAs the title of this paper suggests, it reviews the ultimate drivers of biodiversity change in agricultural systems using examples from eastern Australia and New Zealand. The authors are well-respected for their contribution to ecological research, and particularly for informing the options for the conservation of biological diversity in these landscapes. Therefore, the review is as I’d expect it to be—authoritative and conceptually sound. In particular, Figure 1 will be a useful framework to prompt discussions among land managers and other stakeholders. An example of a decision informed by the framework would be a valuable addition to the paper.More generally, however, the review paints a fairly pessimistic view for biodiversity conservation in agricultural landscapes and reading it left me wanting to know more about the strategies mentioned in the final sentence of the abstract. The authors deliberately reviewed the ultimate drivers of biodiversity change, because unlike proximate drivers, these can be managed—but how? For example, what ecologists might do to fill the knowledge gaps? How might the emerging carbon market be developed to benefit biodiversity and farmers in agricultural landscapes of Australia and New Zealand 1? What are some other examples of potential win-win farming enterprises? How might trade-offs among ecosystem services, including both ecological and socio-economic perspectives, affect outcomes for biodiversity in these landscapes? There is some evidence that traditional, as opposed to novel, approaches such as fencing to exclude livestock and unassisted regeneration of abandoned farmlands can help biodiversity conservation in agricultural landscapes2. What are the incentives for farmers to adopt such practices?These are my other, more minor points:Historical legacies are particularly important in the ancient landscapes of south-western Australia 34. Land-use legacies combine with seed dispersal and recruitment limitations to form ecological thresholds to recovery 5 6. The socio-economic drivers of land-use change are also documented for this region 78. I agree that land-use intensification generally results in the loss of native species, but what about the options for biodiversity conservation at larger scales if intensification is paired with land-sparing? Indeed, the current global debate about land-sparing vs sharing would be relevant to mention here. Nested within social values and awareness are the management goals we as a society set for biodiversity conservation. I think this section of the paper could introduce some of the novel approaches mentioned in the discussion. For example, where and when might it be appropriate to consider novel ecosystems as goals for management in place of historic ecosystems? While it is probably true that agricultural landscapes are understudied compared with other landscapes in Australia as well as New Zealand, this point seems to detract from the arguably more valid point that there’s decades worth of research from which to base good decisions 9. Thank you for the invitation to review this paper. I enjoyed thinking about it.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-214
|
https://f1000research.com/articles/2-213/v1
|
11 Oct 13
|
{
"type": "Case Report",
"title": "Nodular clear cell hidradenoma: a rare case with similarities to metastatic clear renal cell carcinoma",
"authors": [
"José M Mir-Bonafé",
"Ángel Santos-Briz",
"Marta González",
"Emilia Fernández-López",
"Ángel Santos-Briz",
"Marta González",
"Emilia Fernández-López"
],
"abstract": "Hidradenoma is presented as a benign small, firm and solitary nodule. Cytological study reveals a lesion mainly composed by two types of cells. The majority of the cases show a greater amount of a polyhedral and slightly basophilic cell type. Here we present a case with a predominance of a larger, rounded-shaped pale to clear cell type, which is characteristic of clear cell hidradenoma. Our case is of interest as, to the authors’ knowledge, no other related publication has described such striking similarities to metastatic clear renal cell carcinoma. Since the prognosis of these two conditions differs dramatically, a differential diagnosis based on immunohistochemistry is highlighted.",
"keywords": [
"clear cell hidradenoma",
"renal carcinoma",
"immunochemistry",
"cutaneous metastasis."
],
"content": "Introduction\n\nWe read with interest a review of 15 cases of nodular hidradenoma published recently in the American Journal of Dermat opathology1, in which Nandeesh and Rajalakshmi did not demonstrate a preponderance of clear cell populations in any of the cases. Additionally, they stated that clear cells constituted no more than 30% of the total cells in all cases1. Interestingly, here we report a rare case of nodular hidradenoma with a vast preponderance of large clear cells. To date, none of the small number of similar publications2 have described the striking similarities to metastatic cutaneous renal cell carcinoma presented in our patient.\n\n\nCase report\n\nA 60-year-old female patient was referred to the Department of Dermatology, University Hospital of Salamanca, Spain, after a growing lesion appeared on her face over the preceding 6 months. Physical examination revealed a well-demarcated reddish nodule of 0.7cm in size located on the glabellar prominence. No ulceration was noted. On palpation, a firm, dermal round lesion was noticed. Total surgical excision was performed. The specimen was fixed with paraffin. Histopathological examination (hematoxylin and eosin, viewed at ×40) demonstrated a well-circumscribed but non-encapsulated, expansive-growing nodule within the deep and mid-dermis (Figure 1a). This tumor was mainly composed of clear cells separated by fine conjunctive-vascular septae. These cells showed a large empty cytoplasm, medium-small eccentric nuclei and coarse chromatin (Figure 1b). Atypia, necrosis and mitotic figures were absent. The periphery of the lesion presented papillae and tubular formations accompanied by two alternating types of cells: those that were smaller and basophilic and those with abundant clear cytoplasm (Figure 1c). Tubular ducts were covered by cubical to cylindrical cells. Cystic areas filled by eosinophilic fluid were also present. Immunohistochemistry was positive for CK7, AE1/AE3, and slightly for p63. Conversely, negativity for CK20, CD10, Napsin-A, Vimentin and Actin was demonstrated. On account of these findings, our patient was diagnosed with a nodular variant of clear cell hidradenoma (CCH). In addition, renal ultrasonography examinations ruled out any neoplasm.\n\na) Microphotograph showing a well-circumscribed but non-encapsulated, expansive-growing nodule located in deep and mid-dermis (hematoxylin and eosin, ×40). b) Microphotograph showing clear cells with a large empty cytoplasm, medium-small eccentric nuclei and coarse chromatin (hematoxylin and eosin, ×400). c) Microphotograph showing the periphery of lesion, which presented papillae and tubular formations accompanied by two alternating types of cells: those smaller and basophilic and those with abundant clear cytoplasm (hematoxylin and eosin, ×400).\n\n\nDiscussion\n\nHidradenoma is presented as a benign small, firm and solitary nodule. On microscopic examination, spared epidermis may be separated with a dermal well-circumscribed tumor by a Grenz zone. It may be encapsulated and involvement of subcutaneous fat tissue is uncommon3. Cytological study reveals a lesion mainly composed by two types of cells2,4. Firstly, hidradenoma may show a greater amount of a polyhedral and slightly basophilic cell type. On the other hand, a predominance of larger, rounded-shaped pale to clear cell type is characteristic of CCH, as in our case. In the latter form, nuclei are frequently located eccentrically1. CCH shows small ductular lumens accompanied by changes from apocrine glands or from epidermoid cells. Glandular structures compounded by columnar cells are frequently appreciable. Clear cells contain a variable amount of glycogen within their cytoplasm, unaccompanied by lipid material, which is responsible of their clear component. This material is periodic acid-Schiff–positive and diastase-labile1. Solid portions may alternate with cystic areas, which might be materialized as a result of tumor degeneration.\n\nDifferential diagnosis between metastatic clear renal cell carcinoma (MCRCC) and CCH may be confusing as both display clear cell preponderance, and it is essential to exclude metastatic disease because of its poor prognosis. Because of two recently reported cases of renal medullary carcinoma metastatic to cranial skin5,6, we were also obliged to rule out this possibility. Thus, immunohistochemistry becomes absolutely necessary in order to rule out its mimics2. Dorairajan et al.7 stated that skin metastasis may vary from 2.8% to 6.3% in renal cell carcinoma patients. A remarkable case of a patient with both an axillary CCH and a renal cell carcinoma was reported by Volmar et al.2. These authors emphasized the importance of immunohistochemistry in distinguishing between these entities. MCRCC has positive staining for CD10, Napsin-A and Vimentin, all of which were negative in our patient. In contrast, CCH expresses CK7+, AE1/AE3+ and CK20-2. These results were consistent with those observed in our patient. Some authors have reported a variable range of staining for CK6/18, CK8/18, CK10, CK198, 34bE12, carcinoembryonic antigen and CAM 5.29.\n\nIn summary, we report a rare case of nodular clear cell hidradenoma on glabellar prominence. This case is of interest since none of the 15 cases of nodular hidradenoma recently reviewed showed a preponderance of clear cells1. Additionally, it is also remarkable that no related publication2,5–7 have exhibited the striking similarities to renal cell carcinoma presented in our patient.\n\nIn order to avoid serious pitfalls, since the prognosis of these two conditions differs dramatically, a differential diagnosis based on immunohistochemistry is highlighted.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nJosé M. Mir-Bonafé: drafting the initial article Ángel Santos-Briz: clinical images and histologic description Marta González: patient's doctor and revising the manuscript Emilia Fernández-López: revising the whole manuscript and corrected errors.\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\nNandeesh BN, Rajalakshmi T: A Study of Histopathologic Spectrum of Nodular Hidradenoma. Am J Dermatopathol. 2012; 34(5): 461–470. PubMed Abstract | Publisher Full Text\n\nVolmar KE, Cummings TJ, Wang WH, et al.: Clear cell hidradenoma: a mimic of metastatic clear cell tumors. Arch Pathol Lab Med. 2005; 129(5): e113–6. PubMed Abstract | Publisher Full Text\n\nHernandez-Perez E, Cestoni-Parducci R: Nodular hidradenoma and hidradenocarcinoma. A 10-year review. J Am Acad Dermatol. 1985; 12(1 Pt 1): 15–20. PubMed Abstract | Publisher Full Text\n\nGupta R, Singh S, Gupta K, et al.: Clear-cell hidradenoma in a child: a diagnostic dilemma for the cytopathologist. Diagn Cytopathol. 2009; 37(7): 531–3. PubMed Abstract | Publisher Full Text\n\nJohnson RP, Krauland K, Owens NM, et al.: Renal medullary carcinoma metastatic to the scalp. Am J Dermatopathol. 2011; 33(1): e11–3. PubMed Abstract | Publisher Full Text\n\nNava VE, Mullin BR, Schuldenfrei JA: Renal medullary carcinoma metastatic to forehead. Am J Dermatopathol. 2011; 33(7): 757. PubMed Abstract | Publisher Full Text\n\nDorairajan LN, Hemal AK, Aron M, et al.: Cutaneous metastases in renal cell carcinoma. Urol Int. 1999; 63(3): 164–7. PubMed Abstract | Publisher Full Text\n\nBiernat W, Kordek R, Wozniak L: Phenotypic heterogeneity of nodular hidradenoma. Immunohistochemical analysis with emphasis on cytokeratin expression. Am J Dermatopathol. 1996; 18(6): 592–6. PubMed Abstract\n\nHaupt HM, Stern JB, Berlin SJ: Immunohistochemistry in the differential diagnosis of nodular hidradenoma and glomus tumor. Am J Dermatopathol. 1992; 14(4): 310–4. PubMed Abstract"
}
|
[
{
"id": "3657",
"date": "19 Feb 2014",
"name": "David M Nanus",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA well written case report on a rare skin lesion. This is an original report, with sufficient detail and literature review. This is extremely rare, and would be of most relevance to the dermatologist, in contrast to the oncologist.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-213
|
https://f1000research.com/articles/2-212/v1
|
11 Oct 13
|
{
"type": "Research Article",
"title": "Renal corpuscle and tubule morphology in ephrin-A2-/-, ephrin-A5-/- and ephrin-A2A5-/- mice",
"authors": [
"Andrea E. Bertram",
"Robert J. Dugand",
"Clodagh Guildea",
"Samantha Lostrom",
"Gastor Lyakurwa",
"Alexandra Windsor",
"Marissa Penrose-Menz",
"Tom Stewart",
"James E. O’Shea",
"Jennifer Rodger",
"Andrea E. Bertram",
"Robert J. Dugand",
"Clodagh Guildea",
"Samantha Lostrom",
"Gastor Lyakurwa",
"Alexandra Windsor",
"Marissa Penrose-Menz",
"Tom Stewart",
"James E. O’Shea"
],
"abstract": "The B family of Eph receptor tyrosine kinases and their ephrin ligands, best known for their role in the development of the nervous and vascular systems, have recently been implicated in mammalian kidney development and maintenance. However, the renal expression and function of the EphA and ephrin-A families have not been investigated. We performed immunohistochemistry for ephrin-A2 and ephrin-A5 in kidneys of normal adult wildtype (WT) mice and carried out quantitative morphological analysis of renal corpuscles and tubules in haematoxylin- and eosin-stained sections of WT, ephrin-A2-/-, ephrin-A5-/- and ephrin-A2A5-/- (knockout) mice. Ephrin-A2 and ephrin-A5 were strongly expressed in the tubules and glomeruli of the adult mouse kidney. Despite the significant overlap in expression between the two proteins, only the lack of ephrin-A5 had an effect on kidney morphology with glomerular size being mildly reduced in mice lacking the gene for ephrin-A5. However, the magnitude of this change was very small and could only be detected when animals were pooled across genotypes lacking ephrin-A5. The subtle phenotype, together with the relatively infrequent incidence of kidney failure in our breeding colony, suggest that ephrin-A2 and ephrin-A5 play only minor roles in kidney development and function. It is likely that other members of the ephrin-A family are expressed in the mouse kidney and redundancy within this large family of “promiscuous” signalling molecules may compensate for the loss of individual proteins in knockout mice.",
"keywords": [
"Normal kidney development and function requires coordinated cell signalling through complex molecular pathways1. The Eph receptor tyrosine kinases and their ephrin ligands",
"best known for their role in the development of the nervous and vascular systems",
"have recently been implicated in mammalian kidney development and maintenance2–4. Ephs and ephrins are divided into A and B subclasses",
"with ephrin-As typically binding to EphAs and ephrin-Bs to EphBs. Exceptions to this rule are ephrin-B2 and B3",
"which can bind to EphA4",
"and ephrin-A5",
"which can bind to EphB25",
"6. Within the kidney",
"EphBs and ephrin-Bs have been implicated in the development and maintenance of the slit diaphragm2",
"a component of the filtration barrier in the glomerulus",
"and also in renal tubule development4. However",
"the expression and function of the other family of ligands",
"the ephrin-As",
"has not been investigated."
],
"content": "Introduction\n\nNormal kidney development and function requires coordinated cell signalling through complex molecular pathways1. The Eph receptor tyrosine kinases and their ephrin ligands, best known for their role in the development of the nervous and vascular systems, have recently been implicated in mammalian kidney development and maintenance2–4. Ephs and ephrins are divided into A and B subclasses, with ephrin-As typically binding to EphAs and ephrin-Bs to EphBs. Exceptions to this rule are ephrin-B2 and B3, which can bind to EphA4, and ephrin-A5, which can bind to EphB25,6. Within the kidney, EphBs and ephrin-Bs have been implicated in the development and maintenance of the slit diaphragm2, a component of the filtration barrier in the glomerulus, and also in renal tubule development4. However, the expression and function of the other family of ligands, the ephrin-As, has not been investigated.\n\nA possible role for ephrin-As in kidney development and/or function was suggested by sporadic unexpected deaths in a breeding colony of mice lacking ephrin-A2 and/or ephrin-A5 (6.25% over a period of 4 years). Autopsy revealed that these mice had a single remaining kidney that had partially or fully degenerated to a fluid-filled sac. Subsequent dissections of mice used for other research purposes confirmed that mice lacking ephrin-A5 (both ephrin-A5-/- single knockouts and ephrin-A2A5-/- double knockouts, but not ephrin-A2-/- single knockout mice) often had only one kidney with the other either absent, abnormally small, or degenerating. To further investigate a possible role for ephrin-A5, we performed immunohistochemistry for ephrin-A2 and ephrin-A5 in kidneys of normal adult WT mice and carried out quantitative morphological analysis of renal corpuscles and tubules in haematoxylin and eosin stained sections of WT, ephrin-A2-/-, ephrin-A5-/- and ephrin-A2A5-/- (knockout) mice.\n\n\nMaterials and methods\n\nEphrin-A2-/-, ephrin-A5-/- and ephrin-A2A5-/- knockout mice were a generous gift from Prof David Feldheim7. These mice were backcrossed to C57Bl/6j strain for more than 13 generations and were maintained in a breeding colony at the University of Western Australia. Wild-type (WT) mice from the same genetic background (C57Bl/6j) were used as controls. Mice were housed in standard cages with clear plastic walls (17 cm × 19 cm base, 16 cm high) in a controlled environment (12/12 light/dark cycle; temperature 22°C±2°C) with food and water ad libitum. Kidneys were collected from terminally euthanased mice (160 mg/kg pentobarbitone, i.p.; Lethabarb, Virbac Australia) after transcardial perfusion with 4% paraformaldehyde (Sigma Aldrich, St Louis, Montana USA) and preserved in 4% paraformaldehyde for up to 24 months following sacrifice. The histology of renal corpuscles and tubules was examined within one kidney from six ephrin-A5 knockout mice, five ephrin-A2A5 knockout mice, three ephrin-A2 knockout mice, and seven wild type mice. Mice were aged 50–189 days at the time of sacrifice and age was included as a factor in our analysis. All procedures in this study were conducted in accordance with US NIH guidelines and approved by The University of Western Australia Animal Ethics Committee.\n\nFor immunohistochemistry, kidneys were dissected through the mid-sagittal line and were cryopreserved in 30% sucrose (VWR international BVBA, Belgium, 27480.360) in PBS overnight before freezing and cryosectioning on a Leica Cryostat CM1900 at -19°C. Sections were cut at 30 µm free-floating into PBS (NaCl, VWR international BVBA, Belgium, 27810.364, KCl, Chemsupply, Australia, Na2HPO4, VWR international BVBA, Belgium, 28026.36, H2PO4, BDH chemicals, Australia, 10203.4B) with 0.02% Sodium Azide (Sigma, USA, S-2002). Endogenous peroxidises were quenched with 10% MeOH-3% H2O2 in PBS for 20 minutes at RT, followed by 2×10 minute washes with PBS. Cells were permeabilised with Triton-X100 (BDH chemicals, Australia, prod 30632) (0.3% in PBS) for 15 min, and incubated in blocking solution (10% Normal Donkey Serum (Millipore, USA, S30–100 ml) and 5% Bovine Serum Albumin (Sigma, St Louis, USA, A-7888) in PBS) for 3 hours at RT. Sections were then incubated in primary antibodies (anti-Ephrin-A2 rabbit polyclonal, Santa Cruz Biotechnologies (California, USA) SC-912, diluted 1:200 in blocking solution; anti-Ephrin-A5 rabbit polyclonal, Santa Cruz Biotechnologies SC-20722 diluted 1:75 in blocking solution) at 4ºC overnight with agitation. Sections were washed for 3×10 minutes in blocking solution at RT and detection was carried out using the Vectastain ABC kit (Vector, USA, PK6101) following the manufacturer’s instructions. Briefly, anti–rabbit-biotinylated secondary antibody (Vector BA-1100) was diluted 1:300 in blocking solution and applied to sections for 3 hours at RT. Sections were washed 10 minutes in blocking solution, 3×10 minutes in PBS and the ABC solution was applied for 1 hour at RT. Sections were washed 3×10 minutes in PBS and DAB (Thermo Scientific, USA, 34065) applied for 3 minutes. Sections were washed in PBS for 10 minutes, dehydrated in increasing concentrations of ethanol, defatted in xylene and mounted in Entellan (Merck, Germany).\n\nFor haematoxylin and eosin (H&E) staining, kidneys dissected through the mid-sagittal line were processed through dehydration and wax infiltration procedures and then sectioned at 6 µm using a microtome. Wax sections were floated onto slides in a hot distilled water bath, de-waxed using xylene and decreasing concentrations of ethanol, stained with haematoxylin (2%; Sigma Aldrich) and eosin (1%; Sigma Aldrich), dehydrated in increasing concentrations of ethanol and mounted with Entellan.\n\nH&E stained sections photographed using an Olympus DP70 digital camera. Structural aspects of renal corpuscles and tubules within the cortex of each kidney were measured using ImageJ software. Three images were captured under light microscopy at evenly spaced positions across the cortex of each kidney section. Images were captured at 100× magnification for examining renal corpuscles and at 400× magnification for tubules.\n\nRenal corpuscles: In order to determine cell density within glomeruli, nuclei were counted using the cell counter add-on in ImageJ. The number of nuclei within each glomerulus was then divided by the cross-sectional area of each glomerulus. In order to determine relative glomerular size, the cross-sectional area of each glomerulus was divided by the cross-sectional area of each associated renal corpuscle and was converted to a percentage. An average value for glomerular cell density and relative glomerular size were calculated for each mouse.\n\nRenal tubules: To quantify cell density within tubule tissue, nuclei were counted using the cell counter add-on in ImageJ. The number of nuclei within each tubule was then divided by the cross-sectional area of tubule tissue. In order to determine the cross-sectional area of tubule tissue, the difference between the cross-sectional area of each tubule and the cross-sectional area of each associated tubule lumen was converted to a percentage. Only latitudinally sectioned tubules were analysed. Average values for nuclei density within tubule tissue and the cross-sectional area of tubule tissue were calculated for each mouse.\n\nThe averaged values for each renal corpuscle and tubule measurement were compared between strains using a multivariate permutational ANOVA (MANOVA) in PERMANOVA+ for PRIMER8. PERMANOVA+ is a highly robust non-parametric testing program and therefore does not require agreement with the usual ANOVA assumptions of parametric tests. The MANOVA was computed with age at sacrifice as a covariate to account for the effects of aging on kidney morphology. Each variable was then analysed univariately in PERMANOVA+ with age at sacrifice as a covariate. The four variables were also analysed between mice with and without the ephrin-A5 gene using a MANOVA in PERMANOVA+ such that ephrin-A5/ephrin-A2A5 knockout mice and ephrin-A2 knockout/wild type mice were combined to form two groups. The measured variables were then analysed between the two groups univariately in PERMANOVA+ with age at sacrifice as a covariate. These groups were formed to increase sample sizes and were considered valid because mice homozygous for the gene for ephrin-A2 were unaffected by renal failure. Data were normalised prior to conducting each MANOVA. All analyses were computed using 9999 permutations from a resemblance matrix based on Euclidian distance.\n\n\nResults\n\nEphrin-A2 and ephrin-A5 expression patterns were examined in the adult mouse kidney using immunohistochemistry (Figure 1). For both proteins, expression was strongest in the medulla in the Loops of Henle with weaker staining in the pyramids (Figure 1A,D). In the cortex, cells within the proximal and distal tubules were strongly labelled and this was most prominently visualised when tubules were cut in cross section (Figure 1B,E). Within the glomeruli, expression was detected in podocytes and in the squamous cells lining the Bowman’s capsule (Figure 1C,F). The major difference in expression pattern was that ephrin-A5, but not ephrin-A2 expression, was detected in cells along the length of the tubules.\n\nA,D: low power images showing strong expression in the medulla. P: Pyramids. B,E: High power image showing expression in cells within the proximal and distal tubules, most prominently visualised when tubules were cut in cross section (open arrows). Note expression of ephrin-A5, but not ephrin-A2 in cells along the length of the tubules (closed arrow, E). C,F: Expression was also detect in glomeruli, in podocytes and in the squamous cells lining Bowman’s capsule. Scale bars: A,D: 250 µm; B,C,E,F: 50 µm. B: Bowman’s capsule; G: glomerulus; P: pyramid.\n\nCell density within glomeruli and tubule tissue, glomerular size, and the cross-sectional area of tubule tissue, did not differ significantly between the four strains (multivariate analysis: p = 0.2684; pairwise comparisons are shown in Table 1). The relationship between age at sacrifice and each dependent variable was not significant (Table 1).\n\nAn alpha level of 0.05 was used to determine significance. All p-values are not significant.\n\nNo differences in cell density within glomeruli or tubules, or in cross-sectional area of tubule tissue, were observed across the four strains (Figure 2A). Although not significant, there was a trend for glomerular size to be lower in ephrin-A2A5-/- and ephrin-A5-/- mice compared with mice from the other two strains, however this was a subtle difference that was not obvious in histological sections (Figure 2B–D). When mice were pooled based on the presence or absence of the ephrin-A5 gene to form two groups (WT and ephrin-A2-/- mice vs ephrin-A5-/- and ephrin-A2A5-/- mice), the trend for reduced glomerular size in mice lacking ephrin-A5 reached significance (p = 0.033) but no other significant differences in the other measurements were detected between the two groups.\n\nA: Histograms showing morphological parameters measured in kidney sections (left hand axis) with average age at sacrifice (black points; right hand axis), for each strain. A2A5 = ephrin-A2A5-/- knockout mice, A5 = ephrin-A5-/- knockout mice, A2 = ephrin-A2-/- knockout mice, and WT = wild type mice. Error bars are standard error. B–D: Haematoxylin and eosin stained sections of WT (B,C) and ephrin-A2A5-/- (D,E) knockout mice. Scale bars: 100 µm.\n\n\n\n\nDiscussion\n\nIn summary, we show strong expression of ephrin-A2 and ephrin-A5 in the tubules and glomeruli of the adult mouse kidney. Despite the significant overlap in expression between the two proteins, lack of ephrin-A5 appears to have a stronger effect on kidney morphology with glomerular size being mildly reduced in ephrin-A5-/- mice. However, the magnitude of this change was very small and could only be detected when animals were pooled across genotypes lacking ephrin-A5.\n\nInterestingly, the small reduction in glomerular size in mice lacking ephrin-A5 was not related to age and is therefore likely to be a consequence of abnormal development. Glomeruli consist of a capillary tuft surrounded by highly specialised epithelial cells called podocytes and are formed during development through coordinated interactions between vascular and epithelial tissues9. Members of the EphB and ephrin-B families have been shown to be implicated in this process2–4,10. Although Eph-ephrin interactions are most often restricted within the A or B classes, there is evidence that ephrin-A5, but not ephrin-A2, can bind to EphB26, providing a possible explanation for the ephrin-A5-/- phenotype we report. Because ephrin-A5 and EphB2 have been detected within glomeruli (ephrin-A5: present study; EphB22), and both proteins have been implicated in vascular development in other systems11,12, it is possible that EphB2-ephrin-A5 interactions may contribute to the vascular-epithelial signalling events occurring during kidney development. Our data suggesting reduced glomerular size may be secondary to a vascular phenotype that was not detected using our histological methods.\n\nAlthough we cannot rule out the possibility of other cellular phenotypes in ephrin-A5-/- mice, reduced glomerular size alone may contribute to the increased incidence of kidney failure observed in these mice. The fine scale structure of a glomerulus is important for kidney function as it allows for high molecular weight proteins and red blood cells to be retained, while small molecules such as water, sugars, and electrolytes, are able to pass through to the nephron9. Reduced glomerular size would likely result in an abnormally low glomerular filtration rate in mice lacking the gene for ephrin-A5. A recent study found that age-related declines in nephron number led to a proportional increase in glomerular size relative to Bowman’s capsule, probably to compensate for the associated loss in glomerular filtration rate13. It is possible that mice lacking ephrin-A5 may be unable to compensate for the age-related reduction in nephron number. This might lead to a further reduction in glomerular filtration rate, increasing waste concentrations in the blood, and/or fluid retention which can result in hypertension and eventually renal failure14. Future studies investigating kidney function in ephrin-A5-/- mice are needed to better understand the impact of ephrin-A5 on kidney development and function.\n\n\nConclusion\n\nThe minimal phenotype, together with the relatively infrequent incidence of kidney failure, suggest that ephrin-A2 and ephrin-A5 play only minor roles in kidney development and function. This is surprising given the relatively strong and widespread expression patterns of these proteins detected immunohistochemically. A previous study showed upregulation of ephrin-A5 within a model system of the ureteric bud and hypothesised a role of this protein in the branching of the collecting duct system and/or segmentation of the nephron15. However, the normal morphology described in our study suggests that any role played by ephrin-A5 in the growth and branching of the UB is probably very minor. It is also likely that other members of the ephrin-A family are expressed in the mouse kidney and redundancy within this large family of “promiscuous” signalling molecules may compensate for the loss of individual members16,17.",
"appendix": "Author contributions\n\n\n\nJR conceived the study, JR, JEO and TS designed the experiments, AB, RD, CG, SL, GL, AW, MS and TS carried out the research, AB, RD, CG, SL, GL and AW analysed results, JR, AB, RD, CG, SL, GL and AW wrote the manuscript, and all authors were involved in the revision of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nExperiments were funded by project funds to AB, RD, CG, SL, GL and AW from the University of Western Australia (School of Animal Biology). Animal tissue was obtained from studies funded by a project grant from the NHMRC (no 634386) and the Neurotrauma Program of Western Australia. JR is a NHMRC Senior Research Fellow (APP1002258).\n\n\nAcknowledgements\n\nWe are grateful to Leah Attwood and Helen Moulder (Animal Care Services, UWA) for expert animal care and to Marilyn Davies (Animal Care Services, UWA) for autopsy expertise.\n\n\nReferences\n\nUhlenhaut NH, Treier M: Transcriptional regulators in kidney disease: gatekeepers of renal homeostasis. Trends Genet. 2008; 24(7): 361–71. PubMed Abstract | Publisher Full Text\n\nHashimoto T, Karasawa T, Saito A, et al.: Ephrin-B1 localizes at the slit diaphragm of the glomerular podocyte. Kidney Int. 2007; 72(8): 954–64. PubMed Abstract | Publisher Full Text\n\nOgawa K, Wada H, Okada N, et al.: EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases. J Cell Sci. 2006; 119(Pt 3): 559–70. PubMed Abstract | Publisher Full Text\n\nTakahashi T, Takahashi K, Gerety S, et al.: Temporally compartmentalized expression of ephrin-B2 during renal glomerular development. J Am Soc Nephrol. 2001; 12(12): 2673–82. PubMed Abstract\n\nMiao H, Wang B: EphA receptor signaling--Complexity and emerging themes. Semin Cell Dev Biol. Elsevier. 2012; 23(1): 16–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHimanen JP, Chumley MJ, Lackmann M, et al.: Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling. Nat Neurosci. 2004; 7(5): 501–9. PubMed Abstract | Publisher Full Text\n\nFeldheim DA, Kim YI, Bergemann AD, et al.: Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping. Neuron. 2000; 25(3): 563–74. PubMed Abstract | Publisher Full Text\n\nAnderson M, Gorley R, Clarke K: PERMANOVA+ for PRIMER: Guide to software and statistical methods. Plymouth, UK. 2008. Reference Source\n\nLittle MH, McMahon AP: Mammalian kidney development: principles, progress, and projections. Cold Spring Harb Perspect Biol. 2012; 4(5); a008300. PubMed Abstract | Publisher Full Text\n\nKawachi H, Suzuki K, Miyauchi N, et al.: Slit diaphragm dysfunction in proteinuric states: identification of novel therapeutic targets for nephrotic syndrome. Clin Exp Nephrol. 2009; 13(4): 275–80. PubMed Abstract | Publisher Full Text\n\nHara Y, Nomura T, Yoshizaki K, et al.: Impaired Hippocampal Neurogenesis and Vascular Formation in Ephrin-A5-Deficient Mice. Stem Cells. 2010; 28(5): 974–83. PubMed Abstract | Publisher Full Text\n\nNakamoto M, Bergemann AD: Diverse roles for the Eph family of receptor tyrosine kinases in carcinogenesis. Microsc Res Tech. 2002; 59(1): 58–67. PubMed Abstract | Publisher Full Text\n\nMurawski IJ, Maina RW, Gupta IR: The relationship between nephron number, kidney size and body weight in two inbred mouse strains. Organogenesis. 2010; 6(3): 189–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeck LH Jr, Salant DJ: Glomerular and tubulointerstitial diseases. Prim Care: Clinics in Office Practice. 2008; 35(2): 265–96. PubMed Abstract | Publisher Full Text\n\nPavlova A, Stuart RO, Pohl M, et al.: Evolution of gene expression patterns in a model of branching morphogenesis. Am J Physiol. 1999; 277(4 Pt 2): F650–F63. PubMed Abstract\n\nKlein R: Eph/ephrin signalling during development. Development. 2012; 139(22): 4105–9. PubMed Abstract | Publisher Full Text\n\nHimanen JP, Nikolov DB: Eph signaling: a structural view. Trends Neurosci. 2003; 26(1): 46–51. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2065",
"date": "09 Dec 2013",
"name": "Hiroshi Kawachi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 analyzed the phenotype of ephrin-A2-/-, ephrin-A5-/- and ephrin-A2A5 double knockout mice. The authors reported that no obvious differences of the phenotype were detected in the kidney if compared with wild type mice. However, the authors showed that the glomerular size was reduced in mice lacking ephrin-A5 gene (ephrin-A5-/- mice and ephrin-A2A5 double knockout mice). This finding is very interesting. The experimental protocols were well designed, and the analyses for phenotype were well performed. The title is appropriate for this study, and the text is well written.",
"responses": []
},
{
"id": "3343",
"date": "20 Feb 2014",
"name": "Andrew Boyd",
"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 potentially important negative data. However, the report is dependent on the quality of the IHC antibodies used to detect ephrin A2 and A5. Given the availability of the KO mice, it would be important to show that the antibodies do not stain the relevant KO glomeruli. If this is confirmed and added to the figure the report would be much improved.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-212
|
https://f1000research.com/articles/2-134/v1
|
31 May 13
|
{
"type": "Research Article",
"title": "FAM129B is a novel regulator of Wnt/β-catenin signal transduction in melanoma cells",
"authors": [
"Willliam Conrad",
"Michael B Major",
"Michele A Cleary",
"Marc Ferrer",
"Brian Roberts",
"Shane Marine",
"Namjin Chung",
"William T Arthur",
"Andy J Chien",
"Jason D Berndt",
"Randall T Moon",
"Willliam Conrad",
"Michael B Major",
"Michele A Cleary",
"Marc Ferrer",
"Brian Roberts",
"Shane Marine",
"Namjin Chung",
"William T Arthur",
"Andy J Chien",
"Jason D Berndt"
],
"abstract": "The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/β-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/β-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of β-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/β-catenin signaling. Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a β-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/β-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/β-catenin signaling to apoptosis in melanoma.",
"keywords": [
"apoptosis",
"FAM129B",
"Wnt/β-catenin",
"siRNA",
"melanoma",
"tumor"
],
"content": "Introduction\n\nThe incidence of melanoma continues to rise across the U.S. at a rate faster than any other cancer2. Malignant melanoma has a poor prognosis with a 5-year survival rate of only 15%3. The recently approved therapeutic, vemurafenib, extends median patient survival by 7 months4–6. This major advance raises expectations that even greater rates of survival might be attainable with combination therapies.\n\nActivation of the Wnt/β-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma1,7–13. Analysis of melanoma tumor samples show a positive correlation between nuclear β-catenin staining and decreased tumor depth, increased patient survival and increased time to metastasis14–17. Moreover, treatment with WNT3A-containing conditioned media or stable overexpression of WNT3A in mouse B16 or human A375 melanoma cells reduces cell number in vitro10–12. Allografts of mouse B16 or mouse xenografts of human A375 cells overexpressing WNT3A decrease tumor size compared to control17,18. Recently, we found that activation of Wnt/β-catenin signaling concurrent with the inhibition of the ERK/MAPK pathway synergistically elevates apoptosis in a subset of BRAF- and NRAS-mutant cultured human melanoma cells18,19. Given the interaction between Wnt/β-catenin signaling and pathways known to be critical for melanoma pathogenesis, the identification of Wnt/β-catenin regulators might prove to be informative in developing novel approaches to treat this disease.\n\nIn the present study, we identify novel regulators of Wnt/β-catenin signaling in melanoma by performing a large-scale small-interfering RNA (siRNA) screen of a Wnt/β-catenin responsive reporter in human HT1080 fibrosarcoma cells, and by identifying siRNA targets that are also regulated by ERK/MAPK signaling and that have been previously associated with melanoma. By integrating these three approaches, we identified FAM129B as a potential regulator of Wnt/β-catenin signaling. FAM129B is a 746 amino acid protein that contains an amino-terminal pleckstrin homology (PH) domain and a differentially phosphorylated carboxy-terminal region20. FAM129B is known to inhibit TNFα-dependent apoptosis in HeLa cells21. FAM129B is expressed in melanoma and promotes tumor cell invasion into collagen matrices in an ERK/MAPK phosphorylation-dependent manner20. In the present study we demonstrate that FAM129B promotes Wnt/β-catenin signal transduction in melanoma and that reducing levels of FAM129B with siRNA reduces the ability of WNT3A to increase apoptosis in melanoma cells.\n\n\nResults\n\nIn order to identify novel regulators of Wnt/β-catenin signaling, we performed a siRNA screen. We used HT1080 cells stably transduced with a luciferase reporter of β-catenin-mediated transcription (BAR)22. We screened 28,044 pools of siRNAs. 19,490 gene products were targeted by one or more siRNA pool. Cells were transfected with siRNAs and treated with WNT3A-conditioned media to activate the reporter. BAR activity was normalized to the activity of Renilla luciferase driven by the constitutive TK promoter to control for total cell number. siRNAs targeting positive control proteins such as the known Wnt/β-catenin inhibitor, AXIN2, modulated BAR activity by at least 2.0 fold with a p-value less than 0.01 (Figure 1b). Using this as a criterion, we found that 10,215 siRNA pools regulated BAR activity. Of the 19,490 gene products targeted by one or more siRNA in our screen, we identified 5189 gene products for which every given siRNA significantly regulated BAR activity (Data File 1).\n\n(a) Volcano plots depicting siRNA screen hits overlaid with phosphoproteomic data or bioinformatic data (left panel). Median effect of each siRNA treatment as a percent of control siRNAs were plotted against the p-value of that treatment. If, for a given gene, all siRNAs targeting that gene showed a twofold change in normalized reporter activity and a p-value <0.01, that gene was classified as a hit. This screen identified 5,189 gene products as hits, which are depicted in dark grey. Overlapping phosphoproteomic data from Old WM et al. (2009)15 are depicted in light grey. The known regulator of Wnt/β-catenin signaling, AXIN2, is indicated, as is FAM129B (right panel). Data plot is the same as the left panel with melanoma-associated genes plotted in light grey instead. (b) Venn diagram depicting overlaps between phosphoproteomic dataset, siRNA screen and melanoma-associated genes. 12 protein targets overlap between the phosphoproteomic hits and the siRNA screen, 1 protein target overlaps between the phosphoproteomic hits and melanoma-associated protein targets, and 123 proteins overlap between the siRNA screen hits and melanoma associated protein targets. Only FAM129B overlaps with all three datasets.\n\nTo refine the results of our large-scale siRNA screen, we performed an integrative analysis of our siRNA screen regulators by cross-referencing these regulators with a list of genes previously identified in melanoma, and a list of gene products phosphorylated downstream of MEK and ERK in melanoma. First, we identified 12 proteins in common between the siRNA and phosphoproteomic screens (Figure 1a and Data File 3a). Next, we generated a list of melanoma-associated genes using a custom biopython script (Data File 2 and Query Script). We identified 745 melanoma-associated genes by querying the NCBI gene database. Of these, one gene (FAM129B) encoded a protein that was differentially phosphorylated following MEK inhibition (Figure 1 and Data File 3a) and 123 were gene targets of siRNA pools that regulated Wnt/β-catenin signaling (Figure 1b and Data File 3b). Finally, we discovered FAM129B as the only melanoma-associated gene that both modulated Wnt/β-catenin signaling and was phosphorylated following MEK activation, (Figure 1b and Data File 3a).\n\n\n\nThe siRNA screen suggested that FAM129B is a regulator of Wnt/β-catenin signaling. In order to confirm this possibility, we designed three independent siRNAs targeting FAM129B. First, we confirmed that all three siRNAs inhibit expression of FAM129B protein in HT1080 fibrosarcoma, A2058 melanoma and A375 melanoma cells (Figure 2a). Next, we asked whether the siRNAs inhibited the ability of WNT3A to activate BAR. Indeed, we found that each FAM129B siRNA reduced the ability of WNT3A to activate BAR in all three cell lines (Figure 2b). We also tested whether FAM129B siRNAs reduce the ability of WNT3A to elevate expression of the endogenous β-catenin target gene, AXIN2. Similar to inhibition of BAR, FAM129B siRNAs significantly reduced levels of AXIN2 transcript relative to control siRNA (Figure 2c). From these data, we conclude that FAM129B knockdown inhibits the ability of WNT3A to promote β-catenin mediated transcriptional activation.\n\n(a) Immunoblots show three independent siRNAs reduce steady-state levels of FAM129B following 72 hr treatment with 20 nM siRNA. The beta-tubulin immunoblot serves as a control. Three independent siRNAs targeting FAM129B inhibit FAM129B expression in HT1080 (left), A2058 (middle), and A375 cells (right). (b) FAM129B siRNA inhibit WNT3A-dependent luciferase reporter activity (BAR reporter) normalized to constitutively expressed Renilla luciferase in HT1080 (left), A2058 (middle), and A375 cells (right). (c) FAM129B siRNA inhibit Wnt-dependent AXIN2 expression in HT1080 (left), A2058 (middle), and A375 cells (right) relative to beta-actin mRNA expression by qPCR. Columns and error bars represent mean and SEM, respectively. Data are representative of at least three separate biological replicates. *p<0.05 by unpaired, two-tailed T-test.\n\nWhile FAM129B modulates Wnt/β-catenin signaling in the above assays, these experiments do not rule out the formal possibility that reducing levels of FAM129B might affect other signaling pathways. We therefore generated A375 melanoma cell lines stably transduced with a luciferase-based reporter to the TNFα pathway. We then transfected cells with FAM129B siRNAs and stimulated the reporters with cognate ligands. While FAM129B siRNAs inhibit activation of the BAR reporter by WNT3A across a wide range of doses (Figure 3a), FAM129B siRNA has only negligible effects on TNFα-dependent NFκB reporter activity (Figure 3b). While this result does not allow the conclusion that FAM129B functions solely as a modulator of β-catenin signaling, this result does suggest that FAM129B is not required for activation of all pathways.\n\n(a, left panel) pooled FAM129B siRNAs inhibit Wnt-dependent BAR reporter activity over a wide range of doses. Increasing doses of WNT3A d increases activation of the BAR reporter (normalized to constitutive Renilla luciferase in control treated cells). WNT3A does not activate the reporter in the presence of FAM129B or CTNNB1 siRNAs. (Right panel) A375 cells were treated with siRNAs as indicated and treated with an EC50 dose of WNT3A (50 ng/ml). All FAM129B siRNA and positive control CTNNB1 siRNA inhibit Wnt-dependent BAR reporter activity. (b) The same experiment was carried out as in (a, left panel) in A375 lines TNFα/NFκB reporter. Data in the left panel indicate dose-dependent activation of the NFκB reporter by TNFα. However, FAM129B siRNAs do not inhibit the activation of the TNFα/NFκB reporter. (Right panel) FAM129BsiRNA do not regulate activity of the NFκB reporter activated by 1.5 ng TNFα/ml in A375 cells. High dose TNFα (10 ng/ml) does differentially activate the reporter. Data represent 3 separate biological replicates. *p<0.05 by unpaired, two-tailed T-test.\n\nThe combined treatment with WNT3A protein and compounds that inhibit ERK/MAPK signaling synergizes to induce robust apoptosis in cultured melanoma cells18,19. If FAM129B is required for Wnt/β-catenin signaling, then FAM129B loss of function should inhibit this synergy. We monitored apoptosis in A375 melanoma cells by western blot for cleaved caspase-3 and immunofluorescence staining for TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling). As previously reported18,19, A375 cells treated with control siRNA and the combination of WNT3A and PLX4720 exhibit robust levels of cleaved caspase-3 (Figure 4a). siRNA mediated knockdown of FAM129B decreases the levels of cleaved caspase-3 in response to WNT3A siRNA (Figure 4a–4c). Moreover, when measuring WNT and PLX4720-dependent apoptosis by TUNEL staining, we found that siRNA mediated FAM129B knockdown reduced the number of TUNEL positive cells as compared to control siRNAs. Collectively, these results show that FAM129B is required for the synergy between Wnt3A and PLX4720 to induce melanoma apoptosis.\n\n(a) FAM129B siRNA inhibits Wnt-dependent apoptosis as monitored by cleaved caspase-3 immunoblot. A375 cells were treated with pooled control, pooled FAM129B siRNA, or CTNNB1 siRNA as indicated for 48 hr. Cells were subsequently treated with DMSO or 2 µM PLX4720, and L-conditioned or WNT3A-conditioned media for 24 hr as indicated. Knockdown of FAM129B was monitored by FAM129B immunoblot, inhibition of ERK/MAPK signaling by phospho-ERK immunoblot, and total ERK was used as normalization. Relative levels of cleaved caspase-3 were quantitated by normalizing cleaved caspase-3 pixel density to ERK1/2 for each condition relative to the maximum cleaved caspase-3 level. Data are representative of at least 3 biological replicates. FAM129B siRNA inhibit cleaved caspase-3 levels to between 16 and 41% of maximum. (b) FAM129B siRNA inhibits Wnt-dependent apoptosis as quantified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) immunofluorescence (IF). A375 melanoma cells were treated as above, fixed and stained using TUNEL. Percent TUNEL positive cells calculated as a percent of DAPI positive cells. (c) Representative immunofluorescence of A375 cells treated with the indicated conditions. TUNEL staining is depicted in red and DAPI staining is depicted in blue. Columns and error bars represent the mean and SEM of three separate biological replicates. *p<0.05 by student’s T-test.\n\n\nDiscussion\n\nWe combined phosphoprotoemics and siRNA screening to identify novel regulators of Wnt/β-catenin signaling in human melanoma. We focused on FAM129B, a previously identified protein that has not formerly been linked to Wnt/β-catenin signaling. Using independent siRNAs, we confirmed that FAM129B is required for Wnt3A to activate a β-catenin dependent reporter and reduces the ability of Wnt3A to enhance the expression of the β-catenin target gene AXIN2. We demonstrated that loss of function of FAM129B inhibits the apoptosis of melanoma cells induced by the combined treatment with WNT3A and PLX4720.\n\nFAM129B siRNAs suppress apoptosis in melanoma cells treated with WNT3A and PLX4720. This result was surprising given that the transfection of FAM129B siRNA in HeLa cells promotes increased apoptosis in response to TNFα and cyclohexamide21. The discrepancy between the ability of FAM129B siRNAs to suppress Wnt-dependent apoptosis in melanoma and the ability of these siRNA to promote TNFα-mediated apoptosis in HeLa remains unresolved, although it does suggest that FAM129B may function in a manner that is dependent on cellular context. Alternatively, the differences in apoptotic response with FAM129B loss of function may merely reflect the regulation of Wnt/β-catenin signaling in these two cell types. Uncovering the underlying roles of FAM129B in the cell may well illuminate how FAM129B exerts these opposing effects on apoptosis in response to different stimuli. Future studies should probe the role, if any, of TNFα/NFκB in melanoma apoptosis and the cross-talk between Wnt/β-catenin and TNFα/NFκB signaling in cell lines, such as HeLa, that respond to TNFα by apoptosis.\n\n\nMaterials and methods\n\nDetailed information on the β-catenin activated reporter plasmid (pBARLS) has been previously described23,24. Briefly, the reporters are generated from lentiviral plasmids that contain 12 TCF/LEF binding sites (5´-AGATCAAAGG-3´) or Nuclear Factor Kappa B (5´-GGGAATTTCC-3´) signaling pathways separated by distinct 5-base pair linkers upstream of a minimal promoter and the firefly luciferase open reading frame. The reporters also contain a separate PGK (phosphoglycerate kinase) promoter that constitutively drives the expression of a puromycin resistance gene for mammalian cell selection. These reporters were generated by Travis L. Biechele in the lab of Randall T Moon as previously published23,24.\n\nHuman A375 and A2058 cells were a generous gift from Cassian Yee (Fred Hutchinson Cancer Research Institute, Seattle, WA). HT1080 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Stable reporter lines were generated as previously described22. Cell lines were cultured in a Thermo Forma steri-cult humidified incubator (#3310, Thermo Scientific, Rockford, IL) at 37°C and 5% CO2. All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, #11965–084 Invitrogen, St. Louis, MO) containing 10% fetal bovine serum and 1% Penicillin/Streptomycin (Invitrogen, Grand Island, NY), except A375 cells, which were grown in DMEM containing 5% FBS and 1% P/S.\n\nControl (LCM) and WNT3A-conditioned media (WNT3A CM) used to activate the Wnt/β-catenin signaling pathway were prepared as previously described25. To monitor reporter activity and transcript activity, cells were treated with 10% WNT3A CM or LCM overnight before proceeding to subsequent assays. To monitor effects on apoptosis, cells were treated with 1% LCM or WNT3A and DMSO (Sigma St. Louis, MO, product 472301) or 2 uM PLX4720 (Symansis, Timaru New Zealand SY-PLX4720).\n\nThe large-scale siRNA screen was performed as previously described26, with minor modifications. Briefly, HT1080 cells stably transduced with BAR firefly luciferase and Renilla luciferase lentivirus were reverse-transfected in 1536-well plates, with a final concentration of pooled siRNA at 25 nM. 48 hours after reverse transfection, cells were treated with WNT3A-conditioned media. Following overnight incubation, β-catenin dependent transcription was measured by assaying firefly luciferase activity and normalized by monitoring constitutively expressed Renilla luciferase activity as described in the Promega Dual glo luciferase assay system technical manual (Promega, Madison Wi). All siRNAs were designed with a proprietary algorithm27.\n\nApproximately 200,000 A375, A2058, or HT1080 cells (as estimated by hemocytometer counts) were reverse transfected at a final dose of 20 nM siRNA in 6-well format using 5 µl RNAi max/well (Invitrogen, Grand Island, NY). Medium GC universal stealth control siRNA was used as a negative control (Cat. No. 12935–112, Invitrogen, Grand Island, NY). Invitrogen’s stealth siRNA targeting FAM129B were designed using the BLOCK-iT RNAi designer and are described below. The sequence for \"FAM129B A\" is UCACGGACAUGAACCUGAACGUCAU. The sequence for \"FAM129B B\" is ACUGAGGUGCGAGAUGUCUUCUUCA. The sequence for \"FAM129B C\" is CAGCAGCGAUUUGAUGUGUCCAGCA. As a positive control for inhibition of Wnt/β-catenin signal transduction by siRNA, we used silencer select siRNA targeting CTNNB1 with the sequence GGUGGUGGUUAAUAAGGCUTT (Invitrogen, Grand Island, NY).\n\n24 hr after siRNA transfection, cells were plated in 96-well plates at a density of 20,000 cells/well. Twenty-four hours after plating, cells were treated with the indicated conditions, and luciferase activity was measured 15 hours later with a Dual-Luciferase Reporter Assay kit (Promega, Madison, Wi) and an Envision multilabel plate reader (PerkinElmer, Waltham, MA) according to the manufacturer’s suggestions.\n\n24 hr after siRNA transfection, cells were split into a 12-well cluster plate at approximately 50% confluency. 24 hr later, cells were treated with WNT3A- or L-conditioned media. After overnight treatment, RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen). 1 µg of RNA was reverse transcribed using Fermentas’ RevertAid M-MuLV Reverse Transcriptase (Fermentas, Glen Burnie, MD). QPCR was performed on a Lightcycler 480 (Roche, Indianapolis, IN) using Lightcycler 480 DNA SYBR Green 1 master mix (04707516001 Roche, Indianapolis, IN). The following primers were used for qPCR: \"AXIN2 F\" CTCCCCACCTTGAATGAAGA and \"AXIN2 R\" TGGCTGGTGCAAAGACATAG; and, \"ACTB F\" AGAGCAAGAGAGGCATCCTC and \"ACTB R\" CTCAAACATGATCTGGGTCA.\n\nTo test for siRNA knockdown, replicate cell lysates from low throughput reporter assays were pooled and treated with 10× RIPA lysis buffer (500 mM Tris, pH 7.5, 1.5 M NaCl, 10 mM EDTA, 10% Igepal CA-630, 1% SDS, and 2% sodium deoxycholate all purchased from Sigma, St. Louis, MO). For monitoring cleaved caspase-3, 90% confluent 12-well plates were treated for 24-hr with the indicated conditions described in the \"cell lines and cell culture\" section. Media were collected and cells were rinsed once (gently) with PBS. Cells were lysed on-plate in 100 µl 1× RIPA buffer containing protease and phosphatase inhibitors (Complete EDTA-free and PhoStop by Roche, Indianapolis, IN). Cells were disrupted by scraping of a 1000 µl pipette tip against the plate. Apoptotic cells present in the media and PBS wash were centrifuged at 300 g, rinsed once with PBS, and lysed with the RIPA buffer collected from the plate lysis. Cell lysates were cleared by centrifugation at 20,000 g at 4°C for 10 minutes. Protein lysates were separated by SDS-PAGE using NuPAGE 4%–12% Bis-Tris gels (NP0336BOX, Invitrogen, Grand Island, NY) in MES buffer, and transferred onto a nitrocellulose membrane (162–0115, Bio-Rad, Hercules, CA) using IDEA scientific GENIE transfer apparatuses (Idea Scientific, Minneapolis, MN). Blots were probed using polyclonal rabbit anti FAM129B (#HPA023261 Sigma, St. Louis, MO), monoclonal mouse anti Tubulin (#T7816 Sigma, St. Louis, MO), monoclonal mouse anti β-catenin (C2206 Sigma, St. Louis, MO), polyclonal Rabbit anti cleaved-caspase-3 (#9661 cell signaling), Rabbit anti ERK1/2 (#9102 cell signaling, Danvers, MA), Rabbit anti phospho ERK1/2 (#9211 cell signaling, Danvers, MA).\n\nGlass coverslips were coated with poly-l-lysine in a 24-well dish, rinsed with PBS, and dried. Following reverse transfection as described above, cells were seeded at a density to achieve 90 to 100% confluency at harvest. Twenty-four hours after seeding, cells were treated with the indicated conditions and incubated for 24 hours with the indicated conditions as described above in the \"cell lines and cell culture\" section. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using an in situ cell death detection kit (Roche, Indianapolis, IN). Briefly, the medium was gently aspirated, to keep apoptotic bodies on the slide, and cells were fixed in 4% paraformaldehyde for 1 hour at room temperature. Cells were gently rinsed twice with PBS and permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MO) in 0.1% sodium citrate (Sigma, St. Louis, MO) for 2 min on ice. Cells were rinsed twice with PBS and 40 ml of TUNEL reaction mixture was added directly on top of the slide; cells were incubated for 1 hour at 37°C in a humidified incubator. Slips were rinsed three times with PBS and mounted on Superfrost Plus glass slides with ProLong Gold anti-fade mounting medium containing 4´,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Grand Island, NY). Images were obtained on a Nikon TiE inverted wide-field high-resolution microscope. DAPI and TUNEL positive nuclei were quantified blinded for 5 fields per slide using NIS elements (Nikon Instruments Inc, Melville, NY).\n\nExcept where indicated, a student’s t test was used to assess the statistical significance of the differences between the different groups; a p value of <0.05 was considered significant.",
"appendix": "Author contributions\n\n\n\nWC, RTM, AC and JB conceived the study. MF, SM, and NC automated the large-scale screen. MBM, MAC, BR, and WTA designed and carried out the large scale screen. WC carried out the research in melanoma cells and identified melanoma-associated genes. WC prepared the first draft of the manuscript. AC, JB, and RTM contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWC was supported by a Pharmaceutical Sciences Training grant (5T32GM007750). AJC is funded by the NIH/National Cancer Institute (NCI) (K08CA128565).\n\n\nAcknowledgements\n\nWe thank Cassian Yee for contributing the A375 and A2058 cells. Thanks to Travis Biechele for providing the beta-catenin reporter constructs. MC and WA were employees of Rosetta/Merck. JB is an associate of the Howard Hughes Medical Institute. RTM is an investigator of the Howard Hughes Medical Institute. We are indebted to these funding agencies for their continued support of our work. The contents of this manuscript are the sole responsibility of the authors and do not necessarily represent the official views of the NIAMS, NCI, NIH, or the Howard Hughes Medical Institute.\n\n\nReferences\n\nKageshita T, Hamby CV, Ishihara T, et al.: Loss of beta-catenin expression associated with disease progression in malignant melanoma. Br J Dermatol. 2001; 145(2): 210–6. PubMed Abstract | Publisher Full Text\n\nKohler BA, Ward E, McCarthy BJ, et al.: Annual Report to the Nation on the Status of Cancer, 1975–2007, Featuring Tumors of the Brain and Other Nervous System. J Natl Cancer Inst. 2011; 103(9): 714–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican Cancer Society. Cancer Facts & Figures 2011 [Internet], American Cancer Society; 2011. Reference Source\n\nChapman PB, Hauschild A, Robert C, et al.: Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364(26): 2507–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlaherty KT, Puzanov I, Kim KB, et al.: Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010; 363(9): 809–19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSosman JA, Kim KB, Schuchter L, et al.: Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012; 366(8): 707–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSosman JA, Kim KB, Schuchter L, et al.: Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012; 366(8): 707–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaelandsmo GM, Holm R, Nesland JM, et al.: Reduced beta-catenin expression in the cytoplasm of advanced-stage superficial spreading malignant melanoma. Clin Cancer Res. 2003; 9(9): 3383–8. PubMed Abstract\n\nBachmann IM, Straume O, Puntervoll HE, et al.: Importance of P-cadherin, beta-catenin, and Wnt5a/frizzled for progression of melanocytic tumors and prognosis in cutaneous melanoma. Clin Cancer Res. 2005; 11(24 Pt 1): 8606–14. PubMed Abstract | Publisher Full Text\n\nChien AJ, Moore EC, Lonsdorf AS, et al.: Activated Wnt/beta-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model. Proc Natl Acad Sci U S A. 2009; 106(4): 1193–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiechele TL, Camp ND, Fass DM, et al.: Chemical-genetic screen identifies riluzole as an enhancer of Wnt/β-catenin signaling in melanoma. Chem Biol. 2010; 17(11): 1177–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiechele TL, Kulikauskas RM, Toroni RA, et al.: Wnt/β-catenin signaling and AXIN1 regulate apoptosis triggered by inhibition of the mutant kinase BRAFV600E in human melanoma. Sci Signal. 2012; 5(206): ra3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConrad WH, Swift RD, Biechele TL, et al.: Regulating the response to targeted MEK inhibition in melanoma: Enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/β-catenin activation. Cell Cycle. 2012; 11(20): 3724–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKageshita T, Hamby CV, Ishihara T, et al.: Loss of beta-catenin expression associated with disease progression in malignant melanoma. Br J Dermatol. 2001; 145(2): 210–6. PubMed Abstract | Publisher Full Text\n\nMaelandsmo GM, Holm R, Nesland JM, et al.: Reduced beta-catenin expression in the cytoplasm of advanced-stage superficial spreading malignant melanoma. Clin Cancer Res. 2003; 9(9): 3383–8. PubMed Abstract\n\nBachmann IM, Straume O, Puntervoll HE, et al.: Importance of P-cadherin, beta-catenin, and Wnt5a/frizzled for progression of melanocytic tumors and prognosis in cutaneous melanoma. Clin Cancer Res. 2005; 11(24 Pt 1): 8606–14. PubMed Abstract | Publisher Full Text\n\nChien AJ, Moore EC, Lonsdorf AS, et al.: Activated Wnt/beta-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model. Proc Natl Acad Sci U S A. 2009; 106(4): 1193–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiechele TL, Kulikauskas RM, Toroni RA, et al.: Wnt/β-catenin signaling and AXIN1 regulate apoptosis triggered by inhibition of the mutant kinase BRAFV600E in human melanoma. Sci Signal. 2012; 5(206): ra3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConrad WH, Swift RD, Biechele TL, et al.: Regulating the response to targeted MEK inhibition in melanoma: Enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/β-catenin activation. Cell Cycle. 2012; 11(20): 3724–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOld WM, Shabb JB, Houel S, et al.: Functional proteomics identifies targets of phosphorylation by B-Raf signaling in melanoma. Mol Cell. 2009; 34(1): 115–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen S, Evans HG, Evans DR: FAM129B/MINERVA, a novel adherens junction-associated protein, suppresses apoptosis in HeLa cells. J Biol Chem. 2011; 286(12): 10201–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiechele TL, Moon RT: Assaying beta-catenin/TCF transcription with beta-catenin/TCF transcription-based reporter constructs. Methods Mol Biol. 2008; 468: 99–110. PubMed Abstract | Publisher Full Text\n\nBiechele TL, Moon RT: Assaying beta-catenin/TCF transcription with beta-catenin/TCF transcription-based reporter constructs. Methods Mol Biol. 2008; 468: 99–110. PubMed Abstract | Publisher Full Text\n\nFass D, Haggarty S, Scolnick E, et al.: Compositions of modulators of the wnt/beta-catenin pathway and benzamide and/or hydroxamic acid derivatives to treat bipolar disorde. 2013. Reference Source\n\nWillert K, Brown JD, Danenberg E, et al.: Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature. 2003; 423(6938): 448–52. PubMed Abstract | Publisher Full Text\n\nBartz SR, Zhang Z, Burchard J, et al.: Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions. Mol Cell Biol. 2006; 26(24): 9377–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMajor MB, Roberts BS, Berndt JD, et al.: New regulators of Wnt/beta-catenin signaling revealed by integrative molecular screening. Sci Signal. 2008; 1(45): ra12. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1167",
"date": "19 Jul 2013",
"name": "Madelon Maurice",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report describes the identification of FAM129B as a novel regulator of Wnt/β-catenin signalling and apoptosis in melanoma cells. The FAM129B gene was identified via integration of 3 datasets, including Wnt signaling modifiers, melanoma-associated genes and the MEK/ERK phosphoproteome. FAM129B was the only gene that overlapped with all three datasets, making it a promising candidate for regulation of both Wnt and MEK/ERK pathways in melanoma. I find this an interesting report which will provide a good starting point for further studies. Overall, the data are convincing and support the conclusions drawn. Experiments which validate FAM129B as a Wnt pathway modifier (Fig. 1-3) appear solid and display robust effects. The effects of FAM129B on apoptosis (Fig. 4b), however, are somewhat lower than expected based on the effects of FAM129B knockdown in reporter assays and on the inhibition of caspase cleavage. I feel that the authors could provide a more in-depth discussion on their findings and place them in the context of the background information provided in the introduction part; How do the authors consider this candidate relevant for melanoma treatment? How do the authors integrate the beneficial effects of enhanced Wnt responsiveness (this report) and the previously described enhanced tumour cell invasion (ref 20) of FAM129B expressing melanoma cells?It would be helpful for the reader to include a model which shows how Wnt and ERK pathways modulate apoptosis in melanoma cells and where FAM129B would be placed in these events. Also in the legend of Fig. 2a it remains unclear whether endogenous levels of FAM129B are depicted.",
"responses": [
{
"c_id": "575",
"date": "10 Oct 2013",
"name": "William Conrad",
"role": "Author Response",
"response": "Dear Dr. Maurice,We appreciate your thorough reading of our manuscript, as well as the thoughtful questions that you have raised in your review. We have outlined a response to specific questions below:“How do the authors consider this candidate relevant for melanoma treatment?”It is unclear how FAM129B is relevant for melanoma treatment at this point. Our data identifies FAM129B as a regulator of Wnt/β-catenin dependent phenotypes in melanoma cells, while previous studies had already identified FAM129B as a downstream protein regulated by ERK/MAPK signaling. Given the importance of both of these signaling pathways in melanoma biology, we hypothesize that FAM129B could be an important regulatory nexus between Wnt/β-catenin and ERK/MAPK signaling, although at this point we have been unable to draw a direct connection in patient tumors. Thus, at this point it is difficult to make any conclusions on the role of FAM129B’s role in melanoma treatment without being overly speculative. “I feel that the authors could provide a more in-depth discussion on their findings and place them in the context of the background information provided in the introduction part”We have added some more content to the discussion, and we hope that these changes have improved the manuscript. “How do the authors integrate the beneficial effects of enhanced Wnt responsiveness (this report) and the previously described enhanced tumour cell invasion (ref 20) of FAM129B expressing melanoma cells?” You have raised a very interesting and relevant point regarding the difficulties in broadly generalizing results obtained in cultured cell systems across a disease. Our evaluation of published data (presented in this revision) also shows increased expression of FAM129B in a cohort previously defined as more invasive, and with lower activation of Wnt/β-catenin signaling (Figure 5a). These data would suggest that in patients, the effect of FAM129B on melanoma cell invasion may supersede any effects on regulating Wnt/β-catenin signaling. In response to your helpful suggestion, we have added a model which shows how Wnt/β-catenin and ERK/MAPK pathways modulate apoptosis in melanoma cells (Figure 5c). Thank you for your careful reading of the figure legends, we have edited the legend of Figure 2a to clarify that our western blot of FAM129B is for endogenous FAM129B protein levels."
}
]
},
{
"id": "1185",
"date": "24 Jul 2013",
"name": "Udayan Apte",
"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\n1. The title is appropriate for the study.2. The experimental design and methods are appropriate for the topic being studied.3. The information and data provided is enough for replicating the experiment4. The conclusions are correct but need additional data to substantiate it. In this article, the authors have identified FAM129B as a positive regulator of Wnt/beta-catenin signalling in melanoma cells. They also demonstrate that FAM129B is required for Wnt3a-induced apoptosis in vitro.However; there are three important pieces of information missing that the authors should address:Can this effect be replicated in vivo in animal models?Does FAM129B expression correlate with beta-catenin activation and disease progression in melanoma patients?What is the mechanism by which FAM129B regulates Wnt signalling? I would like the authors to do a bit more work on answering at least one of the three questions raised. They should also discuss the other two questions in the discussion section. From my standpoint it seems the easiest thing to do would be to compare FAM129 expression in various tumor samples and correlate them to Beta-catenin activation (nuclear localization) and growth pattern (tumor depth).",
"responses": [
{
"c_id": "574",
"date": "10 Oct 2013",
"name": "William Conrad",
"role": "Author Response",
"response": "Dear Dr. Apte,We appreciate your thorough reading of our manuscript, as well as the thoughtful questions that you have raised in your review. We have outlined a response to specific questions below:“Does FAM129B expression correlate with beta-catenin activation and disease progression in melanoma patients?” This is a very interesting question, and one which we had sought to address using previously published data from defined cohorts of melanoma patients. We analyzed the abundance of FAM129B present in three different cohorts of melanoma (Figure 5a in version 2) and observed a significantly higher abundance of FAM129B mRNA in a melanoma cohort associated with higher invasiveness and relatively lower Wnt/β-catenin signaling. From this observation, we concluded that FAM129B abundance did not predict Wnt/β-catenin signaling in melanoma patients. This conclusion was supported by a lack of significant correlation between levels of FAM129B and expression of the established Wnt/β-catenin target gene, AXIN2, within this cohort (data not shown). We have included a brief discussion of these results in our revision in order to clarify the fact that we do not have data that supports a clear role of direct regulation of Wnt/β-catenin signaling by FAM129B in melanoma patients. “Can this effect be replicated in vivo in animal models?” We did attempt to address this question, but despite repeated attempts we were unable to stably knock down FAM129B using lentiviral shRNA in melanoma cells. As a result, we were not able to generate cell lines that could be used to address this question in murine xenografts. “What is the mechanism by which FAM129B regulates Wnt/β-catenin signaling?” Unfortunately, we have been unable to solve the specific mechanism by which FAM129B regulates Wnt/β-catenin signaling. We had hoped that a proteomics-based approach identifying proteins that interact with FAM129B might prove informative, but to date we have not solidified any mechanistic links based on the hits identified with this strategy. While our efforts to validate this network are currently too preliminary for publication, we have (in the spirit of open access) included our FAM129B protein interaction network in its entirety in order to allow other readers to potentially gain insight and generate hypotheses regarding the role of this protein in regulating cellular signaling (Figure 5b)."
}
]
}
] | 1
|
https://f1000research.com/articles/2-134
|
https://f1000research.com/articles/2-209/v1
|
09 Oct 13
|
{
"type": "Short Research Article",
"title": "Synaptic Vesicle Protein 2 (SV2) does not hydrolyze ATP",
"authors": [
"Jia Yao",
"Sandra M. Bajjalieh",
"Jia Yao"
],
"abstract": "Synaptic vesicle protein 2 (SV2) is a transporter-like protein specifically expressed in endocrine cells and neurons, where it is localized to vesicles that undergo regulated secretion and plays an essential role in regulating neurotransmitter release. SV2 binds adenine nucleotides including ATP. Analysis of ATP transport revealed that SV2 is not an ATP transporter, nor does it affect ATP transport. As a further step toward understanding how ATP binding contributes to SV2 function, we investigated whether SV2 is an ATPase using an in vitro measure of ATPase activity. The study reported here indicates that SV2 does not have ATPase activity. Thus, binding to adenine nucleotides likely modulates other actions of SV2.",
"keywords": [
"SV2",
"nucleotide binding",
"ATP hydrolysis"
],
"content": "Introduction\n\nSynaptic vesicle protein 2 (SV2) is a membrane protein expressed exclusively in neurons and endocrine cells where it is localized to vesicles that undergo regulated secretion1. Most mammals have three SV2 genes that encode three related proteins termed SV2A, SV2B, and SV2C2,3. Of these, SV2A is the most broadly expressed, and is present in nearly all neurons, whereas SV2C demonstrates the most restricted expression2,4,5. SV2 is essential for normal neurotransmission, as evidenced by epileptic seizures and premature death in SV2A or SV2A/B knockout mice6,7. Electrophysiological studies assessing neurotransmitter release in SV2 mutants indicate that SV2 acts as a positive modulator of calcium-dependent exocytosis8–10. Neuron or endocrine cells lacking SV2 contain fewer vesicles competent for Ca2+-triggered secretion.\n\nDespite our understanding of the physiological role of SV2, its molecular action remains unknown. We previously reported that SV2 binds adenine nucleotides including ATP11. Analysis of ATP transport by vesicles isolated from wild-type and SV2 mutants revealed that SV2 is not an ATP transporter, nor does it affect ATP transport11. Thus it is not clear how ATP binding relates to SV2 function. One untested possibility is that SV2 is an enzyme that hydrolyzes ATP. To address this, we investigated whether SV2 has ATPase activity. Our results show that recombinant SV2A and SV2B do not hydrolyze ATP. Thus, binding to adenine nucleotides likely modulates other actions of SV2.\n\n\nMethods\n\nHEK293T cell (ATCC, Manassas, VA) culture and transfection with Lipofectamine™ 2000 reagent (Invitrogen, Grand Island, NY) were performed following the manufacturer's protocol11. To transfect one 15 cm plate of cells, 48 µg of plasmid DNA and 96 µl of Lipofectamine™ 2000 reagent were mixed with 3 ml of serum and antibiotic-free minimum essential media (MEM) (Invitrogen, Grand Island, NY). After incubation at room temperature for 5 min, the DNA and Lipofectamine™ 2000 reagent mixture was combined and allowed to sit for 20 min at room temperature. The transfection mixture was then added drop-wise to cells cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in a humidified incubator containing 5% CO2. After 48 h, medium was removed, cells were harvested, and rinsed with ice-cold phosphate-buffered saline (PBS) two times. Cell pellets were stored at -80°C until use. Rat SV2A and SV2B were expressed with a C-terminal FLAG epitope (DYKDDDK) in the mammalian expression vector pIRES2-EGFP (Clontech, Mountain View, CA).\n\nRecombinant SV2A and SV2B -FLAG fusion proteins were purified using anti-FLAG M2 affinity agarose beads (Sigma; catalog number: A2220, mouse IgG1 monoclonal antibody) as described previously11. Briefly, transfected cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). Cell extracts were made by adding extraction buffer (150 mM potassium acetate (J.T. Baker, Phillipsburg, NJ), 10 mM HEPES-KOH (pH 7.4) (Sigma, St. Louis, MO), 1% Triton X-100 (Roche Applied Science, Indianapolis, IN), with 1× fresh protease inhibitor mixture added before each use (Roche Applied Science, Indianapolis, IN)) and incubated at 4°C for 1 h. After extraction, insoluble material was removed by centrifugation (Beckman, Indianapolis IN) at 19,000 × g for 20 min. The resulting extract was mixed with pre-equilibrated anti-FLAG M2 agarose beads (Sigma, St. Louis, MO) and incubated with agitation at 4°C for 3–4 h. The beads were washed four times with 20 volumes of extraction buffer. FLAG fusion protein was eluted from the beads with 3× FLAG peptide (Sigma, St. Louis, MO) in buffer containing 150 mM potassium acetate, 10 mM HEPES-KOH (pH 7.4) and 0.5% Triton X-100. Final preparations were checked by silver staining (Thermo, Waltham, MA) of SDS polyacrylamide gels (Bio-Rad, Hercules, CA) and immunoblot with anti-SV2 (mouse monoclonal antibody against an epitope located on the N-terminus of SV2)12 or anti-FLAG antibodies (Sigma, catalog number: F3165, mouse IgG1 monoclonal antibody). Protein concentration was determined by comparison to bovine serum albumin (BSA) (Fisher, Pittsburgh, PA) standards in PAGE gels. This method was used because the 3× FLAG peptide in the eluate will affect solution-based protein assays. BSA standards, ranging from 0.5 to 4 µg were loaded on the same gel along with purified SV2-FLAG fusion proteins. Gels stained with coomassie blue were scanned with a Kodak Image Station 440CF (Kodak, Rochester, NY). Net intensities of protein bands were determined by Kodak Molecular Imaging software (Kodak, Rochester, NY) and protein concentration of recombinant SV2-FLAG was calculated according to BSA standards.\n\nATP hydrolysis reactions were conducted at 25°C in a buffer consisting of 150 mM potassium acetate, 10 mM HEPES (pH 7.4), 2 mM ATP, 3 MgCl2, 0.1% Triton X-100, 1 mM DTT and 10–20 µg/ml purified recombinant SV2-FLAG protein. The ATP hydrolysis reaction was set up on ice and recombinant SV2-FLAG protein was added last. Reaction mixtures were prepared on ice, and an aliquot of reaction was immediately withdrawn as the 0°C, 0 min sample. The reaction was shifted to 25°C, and at designated intervals (1 min, 2 min, 5 min and 10 min), aliquots were removed from the reaction for analysis by the malachite green assay described below. Duplicates were assayed for each time point and the average value of the duplicates was used for quantification.\n\nCommercially available calf intestinal alkaline phosphatase (CIP) (New England Biolabs, Ipswich, MA) was used as a positive control for the ATP hydrolysis reaction. As a negative control, an aliquot of CIP was heated at 95°C for 20 min to inactivate the enzyme.\n\nTo prepare the malachite green solution, one volume of 4.2% ammonium molybdate (Sigma, St. Louis, MO) in 4 M HCl was added to 3 volumes of 0.045% malachite green (Sigma, St. Louis, MO). The solution was stirred at room temperature for a minimum of 30 min before being filtered through a 0.22 µm membrane (Millipore, Billerica, MA). Malachite green solutions were kept at 4°C in the dark. Before each assay, 0.01% Tween-20 was added to the malachite green solution.\n\n50 µl aliquots of ATP hydrolysis reaction were added to 950 µl of malachite green solution and color was allowed to develop for 20 min at room temperature. Absorbance at 620 nm was measured in a cuvette with the malachite green solution as a blank in a SmartSpec3000 spectrometer (Bio-Rad, Hercules, CA). A 0.02 mM KH2PO4 solution (Fisher, Pittsburgh, PA) was used as an inorganic phosphate standard. Phosphate release in ATP hydrolysis reactions was quantified by comparison to inorganic phosphate standards.\n\n\nResults and discussion\n\nTo determine if SV2 is an ATPase we used a fast and reproducible method for measuring inorganic free phosphate released by ATP hydrolysis in aqueous solutions13,14. The assay is based on the change of absorbance at 620 nm after free phosphate forms a colored complex with molybdate/malachite green15. Figure 1 shows a standard curve testing 0–400 pmol of KH2PO4 in the reaction. It indicates that the cuvette-based assay produced a reliable linear standard curve.\n\nShown is a standard curve for inorganic phosphate (Pi) in the malachite green assay. The data are representative of 5 independent experiments. Inorganic phosphate standards were prepared from KH2PO4. Each point represents the mean value of duplicate measurements. 50 µl of standard solution containing different amounts of phosphate (Pi) was mixed with 950 µl of malachite green solution and allowed to develop color at room temperature for 20 min. The absorbance of each sample was measured at 620 nm and plotted against pmol of phosphate in the reaction.\n\nAs a positive control for phosphate production, we used CIP16, a type of alkaline phosphatase that catalyzes the hydrolysis of 5´-phosphate groups from various substrates, including 5´-nucleotides, RNA and DNA. Figure 2 shows that CIP catalyzed ATP hydrolysis in a time-dependent manner. Heat-inactivated CIP produced little activity that did not increase with time. These results attest to the effectiveness of the ATP hydrolysis reaction and the malachite green assay in this study.\n\nShown is a time course of one calf intestinal alkaline phosphatase (CIP) reaction with ATP as a substrate. The data are representative of four independent experiments. The CIP reaction was carried out at 25°C. At different time points, aliquots were withdrawn and subjected to the malachite green assay as described in Figure 1. Pi in each sample was quantified by comparing to the standard curve generated for inorganic phosphate. Each point represents the mean value of duplicate measurements. CIP exhibited time-dependent ATP hydrolysis activity while heat-inactivated CIP produce much lower activity that was not time-dependent.\n\nTo test for ATPase activity in SV2, we performed the assay with purified recombinant SV2. SV2A or SV2B fused to a FLAG epitope (for affinity purification) is the same preparation that was used to characterize ATP binding activity in vitro in our previous study11. In the malachite green assay neither SV2A nor SV2B produced ATP hydrolysis activity (Figure 3).\n\nShown is a representative plot of ATP hydrolysis activity by recombinant SV2A (Panel A) or SV2B (Panel B). ATP hydrolysis reactions and the malachite green assay were performed as described under \"Methods\". The data represent two (SV2A) and three (SV2B) separate measures. For comparison, results of calf intestinal alkaline phosphatase (CIP) reactions run on the same day are included in the plots.\n\nThis result indicates that SV2 does not hydrolyze ATP. The concern that the FLAG epitope included at the carboxy terminus of recombinant SV2 used in these assays might affect activity is not likely given that SV2-EGFP fusion proteins can rescue normal neurotransmission in neurons from SV2 knockouts17. Together with previous studies reporting that SV2 is not likely to be an ATP transporter11, these findings exclude the possibility that SV2 affects ATP levels either by transport or hydrolysis. Therefore SV2 binding of ATP likely regulates its conformation or its molecular interactions, possibilities that suggest SV2 action is modulated by synaptic energy levels.\n\nWe previously show that a tyrosine-based endocytosis motif (Y46SRF) in the N-terminus of SV2 is required for its trafficking to synaptic vesicles, and that this motif mediates binding of multiple endocytosis-related proteins18. Interestingly, one of the two ATP binding sites in SV2A (a.a. 58–104) is located in the same region11. This suggests a possible link between ATP binding and SV2 endocytosis.\n\n\nAbbreviations\n\nSV2, synaptic vesicle protein 2; CIP, calf intestinal alkaline phosphatase; ATP, adenosine-5´-triphosphate",
"appendix": "Author contributions\n\n\n\nJY and SB conceived and designed the study. JY performed the experiments and analyzed the data. JY and SB prepared the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors have no conflicts of interests to disclose.\n\n\nGrant information\n\nThis work was supported by National Institutes of Mental Health Grant R01 MH 059842 to SB.\n\n\nAcknowledgements\n\nWe thank Dr Alex Merz for help with the malachite green assay and Mario Rosasco for editorial review of the manuscript.\n\n\nReferences\n\nBajjalieh SM, Peterson K, Shinghal R, et al.: SV2, a brain synaptic vesicle protein homologous to bacterial transporters. Science. 1992; 257(5074): 1271–1273. PubMed Abstract | Publisher Full Text\n\nBajjalieh SM, Peterson K, Linial M, et al.: Brain contains two forms of synaptic vesicle protein 2. Proc Natl Acad Sci U S A. 1993; 90(6): 2150–2154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJanz R, Sudhof TC: SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family. Neuroscience. 1999; 94(4): 1279–1290. PubMed Abstract | Publisher Full Text\n\nDardou D, Dassesse D, Cuvelier L, et al.: Distribution of SV2C mRNA and protein expression in the mouse brain with a particular emphasis on the basal ganglia system. Brain Res. 2011; 1367: 130–145. PubMed Abstract | Publisher Full Text\n\nBajjalieh SM, Frantz GD, Weimann JM, et al.: Differential expression of synaptic vesicle protein 2 (SV2) isoforms. J Neurosci. 1994; 14(9): 5223–5235. PubMed Abstract\n\nJanz R, Goda Y, Geppert M, et al.: SV2A and SV2B function as redundant Ca2+ regulators in neurotransmitter release. Neuron. 1999; 24(4): 1003–1016. PubMed Abstract | Publisher Full Text\n\nCrowder KM, Gunther JM, Jones TA, et al.: Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A). Proc Natl Acad Sci U S A. 1999; 96(26): 15268–15273. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCuster KL, Austin NS, Sullivan JM, et al.: Synaptic vesicle protein 2 enhances release probability at quiescent synapses. J Neurosci. 2006; 26(4): 1303–1313. PubMed Abstract | Publisher Full Text\n\nChang WP, Sudhof TC: SV2 renders primed synaptic vesicles competent for Ca2+ -induced exocytosis. J Neurosci. 2009; 29(4): 883–897. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWan QF, Zhou ZY, Thakur P, et al.: SV2 acts via presynaptic calcium to regulate neurotransmitter release. Neuron. 2010; 66(6): 884–895. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYao J, Bajjalieh SM: Synaptic vesicle protein 2 binds adenine nucleotides. J Biol Chem. 2008; 283(30): 20628–20634. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBuckley K, Kelly RB: Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J Cell Biol. 1985; 100(4): 1284–1294. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaehama T, Taylor GS, Slama JT, et al.: A sensitive assay for phosphoinositide phosphatases. Anal Biochem. 2000; 279(2): 248–250. PubMed Abstract | Publisher Full Text\n\nTaylor GS, Maehama T, Dixon JE: Myotubularin, a protein tyrosine phosphatase mutated in myotubular myopathy, dephosphorylates the lipid second messenger, phosphatidylinositol 3-phosphate. Proc Natl Acad Sci U S A. 2000; 97(16): 8910–8915. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLanzetta PA, Alvarez LJ, Reinach PS, et al.: An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979; 100(1): 95–97. PubMed Abstract | Publisher Full Text\n\nChen KT, Malo MS, Moss AK, et al.: Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase. Am J Physiol Gastrointest Liver Physiol. 2010; 299(2): G467–475. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNowack A, Yao J, Custer KL, et al.: SV2 regulates neurotransmitter release via multiple mechanisms. Am J Physiol Cell Physiol. 2010; 299(5): C960–967. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYao J, Nowack A, Kensel-Hammes P, et al.: Cotrafficking of SV2 and synaptotagmin at the synapse. J Neurosci. 2010; 30(16): 5569–5578. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2051",
"date": "11 Oct 2013",
"name": "Yoshinori Moriyama",
"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\nSV2 is a transporter-like synaptic vesicle membrane protein with unknown function. In the previous work, this group has shown that SV2 can bind ATP. In this paper, the group extended characterization of SV2 protein and found that SV2 does not hydrolyze ATP. This is important information about function of SV2. It is very difficult, however, to give any conclusion from negative results. Although I almost agree that SV2 does not possess ATP hydrolytic activity, the results shown here only mean that ATP hydrolytic activity by SV2 is under the detection limit under the conditions employed. The title and some sentences in the text are thus overstated. Further characterization on the SV2-dependent ATP hydrolytic activity, that is the effects of phospholipids and detergents, temperature and dose dependence on the ATP hydrolytic activity, will be required to support this conclusion. In addition, it is helpful for readers to evaluate the suitability of the assay system used in the paper if the authors use any kinds of ATPase as another positive control.Furthermore, some important properties for enzymatic reaction by ATPase are lacking, some of which are as follows:Indicate evidence that SV2A and B were purified. Use of CIP as positive control is inappropriate. Please use ATPase such as ABC-type transporter for positive control. Please show the effect of phospholipids on ATP hydrolysis by SV2. Please show the dose dependence of SV2 protein. Temperature dependence.",
"responses": []
},
{
"id": "2050",
"date": "21 Oct 2013",
"name": "Liangyi Chen",
"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\nSV2 is an important protein essential for normal synaptic transmission. However, its molecular action remains unclear. Previously these researchers have identified SV2 as a ATP binding protein. In this study, they proceed to test whether ATP binding to SV2 leads to the hydrolysis of ATP. However, the evidence presented here is not sufficient enough for such a strong assertion.Main problem:Although purified SV2 protein can bind ATP, it is unclear whether it has the correct structural conformation to mediate ATP hydrolysis. To prove such a method can work in vitro, control experiments with similar purified transmembrane proteins that catalyze ATP hydrolysis should be done. Most importantly, it is unclear the local environment of SV2 proteins in vesicles in vivo, such as the concentration of ATP, pH and other factors. Therefore it will be ideal if the authors can measure ATP/ADP level in vivo in cells with overexpressed SV2 or not, using fluorescence indicators such as Perceval (Berg et al., Nat Methods. 2009; Tantama et al., Nat Commun. 2013).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-209
|
https://f1000research.com/articles/2-136/v1
|
06 Jun 13
|
{
"type": "Review",
"title": "Clinical applications of microRNAs",
"authors": [
"Per Hydbring",
"Gayane Badalian-Very",
"Per Hydbring"
],
"abstract": "MicroRNAs represent a class of small RNAs derived from polymerase II controlled transcriptional regions. The primary transcript forms one or several bulging double stranded hairpins which are processed by Drosha and Dicer into hetero-duplexes. The targeting microRNA strand of the duplex is incorporated into the RNA Induced Silencing Complex from where it silences up to hundreds of mRNA transcript by inducing mRNA degradation or blocking protein translation. Apart from involvement in a variety of biological processes, microRNAs were early recognized for their potential in disease diagnostics and therapeutics. Due to their stability, microRNAs could be used as biomarkers. Currently, there are microRNA panels helping physicians determining the origins of cancer in disseminated tumors. The development of microRNA therapeutics has proved more challenging mainly due to delivery issues. However, one drug is already in clinical trials and several more await entering clinical phases. This review summarizes what has been recognized pre-clinically and clinically on diagnostic microRNAs. In addition, it highlights individual microRNA drugs in running platforms driven by four leading microRNA-therapeutic companies.",
"keywords": [
"microRNA",
"siRNA",
"cancer",
"diagnosis",
"therapeutics",
"Miravirsen",
"Anti-miRs",
"MRX34"
],
"content": "MicroRNA discovery\n\nTwo decades have passed since the groundbreaking work from the laboratories of Gary Ruvkun and Victor Ambros, which demonstrated that a small temporal non-coding RNA could influence development of Caenorhabditis elegans by base pairing to the 3′ untranslated region (3′UTR) of a coding messenger RNA (mRNA) thereby regulating its translation1,2. For several years these RNA molecules were considered to be specific to C. elegans. A final recognition that they also played this role in additional systems, including human cells, marked the official birth of microRNAs (miRNAs)3–5.\n\nmiRNAs are separated from other small RNAs by the prediction of a hairpin fold-back structure from the precursor transcript together with expression evidence of an approximately 22 nucleotide-long mature sequence. Currently, 1600 human miRNA precursors have been deposited into miRBase v19 based on analyses of RNA deep-sequencing data6. The nomenclature of these miRNAs is based on a “mir” or “miR” prefix with identifying numbers assigned sequentially at the time of discovery. “mir” denotes a precursor miRNA whereas “miR” denotes a mature miRNA sequence. Similar or identical sequences can be given the same number. Distinction is then accomplished by a letter (similar sequences) or numeral ending (identical sequences). For example, miR-15a and miR-15b have identical 5’ ends but differ by four nucleotides in their 3’ regions. In contrast, miR-16-1 and miR-16-2 are identical but encoded on two separate chromosomes, chromosome 13 and 3, respectively7 (miRBase v19).\n\nmiRNA genes reside either in intergenic regions, within introns of coding or non-coding genes or within exons of non-coding genes8. Approximately one third of miRNAs are intergenic and about one third of all miRNA loci contain clustered miRNAs (miRBase v19). Work from the laboratory of David Bartel and others demonstrated that miRNAs that are oriented in the same genomic orientation and separated by less than 50 kb but more than 0.1 kb are highly correlated in their expression patterns. These results suggest that they originated from polycistronic transcripts9,10. Consistent with this, the laboratory of David Fisher showed that intronic miRNAs with independent transcriptional start sites were located substantially further away from their host gene start site, displaying a median distance of 57 kb11. Predicting the number of human miRNA clusters and precursors using such cutoffs in miRBase generates 175 and 538, respectively.\n\nThe majority of miRNAs are transcribed as long primary transcripts by RNA polymerase II and many are capped and polyadenylated12,13. Analysis of miRNAs residing in intergenic primary transcripts indicates that such transcripts are shorter than protein-coding transcripts with transcriptional start sites about 2 kb upstream of the pre-miRNA and polyadenylation signals 2 kb downstream14. A subset of miRNAs is transcribed by RNA polymerase III. This cluster of miRNAs is located among Alu rich regions on chromosome 1915.\n\nWhile most intronic miRNAs seem to be expressed from their host-gene transcriptional machinery, promoters and transcriptional activators of intergenic miRNAs are poorly defined. In order to identify miRNA promoters and their transcription factors, the laboratory of Richard Young set up a score system. This system overlaid genomic coordinates of tri-methylated Lysine 4 of Histone 3 (H3K4me3), their proximity to annotated miRNA sequences, expression sequence tag (EST) data and conservation between species with genome-wide association of transcription factors Oct4, Sox2, Nanog and Tcf3. The results demonstrated that miRNAs are regulated by Oct4, Sox2, Nanog and Tcf3 to the same degree as protein-coding genes16. These findings suggest that most miRNAs are transcriptionally regulated in a “protein-coding like” fashion.\n\nPrimary miRNA transcripts (pri-miRNAs) form hairpin bulges due to sequence complementarity. These hairpins are subject to processing by the Microprocessor complex which takes place in the nucleus20. The Microprocessor consists of the RNase III enzyme Drosha and its partner DiGeorge syndrome critical region gene 8 (DGCR8). Upon binding of DGCR8 to pri-miRNA at the junction of single-stranded to double-stranded RNA, Drosha cuts the pri-miRNA hairpin 11 bp into its double-stranded stem sequence generating slightly smaller hairpins known as pre-miRNAs. Intronic miRNAs are cleaved by Drosha co-transcriptionally preceding splicing of the primary transcript17. Pre-miRNAs are exported into the cytoplasm by the exportin 5 Ran-GTP complex where they are released by GTP hydrolysis and further processed by the RNase III Dicer into an approximate 22 nucleotide duplex. Dicer counts 22 nucleotides from the 5′ end of the pre-miRNA before cleaving, generating a duplex with 3′ end overhangs at each side. RNA helicases facilitate unwinding of the duplex with one strand being incorporated into the targeting miRNA containing RNA Induced Silencing Complex (RISC) together with the Argonate protein. Thermostability of the 5′ ends determines that the strand will be incorporated in the complex and that the strand left out will be degraded8,18.\n\nThe miRNA-RISC complex binds to 3′UTRs of mRNAs via nucleotide complementarity. Upon binding, it either induces mRNA degradation or inhibits protein translation. It is unclear to what extent mRNA degradation and translational inhibition coincide, but steady state levels suggest that mRNA degradation serves as the major final outcome for miRNA targeted transcripts19.\n\nmiRNAs bind their targets through seed- or seed-less pairing. The miRNA “seed” is located in its 5′end and expands from nucleotides 2–7. miRNAs that are interacting with their targets only through the seed region rarely produce efficient targeting. Additional base pairing of either nucleotide one, eight or both results in canonical seed targeting referred to as “7mers” or “8mers” correlating with functional outcomes. Disrupted complementarity, at any of the nucleotides between two to seven, leads to seed-less targeting. Seed-less targeting depend on compensatory complementarity along the mature miRNA sequence. In contrast, canonical seed targeting may be functional without such additional binding. During recent years, a number of prediction algorithms have been developed to track miRNA targeting based on seed pairing, overall complementarity, pairing stability, target site evolutionary conservation and UTR context20.\n\nGenerally, a single miRNA is predicted to have hundreds of targets. Prediction algorithms provide significant information for selecting appropriate miRNAs for targets of interest but there are several shortcomings when one seeks the biological outcome of a specific miRNA. First, expression levels of many miRNAs vary greatly between different cell types/tissues or disease states10,21, and this affects the stoichiometry of the binding of the miRNA to the target, potentially altering its impact. Secondly, the targeting capacity of a certain miRNA can be blunted by the state of its target, for example, shortened 3′ UTRs in cancer cells or point-mutations in the 3′ UTRs22,23.\n\nThe effect of a given miRNA may not necessarily correlate with repression of its targets. 3′ UTRs vary greatly in size, with many harboring binding sites for numerous endogenous miRNAs20,22. Inhibiting a single miRNA is therefore likely to give non-detectable or very modest derepression of an investigated target. Finally, functional assays demonstrating mRNA or protein alterations preceded by the introduction of ‘mimic miRNAs’ or inhibiting endogenous miRNAs could stem from the execution of indirect biological programs and not necessarily reflect miRNA-to-target interaction.\n\nTo combat such issues, miRNA researchers have turned to biochemical methods. Pull-down assays for either the miRNA itself or the RISC-complex followed by RT-PCR, microarray or sequencing could determine if the physical interaction between a given miRNA/mRNA is present or not24–26. By combining biochemical approaches with functional assays, it is possible to build confidence in specific miRNA targets. Given the huge number of predicted targets for any given miRNA, it is plausible to assume that only a fraction of predicted targets are substantially affected27,28. Interestingly, a recent study overlaid interacting transcripts with the targets that were significantly downregulated due to exogenous expression of a mimic miRNA in two different cell lines. They observed that the majority of miRNA to transcript interactions may result in no or very weak functional outcomes25. Such combinatory approaches are likely to be crucial in order to fully dissect miRNA functional targeting and they may ease biological network analyses by substantially reducing the number of targets.\n\n\nMicroRNAs in disease diagnostics\n\nObservations that miRNAs displayed high stability in paraffin-embedded tissues from clinical samples or in human plasma29,30 raised the possibility that miRNA expression analysis may be a useful tool to define disease states. An early key report from the laboratory of Todd Golub covering 217 mammalian miRNAs and several hundred samples, including clinical samples, common cancer cell lines and mouse tumors, demonstrated that miRNA profiles could distinguish a tumor’s developmental origin and that miRNAs are generally downregulated in cancers (129 out of 217 miRNAs). Interestingly, poorly differentiated tumors displayed lower miRNA expression compared to tumors with a higher differentiation21. This report was complemented by a study looking at miRNA expression profiles in human solid tumors31. miRNA signatures were further used to define subtypes of cancers, such as the distinction between basal and luminal breast cancers32,33. Sempere et al. showed that ER+PR+HER2+, ER-PR-HER+ and ER-PR-HER2- breast cancer tumors exhibited distinct miRNA patterns with miR-205 being highly expressed in triple negative breast cancers and miR-145 showing the opposite pattern. Analysis of separate cancers demonstrated miRNA profiles could predict clinical progression34,35. miR-15a/16-1 act as prognostic biomarkers in chronic lymphocytic leukemia (CLL) and let-7a is a marker for lung cancers34,35.\n\nIn addition to cancer, miRNA expression profiles could also be used to distinguish distinct forms of heart disease36, muscular disorders37 and neurodegenerative diseases38. The human miRNA-associated disease database (HMDD) serves as a resource for scientists screening the constantly increasing number of miRNA profiles for a wide range of diseases39. At the time of writing this review, the HMDD-database covered disease-associations from 2741 scientific publications.\n\nThe vast majority of miRNA expression profile reports stem from solid tissues although miRNAs can be readily detected in human plasma, serum or total blood due to their small size and high stability30,40. The potential of circulating miRNAs as biomarkers in serum was indicated early by studies examining patients with diffuse large B-cell lymphoma, highlighting miR-21 as a potential biomarker41. Prostate cancer patients could be distinguished from healthy counterparts by analyzing the expression level of miR-14330. These studies were followed by additional reports of miRNAs in breast cancer (using whole blood)42, colorectal cancer (using plasma)43 and squamous cell lung cancer (using sputum)44 patients. Interestingly, Boeri et al. demonstrated that circulating miRNAs may also be used for predicting purposes. They displayed miRNA signatures with strong predictive value in lung cancer patients years before the onset of disease by analyzing expression in samples taken before diagnosis, at the time of detection by computed tomography and in disease-free smokers45.\n\nIn a massive undertaking Keller et al. analyzed 863 miRNAs from 454 human blood samples. The samples were from patients suffering from 14 separate diseases including lung cancer, prostate cancer, pancreatic ductal adenocarcinoma, melanoma, ovarian cancer, gastric tumors, Wilms tumor, pancreatic tumors, multiple sclerosis, chronic obstructive pulmonary disease, sarcoidosis, periodontitis, pancreatitis and myocardial infarction. On average, more than 100 miRNAs were deregulated in the blood for each disease. By utilizing this data and developing mathematical algorithms and probability plots the authors were able to accurately predict the disease in more than two thirds of individuals involved in the study46. It should be noted that miRNA patterns in blood are unlikely to be the same between different types of blood cells. Distinct hematopoietic lineages display different miRNA-profiles47, possibly suggesting that differences in expression in specific diseases are due to shifts in hematopoietic cell populations. This could be pronounced in diseases directly affecting the blood such as cancers spreading to the bone marrow or multiple sclerosis, an autoimmune disease associated with abnormal levels of white blood cells. Using 27 distinct cell populations with the highest variance, Keller et al. computed that such shifts could account for a maximum of 60% of differences in the observed miRNA profiles46.\n\nHow are miRNAs released into the blood stream? Rechavi et al. demonstrated that synthetic miRNA mimics, viral miRNAs or endogenous miRNAs could be released from B-cells and taken up by T-cells upon cell contact48. Further, Pegtel et al. demonstrated secretion of miRNAs from Epstein-Barr virus (EBV)-infected B-cells via exosomes, providing protection of the miRNA from RNases49 and Yuan et al. reported the transfer of miRNAs from embryonic stem cells to fibroblasts via embryonic stem cell microvesicles50. Finally, Kosaka et al. demonstrated that miRNA secretion occurs in a ceramide-dependent fashion, which could be blunted by knocking down nSmase2, an enzyme required for ceramide biosynthesis51.\n\nIn addition to serum/plasma, the presence of miRNAs has also been demonstrated in urine52 and saliva53, with two miRNAs, miR-125a and miR-200a, displaying lower expression in patients with oral squamous cell carcinoma compared to healthy subjects54.\n\nmiRNA expression profiling and disease association studies are conducted using a set of different methods including miRNA microarray platforms, quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization and high throughput sequencing. Both qRT-PCR and hybridization methods are highly sensitive and quantitative. This makes them useful for analyzing small sets of miRNAs55. It is worth noting that there are limitations with hybridization methods due to potential hybridization of the probe to pri-, pre- and mature miRNAs. Detection of mature miRNA sequences through qRT-PCR demands a stem-loop RT primer, specific primers for amplification of the cDNA and a TaqMan probe (Roche Molecular Diagnostics, Applied Biosystems)56. In situ hybridization can either be represented by fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) and detection of miRNAs as well as other non-coding RNAs is possible without protease-treatment of tissues57.\n\nFor high-throughput studies, miRNAs are profiled using array platforms or sequencing. Next-generation sequencing technologies offer lower costs and shorter processing time making this platform highly attractive. Today, researchers frequently use sequencing techniques to profile miRNA signatures in distinct sets of tissues/diseases.\n\nA major challenge in clinical diagnostics is cancers with poor differentiation. Even though these cancers account for only a few percent of malignancies, they display substantially distinct gene-signatures making it notoriously hard to trace the cell of origin despite the availability of the latest microRNA platforms58.\n\nPerhaps the most exciting potential of miRNAs in current diagnostics started with a study comparing miRNA expression using miRNA microarrays in 205 primary versus 131 metastatic tumors covering 22 different tumor origins. The authors developed a binary decision tree classifier based on miRNA expression with tissues displaying the highest specificity of certain miRNAs placed at the top of the tree. Following this classifier, a remarkably low number of 48 miRNAs predicted tissue origin at close to 90% accuracy when tested on a blinded set59 (Table 1). This study, together with subsequent studies confirming the accuracy of using miRNAs as diagnostics for tumors of unknown origin60,61, was partly driven by the miRNA diagnostics company Rosetta Genomics, Israel.\n\nEach node uses 1 to 5 miRNAs for classification. Nodes giving no defined cancer types make broader distinctions. For example, Node #3 distinguishes epithelial tissues from mixed origin. Table is adapted from Rosenfeld et al. 200859.\n\nBased on these reports, Rosetta Genomics is now offering a panel (miRview-mets2) to clinicians so that the origin of metastatic cancers can be identified where the primary origin of metastasis is questionable. The panel consists of a test of 64 miRNA biomarkers validated on 489 samples of which 146 represent metastatic tumors covering 42 tissues of origin62. In addition to the “miRview-mets2” panel, Rosetta Genomics also offers four additional clinical tests: “miRview-lung”, “miRview-squamous”, “miRview-meso” and “miRview-kidney”. MiRview-lung differentiates four types of primary lung cancer (small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC and carcinoid) using eight separate miRNA biomarkers63. MiRview-squamous separates non-small cell lung cancers (NSCLC) into squamous cell carcinomas and adenocarcinomas using a single miRNA, hsa-miR-20564. MiRview-meso defines samples into a mesothelioma or non-mesothelioma origin based on three separate miRNAs65 and miRview-kidney separates kidney cancer into its four primary types (benign oncocytoma, clear cell renal carcinoma, papillary renal carcinoma and chromophobe renal carcinoma) using six miRNAs. All clinical panels rely on the same tree-classifier as in the original paper59,66.\n\nThe move of miRNA diagnostics into the clinics led by Rosetta Genomics and others (including Asuragen Austin TX, USA) may aid the use of personalized medicine in the treatment of cancers. Importantly, miRNA diagnostics are not just an additional approach for differentiated cancers but a potential breakthrough for cancers initially defined as of unknown origin.\n\nIn addition to more well-established approaches of analyzing miRNA expression profiles, predisposition to certain cancer types may be predicted by the existence of single nucleotide polymorphisms (SNPs). SNPs can reside in either precursor miRNAs, mature miRNAs or in 3′ UTRs disrupting or creating miRNA binding sites67.\n\nScreening of 42 miRNA expression profiles in chronic lymphocytic leukemia (CLL) revealed the presence of mutations in 5 miRNAs; all SNPs were detected in the pri- or pre-miRNA structure. For one of these, miR-16-1, a mutation in the precursor transcript led to a significant reduction in mature miRNA expression35. Other reports of SNP variants in miRNA precursors where the mutant variant correlates with an increased risk of cancer include miR-196a-2 and miR-499 in breast cancer68, miR-196a-2 in head and neck cancer69, whilst increased mature levels of miR-146a can lead to an earlier onset of breast and ovarian cancers70. In addition, SNPs affecting factors in the miRNA processing machinery lead to either an increased or reduced risk of renal cell carcinoma depending on which factors were affected71.\n\nExamples of SNPs located in 3′ UTRs of miRNA target sites include the binding site of miR-221/222 and miR-146 in Kit72 as well as the let-7 binding site in the 3′ UTR of K-Ras. The latter displays an alteration associated with an increased risk of NSCLC23. Further, a SNP in the miR-184 binding site in the 3′ UTR of tumor necrosis factor alpha-induced protein 2 (TNFAIP2) resulted in an increased risk of squamous cell carcinoma of the head and neck73.\n\nUsing available SNP data from “The 1000 Genomes Project”74, Richardson et al. concluded that approximately 5% of all SNPs maps to miRNA recognition elements (MREs). Among these SNPs, 22% associated with disease phenotype75. Interestingly, these numbers suggest that SNPs residing within MREs are likely to be under selective pressure.\n\n\nMicroRNAs in therapeutics\n\nThe idea of using miRNAs in therapeutics is highly appealing after observing the outcomes from manipulating these molecules. Rather than intercepting a single target as in the case of selective protein inhibitors, miRNAs can modulate entire gene programs. Given the notion of deregulation of these molecules in a wide range of diseases and the robust degree of accurate profiling, one would estimate the chances of mapping specific miRNAs for manipulation of a given disease with minimal side-effects to be fairly good.\n\nmiRNAs fall into the class of RNAi-based therapeutics, a concept that is not completely new, having given birth to more than 20 clinical trials so far76, but unlike short interfering RNAs/short hairpin RNAs (siRNAs/shRNAs), which are designed to target a single transcript, modulation of miRNAs, will affect hundreds of transcripts and so would potentially be capable of shutting down entire deregulated pathways. Of course, specific targeting of disease associated transcripts is probably required since a completely random modulation of hundreds of transcripts would be too harmful for the patient. However, such issues can be relatively easily investigated from in vitro studies conducting genome-wide mRNA expression together with pathway enrichment analysis. To date there is only one miRNA drug in clinical trials (SPC3649: inhibitor of miR-122, Santaris Pharma, Denmark) (Figure 1). The slow progress stems from the general technical challenges with RNAi-based therapeutics including delivery, stability and avoidance of activating immune responses.\n\nThe four stages at the top denote the progress of individual miRNA drugs. Red colored miRNAs indicate replacement therapy with drug mimicking the endogenous miRNA. Blue colored miRNAs indicate inhibition therapy with drug targeting the endogenous miRNA. *Santaris Pharma has completed two Phase I clinical trials and is currently in Phase II with their miR-122 targeting drug miravirsen.\n\nAs discussed earlier in this review, endogenous miRNAs remain highly stable even when secreted into circulation. They resist nucleases by being enclosed into microvesicles or exosomes. Creating complex delivery-vehicles for miRNA therapeutics is feasible but could be time-consuming and costly. To minimize stability issues, a range of chemical modifications have been developed for siRNAs, modifications likely to be transferrable to miRNAs due to their similar structures. These include Phosphorothioate, 2′-O-methyl RNA, 2′-Fluoro-RNA and 2′O-methoxy-ethyl RNA, all of which provide greater nuclease resistance76,77. A phosphorothioate modification exchanges a nonbridging oxygen atom with a sulfur atom at the phospho-backbone of the unmodified RNA. In addition to providing nuclease resistance this also promotes RNase H-mediated cleavage of targeted transcripts. The 2′O-methyl modification adds a methyl group (-CH3) to the second carbon of the ribose. Likewise, 2′Fluoro adds a fluorine atom at this position and 2′O-methoxy-ethyl adds a methoxy-ethyl group (-C3H7O). All 2′-modifications increase Watson-Crick base pairing to targeted transcripts and substantiate nuclease resistance due to closer proximity between the 2′ group and 3′ phospho-group. In addition, a modification known as locked nucleic acid (LNA) is widely used in synthesis for miRNA inhibiting drugs, sometimes referred as antagomirs. This modification utilizes a bridge between the 2′O group and 4′ carbon atom also referred to as 2′O-4′C-methylene linked ribonucleotides77.\n\nEven though siRNAs, and in a similar way miRNA mimics, can be stabilized through chemical modifications, their size still demands complex vehicles in order to ensure delivery to different tissues in vivo78. In contrast, miRNA inhibiting drugs can be synthesized as short single-stranded oligonucleotides, their small size together with their potency/stability provided by the LNA-modifications make delivery possible without vehicle-systems. For the rest of this review we will summarize miRNA drugs currently in clinical trials or likely to enter clinical trials in the near future.\n\nMiravirsen (or SPC3649) is a LNA-modified oligonucleotide designed to inhibit miR-122 developed by Danish firm Santaris Pharma A/S79. This liver specific and highly abundant miRNA accounts for more than 70% of all miRNAs in the liver and has been shown to be crucial for the functional infection of Hepatitis C virus (HCV). The exact mechanism of how this miRNA facilitates viral replication is not clearly understood. It is suggested that interaction between miR-122 and two seed-sites in the 5′ noncoding region of HCV induces viral transcripts, giving this miRNA a non-classical function of inducing rather than inhibiting the function of its target80.\n\nTreatment of chronic Hepatitis C-infected chimpanzees with miravirsen led to suppression of HCV without any obvious side-effects. Further, liver transcriptome analysis revealed 259 mRNAs containing full 8mer-seed miR-122 predicted binding sites to display increased expression following treatment, thus indicating such transcripts to be targeted by endogenous miR-122 in Hepatitis C. It is likely that some of those transcripts play additional therapeutic roles. Total serum cholesterol was reduced with downregulation of cholesterol metabolism genes79.\n\nSince reporting their non-human primate study, miravirsen has gone through two phase I clinical trials, successfully proving that the drug is safe even in humans (NCT00688012, NCT00979927), and a Phase IIa clinical trial (NCT01200420) (Figure 1). This Phase IIa trial enrolled 38 patients with treatment-naïve chronic HCV infection to monitor safety, tolerability, pharmacokinetics and efficacy on HCV viral titer. Multiple dosage of miravirsen administered subcutaneously to patients gave promising outcomes with a mean reduction of HCV RNA levels by two to three logarithmic levels. Further, almost half of the patients treated by the highest dose displayed undetectable levels of HCV RNA within 4 weeks. These results are encouraging and highlight miravirsen as a potential future replacement therapy for patients with chronic HCV infection.\n\nDiscovering the general potential of miRNA inhibitory therapeutics using LNA-based or similar platforms for a wide range of diseases is a major task for the future. One might imagine that the tissue specificity of miR-122, its high abundance in this tissue compared to other miRNAs and the general convenience of delivering drugs systemically to the liver makes inhibition in this case easier than most other miRNAs. Despite such possible concerns, the development of miravirsen by Santaris Pharma provides a landmark breakthrough for miRNA based therapeutics.\n\nRegulus Therapeutics is a San Diego based company with a wide-range program focusing on targeting endogenous miRNAs through inhibitory oligonucleotides in hepatocellular carcinoma, kidney fibrosis, atherosclerosis, HCV infection and glioblastoma. To keep such a platform running, Regulus has partnered with the corporate giants Sanofi, GlaxoSmithKline and AstraZeneca. In 2010, Regulus signed a deal with GlaxoSmithKline for the development of a miR-122 inhibitor drug, thus providing direct competition with Santaris Pharma (see above).\n\nNone of Regulus’ programs have reached clinical phases but substantial preclinical work has been completed including a non-human primate study of inhibiting miR-33a/b for the treatment of atherosclerosis81 (Figure 1). miR-33b and miR-33a are encoded in the introns of the transcription factor loci SREBP1 and SREBP2, respectively, and are involved in the regulation of cholesterol and fatty acid homeostasis. This puts them as potential targets for treatment of cardiovascular diseases81. By treating African Green monkeys subcutaneously with 2′-fluoro-methoxyethyl-phosphorothioate modified antisense-miR-33 oligonucleotides (anti-miR-33), the study demonstrated a decrease in very-low-density lipoprotein (VLDL)-triglycerides and an increase in high-density lipoprotein (HDL)81. Mechanistically, there was a significantly reduced repression of miR-33 predicted target genes and primates went through treatment without displaying significant side-effects81.\n\nUsing another anti-miR (anti-miR-21), Regulus convincingly demonstrated that their technology could be used for the inhibition of migration/invasion of glioma in mice82, attenuating cardiac dysfunction in a mouse model of cardiac disease83 and executing an anti-fibrotic response in mice exposed to kidney injury84. Results from additional mouse-models exposed to Regulus-developed anti-miRs include suppression of lung-metastasis originating from breast tumors (anti-miR-10b)85, inhibition of neuroblastoma (anti-miR-380-5p)86, antagonizing liver metastasis sourcing from melanoma (anti-miR-182)87 and improved glucose homeostasis and insulin sensitivity (anti-miR-103/107)88 (Figure 1).\n\nIn contrast to the companies described above, Mirna Therapeutics is not focusing their platform around the inhibition of endogenous miRNAs but instead on introducing synthetic miRNA mimics as replacement therapies. The idea is to restore expression of certain miRNAs in tumors to a comparable level to surrounding healthy tissues. The platform of Mirna Therapeutics consists of eight drug candidates, solely focused on addressing tumor suppressive roles of miRNAs. Three out of eight candidate mimics represent miRNAs demonstrated by numerous publications to display tumor suppressive properties (miR-34, let-7 and miR-16). Their lead candidate, MRX34, a miR-34a mimic compound, will probably be the first miRNA replacement compound to reach clinical trials (Figure 1). At the time of writing this review, Mirna Therapeutics is recruiting participants to a Phase I study of MRX34 (NCT01829971).\n\nmiR-34a represents one of the most documented tumor suppression-associated miRNAs, being a transcriptional product of the transcription factor and genome guardian p5389. Pre-clinical work by Mirna Therapeutics has demonstrated potent anti-tumor effects by introducing miR-34a mimics into a variety of mice cancer models90,91.\n\nThe usage of miRNA mimics for systemic delivery is challenging compared to anti-miR drugs. miRNA mimics need to be double-stranded in order to be processed correctly by the cellular RNAi-machinery and therefore cannot be administered “naked”. Successful delivery therefore requires complex delivery vehicles mimicking physiological settings where miRNAs reside in microvesicles or exosomes. For MRX34, Mirna therapeutics has developed custom nanoparticle liposomes. According to company information (MIRNA THERAPEUTICS) these liposomes increase stability, enhance delivery and prevent immune response effects. Extensive pre-clinical testing of MRX34 in mouse models of hepatocellular carcinoma using liposomes provided promising outcomes and the upcoming clinical trial is recruiting patients with non resectable primary liver cancer or metastatic cancer with liver involvement (NCT01829971).\n\nmiRagen Therapeutics is an American company founded in 2007 for the development of miRNA based drugs treating cardiac and muscular diseases. A partner alliance with Danish Santaris Pharma gives miRagen commercial rights to the LNA-based technique developed by Santaris. miRagen has programs covering both anti-miR and miRNA mimics. The anti-miR program contains candidate drugs relatively close to clinical stages (Figure 1). This platform includes three drugs: MGN-9103, MGN-1374 and MGN-4893.\n\nThe lead drug, MGN-9103, targets miR-208 with implications for the treatment of chronic heart failure. miR-208 is a heart specific miRNA located in an intron of the alphaMHC gene, which has been demonstrated to be required for cardiac hypertrophy, myosin switching and fibrosis in response to stress92. Such cardiac remodeling was blocked by treating rats subcutaneously with LNA-based anti-miR-208 during hypertension induced heart failure93. Interestingly, MGN-9103 was also recently suggested to play beneficial roles in a mouse model of diabetes/obesity94.\n\nMGN-1374 targets miR-15 and miR-195, miRNAs shown to be upregulated in mouse hearts shortly after birth. Upregulation of miR-15 and miR-195 executes a postnatal cell cycle arrest in the heart generation after myocardial infarction95. By inhibiting these miRNAs using their LNA-based MGN-1374, miRagen enables post-myocardial infarction remodeling. Such remodeling enhanced heart function and induced cardiomyocyte proliferation in mice and pigs96.\n\nMGN-4893, targets miR-451, a miRNA required for the expansion of red blood cells. Inhibition of miR-451 in mice using anti-miR-451 blocked erythrocyte differentiation suggesting that such inhibitors could be useful for the treatment of disorders leading to abnormal red blood cell production such as polycythemia vera97.\n\n\nConcluding remarks\n\nMicroRNAs have come a long way since the initial discoveries two decades ago. Their emerging potential as biomarkers in clinical diagnostics as well as modulators for the treatment of a variety of diseases is truly exciting. In the near future it will become clearer as to whether they have the power to become established as new molecular diagnostic benchmarks and whether microRNA-based therapy can compete with that of selective protein inhibitors.",
"appendix": "Author contributions\n\n\n\nPH wrote the initial draft of the article, GB reviewed and wrote the final draft.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWightman B, Ha I, Ruvkun G: Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993; 75(5): 855–62. PubMed Abstract | Publisher Full Text\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\nLee RC, Ambros V: An extensive class of small RNAs in Caenorhabditis elegans. Science. 2001; 294(5543): 862–4. PubMed Abstract | Publisher Full Text\n\nLau NC, Lim LP, Weinstein EG, et al.: An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science. 2001; 294(5543): 858–62. PubMed Abstract | Publisher Full Text\n\nLagos-Quintana M, Rauhut R, Lendeckel W, et al.: Identification of novel genes coding for small expressed RNAs. Science. 2001; 294(5543): 853–8. PubMed Abstract | Publisher Full Text\n\nGriffiths-Jones S, Saini HK, van Dongen S, et al.: miRBase: tools for microRNA genomics. Nucleic Acids Res. 2008; 36(Database issue): D154–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmbros V, Bartel B, Bartel DP, et al.: A uniform system for microRNA annotation. RNA. 2003; 9(3): 277–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim VN, Han J, Siomi MC: Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009; 10(2): 126–39. PubMed Abstract | Publisher Full Text\n\nBaskerville S, Bartel DP: Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA. 2005; 11(3): 241–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandgraf P, Rusu M, Sheridan R, et al.: A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007; 129(7): 1401–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOzsolak F, Poling LL, Wang Z, et al.: Chromatin structure analyses identify miRNA promoters. Genes Dev. 2008; 22(22): 3172–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee Y, Kim M, Han J, et al.: MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 2004; 23(20): 4051–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCai X, Hagedorn CH, Cullen BR: Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA. 2004; 10(12): 1957–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaini HK, Griffiths-Jones S, Enright AJ: Genomic analysis of human microRNA transcripts. Proc Natl Acad Sci U S A. 2007; 104(45): 17719–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorchert GM, Lanier W, Davidson BL: RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol. 2006; 13(12): 1097–101. PubMed Abstract | Publisher Full Text\n\nMarson A, Levine SS, Cole MF, et al.: Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell. 2008; 134(3): 521–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPawlicki JM, Steitz JA: Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production. J Cell Biol. 2008; 182(1): 61–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark JE, Heo I, Tian Y, et al.: Dicer recognizes the 5′ end of RNA for efficient and accurate processing. Nature. 2011; 475(7355): 201–5. PubMed Abstract | Publisher Full Text\n\nGuo H, Ingolia NT, Weissman JS, et al.: Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature. 2010; 466(7308): 835–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBartel DP: MicroRNAs: target recognition and regulatory functions. Cell. 2009; 136(2): 215–33. PubMed Abstract | Publisher Full Text\n\nLu J, Getz G, Miska EA, et al.: MicroRNA expression profiles classify human cancers. Nature. 2005; 435(7043): 834–8. PubMed Abstract | Publisher Full Text\n\nSandberg R, Neilson JR, Sarma A, et al.: Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites. Science. 2008; 320(5883): 1643–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChin LJ, Ratner E, Leng S, et al.: A SNP in a let-7 microRNA complementary site in the KRAS 3′ untranslated region increases non-small cell lung cancer risk. Cancer Res. 2008; 68(20): 8535–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarginov FV, Conaco C, Xuan Z, et al.: A biochemical approach to identifying microRNA targets. Proc Natl Acad Sci U S A. 2007; 104(49): 19291–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLal A, Thomas MP, Altschuler G, et al.: Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling. PLoS Genet. 2011; 7(11): e1002363. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChi SW, Zang JB, Mele A, et al.: Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature. 2009; 460(7254): 479–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSelbach M, Schwanhausser B, Thierfelder N, et al.: Widespread changes in protein synthesis induced by microRNAs. Nature. 2008; 455(7209): 58–63. PubMed Abstract | Publisher Full Text\n\nBaek D, Villen J, Shin C, et al.: The impact of microRNAs on protein output. Nature. 2008; 455(7209): 64–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNelson PT, Baldwin DA, Scearce LM, et al.: Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods. 2004; 1(2): 155–61.. PubMed Abstract | Publisher Full Text\n\nMitchell PS, Parkin RK, Kroh EM, et al.: Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008; 105(30): 10513–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVolinia S, Calin GA, Liu CG, et al.: A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006; 103(7): 2257–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlenkiron C, Goldstein LD, Thorne NP, et al.: MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol. 2007; 8(10): R214. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSempere LF, Christensen M, Silahtaroglu A, et al.: Altered MicroRNA expression confined to specific epithelial cell subpopulations in breast cancer. Cancer Res. 2007; 67(24): 11612–20. PubMed Abstract | Publisher Full Text\n\nTakamizawa J, Konishi H, Yanagisawa K, et al.: Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 2004; 64(11): 3753–6. PubMed Abstract | Publisher Full Text\n\nCalin GA, Ferracin M, Cimmino A, et al.: A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med. 2005; 353(17): 1793–801. PubMed Abstract | Publisher Full Text\n\nvan Rooij E, Sutherland LB, Liu N, et al.: A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proc Natl Acad Sci U S A. 2006; 103(48): 18255–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEisenberg I, Eran A, Nishino I, et al.: Distinctive patterns of microRNA expression in primary muscular disorders. Proc Natl Acad Sci U S A. 2007; 104(43): 17016–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHebert SS, De Strooper B: Alterations of the microRNA network cause neurodegenerative disease. Trends Neurosci. 2009; 32(4): 199–206. PubMed Abstract | Publisher Full Text\n\nLu M, Zhang Q, Deng M, et al.: An analysis of human microRNA and disease associations. PLoS One. 2008; 3(10): e3420. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwarzenbach H, Hoon DS, Pantel K: Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011; 11(6): 426–37. PubMed Abstract | Publisher Full Text\n\nLawrie CH, Gal S, Dunlop HM, et al.: Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol. 2008; 141(5): 672–5. PubMed Abstract | Publisher Full Text\n\nHeneghan HM, Miller N, Kelly R, et al.: Systemic miRNA-195 differentiates breast cancer from other malignancies and is a potential biomarker for detecting noninvasive and early stage disease. Oncologist. 2010; 15(7): 673–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang Z, Huang D, Ni S, et al.: Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer. Int J Cancer. 2010; 127(1): 118–26. PubMed Abstract | Publisher Full Text\n\nXing L, Todd NW, Yu L, et al.: Early detection of squamous cell lung cancer in sputum by a panel of microRNA markers. Mod Pathol. 2010; 23(8): 1157–64. PubMed Abstract | Publisher Full Text\n\nBoeri M, Verri C, Conte D, et al.: MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A. 2011; 108(9): 3713–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKeller A, Leidinger P, Bauer A, et al.: Toward the blood-borne miRNome of human diseases. Nat Methods. 2011; 8(10): 841–3. PubMed Abstract | Publisher Full Text\n\nPetriv OI, Kuchenbauer F, Delaney AD, et al.: Comprehensive microRNA expression profiling of the hematopoietic hierarchy. Proc Natl Acad Sci U S A. 2010; 107(35): 15443–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRechavi O, Erlich Y, Amram H, et al.: Cell contact-dependent acquisition of cellular and viral nonautonomously encoded small RNAs. Genes Dev. 2009; 23(16): 1971–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPegtel DM, Cosmopoulos K, Thorley-Lawson DA, et al.: Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A. 2010; 107(14): 6328–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYuan A, Farber EL, Rapoport AL, et al.: Transfer of microRNAs by embryonic stem cell microvesicles. PLoS One. 2009; 4(3): e4722. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKosaka N, Iguchi H, Yoshioka Y, et al.: Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem. 2010; 285(23): 17442–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHanke M, Hoefig K, Merz H, et al.: A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer. Urol Oncol. 2010; 28(6): 655–61. PubMed Abstract | Publisher Full Text\n\nMichael A, Bajracharya SD, Yuen PS, et al.: Exosomes from human saliva as a source of microRNA biomarkers. Oral Dis. 2010; 16(1): 34–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPark NJ, Zhou H, Elashoff D, et al.: Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. an official journal of the American Association for Cancer Research. Clin Cancer Res. 2009; 15(17): 5473–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIorio MV, Croce CM: MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med. 2012; 4(3): 143–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen C, Ridzon DA, Broomer AJ, et al.: Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005; 33(20): e179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Planell-Saguer M, Rodicio MC, Mourelatos Z: Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat Protoc. 2010; 5(6): 1061–73. PubMed Abstract | Publisher Full Text\n\nRamaswamy S, Tamayo P, Rifkin R, et al.: Multiclass cancer diagnosis using tumor gene expression signatures. Proc Natl Acad Sci U S A. 2001; 98(26): 15149–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRosenfeld N, Aharonov R, Meiri E, et al.: MicroRNAs accurately identify cancer tissue origin. Nat Biotechno. 2008; 26(4): 462–9. PubMed Abstract | Publisher Full Text\n\nVaradhachary GR, Spector Y, Abbruzzese JL, et al.: Prospective gene signature study using microRNA to identify the tissue of origin in patients with carcinoma of unknown primary. Clin Cancer Res. 2011; 17(12): 4063–70. PubMed Abstract | Publisher Full Text\n\nMueller WC, Spector Y, Edmonston TB, et al.: Accurate classification of metastatic brain tumors using a novel microRNA-based test. Oncologist. 2011; 16(2): 165–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeiri E, Mueller WC, Rosenwald S, et al.: A second-generation microRNA-based assay for diagnosing tumor tissue origin. Oncologist. 2012; 17(6): 801–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGilad S, Lithwick-Yanai G, Barshack I, et al.: Classification of the four main types of lung cancer using a microRNA-based diagnostic assay. J Mol Diagn. 2012; 14(5): 510–7. PubMed Abstract | Publisher Full Text\n\nLebanony D, Benjamin H, Gilad S, et al.: Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol. 2009; 27(12): 2030–7. PubMed Abstract | Publisher Full Text\n\nBenjamin H, Lebanony D, Rosenwald S, et al.: A diagnostic assay based on microRNA expression accurately identifies malignant pleural mesothelioma. J Mol Diagn. 2010; 12(6): 771–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFridman E, Dotan Z, Barshack I, et al.: Accurate molecular classification of renal tumors using microRNA expression. J Mol Diagn. 2010; 12(5): 687–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRyan BM, Robles AI, Harris CC: Genetic variation in microRNA networks: the implications for cancer research. Nat Rev Cancer. 2010; 10(6): 389–402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu Z, Liang J, Wang Z, et al.: Common genetic variants in pre-microRNAs were associated with increased risk of breast cancer in Chinese women. Hum Mutat. 2009; 30(1): 79–84. PubMed Abstract | Publisher Full Text\n\nChristensen BC, Avissar-Whiting M, Ouellet LG, et al.: Mature microRNA sequence polymorphism in MIR196A2 is associated with risk and prognosis of head and neck cancer. Clin Cancer Res. 2010; 16(14): 3713–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen J, Ambrosone CB, DiCioccio RA, et al.: A functional polymorphism in the miR-146a gene and age of familial breast/ovarian cancer diagnosis. Carcinogenesis. 2008; 29(10): 1963–6. PubMed Abstract | Publisher Full Text\n\nHorikawa Y, Wood CG, Yang H, et al.: Single nucleotide polymorphisms of microRNA machinery genes modify the risk of renal cell carcinoma. Clin Cancer Res. 2008; 14(23): 7956–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHe H, Jazdzewski K, Li W, et al.: The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci U S A. 2005; 102(52): 19075–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu Z, Wei S, Ma H, et al.: A functional variant at the miR-184 binding site in TNFAIP2 and risk of squamous cell carcinoma of the head and neck. Carcinogenesis. 2011; 32(11): 1668–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\n1000 Genomes Project ConsortiumAbecasis GR, Altshuler D, Auton A, et al.: A map of human genome variation from population-scale sequencing. Nature. 2010; 467(7319): 1061–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRichardson K, Lai CQ, Parnell LD, et al.: A genome-wide survey for SNPs altering microRNA seed sites identifies functional candidates in GWAS. BMC genomics. 2011; 12: 504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurnett JC, Rossi JJ: RNA-based therapeutics: current progress and future prospects. Chem Biol. 2012; 19(1): 60–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShukla S, Sumaria CS, Pradeepkumar PI: Exploring chemical modifications for siRNA therapeutics: a structural and functional outlook. Chem Med Chem. 2010; 5(3): 328–49. PubMed Abstract | Publisher Full Text\n\nSiegwart DJ, Whitehead KA, Nuhn L, et al.: Combinatorial synthesis of chemically diverse core-shell nanoparticles for intracellular delivery. Proc Natl Acad Sci U S A. 2011; 108(32): 12996–3001. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLanford RE, Hildebrandt-Eriksen ES, Petri A, et al.: Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science. 2010; 327(5962): 198–201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJopling CL, Yi M, Lancaster AM, et al.: Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science. 2005; 309(5740): 1577–81. PubMed Abstract | Publisher Full Text\n\nRayner KJ, Esau CC, Hussain FN, et al.: Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature. 2011; 478(7369): 404–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGabriely G, Wurdinger T, Kesari S, et al.: MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol. 2008; 28(17): 5369–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThum T, Gross C, Fiedler J, et al.: MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature. 2008; 456(7224): 980–4. PubMed Abstract | Publisher Full Text\n\nChau BN, Xin C, Hartner J, et al.: MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways. Sci Transl Med. 2012; 4(121): 121ra18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMa L, Reinhardt F, Pan E, et al.: Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model. Nat Biotechnol. 2010; 28(4): 341–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwarbrick A, Woods SL, Shaw A, et al.: miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma. Nat Med. 2010; 16(10): 1134–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuynh C, Segura MF, Gaziel-Sovran A, et al.: Efficient in vivo microRNA targeting of liver metastasis. Oncogene. 2011; 30(12): 1481–8. PubMed Abstract | Publisher Full Text\n\nTrajkovski M, Hausser J, Soutschek J, et al.: MicroRNAs 103 and 107 regulate insulin sensitivity. Nature. 2011; 474(7353): 649–53. PubMed Abstract | Publisher Full Text\n\nHe L, He X, Lim LP, et al.: A microRNA component of the p53 tumour suppressor network. Nature. 2007; 447(7148): 1130–4. PubMed Abstract | Publisher Full Text\n\nLiu C, Kelnar K, Liu B, et al.: The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat Med. 2011; 17(2): 211–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrang P, Wiggins JF, Daige CL, et al.: Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol Ther. 2011; 19(6): 1116–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Rooij E, Sutherland LB, Qi X, et al.: Control of stress-dependent cardiac growth and gene expression by a microRNA. Science. 2007; 316(5824): 575–9. PubMed Abstract | Publisher Full Text\n\nMontgomery RL, Hullinger TG, Semus HM, et al.: Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure. Circulation. 2011; 124(14): 1537–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrueter CE, van Rooij E, Johnson BA, et al.: A cardiac microRNA governs systemic energy homeostasis by regulation of MED13. Cell. 2012; 149(3): 671–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPorrello ER, Mahmoud AI, Simpson E, et al.: Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family. Proc Natl Acad Sci U S A. 2013; 110(1): 187–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHullinger TG, Montgomery RL, Seto AG, et al.: Inhibition of miR-15 protects against cardiac ischemic injury. Circ Res. 2012; 110(1): 71–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatrick DM, Zhang CC, Tao Y, et al.: Defective erythroid differentiation in miR-451 mutant mice mediated by 14-3-3zeta. Genes Dev. 2010; 24(15): 1614–9. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1008",
"date": "18 Jun 2013",
"name": "Florian Karreth",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Hydbring and Badalian-Very summarizes our current knowledge of microRNAs and outlines their clinical value. The authors provide a comprehensive overview of microRNA biology and target prediction and discuss the utility of miRNAs as prognostic and diagnostic markers and therapeutic targets. Finally, Hydbring and Badalian-Very highlight the various strategies adopted by leading biotechnology companies and the state of their current clinical trials. I have two minor suggestions regarding the biology of miRNAs that the authors may wish to include in their manuscript: A recent paper by paper by Tollervey and colleagues (Helwak et al., Cell 2013) describes that a large fraction of miRNAs bind their targets independent of seed match complementarity at nucleotides 2-7. This observation greatly affects our ability to accurately predict miRNA targets using existing algorithms.Besides the 3’UTR, miRNAs have been demonstrated to target the 5’UTR and coding sequence of mRNAs as well as other RNA species such as lncRNAs, pseudogenes, rRNAs and tRNAs.",
"responses": [
{
"c_id": "528",
"date": "15 Aug 2013",
"name": "Per Hydbring",
"role": "Author Response",
"response": "We thank reviewer Dr. Florian Karreth for very valuable comments helping to improve our manuscript. We have adjusted our text according to the comments from Dr. Karreth. For clarification, we include a bullet-point response to each comment below. We thank the reviewer for pointing out this paper which brings the understanding of microRNA targeting to a higher level. We have included this reference and commented on it in the text (paragraph “MicroRNA functional targeting”).The reviewer makes a very important point and to cover some of these mechanisms we have added three references and commented shortly on them in the text (paragraph “MicroRNA functional targeting”). However, since our main focus is on the clinical aspects of microRNAs we believe a deeper description of non-canonical microRNA targeting is out of scope for this review."
}
]
},
{
"id": "1021",
"date": "24 Jun 2013",
"name": "Gustavo J. Gutierrez Gonzalez",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHydbring and Badalian-Very summarize in this review, the current status in the potential development of clinical applications based on miRNAs’ biology. The article gives an interesting historical and scientific perspective on a field that has only recently boomed; focusing mostly on the two main products in the pipeline of several biotech companies (in Europe and USA) which work with miRNAs-based agents, disease diagnostics and therapeutics. Interestingly, not only the specific agents that are being produced are mentioned, but also clever insights in the important cellular pathways regulated by key miRNAs are briefly discussed.Minor points to consider in subsequent versions:Page 2; paragraph ‘Genomic location and transcription of microRNAs’: the concept of miRNA clusters and precursors could be a bit better explained.Page 2; paragraph ‘Genomic location and transcription of microRNAs’: when discussing the paper by the laboratory of Richard Young (reference 16); I think it is important to mention that that particular study refers to stem cells.Page 2; paragraph ‘Processing of microRNAs’: “Argonate” should be replaced by “Argonaute”.Page 3; paragraph ‘MicroRNAs in disease diagnostics’: are miR-15a and 16-1 two different miRNAs? I suggest mentioning them as: miR-15a and miR-16-1 and not using a slash sign (/) between them.Page 4; paragraph ‘Circulating microRNAs’: I am a bit bothered by the description of multiple sclerosis (MS) only as an autoimmune disease. Without being an expert in the field, I believe that there are other hypotheses related to the etiology of MS.Page 5; paragraph ‘Clinical microRNA diagnostics’: Does ‘hsa’ in hsa-miR-205 mean something?Page 5; paragraph ‘Clinical microRNA diagnostics’: the authors mention the company Asuragen, Austin, TX, USA but they do not really say anything about their products. I suggest to either remove the reference to that company or to include their current pipeline efforts.Page 6; paragraph ‘MicroRNAs in therapeutics’: in the first paragraph the authors suggest that miRNAs-based therapeutics should be able to be applied with “minimal side-effects”. Since one miRNA can affect a whole gene program, I found this a bit counterintuitive; I was wondering if any data has been published to support that statement. Also, in the same paragraph, the authors compare miRNAs to protein inhibitors, which are described as more specific and/or selective. I think there are now good indications to think that protein inhibitors are not always that specific and/or selective and that such a property actually could be important for their evidenced therapeutic effects.Page 6; paragraph ‘MicroRNAs in therapeutics’: I think the concept of “antagomir” is an important one and could be better highlighted in the text.Throughout the text (pages 3, 5, 6, and 7): I am a bit bothered by separating the word “miRNA” or “miRNAs” at the end of a sentence in the following way: “miR-NA” or “miR-NAs”. It is a bit confusing considering the particular nomenclature used for miRNAs. That was probably done during the formatting and editing step of the paper.I was wondering if the authors could develop a bit more the general concept that seems to indicate that in disease (and in particular in cancer) the expression and levels of miRNAs are in general downregulated. Maybe some papers have been published about this phenomenon?",
"responses": [
{
"c_id": "527",
"date": "15 Aug 2013",
"name": "Per Hydbring",
"role": "Author Response",
"response": "We thank reviewer Dr. Gustavo Gutierrez Gonzalez, for very valuable comments helping to improve our manuscript. We have adjusted our text according to the comments from Dr. Gutierrez Gonzalez. For clarification, we include a bullet-point response to each comment below. In response to this comment we have added a few words in this paragraph. Further, we added a sentence in the previous paragraph “MicroRNA discovery”.We have clarified in this paragraph that the study refers to stem cells.We thank the reviewer for pointing this out. This typo has been adjusted.miR-15a and 16-1 originate from the same polycistronic transcript and have identical seed sequences. However, as the reviewer point out, they are still two separate mature miRNAs. We have therefore adjusted the text according to the reviewer’s suggestion to avoid confusion.We agree with the reviewer of this misleading description and have adjusted the text accordingly.This means “homo sapiens” but to avoid confusion we have removed it from miR-205.In accordance to the reviewer’s suggestion we have removed this company reference.Since there is currently only one miRNA drug in clinical trials, there is little direct evidence of that miRNAs can be applied with minimal side-effects. In support of our hypothesis, their mechanism of action is tuning expression rather than blunting their targets which reasonably should be less detrimental to healthy tissues. We have added one sentence in this paragraph to clarify our hypothesis. We further agree with the reviewer that a lot of enzymatic protein inhibitors could be more selective. However, the purpose of these drugs is selectivity. In contrast, miRNA drugs are developed with the idea of controlling multiple gene-components in the same or overlapping signaling-pathways. Such gene-products are not limited to proteins with enzymatic activity but could include any deregulated genes or proteins in a given disease.We agree with the reviewer and have made slight adjustments in the text to better highlight this concept. This has been an editing/formatting mistake. In our original manuscript there are no separations of the word “miRNA” or “miRNAs”.We describe downregulation of miRNAs in the section “MicroRNAs in disease diagnostics” referring to a few publications (25, 35-39). To better clarify a general impact of reduced miRNA expression in cancers we included an additional reference (40), describing a conditional knockout mice model for miRNA biogenesis leading to reduced survival in affected mice."
}
]
}
] | 1
|
https://f1000research.com/articles/2-136
|
https://f1000research.com/articles/2-156/v1
|
16 Jul 13
|
{
"type": "Review",
"title": "Did the elimination of lead from petrol reduce crime in the USA in the 1990s?",
"authors": [
"Wayne Hall"
],
"abstract": "This article assesses the evidence for the hypothesis that a decline in all types of crime since the early 1990s in the USA was an unintended consequence of removing lead from petrol between 1975 and 1985. It describes ecological and econometric studies that have generally but not always found correlations between lead exposures in childhood and some types of crime 20 years later; a small number of epidemiological studies that have found a dose-response relationship between lead exposure in childhood and self-reported and officially recorded criminal offences in young adulthood; and evidence for the biological plausibility of a causal relationship. The major anomaly in the evidence is that the associations reported in ecological studies are much stronger (explaining 56-90% of the variation in crime rates) than the weaker relationships found in the cohort studies (that typically explain less than 1% of the variance in offending). Suggestions are made for research that will better assess the contribution that reduced lead exposure has made to declining crime rates in the USA.",
"keywords": [
"In the early 1990s the USA experienced a decline in crime that was unprecedented",
"and contrary to a predicted increase in crime1",
"2. Rates of all types of crime declined steeply",
"across all demographic groups and in all geographic regions in the USA2. The decline may have been unexpected and unpredicted but",
"after the fact",
"there has been no shortage of possible explanations",
"the merits of which criminologists",
"economists and sociologists have debated (e.g. Blumstein and Wallman1",
"Levitt2",
"Drum3",
"Nevin4 and Reyes5)."
],
"content": "Introduction\n\nIn the early 1990s the USA experienced a decline in crime that was unprecedented, and contrary to a predicted increase in crime1,2. Rates of all types of crime declined steeply, across all demographic groups and in all geographic regions in the USA2. The decline may have been unexpected and unpredicted but, after the fact, there has been no shortage of possible explanations, the merits of which criminologists, economists and sociologists have debated (e.g. Blumstein and Wallman1, Levitt2, Drum3, Nevin4 and Reyes5).\n\nA number of factors have been accepted as contributing to the decline. These include: increased numbers of police and increased effectiveness of policing2; the incapacitation of recidivist offenders who commit violent crimes by long prison sentences2,6; and a reduction in drug-related violence that followed the decline in crack cocaine use in major US cities in the late 1980s7,8.\n\nA more controversial hypothesis was that the crime decline was a delayed demographic effect of more liberal abortion laws introduced in the USA in the early 1970s after the Roe vs Wade case in the Supreme Court2,9. Liberal abortion laws in the early 1970s, Donohue and Levitt9 argued, reduced the number of unwanted male children who were born to unmarried mothers in US cities over subsequent decades. The absence of these children reduced crime rates 20 years later because there were fewer most-at-risk young men to commit violent offences.\n\nAnother explanation of the crime decline has recently been proposed2,9, which has one major advantage over other explanations, namely, that it claims to explain not only the steep decline in crime that began in the early 1990s but also the steep rise in crime that occurred between 1960 and 19903,4,10,11. According to this hypothesis, the rise in crime between 1960 and 1990 was driven by increased exposure of young children to environmental lead (primarily from leaded petrol and secondarily from leaded paint in old inner city housing) between the 1940s and the 1980s4,11. Crime increased 20 years later when the children exposed to lead entered young adulthood, the age at which the majority of crime is committed. The decline in crime after 1990 was an unexpected social benefit of removing lead from petrol in the USA over the period 1975 to 19855.\n\n\nWhy was reduced lead exposure a candidate explanation for the crime reduction?\n\nThe rationale for the hypothesis that lead exposure was criminogenic was that (1) lead exposure in childhood has been shown to reduce IQ and increase impulsiveness, aggressiveness and conduct problems in late childhood; and (2) these characteristics increase the risk that young people will commit criminal offences3,5.\n\nThe first observations of the neurological effects of childhood lead exposure were made in Brisbane in the early 1890s12. For much of the 20th century, lead-based paint was the most common source of lead poisoning in children13. In the 1970s, epidemiologists found that elevated blood lead levels were associated with lower IQ and increased rates of behavioural disturbance14–16. Lead in petrol was then identified as the primary source of environmental lead exposure14,16.\n\nThe peak body of the lead industry contested these findings. The industry argued that: lead only caused harm at the high blood levels (80 g/dL) found in cases of lead poisoning; that lower lead levels did not cause harm because lead was a \"natural\" substance that the human body could efficiently excrete; that lead from paint was the major source of exposure in childhood, not lead from petrol; and that lead poisoning was a disease of poverty that occurred in the minority of children who ingested lead12,14,16.\n\nIn 1975, the EPA specified that over a five year period, the average lead content of petrol would be reduced from 2 g to 0.5 g per gallon. In 1985, the EPA further reduced the allowable level, effectively removing all lead from petrol14. The ban on the use of lead in petrol produced very steep declines in the amount of lead in petrol and blood lead levels in children between 1976 and 198015. The fact that the steepest decline in blood lead levels coincided with elimination of leaded petrol made it hard for the lead industry to argue that leaded petrol was not a major source of environmental lead exposure in children.\n\n\nEvaluating the role of lead exposure in crime\n\nIn order to evaluate the hypothesis that lead exposure in childhood explains crime in young adulthood we need evidence that: (1) there is an association between lead exposure and crime; (2) the direction of the relationship is from lead exposure to crime rather than vice versa; and (3) enables us to exclude plausible alternative explanations of any association between childhood lead exposure and crime in young adulthood. If all of these conditions can be met, the case for a causal explanation is strengthened by the existence of a plausible mechanism by which lead exposure could increase crime.\n\nEcological correlations. Nevin4 reported ecological correlations between lead levels in the childhood environment, blood lead levels in children, and rates of crimes committed 23 years later. He showed that the time series curves for lead exposure and various types of crime (murders, rapes, aggravated assaults, and robberies) very closely followed each other when a 23 year lag was allowed between lead exposures in childhood and criminal activity in young adulthood. Regression analyses indicated that variations in childhood lead exposure explained large proportions of the variation in crime rates 23 years later (46% to 90%).\n\nMcCall and Land17 tested Nevin’s4 hypothesis by performing an age-period-cohort analysis that compared murder rates between 1960 and 1995 in 11 birth cohorts whose members differed in their lead exposure during childhood. They failed to find any relationship between lead exposure in childhood and murder rates, although the small number of birth cohorts that were available for analysis probably did not provide a powerful test of the hypothesis.\n\nNevin11 extended his earlier analyses by analysing relationships between lead and crime in the USA and eight other developed countries (Australia, Canada, Finland, France, Italy, New Zealand, United Kingdom, West Germany) over the period 1960 to the early 1990s. He reported similar declines in crime rates in all these countries for all categories of crime (burglary and theft; aggravated assault, sexual assault, and murder). He identified an optimum lag between the elimination of lead in petrol and the onset of crime declines in all these countries as 19 years. The lag varied by type of offence, from 18 years for burglary to 23 years for robbery11. Nevin also reported that lead levels during the 1970s predicted murder rates in large US cities in the period 1985–1994. The major weakness of Nevin’s analyses was his limited ability to control for the effects of other factors, apart from unemployment. He did have a crude control for national differences in crime rates between countries by including country as a dummy variable in his regressions.\n\nAn econometric study of national US data. Reyes5 attempted to address the confounding issue by conducting a detailed econometric analysis of relationships between lead exposure and violent crime over 50 US states during the period 1985–2002. She controlled for state differences in potential confounders and tested the robustness of her results to different ways of measuring lead exposure and different statistical models. Reyes’ estimated the lead content of petrol (g per gallon) in each state in each year and compared this measure with blood lead levels for the years in which these data were collected in the National Health and Nutrition Examination Survey (NHANES) (1976–1980) together with measures of ambient air lead within each state (1960–2000). Crime data were obtained from uniform crime statistics on property crimes, violent offences (assaults and robberies), and murders.\n\nReyes examined relationships between state crime levels and estimates of lead exposure in children in that state at lags of 20 and 30 years earlier. She controlled for differences between states in: rates of legalised abortion, unemployment, police numbers and incarceration rates, and also controlled for national trends in these factors over time (by including the study year as a variable in her model). She also tested the robustness of her findings to the choice of measure of lead exposure (namely, ambient lead vs lead in gasoline), the choice of statistical model (log-linear vs log-log), and the inclusion or exclusion of data from the states and territory whose large populations and high crime rates may have unduly affected the results (California, New York and Washington, DC).\n\nReyes found that the average levels of lead in gasoline and blood lead levels in children were correlated (r = 0.54) and the correlation increased to 0.84 if data from CA, NY and DC were excluded. She also found a close relationship, with a 20 year time lag, between the decline in lead exposure and the decline in violent crime. And she found that the largest declines in violent crime rates occurred in the states with the largest declines in lead exposure.\n\nUnlike Nevin, Reyes did not find any relationship between lead levels and property crime. The relationship between lead levels and murder were also weak. There was no relationship between lead levels and murder rates when all 50 states were included in the analysis but there was a small effect when CA, NY and DC were excluded from the analysis. Reyes conjectured that this may reflect the contribution made to murder rates by gang-related drug violence in the major cities in these states and territories in the late 1980s.\n\nReyes estimated that the decline in lead exposure over the study period explained about 56% of the drop in violent crime between 1992 and 2002. She also estimated that legalized abortion accounted for 29% of the drop and that increased police numbers and decreased alcohol use accounted for a smaller proportion of the decline.\n\nReyes concluded that lead exposure in childhood was causally related to rates of violent crime (assaults and robberies) committed 20 years later when children exposed to lead in childhood entered the peak period of criminal offending. She concluded that \"lead exposure was likely an important factor in both the rise and the decline of violent crime in the last 30 years\" and that \"two major acts of government, the Clean Air Act and Roe v Wade, neither intended to have any effect on crime, may have been the largest factors affecting violent crime trends at the turn of the century\" (p 36).\n\nReyes’ model of the effects of lead exposure on crime over-predicted the observed crime decline, that is, according to her model, crime rates should have declined by 56% as a result of the decline in environmental lead but the observed rate of decline was only 34%. She hypothesized that there were other countervailing factors at work that increased crime rates at the same time that declining lead exposure was driving them down.\n\nEcological studies of large cities. The ecological analyses of Nevin and Reyes suffer from the limitation that they use a single estimated lead exposure for the whole of a state. This ignores the fact that lead exposures differ greatly between the populations of large cities, small cities and rural areas within states. Mielke and Zahran18 attempted to address this problem by analysing relationships between measures of ambient lead and aggravated assaults per 100,000 people in six large US cities (Atlanta, Chicago, Indianapolis, Minneapolis, New Orleans and San Diego)18. They argued that aggravated assault was more likely to be impulsive and hence reflect the effects of childhood lead exposure. They estimated annual city burdens of air lead from data on vehicle traffic in metric tons, state gasoline usage, city traffic volume, average miles per gallon and lead per gallon of different types of fuel. They followed Reyes in forward lagging effects of lead exposure on crime rates by 22 years and they controlled for income per capita, and the percentage of city population age group most at risk of committing these offences (15–24 years).\n\nMielke and Zahran found that ambient lead levels in these cities predicted aggravated assault rates, after controlling for income and demography and baseline differences in crime rates between cities. They estimated that between 66% and 89% of the variations in aggravated assault rates in these cities were explained by variations in ambient lead levels. They also estimated (making worst case assumptions about the contribution of lead paint to ambient lead) that leaded petrol accounted for at least 85% of the lead to which children were exposed in New Orleans, the city with highest ambient lead.\n\nSceptical researchers (e.g. Fergusson19 and Firestone20) have argued that it is hazardous to draw causal inferences about lead exposure and crime from correlations between time series data on lead exposures and crime rates. To do so is to run the risk of committing the ecological fallacy, that is, mistakenly assuming that a correlation between two time series necessarily means that the people committing the crimes are those who were exposed to lead in childhood.\n\nEpidemiological studies rule out the ecological fallacy by examining the relationship between lead exposure and rates of crime in individuals. They thereby enable us to see if it is the individuals with highest lead exposure who commit the most crimes. They also can address the contributions of potential confounders to the relationship by assessing whether lead exposure in childhood predicts crime rates in young adulthood, after controlling for other risk factors for lead exposure and crime.\n\nNeedleman et al.13 reported a case-control study on lead exposure and crime. They compared lead levels in 194 youths aged 12–18 who were \"adjudicated\" delinquents (i.e. had been found guilty of criminal offences) and 146 non-delinquent control subjects from high schools in the same city. They also measured factors that predict delinquency such as: race, parental education and occupation, number of parents in the home, number of children in the home, and neighbourhood crime rates. They found a large difference in lead concentrations between delinquents and controls in both black and white participants (11 vs 1.5 parts per million (ppm)). The delinquent participants were 1.9 times more likely to have a lead level above 25 ppm than controls. Delinquency was confounded by race: fewer black than white controls were recruited, and more black delinquents came from single parent families and lived in higher crime areas than white delinquents. When potential confounders were included in the model, the strength of the relationship between lead level and delinquency increased to an odds ratio of 4.0 (95% CI: 1.4, 11.1) and lead level was the second strongest predictor of delinquency after race. Lead was the second strongest predictor of delinquency (after being raised by a single parent) when separate analyses were carried out within the black and white samples.\n\nWright et al.21 conducted a prospective study of the relationship between lead exposure before birth and until age 6.5 years and records of arrest in 250 primarily African American individuals (90%) who were followed from age 19 to 24 years. Participants were in a cohort study of children born to 376 pregnant women recruited from four prenatal clinics in socially disadvantaged areas of Cincinnati, Ohio. In an earlier follow up of 195 members of the cohort, Dietrich et al.22 reported a dose-response association between measures of childhood lead exposure and acts of delinquency from self-report and parental report by age 18 years. These relationships persisted after adjustment for confounders.\n\nWright et al. examined delinquent acts that resulted in arrests in the county in which the participants lived. They found elevated lead levels (13.4 µg/dL on average during childhood) and an elevated risk of arrest in their sample (54% had been arrested a total of 800 times, 14% for violent offences). The risk of arrest increased with blood lead, after adjustment for: sex; quality of home environment; maternal alcohol, tobacco and illicit drug use during pregnancy; maternal arrests rates, socioeconomic status (SES), number of children in the home and whether the mother was on welfare. The risk of any arrest increased 1.40 times for each increase of 5 µg/dL in prenatal blood and 1.27 for each increment in average blood lead level during childhood. The risk of arrest for violent offences increased 1.34 times for each 5 µg/dL increase in antenatal blood lead, and 1.48 for each 5 µg/dL increase in average blood lead during childhood.\n\nFergusson et al. reported a prospective study of the relationship between childhood lead exposure between 6 and 9 years, measured in deciduous teeth, and (i) convictions and (ii) self-reported criminal activity between the ages of 14 and 21 years in a New Zealand birth cohort of 1011 individuals. They found dose-response relationships between lead in teeth and both convictions for property and/or violent offences and self-reported rates of property and/or violent crimes. The relationship was stronger for self-reported crime, probably because conviction rates were much lower. Relationships persisted after adjustment for confounders (maternal education, ethnicity, family conflict, maternal smoking during pregnancy, exposure to childhood physical abuse, parental alcohol and drug use and offending).\n\nFergusson et al. also tested a plausible causal pathway for the effects of lead on crime. They assessed whether the relationship between lead dentine levels and crime could be explained by a decline in IQ produced by lead exposure. Adjustment for educational failure eliminated the association between self-reported crime and lead levels but the association with convictions persisted, albeit much attenuated. They estimated lead exposure explained less than 1% of the variance in convictions and self-reported crime and argued that this was too weak to explain Nevin’s very high estimates of the percentage variation in crime rates attributed to lead exposure (63–90%)11.\n\nLead exposure and violent crime are correlated in ecological studies and in a small number of epidemiological studies in the USA and New Zealand. The prospective epidemiological studies indicate that lead exposure in childhood precedes criminal acts in young adulthood but it is much more difficult to exclude other plausible explanations of the association. Foremost among these is the possibility that the association can be explained by uncontrolled factors that increase the likelihood of being exposed to lead and of committing crimes (e.g. living in disadvantaged high crime inner city areas where lead exposure is common). This is a special challenge in the USA because the most socially disadvantaged African-American populations live in inner city areas with high levels of exposure to dust from lead-pigmented paints and, until the late 1970s, high levels of lead from leaded petrol from the freeways that ran through inner city housing in large US cities11.\n\nNevin’s ecological studies had limited ability to address this confounding. Reyes controlled for some confounding factors using state data on unemployment, number of police officers and so on. Potential confounders were better controlled in two of the prospective epidemiological studies. In the Wright et al. study, the whole sample came from a very socially disadvantaged population and had high average lead exposure throughout childhood. Yet Wright et al. found a relationship between lead exposure during childhood and arrest for all crimes, and for violent crimes in adolescence and young adulthood. These relationships persisted after controlling for potential confounders.\n\nThe Fergusson et al. study was conducted in a very different cultural setting. Christchurch in New Zealand has much lower rates of social disadvantage than Cincinnati in the USA, a good social welfare safety net, and a very different minority ethnic group with a higher rate of criminal convictions. There were also much lower average lead levels in Christchurch than in Cincinnati but Fergusson et al. nonetheless found a dose-response relationship between lead exposure in childhood and rates of criminal convictions and self-reported criminal offences committed by age 21. These relationships persisted after adjustment for confounders. There was also a plausible mechanism in that controlling for IQ reduced the strength of the relationship between lead and rates of self-reported criminal offences.\n\nThere is a consilience of evidence in support of the hypothesis that lead exposure in childhood increases the risk of criminal offending in young adulthood. Low intelligence increases the risk of offending, and there is a dose-response relationship between lead exposure in childhood and a decline in average IQ13,19,23. Fergusson et al.19 also showed that the relationship between dentine lead levels and self-reported crime was no longer significant after adjusting for school completion.\n\nThere is also support for a second way that lead exposure may increase offending, namely, that lead exposure in childhood makes adolescents more impulsive, hyperactive and aggressive. Meta-analyses find small but consistent correlations between blood and dentine measures of lead exposure and symptoms of conduct problems (r = 0.15)24, inattentiveness (r = 0.14) and hyperactivity (r = 0.12)25. These patterns of behaviour in turn increase the risk of antisocial behaviour in adolescence and young adulthood26. A recent neuroimaging study of the Cincinnati cohort reported correlations between childhood lead exposure and the size of frontal areas in the brain implicated in executive functioning, mood regulation and decision-making27.\n\n\nDoes the decline in lead exposure explain the decline in crime in the USA?\n\nEliminating lead from petrol in the USA between 1975 and 1985 dramatically reduced lead exposure in childhood and this occurred over a period that could explain the post-1990 decline in crime in the USA. Ecological studies have generally but not always found correlations between lead exposures in childhood and crime rates 20 years later. But these studies have not found the same relationships between lead exposure and crime. Nevin11 found a relationship for all types of crime (violence, sexual assault, murder and property crime) but Reyes only found a relationship for violent offences and there was only a suggestive relationship for murder. Meilke and Zahran’s supported Reyes findings in their analysis of aggravated assault in six US cities. McCall and Land’s17 age-period-cohort analysis found no association between lead exposure and rates of murder in their age-period-cohort analysis.\n\nThere are a small number of epidemiological studies that find dose-response relationships between lead exposure in childhood and both self-reported and officially recorded criminal offences in young adulthood. A causal relationship is biologically plausible because lead is neurotoxic in animals, produces hyperactivity, and impairs learning28. There is now suggestive human neuroimaging evidence that lead exposure is related to size reductions in brain regions involved in executive functioning and decision making27.\n\nBut the evidence is not sufficient to conclude that variations in environmental lead exposure in childhood over the past 50 or so years in the USA explain, first the rise, and then the decline in crime rates. The major reason for doubt is that the associations in ecological studies are much stronger (explaining 60–90% of the variation in crime rates) than the weaker relationships in the cohort studies (that explain less than 1% of the variance in offending)19. Lead exposure in childhood may have played a small role in rising and falling crime rates in the USA but it is unlikely to account for the very high percentage of the decline suggested by Nevin11 and Reyes5.\n\nThis issue is not solely a matter of interest only to criminologists. Understanding the reasons for the US crime decline has important implications for social policy. If lead exposure is a major cause of the decline, then we could avoid mistaken investments in crime reduction strategies that have been erroneously given the credit for the crime decline. We would also need to weigh the costs and benefits of different ways of reducing residual childhood lead exposures in inner city areas.\n\nThe contribution of lead exposure to crime rates deserves serious research attention. We need more cohort studies in environments where lead exposure remains high, particularly in developing countries such as China. The results of these studies could inform epidemiological modelling to estimate the likely population level effects on crime of observed reductions in lead exposure. This modelling would test the plausibility of the lead hypothesis. So too would controlled evaluations of the costs and benefits of reducing environmental lead exposure in high crime inner city areas or moving highly exposed populations from areas of high lead burden.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nI would like to acknowledge Sarah Yeates for assistance in searching the literature and preparing the manuscript for publication and Dr Don Weatherburn, Director of the Bureau of Crime Statistics and Research in the NSW Attorney General’s Department, for commenting on a draft article.\n\n\nReferences\n\nBlumstein A, Wallman J: The recent rise and fall of American violence. In: Blumstein A, Wallman J, eds. The crime drop in America. Cambridge; New York: Cambridge University Press, 2000; 1–12. Publisher Full Text\n\nLevitt S: Understanding why crime fell in the 1990s: four factors that explain the decline and six that do not. J Econ Perspect. 2004; 18: 163–90. Publisher Full Text\n\nDrum K: America's real criminal element: lead.Mother Jones. 2013. Reference Source\n\nNevin R: How lead exposure relates to temporal changes in IQ, violent crime, and unwed pregnancy. Environ Res. 2000; 83: 1–22. PubMed Abstract | Publisher Full Text\n\nReyes J: Environmental policy as social policy? The impact of childhood lead exposure on crime. B E J Econom Anal Policy. 2007; 7 (1). Publisher Full Text\n\nSpelman W: The limited importance of prison expansion. In: Blumstein A, Wallman J, eds. The crime drop in America. Cambridge; New York: Cambridge University Press, 2000; 97–129. Publisher Full Text\n\nFryer R, Heaton P, Levitt S, et al.: Measuring crack cocaine and its impact. Econ Inq. 2013; 51: 1651–81. Publisher Full Text\n\nJohnson B, Golub A, Dunlap E: The rise and decline of hard drugs, drug markets and violence in inner-city New York. In: Blumstein A, Wallman J, eds. The crime drop in America. Cambridge; New York: Cambridge University Press, 2000; 164–206. Publisher Full Text\n\nDonohue JJ III, Levitt S: The impact of legalized abortion on crime. Q J Econ. 2001; 116: 379–420. Publisher Full Text\n\nMonbiot G: Yes, lead poisoning could really be a cause of violent crime. It seems crazy, but the evidence about lead is stacking up. Behind crimes that have destroyed so many lives, is there a much greater crime? The Guardian. 2013. Reference Source\n\nNevin R: Understanding international crime trends: the legacy of preschool lead exposure. Environ Res. 2007; 104: 315–36. PubMed Abstract | Publisher Full Text\n\nRosner D, Markowitz G, Lanphear B: J. Lockhart Gibson and the discovery of the impact of lead pigments on children's health: a review of a century of knowledge. Public Health Rep. 2005; 120: 296–300. PubMed Abstract | Free Full Text\n\nNeedleman H, McFarland C, Ness RB, et al.: Bone lead levels in adjudicated delinquents. A case control study. Neurotoxicol Teratol. 2002; 24: 711–7. PubMed Abstract | Publisher Full Text\n\nMarkowitz G, Rosner D: Lead wars: the politics of science and the fate of America's children. Berkeley; New York: University of California Press; Milbank Memorial Fund, 2013. Reference Source\n\nNeedleman H: Lead poisoning. Annu Rev Med. 2004; 55: 209–22. PubMed Abstract | Publisher Full Text\n\nNeedleman H: The removal of lead from gasoline: historical and personal reflections. Environ Res. 2000; 84: 20–35. PubMed Abstract | Publisher Full Text\n\nMcCall P, Land K: Trends in environmental lead exposure and troubled youth, 1960–1995: an age-period-cohort-characteristic analysis. Soc Sci Res. 2004; 33: 339–59. PubMed Abstract\n\nMielke HW, Zahran S: The urban rise and fall of air lead (Pb) and the latent surge and retreat of societal violence. Environ Int. 2012; 43: 48–55. PubMed Abstract | Publisher Full Text\n\nFergusson DM, Boden JM, Horwood LJ: Dentine lead levels in childhood and criminal behaviour in late adolescence and early adulthood. J Epidemiol Community Health. 2008; 62: 1045–50. PubMed Abstract | Publisher Full Text\n\nFirestone S: Does lead exposure cause violent crime? The science is still out.Discover: The Magazine of Science, Technology, and the Future. 2013. Reference Source\n\nWright J, Dietrich K, Ris M, et al.: Association of prenatal and childhood blood lead concentrations with criminal arrests in early adulthood. PLoS Med. 2008; 5: e101. Publisher Full Text | Free Full Text\n\nDietrich K, Ris M, Succop P, et al.: Early exposure to lead and juvenile delinquency. Neurotoxicol Teratol. 2001; 23: 511–8. PubMed Abstract | Publisher Full Text\n\nLanphear B, Hornung R, Khoury J, et al.: Low-level environmental lead exposure and children's intellectual function: an international pooled analysis. Environ Health Perspect. 2005; 113: 894–9. PubMed Abstract | Free Full Text\n\nMarcus D, Fulton J, Clarke E: Lead and conduct problems: a meta-analysis. J Clin Child Adolesc Psychol. 2010; 39: 234–41. PubMed Abstract | Publisher Full Text\n\nGoodlad JK, Marcus DK, Fulton JJ: Lead and attention-deficit/hyperactivity disorder (ADHD) symptoms: a meta-analysis. Clin Psychol Rev. 2013; 33: 417–25. PubMed Abstract | Publisher Full Text\n\nRaine A: The anatomy of violence: the biological roots of crime. London: Allen Lane, 2013. Reference Source\n\nCecil KM, Brubaker CJ, Adler C, et al.: Decreased brain volume in adults with childhood lead exposure. PLoS Med. 2008; 5: e112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilbergeld E: Experimental studies of lead toxicity: implications for mechanisms, dose response and reversibility In: Rutter M, Russell Jones R, eds. Lead versus health: sources and effects of low level lead exposure. Chichester: Wiley, 1983; 191–216."
}
|
[
{
"id": "1532",
"date": "19 Aug 2013",
"name": "David Carpenter",
"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 good review of the published literature, but the conclusions drawn by the author are not consistent with the data presented, specifically the statement “the evidence is not sufficient to conclude that variations in environmental lead exposure in childhood over the past 50 or so years in the USA explain, first the rise, and then the decline in crime rates”. The evidence used to argue against this conclusion is primarily a study done in New Zealand, not in the US, plus a very limited cohort study by McCall and Land. The issue of concern is whether lead plays a role in rates of crime, and while one can argue about the percentage due to lead, the overall information certainly indicates that lead exposure is particularly important. This needs to be stated in a much more balanced fashion. Because this is the conclusion it is important that it be both balanced and supported by the data. If you discount lead exposure then there must be presentation of a credible alternative explanation, and none of the others are well supported by evidence.",
"responses": []
},
{
"id": "1690",
"date": "05 Sep 2013",
"name": "Howard Mielke",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a good summary of the existing evidence on lead and violence and there's not much to dispute about it. However, a change in the perspective would improve it and at least one correction is needed. In the abstract the word 'unintended' is not correctly placed or in context of the problem. If anything, the unintended consequence is the plausible link between the toxic response due to rapid increase in the commercial consumption of fuel containing lead additives (especially in the 1950’s and 1960’s) and the unintended cascade of societal health costs, including learning disabilities and criminal behaviour, that was triggered around 20 years later. These consequences are only becoming appreciated recently, after the use of lead additives in fuels has decreased 1. Ecological studies like ours provide suggestive evidence, however when coupled with neuroimaging studies, animal studies and individual-level analyses, the current state of the science on lead provides strong evidence for a serious societal problem in need of attention to better protect future generations 1. The lead story is rich and so well-documented that the benefits of using resources for cleaning up play areas and improving the environment for children is becoming recognized by progressive nations as a cost effective and important activity 2. Correction: Page 5: “Meilke”--Mielke is misspelled.",
"responses": []
},
{
"id": "1933",
"date": "02 Oct 2013",
"name": "Kenneth Land",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle and Abstract: The title and abstract are appropriate to the content of the article. The abstract represents a suitable summary of the work.Article content: The article is a survey and review of prior research on the topic of whether or not the decline in all types of crime since the early 1990s in the USA was an unintended consequence of the elimination of lead from petrol between 1975 and 1985. This review is competently and adequately done for the most part. Its conceptual orientation comes from epidemiology, which is good, but the incorporation of the body of research done on this topic from a criminology perspective would add greater substance and depth to the article. The synthesis of these two perspectives would likely produce the inference that although the presence of lead in petrol was an influencing factor on the rise and subsequent decline of crime rates in the USA, it was but one of many factors and cannot account for these secular trends alone.Conclusions: The conclusions are sensible, balanced and justified on the basis of the content of the survey and review of the existing research.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-156
|
https://f1000research.com/articles/2-207/v1
|
08 Oct 13
|
{
"type": "Research Article",
"title": "A pilot study of basal ganglia and thalamus structure by high dimensional mapping in children with Tourette syndrome",
"authors": [
"Alton C. Williams",
"Marie E. McNeely",
"Deanna J. Greene",
"Jessica A. Church",
"Stacie L. Warren",
"Johanna M. Hartlein",
"Bradley L. Schlaggar",
"Kevin J. Black",
"Lei Wang",
"Alton C. Williams",
"Marie E. McNeely",
"Deanna J. Greene",
"Jessica A. Church",
"Stacie L. Warren",
"Johanna M. Hartlein",
"Bradley L. Schlaggar",
"Lei Wang"
],
"abstract": "Background: Prior brain imaging and autopsy studies have suggested that structural abnormalities of the basal ganglia (BG) nuclei may be present in Tourette Syndrome (TS). These studies have focused mainly on the volume differences of the BG structures and not their anatomical shapes. Shape differences of various brain structures have been demonstrated in other neuropsychiatric disorders using large-deformation, high dimensional brain mapping (HDBM-LD). A previous study of a small sample of adult TS patients demonstrated the validity of the method, but did not find significant differences compared to controls. Since TS usually begins in childhood and adult studies may show structure differences due to adaptations, we hypothesized that differences in BG and thalamus structure geometry and volume due to etiological changes in TS might be better characterized in children.Objective: Pilot the HDBM-LD method in children and estimate effect sizes.Methods: In this pilot study, T1-weighted MRIs were collected in 13 children with TS and 16 healthy, tic-free, control children. The groups were well matched for age. The primary outcome measures were the first 10 eigenvectors which are derived using HDBM-LD methods and represent the majority of the geometric shape of each structure, and the volumes of each structure adjusted for whole brain volume. We also compared hemispheric right/left asymmetry and estimated effect sizes for both volume and shape differences between groups.Results: We found no statistically significant differences between the TS subjects and controls in volume, shape, or right/left asymmetry. Effect sizes were greater for shape analysis than for volume.Conclusion: This study represents one of the first efforts to study the shape as opposed to the volume of the BG in TS, but power was limited by sample size. Shape analysis by the HDBM-LD method may prove more sensitive to group differences.",
"keywords": [
"Tourette syndrome (TS) is a chronic idiopathic syndrome characterized by the appearance of both vocal and motor tics during childhood or adolescence1",
"2. Tics are repetitive",
"stereotyped",
"suppressible movements or vocalizations that may include blinking",
"abdominal tensing",
"sniffing",
"or throat clearing3. TS affects approximately 0.5% of school-age children",
"but its causes and pathophysiology are not yet well understood4."
],
"content": "Introduction\n\nTourette syndrome (TS) is a chronic idiopathic syndrome characterized by the appearance of both vocal and motor tics during childhood or adolescence1,2. Tics are repetitive, stereotyped, suppressible movements or vocalizations that may include blinking, abdominal tensing, sniffing, or throat clearing3. TS affects approximately 0.5% of school-age children, but its causes and pathophysiology are not yet well understood4.\n\nIt has been suggested that problems with activity modulation in the basal ganglia and thalamus may contribute to the inability of TS patients to exercise behavioral inhibition5,6 as a result of these structures’ effects on behavioral inhibition via the prefrontal, parietal, temporal, and cingulate cortices7. The basal ganglia and thalamus modulate cortical activity through cortico-basal ganglia-thalamo-cortical loops, composed of connections from the frontal cortex to the striatum, the striatum to the globus pallidus, substantia nigra, and thalamus, and the thalamus back to the cortex8.\n\nSeveral lines of evidence support the presence of structural abnormalities in basal ganglia nuclei in individuals with TS4. Autopsy studies have found abnormalities within the basal ganglia, including increased number of neurons in the globus pallidus interna, decreased density and number of neurons in the globus pallidus pars externa, and decreased parvalbumin and choline acetyltransferase staining cholinergic interneurons in the caudate nucleus and putamen9,10. However, since TS is rarely a fatal disease, the number of autopsied cases is limited11. Case studies of focal brain lesions have demonstrated new tic onset after lesions to the prefrontal cortex, thalamus, and basal ganglia12. In addition, encephalitis lethargica, frontal lobe degeneration, Huntington disease, Wilson disease, and other degenerative illnesses are associated with tics12. Further, some TS patients have benefitted from deep brain stimulation of the globus pallidus and thalamus in TS13–16. Collectively, these observations suggest a role for the basal ganglia, thalamus, and frontal cortex in tics.\n\nNeuroimaging studies can be especially beneficial for studying structural abnormalities because they allow longitudinal study design, reduced investigator and sampling bias, and are relatively non-invasive. A number of MRI studies have examined anatomical volumes and cortical thickness in children and adults with TS and reported significant differences in various brain regions, including the caudate, sensorimotor and prefrontal cortex, and corpus callosum17. Most consistently, basal ganglia volumes were found to be smaller in TS subjects compared with healthy controls, but neuroanatomical shape differences and asymmetry abnormalities have not yet been consistently described18–24.\n\nLarge-deformation high dimensional brain mapping (HDBM-LD) is a computational anatomy tool that reduces the potential for human error in image analysis by further automating elements of image analysis. It has been successfully employed in characterizing shape and volume abnormalities of the hippocampus in neuropsychiatric disorders such as schizophrenia25–27, dementia of the Alzheimer type28–31, depression32 and epilepsy33. It has also been applied to examine the thalamus in schizophrenia34.\n\nHDBM-LD was applied to assess volume and shape differences in putamen, caudate nucleus, nucleus accumbens, globus pallidus, and thalamus in 15 adults with TS and 15 matched controls. No differences in volume or shape were found35. However, TS begins before adulthood. Several structural imaging studies in TS have found an interaction between regional brain volumes and age21,22. It has been suggested that differences seen in adult studies may reflect adaptations or selection bias rather than changes etiologically relevant to TS20. Thus the present study applied HDBM-LD to investigate the volume and shape of these structures in children. We hypothesized that we would find reduced volume, abnormal shape, or abnormal right-to-left asymmetry in one or more of these structures, compared to age-matched controls. Given that there were no prior studies using the HDBM-LD method to analyze brain structures of children with TS in the literature, another goal of this pilot study was to estimate the effect size of these measures in this population.\n\n\nMaterials and methods\n\nA parent of each subject gave written informed consent to participate in the study, and each subject assented to participation. The study was approved by the Washington University Human Studies Committee (approval # 03-1282).\n\nThis study included 13 children with TS (mean age (SD) = 12.44 (2.22), 3 female, 12 right-handed) and 16 healthy controls (mean age (SD) = 12.39 (1.92), 2 female, 15 right-handed). A movement disorders-trained physician examined all TS subjects and 10 of the control subjects. The remaining control subjects underwent neuropsychological evaluation as described previously36. Exclusion criteria were: inability to give informed consent, contraindication to MRI, currently symptomatic major depression, or lifetime history of mental retardation, autism, psychosis, mania, anorexia, bulimia, or drug abuse. All TS subjects met DSM-IV-TR criteria either for Tourette’s Disorder or Chronic Tic Disorder. Disease duration and severity and other clinical characteristics are summarized in Table 1.\n\n*YGTSS total tic score includes only the motor tic and vocal tic subscores for a maximum of 50 points. R = right-handed, L = left-handed, A = ambidextrous.\n\nA 1.5 T Siemens Vision system with a standard head receiver coil was used to collect T1-weighted MR structural images. Prior to scanning sessions, the transmitter was tuned and the main field was shimmed. Anatomic images used a 3D T1-weighted sequences (MPRAGE, 1x1x1.25 mm3 voxels)37. Individual MPRAGE collections lasted approximately 6.5 minutes.\n\nInitial image processing was done as described previously35,38. Using AnalyzeTM software (Rochester, Minnesota), images were linearly rescaled so that voxels with intensity two standard deviations above the mean in the corpus callosum were mapped to 255, and voxels with intensity levels two standard deviations below the mean in the lateral ventricles were mapped to 0.\n\nWhole-brain volume for each subject, excluding the ventricles, was obtained from FreeSurfer (http://surfer.nmr.mgh.harvard.edu/)39.\n\nHDBM-LD was used to determine the volume and shape of the brain structures of interest in all subject scans, as described in detail elsewhere35. Briefly, on each subject’s brain image, a single rater (MEMcN) marked 27 points on the boundaries of the basal ganglia and thalamus in each hemisphere, which were used as an initial step to roughly align the brain image to a labeled standard brain image (template). From this starting point a differentiable, invertible transformation was computed that mapped all voxels of the subject’s image to the template. Using this transformation, the labels on the template image are automatically assigned to the corresponding voxels of each subject’s image. The authors checked the segmentation of each subject’s MR image by visual inspection. This method is extremely reliable and has been validated against expert manual tracings35.\n\nAll brain structure volume and shape analysis methods were conducted as described previously35. We examined five structures: caudate nucleus, nucleus accumbens, globus pallidus, putamen, and thalamus. Volume for each structure was analyzed using a repeated measures ANCOVA, with diagnostic group as the between-subjects factor, brain hemisphere as the within-subjects factor, and age and whole brain volume as covariates. The degree of volumetric asymmetry was examined with the hemisphere effect, and group differences in volumetric asymmetry were assessed by examining the group-by-hemisphere interactions. We also analyzed the total (left and right hemisphere) structure volumes using an ANCOVA. The volume ANCOVAs were repeated with other covariates and factors, including estimated total intracranial volume, sex and handedness, none of which substantively changed the results.\n\nBrain structure shapes were determined from the inter-subject deformation vector fields provided by the HDBM-LD transformations. Eigenvalues and a complete orthonormal set of eigenvectors representing shape variation were obtained using singular value decomposition (SVD) of the pooled covariance in the population studied. The coefficients (eigenscores) associated with the eigenvalues and eigenvectors were calculated for each subject and for each structure in each hemisphere35,40. We used the eigenscores based on the first ten eigenvectors for each structure in each hemisphere in a multivariate ANCOVA to test for group differences in shape. These first ten eigenscores explained 81–92% of the total variance for each structure.\n\n\n\n\nResults\n\nRepeated-measures ANCOVAs showed no significant group effect for any structure. Structural volumes and ANCOVA statistics are shown in Table 2. Additionally, no significant hemisphere effects or group by hemisphere interactions were seen for any of the five structures examined (see Table 2).\n\nL = left, R = right, T = total volume. Repeated-measures ANOVA of each structure showed no significant group effect. Further, we found no hemisphere effect or group by hemisphere interactions for any of the structures (age and whole brain volume w/out ventricles as covariates).\n\nMANCOVAs (using the first ten eigenscores as dependent variables) for each structure in each hemisphere showed no significant group effect (see Table 3). Effect sizes (Cohen’s ƒ2) for both volume and shape are provided in Table 4; the effect sizes for the shape comparisons were larger than those for the volume comparisons.\n\nL = left, R = right, T = total volume. Multivariate analysis of the first 10 eigenvectors of each structure showed no significant group effect (age as covariate).\n\n\nDiscussion\n\nUsing HDBM-LD, a validated method for automatic, high-dimensional mapping of basal ganglia and thalamic structures, we found no significant differences in basal ganglia volumes or shape between children with TS and matched control children. For most basal ganglia regions, this reflects the conclusions of a recent review17. For instance, two groups found increased putamen volume in TS41,42, but a larger study found decreased volume43. However, the majority of these studies found no abnormality in putamen, similar to the current study. Three other studies, including the HDBM-LD study in adults with TS, found no volumetric change in any basal ganglia structure35,44–46. Possibly there is no true difference in these structures in TS when groups are matched carefully for age, sex and handedness. Alternatively, structural abnormalities in TS may be limited to certain subgroups, such as those with more severe tics or with ADHD.\n\nOn the other hand, the largest published MRI study of basal ganglia volume compared 154 adults and children with TS to 130 tic-free control subjects, and found that the caudate was 4.9% smaller in the TS group (p<0.01)43. Two other groups also found lower caudate volume in samples of 18–23 TS subjects and a similar number of controls23,24,47,48. The possible etiologic relevance of this finding is highlighted by the observation that a smaller caudate nucleus in adolescents with TS predicts more severe symptoms in early adulthood49. The largest of the studies that did not find significant decreases in caudate volume was that of Roessner et al.42, which compared 55 subjects with TS to 42 control subjects. The other studies with negative findings regarding caudate volume, including the present one, had fewer than 20 TS subjects each. It is possible these negative results represent a Type II error.\n\nThe present study and the HDBM-LD study in adults represent some of the first efforts to study the shape (as opposed to the volume) of basal ganglia nuclei in TS, and provide effect size estimates for planning a study with larger samples.",
"appendix": "Author contributions\n\n\n\nLW and KJB designed the study. JAC, BLS, JMH and KJB collected the data. MEMcN, ACW, DJG, SLW and LW analyzed the data. MEMcN, ACW and KJB wrote the first draft of the manuscript. All authors were involved in revising 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 study was funded by a research grant from the Tourette Syndrome Association to LW (Morphological abnormalities of the thalamus and basal ganglia in Tourette syndrome by computational anatomy). Manuscript preparation was supported in part by the National Institutes of Health grant K24 MH087913 to KJB.\n\n\nAcknowledgments\n\nWe thank Mary Creech, RN, MSW, Emily Bihun, M.Ed. Counseling, Audrey Lim, and Jonathan M Koller BSEE BSBME for assistance with subject recruitment, data management and analysis.\n\n\nReferences\n\nAmerican Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision., Washington, DC American Psychiatric Association. 2000; 943. Reference Source\n\nDefinitions and classification of tic disorders. The Tourette Syndrome Classification Study Group. Arch Neurol. 1993; 50(10): 1013–1016. PubMed Abstract | Publisher Full Text\n\nBlack KJ: Tics,. in Encyclopedia of Movement Disorders,. K. Kompoliti, et al., Editors., Elsevier (Academic Press): Oxford. 2010; 231–236.\n\nBlack KJ: Tourette syndrome and other tic disorders. eMedicine. 2009. Reference Source\n\nMink JW: Basal ganglia dysfunction in Tourette's syndrome: a new hypothesis. Pediatr Neurol. 2001; 25(3): 190–8. PubMed Abstract | Publisher Full Text\n\nPeterson BS, Skudlarski P, Anderson AW, et al.: A functional magnetic resonance imaging study of tic suppression in Tourette syndrome. Arch Gen Psychiatry. 1998; 55(4): 326–33. PubMed Abstract | Publisher Full Text\n\nGoldman-Rakic P: Circuitry of primate prefrontal cortex and regulation of behavior by representation memory. in Handbook of Physiology: the Nervous System. V. Mountcastle, F. Plum, and S. Geiger, Editors., American Physiological Socity: Bethesda, MD.1987; 254–373. Publisher Full Text\n\nHaber SN: Neuroanatomy of reward: A view from the ventral striatum. in Neurobiology of Sensation and Reward,. J.A. Gottfried, Editor., CRC Press: Boca Raton, FL. 2011; 235–262. PubMed Abstract\n\nKalanithi PS, Zheng W, Kataoka Y, et al.: Altered parvalbumin-positive neuron distribution in basal ganglia of individuals with Tourette syndrome. Proc Natl Acad Sci U S A. 2005; 102(37): 13307–13312. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKataoka Y, Kalanithi PS, Grantz H, et al.: Decreased number of parvalbumin and cholinergic interneurons in the striatum of individuals with Tourette syndrome. J Comp Neurol. 2010; 518(3): 277–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwerdlow NR, Young AB: Neuropathology in Tourette syndrome: an update. Adv Neurol. 2001; 85: 151–161. PubMed Abstract\n\nKurlan R: The differential diagnosis of tic disorders., in Tourette Syndrome,. D. Martino and J.F. Leckman, Editors., Oxford University Press: New York. 2013; 395–401.\n\nCannon E, Silburn P, Coyne T, et al.: Deep brain stimulation of anteromedial globus pallidus interna for severe Tourette's syndrome. Am J Psychiatry. 2012; 169(8): 860–6. PubMed Abstract | Publisher Full Text\n\nServello D, Porta M, Sassi M, et al.: Deep brain stimulation in 18 patients with severe Gilles de la Tourette syndrome refractory to treatment: the surgery and stimulation. J Neurol Neurosurg Psychiatry. 2008; 79(2): 136–142. PubMed Abstract\n\nMuller-Vahl KR, et al.: European clinical guidelines for Tourette syndrome and other tic disorders. Part IV: deep brain stimulation. Eur Child Adolesc Psychiatry. 2011; 20(4): 209–17. PubMed Abstract | Publisher Full Text\n\nAckermans L, Duits A, van der Linden C, et al.: Double-blind clinical trial of thalamic stimulation in patients with Tourette syndrome. Brain. 2011; 134(Pt 3): 832–44. PubMed Abstract | Publisher Full Text\n\nGreene DJ, Black KJ, Schlaggar BL: Neurobiology and functional anatomy of tic disorders,. in Tourette Syndrome,. D. Martino and J.F. Leckman, Editors., Oxford University Press: Oxford. 2013.\n\nBerthier ML, Bayes A, Tolosa ES: Magnetic resonance imaging in patients with concurrent Tourette's disorder and Asperger's syndrome. J Am Acad Child Adolesc Psychiatry. 1993; 32(3): 633–9. PubMed Abstract | Publisher Full Text\n\nCastellanos FX, Giedd JN, Hamburger SD, et al.: Brain morphometry in Tourette's syndrome: the influence of comorbid attention-deficit/hyperactivity disorder. Neurology. 1996; 47(6): 1581–1583. PubMed Abstract | Publisher Full Text\n\nGerard E, Peterson BS: Developmental processes and brain imaging studies in Tourette syndrome. J Psychosom Res. 2003; 55(1): 13–22. PubMed Abstract | Publisher Full Text\n\nPeterson BS, Staib L, Scahill L, et al.: Regional brain and ventricular volumes in Tourette syndrome. Arch Gen Psychiatry. 2001; 58(5): 427–40. PubMed Abstract | Publisher Full Text\n\nPeterson BS, Thomas P, Kane MJ, et al.: Basal Ganglia volumes in patients with Gilles de la Tourette syndrome. Arch Gen Psychiatry. 2003; 60(4): 415–24. PubMed Abstract | Publisher Full Text\n\nMakki, MI, Behen M, Bhatt A, et al.: Microstructural abnormalities of striatum and thalamus in children with Tourette syndrome. Mov Disord. 2008; 23(16): 2349–56. PubMed Abstract | Publisher Full Text\n\nMüller-Vahl KR, Kaufmann J, Grosskreutz J, et al.: Prefrontal and anterior cingulate cortex abnormalities in Tourette Syndrome: Evidence from voxel-based morphometry and magnetization transfer imaging. BMC Neurosci. 2009; 10: 47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCsernansky JG, Wang L, Jones D, et al.: Hippocampal deformities in schizophrenia characterized by high dimensional brain mapping. Am J Psychiatry. 2002; 159(12): 2000–6. PubMed Abstract | Publisher Full Text\n\nWang L, Joshi SC, Miller MI, et al.: Statistical analysis of hippocampal asymmetry in schizophrenia. Neuroimage. 2001; 14(3): 531–45. PubMed Abstract | Publisher Full Text\n\nSmith MJ, Wang L, Cronenwett W, et al.: Thalamic morphology in schizophrenia and schizoaffective disorder. J Psychiatr Res. 2011; 45(3): 378–85. PubMed Abstract | Publisher Full Text\n\nWang L, Harms MP, Staggs JM, et al.: Donepezil treatment and changes in hippocampal structure in very mild Alzheimer disease. Arch Neurol. 2010; 67(1): 99–106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCsernansky JG, Wang L, Joshi S, et al.: Early DAT is distinguished from aging by high-dimensional mapping of the hippocampus. Dementia of the Alzheimer type. Neurology. 2000; 55(11): 1636–43. PubMed Abstract | Publisher Full Text\n\nWang L, Swank JS, Glick IE, et al.: Changes in hippocampal volume and shape across time distinguish dementia of the Alzheimer type from healthy aging. Neuroimage. 2003; 20(2): 667–82. PubMed Abstract | Publisher Full Text\n\nMamah D, Harms MP, Wang L, et al.: Basal ganglia shape abnormalities in the unaffected siblings of schizophrenia patients. Biol Psychiatry. 2008; 64(2): 111–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPosener JA, Wang L, Price JL, et al.: High-dimensional mapping of the hippocampus in depression. Am J Psychiatry. 2003; 160(1): 83–9. PubMed Abstract | Publisher Full Text\n\nHogan RE, Wang L, Bertrand ME, et al.: MRI-based high-dimensional hippocampal mapping in mesial temporal lobe epilepsy. Brain. 2004; 127(Pt 8): 1731–40. PubMed Abstract | Publisher Full Text\n\nCsernansky JG, Schindler MK, Splinter NR, et al.: Abnormalities of thalamic volume and shape in schizophrenia. Am J Psychiatry. 2004; 161(5): 896–902. PubMed Abstract | Publisher Full Text\n\nWang L, Lee DY, Bailey E, et al.: Validity of large-deformation high dimensional brain mapping of the basal ganglia in adults with Tourette syndrome. Psychiatry Res. 2007; 154(2): 181–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChurch JA, Fair DA, Dosenbach NU, et al.: Control networks in paediatric Tourette syndrome show immature and anomalous patterns of functional connectivity. Brain. 2009; 132(Pt 1): 225–238. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMugler JP, Brookeman JR: Three-dimensional magnetization-prepared rapid gradient-echo imaging (3D MP RAGE). Magn Reson Med. 1990; 15(1): 152–7. PubMed Abstract | Publisher Full Text\n\nHaller JW, Banerjee A, Christensen GE, et al.: Three-dimensional hippocampal MR morphometry with high-dimensional transformation of a neuroanatomic atlas. Radiology. 1997; 202(2): 504–10. PubMed Abstract\n\nDesikan RS, Ségonne F, Fischl B, et al.: An automated labeling system for subdividing the human cerebral cortex on MRI scans into gyral based regions of interest. Neuroimage. 2006; 31(3): 968–80. PubMed Abstract | Publisher Full Text\n\nCsernansky JG, Wang L, Joshi SC, et al.: Computational anatomy and neuropsychiatric disease: probabilistic assessment of variation and statistical inference of group difference, hemispheric asymmetry, and time-dependent change. Neuroimage. 2004; 23(Suppl 1): S56–68. PubMed Abstract | Publisher Full Text\n\nLudolph AG, Juengling FD, Libal G, et al.: Grey-matter abnormalities in boys with Tourette syndrome: magnetic resonance imaging study using optimised voxel-based morphometry. Br J Psychiatry. 2006; 188: 484–485. PubMed Abstract | Publisher Full Text\n\nRoessner V, Overlack S, Schmidt-Samoa C, et al.: Increased putamen and callosal motor subregion in treatment-naïve boys with Tourette syndrome indicates changes in the bihemispheric motor network. J Child Psychol Psychiatry. 2011; 52(3): 306–14. PubMed Abstract | Publisher Full Text\n\nPeterson BS, Thomas P, Kane MJ, et al.: Basal ganglia volumes in patients with Gilles de la Tourette syndrome. Arch Gen Psychiatry. 2003; 60(4): 415–424. PubMed Abstract | Publisher Full Text\n\nMoriarty J, Varma AR, Stevens J, et al.: A volumetric MRI study of Gilles de la Tourette's syndrome. Neurology. 1997; 49(2): 410–415. PubMed Abstract | Publisher Full Text\n\nZimmerman AM, Abrams MT, Giuliano JD, et al.: Subcortical volumes in girls with tourette syndrome: support for a gender effect. Neurology. 2000; 54(12): 2224–9. PubMed Abstract | Publisher Full Text\n\nRoessner V, Overlack S, Baudewig J, et al.: No brain structure abnormalities in boys with Tourette's syndrome: a voxel-based morphometry study. Mov Disord. 2009; 24(16): 2398–403. PubMed Abstract | Publisher Full Text\n\nPeterson BS, Riddle MA, Cohen DJ, et al.: Reduced basal ganglia volumes in Tourette's syndrome using three-dimensional reconstruction techniques from magnetic resonance images. Neurology. 1993; 43(5): 941–949. PubMed Abstract | Publisher Full Text\n\nMakki, MI, Govindan RM, Wilson BJ, et al.: Altered fronto-striato-thalamic connectivity in children with Tourette syndrome assessed with diffusion tensor MRI and probabilistic fiber tracking. J Child Neurol. 2009; 24(6): 669–78. PubMed Abstract | Publisher Full Text\n\nBloch MH, Leckman JF, Zhu H, et al.: Caudate volumes in childhood predict symptom severity in adults with Tourette syndrome. Neurology. 2005; 65(8): 1253–1258. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "3072",
"date": "24 Jan 2014",
"name": "Jeremy S. Stern",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 continues the quest for subtle structural abnormalities in striato-thalamo-cortical circuitry in Tourette syndrome. The technique is innovative and in this paper is negative for a relatively small number of children as it has been for a similar number of adults in the past. The technique has yielded positive results in certain other conditions. The current results are not consistent with some other larger studies of caudate volumes and the reasons for this are explored and are a common issue in this area. Given the small number of subjects with heterogeneity of comorbidities and medication history the negative result here cannot be definitive, as suggested in the discussion.A common group of core authors in a small number of sites published the previous HDBM-LD work and so external replication, particularly of the so far positive results would be valuable. The methadology is described more fully in an earlier paper, but it is hard to confirm if it could be reproduced directly from this alone.The title and abstract are appropriate.",
"responses": [
{
"c_id": "703",
"date": "11 Feb 2014",
"name": "Kevin J Black",
"role": "Author Response",
"response": "We greatly appreciate Prof. Stern's thoughtful comments about the relevance and limitations of this pilot study. The prior report (ref. 35 above) does describe the methods in detail."
}
]
},
{
"id": "4285",
"date": "27 Mar 2014",
"name": "Kirsten R Müller-Vahl",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study a relatively new and sophisticated method (using large-deformation, high dimensional brain mapping (HDBM-LD)) has been used to investigate for the first time structure geometry and volume of basal ganglia and thalamus in 13 children with Tourette syndrome (TS) compared to 16 healthy controls. Comparable to a prior study in adults with TS performed by the same group, no significant differences could be detected in volume, shape, or right/left asymmetry. These findings are in contrast to several other studies suggesting a reduction of the volume of the caudate in both children and adults with TS. The major limitation of this study is the small sample size and the heterogeneity of the patients’ group with respect to comorbidities and medication. In particular, medication with neuroleptic drugs might have influenced the results, since there is evidence that exposure to antipsychotic medication may cause basal ganglia enlargement. However, the study has also several strengths including the well experienced team of researchers, low age range and inclusion of children only, and excellent matching for age, sex, and handedness. I enjoyed reading the discussion, because results obtained in children were not mixed with those in adults. The introduction would benefit from a more precise clinical description. For example: tics are no longer characterized as “stereotyped” movements, abdominal tension is an untypical example for a motor tic, and tics “may” be associated with other disorders such as Wilson’s and Huntington’s disease. The title and abstract are appropriate. Although this study contributes to our understanding of the neurobiology of TS, comparable to most MRI studies in TS, it is limited by the small sample size and possible influences of medication and comorbidities. I would love to read the results of a follow-up study including a larger number of patients soon.",
"responses": [
{
"c_id": "744",
"date": "27 Mar 2014",
"name": "Kevin J Black",
"role": "Author Response",
"response": "We appreciate Dr. Müller-Vahl's thoughtful comments."
}
]
}
] | 1
|
https://f1000research.com/articles/2-207
|
https://f1000research.com/articles/2-173/v1
|
13 Aug 13
|
{
"type": "Research Article",
"title": "Regulation of CYP3A genes by glucocorticoids in human lung cells",
"authors": [
"Jessica K Roberts",
"Chad D Moore",
"Erin G Romero",
"Robert M Ward",
"Garold S Yost",
"Christopher A Reilly",
"Jessica K Roberts",
"Chad D Moore",
"Erin G Romero",
"Robert M Ward",
"Garold S Yost"
],
"abstract": "Inhaled glucocorticoids are the first-line treatment for patients with persistent asthma. However, approximately thirty percent of patients exhibit glucocorticoid insensitivity, which may involve excess metabolic clearance of the glucocorticoids by CYP3A enzymes in the lung. CYP3A4, 3A5, and 3A7 enzymes metabolize glucocorticoids, which in turn induce CYP3A genes. However, the mechanism of CYP3A5 mRNA regulation by glucocorticoids in lung cells has not been determined. In hepatocytes, glucocorticoids bind to the glucocorticoid receptor (GR), which induces the expression of the constitutive androstane receptor or pregnane X receptor; both of which bind to the retinoid X receptor alpha, leading to the induction of CYP3A4, 3A5, and 3A7. There is also evidence to suggest a direct induction of CYP3A5 by GR activation in liver cells. In this study, these pathways were evaluated as the mechanism for CYP3A5 mRNA induction by glucocorticoids in freshly isolated primary tracheal epithelial, adenocarcinomic human alveolar basal epithelial (A549), immortalized bronchial epithelial (BEAS-2B), primary normal human bronchial/tracheal epithelial (NHBE), primary small airway epithelial (SAEC), and primary lobar epithelial lung cells. In A549 cells, beclomethasone 17-monopropionate ([M1]) induced CYP3A5 mRNA through the glucocorticoid receptor. CYP3A5 mRNA induction by five different glucocorticoids was attenuated by inhibiting the glucocorticoid receptor using ketoconazole, and for beclomethasone dipropionate, using siRNA-mediated knock-down of the glucocorticoid receptor. The constitutive androstane receptor was not expressed in lung cells. SAEC cells, a primary lung cell line, expressed CYP3A5, but CYP3A5 mRNA was not induced by glucocorticoid treatment despite evaluating a multitude of cell culture conditions. None of the other lung cells expressed CYP3A4, 3A5 or 3A7 mRNA. These studies demonstrate that CYP3A5 mRNA is induced by glucocorticoids in A549 cells via the glucocorticoid receptor, but that additional undefined regulatory processes exist in primary lung cells.",
"keywords": [
"Inhaled glucocorticoids are the first-line treatment for asthma1–3. Glucocorticoids bind to the glucocorticoid receptor to reduce the expression of genes that produce a variety of pro-inflammatory mediators and mucus in the lung4–6. The most commonly prescribed glucocorticoids are beclomethasone dipropionate (BDP)",
"triamcinolone acetonide (TCL)",
"budesonide (BUD)",
"fluticasone propionate (FLT)",
"and flunisolide (FLN)1. BDP is a pro-drug and requires removal of the C-21 propionate group to become pharmacologically active",
"the active drug is beclomethasone 17-monopropionate",
"referred to as [M1] (Figure 1)7. Pharmacological inactivation and clearance of glucocorticoids",
"such as BDP and its active metabolite [M1]",
"is mediated",
"in part",
"by cytochrome P450 (CYP) enzymes (Figure 1)."
],
"content": "Introduction\n\nInhaled glucocorticoids are the first-line treatment for asthma1–3. Glucocorticoids bind to the glucocorticoid receptor to reduce the expression of genes that produce a variety of pro-inflammatory mediators and mucus in the lung4–6. The most commonly prescribed glucocorticoids are beclomethasone dipropionate (BDP), triamcinolone acetonide (TCL), budesonide (BUD), fluticasone propionate (FLT), and flunisolide (FLN)1. BDP is a pro-drug and requires removal of the C-21 propionate group to become pharmacologically active; the active drug is beclomethasone 17-monopropionate, referred to as [M1] (Figure 1)7. Pharmacological inactivation and clearance of glucocorticoids, such as BDP and its active metabolite [M1], is mediated, in part, by cytochrome P450 (CYP) enzymes (Figure 1).\n\nIn humans, CYP3A4, 3A5, and 3A7 are the primary CYP enzymes involved in glucocorticoid metabolism8–11. CYP3A4 is the most abundant CYP3A enzyme in the liver and intestines8,12,13, CYP3A5 is more prevalent in the lung than the liver12,14–16, and CYP3A7 is expressed in fetal liver, but diminishes after birth when CYP3A4 becomes the dominant adult hepatic CYP3A enzyme17,18. Expression of CYP3A7 in fetal and adult respiratory tissue has also been observed16.\n\nRegulation of CYP3A enzymes in response to glucocorticoid treatment has been extensively characterized in the liver, but less is known about this phenomenon in the lung. In hepatocytes, CYP3A enzyme induction is mediated by the pregnane X receptor (PXR)19,20 (Figure 2A). However, PXR is not expressed in the lung21. Glucocorticoids can also influence CYP3A induction via the glucocorticoid receptor (GR) and the constitutive androstane receptor (CAR) in the liver22,23. Briefly, glucocorticoids bind GR in the cytosol, which forms a homodimer and translocates into the nucleus, leading to increased transcription of CAR. CAR forms a heterodimer with the retinoid X receptor alpha (RXRα), which binds to the RXR-response element and induces the expression of CYP3A enzymes (Figure 2A)22. Previous work by Hukannen et al. demonstrated that CAR was not expressed in A549 (adenocarcinomic human alveolar basal epithelial) cells and suggested that glucocorticoid binding to GR may directly regulate CYP3A gene expression in A549 cells (Figure 2B), based on inhibition using RU-48615,24. However, these pathways have not been evaluated in primary lung cell cultures or lung tissue.\n\n(A) Active glucocorticoid will bind to the glucocorticoid receptor (GR), which forms a homodimer and translocates to the nucleus. The homodimer binds to its response element (GRE) and induces the expression of either the pregnane X receptor (PXR) or the constitutive androstane receptor (CAR). CAR or PXR (though this receptor is not expressed in the lungs) forms a heterodimer with the retinoic X receptor alpha (RXRα) which in turn induces the expression of the CYP3A enzymes via binding of the respective response-elements (CARE and/or PXRE). (B) Active glucocorticoid will bind to the glucocorticoid receptor (GR), which forms a homodimer and translocates into the nucleus. The homodimer binds to its response element (GRE) and induces the expression of CYP3A enzymes.\n\nThe purpose of this study was three fold: to evaluate the changes in the expression of CYP3A mRNA in primary lung cells treated with glucocorticoids; to determine which pathway was responsible for glucocorticoid-induced changes in CYP3A mRNA expression; and to determine the role of BDP metabolism in this phenomenon. The cell lines used in this study were BEAS-2B (immortalized bronchial epithelial cell line), NHBE (normal human bronchial/tracheal epithelial cells), lobar epithelial cells (secondary bronchus epithelial cells), primary cells recovered from tracheal washes of pediatric patients on ventilation, SAEC (small airway epithelial cells), and A549 (human lung adenocarcinoma) cells. It was hypothesized that CYP3A5 mRNA induction in primary cells by BDP11 and other glucocorticoids would occur via a mechanism involving GR/CAR/RXRα, as previously demonstrated using hepatocytes.\n\n\nMethods\n\nBeclomethasone dipropionate (BDP), triamcinolone acetonide (TCL), fluticasone propionate (FLT), flunisolide (FLN), budesonide (BUD), prednisolone, ammonium acetate, eserine, and methanol were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Paraoxon was purchased from Chem Service (West Chester, PA).\n\nA549 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's Modified Eagle Medium (DMEM) fortified with 5% fetal bovine serum (Life Technologies, Grand Island, NY). SAEC cells (LONZA, Walkersville, MD; donor numbers 11662, 14453, 14457) were cultured in small airway epithelial growth medium, supplemented with the SAGM bullet kit (LONZA). Cells were cultured with and without hydrocortisone by adding or not adding the hydrocortisone component from the SAGM bullet kit. NHBE cells (LONZA; donor numbers 15268, 5S03795) were grown in bronchial epithelial cell growth medium (BEGM Bullet kit) (LONZA). BEAS-2B cells (American Type Culture Collection) were cultured in LHC-9 medium (Life Technologies). Lobar cells (donor number 01334) were cultured in BronchiaLife Basal Medium supplemented with the BronchiaLife B/T supplement kit (Lifeline Cell Technology, Walkersville, MD). All cells except A549 cells were plated in 12-well plates pre-coated with LHC basal medium (Life Technologies). Tracheal epithelial cells were recovered from tracheal washes from mechanically ventilated pediatric patients in the neonatal intensive care unit and pediatric intensive care unit at Primary Children’s Medical Center at the University of Utah, with IRB approval (00026839). Briefly, cells were separated from sputum by centrifugation at 900 x g for 30 min in 14 mL of DMEM/F12 media. Cells were plated in a 12-well plate pre-coated with 2% gelatin (Life Technologies) and cultured in DMEM/F12 media + 10% fetal bovine serum (FBS) (Life Technologies). All cells were cultured in an atmosphere of 5% CO2:95% air at 37ºC.\n\nCell treatments were prepared in treatment media with a final concentration of DMSO less than 1%. Cells were treated at ~70% confluence. A549 cells were cultured in OPTIMEM (Life Technologies) and SAEC cells were cultured in growth media either with or without hydrocortisone and with or without heat inactivated and/or charcoal-stripped FBS. All other cell lines were treated in their respective growth medium, and heat inactivated to eliminate esterase activity from the FBS, which would metabolize BDP before it could diffuse into the cells. Cytotoxicity assays were performed using the Dojindo Cell Counting Kit-8 (Dojindo Laboratories, Rockville, MD) to determine glucocorticoid, esterase inhibitor, and ketoconazole concentrations exhibiting <20% cytotoxicity in A549 cells. All other cell lines were treated with the same concentrations as determined with A549 cells. Glucocorticoid treatments were as follows: BDP (10 μM), TCL (1 μM), BUD (10 μM), FLT (1 μM), and FLN (100 nM). Pre-treatments in various experiments included ketoconazole (Sigma-Aldrich Chemical Company) (50 μM, 10 μM, and 1 μM, to antagonize GR), esterase inhibitors (1:1 mixture of eserine and paraoxon, each at 175 μM, to inhibit [M1] formation), and 1-aminobenzotriazole (Sigma-Aldrich Chemical Company) (1-ABT; 200 μM, to inhibit P450-mediated metabolism) for 2 h prior to a 22 h glucocorticoid co-treatment. Controls were treated with an equivalent concentration of DMSO. All A549 cell treatments were carried out in 6-well plates for 24 h (n=6). All other cell lines were cultured in pre-coated 12-well plates and treated for 24 h (n=3).\n\nAfter treatment, BDP and its metabolites were extracted from the collected media by adding 2x volume (6 mL for A549, 4 mL for all other cell lines) methyl tert-butyl ether containing 1 nM prednisolone (internal standard for quantification) and shaking for 25 min. Samples were clarified by centrifugation, the organic fraction was collected, dried under air, reconstituted in 100 µL 1:1 H2O:MeOH, clarified again by centrifugation, and transferred to autosampler vials for analysis by liquid chromatography-mass spectrometry (LC/MS/MS). LC/MS/MS was conducted on a Thermo LCQ Advantage Max ion trap instrument equipped with a Finnigan Surveyor LC pump, Surveyor Autosampler and universal Ion Max source operated with Thermo Xcalibur software version 2.0 (Thermo Fisher Scientific, Waltham, MA) as previously described11.\n\nTotal RNA was isolated from cells using TRIzol reagent (Life Technologies). cDNA was synthesized using iScript Reverse Transcription Supermix for qPCR (BIO RAD, Hercules, CA). qPCR was performed using either LightCycler 480 Probes Master mix (CYP3A5*1) or LightCycler 480 SYBR Green I Master Mix (all other genes) (Roche, Indianapolis, IN) with a LightCycler 480 System. The PCR program for the probe mix consisted of a 5 min incubation at 95ºC, followed by 45 cycles of 95ºC for 10s, 55ºC for 30s, then 72ºC for 1s. The PCR program for SYBR Green I mix consisted of a 5 min incubation at 95ºC, followed by 40 cycles of 95ºC for 10s, 63ºC for 5s for CYP3A4, CYP3A7 and β2-microglobulin. For GR and CAR, annealing was performed at 65ºC for 5s and extension at 72ºC for 10s. mRNA copy number was determined from standard curves for each gene and was normalized using β2-microglobulin. Primer sequences for the various genes are listed in Table 125.\n\nPre-annealed, short interfering “Smart Pool” siRNAs specific to human GR were purchased from Dharmacon (Waltham, MA). siRNA directed against GFP (negative control)26 was synthesized at the University of Utah oligonucleotide synthesis core and annealed by combining 40 µM of each strand and incubating in annealing buffer (100 mM potassium acetate, 30 mM HEPES KOH, 2 mM magnesium acetate adjusted to pH 7.4) for 1 min at 90ºC followed by 1 h at 37ºC, in a final volume of 0.5 mL. A549 cells were plated into 6-well plates containing 20 nM siRNA per well, previously complexed with Lipofectamine 2000 (Life Technologies) using a ratio of 3:2 lipid to siRNA in 100 µL of OPTIMEM (Life Technologies). The cells were grown for 48, 72, and 96 h to determine the time at which maximum decreases in GR mRNA occurred (72 h). In subsequent experiments, cells were treated with DMSO, 10 µM BDP, or 10 μM BDP + 175 µM esterase inhibitors (1:1 eserine:paraoxon) for 24 h to determine the effects of attenuated GR expression on the induction of CYP3A5 in A549 cells.\n\nStatistical analysis was performed using GraphPad Prism 4.02 software for Windows (San Diego, CA). One-way ANOVA and Dunnett’s post-hoc test were used with p<0.05. All data are represented as a mean with error bars representing standard deviation.\n\n\nResults\n\nMedia from A549 cells treated with BDP (10 µM) for 24 h was extracted and analyzed for metabolites of BDP produced by CYP3A enzymes. The only CYP3A-mediated metabolite detected was [M5] (Figure 1 and Figure 3A)11. For the remainder of the studies, [M1], the active metabolite, was used as a marker for esterase activity and [M5] was used as a marker for CYP3A5 activity. Only CYP3A5*1 mRNA was detected in A549 cells. CYP3A4 and CYP3A7 mRNA were not detected in A549 cells, as previously documented11,16. BDP treatment significantly induced the expression of CYP3A5 mRNA (~2-fold) compared to the DMSO control (Figure 3B). Inhibiting the production of [M1] using esterase inhibitors also blocked the induction of CYP3A5 mRNA (Figure 2A and Figure 2B); esterase inhibitor (EI) treatment alone had no effect on CYP3A5 expression. 1-ABT, a mechanism-based inactivator of P450 enzymes, also inhibited esterase activity (i.e. [M1] formation) (Figure 3A), and as a result, prevented the induction of CYP3A5 mRNA (Figure 3B).\n\n(A) Relative quantities of [M1] and [M5] measured by LC/MS/MS in A549 cell culture media following beclomethasone dipropionate (BDP) treatment alone, BDP + esterase inhibitors (EI), 1-aminobenzotriazole (1-ABT) + BDP, and 1-ABT + EI + [M1]. (B) CYP3A5 mRNA detected in A549 cells following DMSO control, BDP treatment alone, BDP + EI, 1-ABT alone, 1-ABT + BDP, and 1-ABT + EI + [M1]. Values are expressed as fold over DMSO controls. Statistics used for data analysis were one-way ANOVA with Dunnett’s post-hoc test. Data are the mean and standard deviation from n=6 replicates. * p<0.05, ** p<0.01.\n\nCells were treated with [M1] in either the absence or presence of 1-ABT and esterase inhibitors. [M1] treatment was sufficient to induce CYP3A5 mRNA (~2-fold), without the requirement of esterases to produce [M1] (Figure 3B), indicating that CYP3A5 mRNA induction in A549 cells was mediated by [M1].\n\nGR and CAR mRNA were quantified in A549 cells. A significant increase in GR mRNA (~2.5-fold) was observed following 24 h treatment with BDP (Table 2), consistent with previous studies15, suggesting that GR, not CAR, was responsible for the induction of CYP3A5 message in A549 cells.\n\nData are represented as fold over DMSO control. Statistics used for data analysis were one-way ANOVA with Dunnett’s post-hoc test. ** p<0.01, N.D. = not detected, GC = glucocorticoid.\n\nKetoconazole is a competitive antagonist of GR25. Ketoconazole alone had no significant effect on CYP3A5 mRNA expression as compared to DMSO controls. As the concentration of ketoconazole was decreased, dose-dependent increases in the expression of CYP3A5 mRNA were observed for BDP, TCL, FLT, BUD, and FLN (Figure 4A–E): BDP caused a ~2-fold induction, BUD caused a ~4-fold induction, TCL caused a ~5.5-fold induction, FLT caused a ~3.5-fold induction, and FLN caused a ~5.5-fold induction, relative to their respective controls. These data confirm the hypothesis that the induction of CYP3A5 mRNA in A549 cells was mediated by GR. BDP or FLT paired with KTZ 1 µM treatment also showed further induction of CYP3A5 mRNA as compared to controls (~3.5 for BDP and ~6.5 for FLT). However, the basis and significance for this enhanced induction are not clear at this time.\n\nCYP3A5 mRNA detected in A549 cells treated with (A) beclomethasone dipropionate (BDP), (B) budesonide (BUD), (C) triamcinolone acetonide (TCL), (D) fluticasone propionate (FLT), and (E) flunisolide (FLN), with and without ketoconazole (KTZ), a competitive antagonist for GR. Results are presented as fold over DMSO controls. Statistics used for data analysis were one-way ANOVA with Dunnett’s post-hoc test. Data are the mean and standard deviation from n=6 replicates. * p<0.05, ** p<0.01, *** p<0.001.\n\nCells were transfected with siRNA and grown for 48, 72, and 96 h to determine the time of maximum GR mRNA knock down (Figure 5A). Maximum suppression occurred as early as 48 h, but the 72 h time point was chosen for further experiments to ensure efficient GR protein depletion. An approximate 2-fold induction of CYP3A5 mRNA was observed in A549 cells following treatment with BDP in control cells transfected with “nonsense” siRNA directed against GFP. Consistent with previous results (Figure 3A and Figure 3B), CYP3A5 mRNA induction was prevented by esterase inhibitors (Figure 5B). Cells transfected with siRNA targeted for GR mRNA showed no change in CYP3A5 mRNA with BDP treatment (Figure 5B), further confirming the role of GR in directly regulating the induction of CYP3A5 mRNA in A549 cells treated with BDP and presumably the other glucocorticoids used in Figure 4.\n\n(A) siRNA-mediated GR knockdown in A549 cells at 48, 72, 96 h compared to \"nonsense\" GFP siRNA (negative control), expressed as fold over designated GFP control for each time point. (B) Cells were exposed to siRNA for 72 h then treated with DMSO, BDP, or BDP + esterase inhibitor (EI). Statistics used for data analysis were one-way ANOVA with Dunnett’s post-hoc test. Data are the mean and standard deviation from three replicates. * p<0.05, ** p<0.01.\n\nNeither CYP3A5*1 mRNA nor any other variant form of CYP3A5 mRNA was detected or induced by glucocorticoids in NHBE, BEAS-2B, lobar, and freshly isolated tracheal wash samples (Table 2).\n\nSAEC cells from three separate donors were evaluated for CYP3A5*1 and other variant forms of CYP3A5 mRNA expression and induction in response to glucocorticoid treatment. Initial experiments demonstrated that mRNA for CYP3A5*1, but not CYP3A4 or 3A7, was expressed in one of the three SAEC samples (donor number 11662), but that expression levels were not altered by glucocorticoid treatment. It was hypothesized that the high concentration of hydrocortisone (500 µM) in the SAEC growth media prevented the induction of CYP3A5 mRNA by substantially lower concentrations of the glucocorticoids used in the treatments. Elimination of hydrocortisone from the media decreased the basal expression of CYP3A5 mRNA (Figure 6). However, no change in mRNA abundance was observed over a 24 h treatment period with BDP. Furthermore, neither increasing the treatment concentration of BDP to 50 µM, nor treatment with [M1] at 150 µM led to an increase in CYP3A5 mRNA in SAEC cells. It was subsequently hypothesized that phthalates or other substances in the FBS might alter GR function and CYP3A5 mRNA induction by glucocorticoids27. However, neither heat inactivation nor charcoal-stripping of the FBS in media with or without hydrocortisone led to CYP3A5 mRNA induction. The various manipulations to SAEC culture conditions and results for CYP3A5 induction are summarized in Table 3.\n\nSAEC cells treated with BDP (10 µM; 24 h) or DMSO matching controls with and without hydrocortisone (HC). There was no significant difference between treatments or matching controls using one-way ANOVA with p<0.05.\n\n\nDiscussion\n\nInhaled glucocorticoids are used to control undesirable symptoms in asthmatic patients. However, about 30% of the population does not benefit from this first-line treatment6. Prior work demonstrated that the five most commonly prescribed glucocorticoids used in the treatment of asthma are metabolized by CYP3A enzymes, specifically CYP3A4, CYP3A5, and CYP3A710,11. Therefore, it has been proposed that unusually high rates of metabolism of glucocorticoids in lung cells by these enzymes might contribute to the decrease and/or lack of efficacy in some individuals. However, it is not understood how the expression of CYP3A enzymes is regulated in the lung in response to glucocorticoid treatment, despite extensive knowledge of this phenomenon in hepatocytes and the liver22.\n\nUsing A549 cells, it was demonstrated that CYP3A5*1 mRNA was induced by glucocorticoid treatment (Figure 3B and Figure 4A–E); neither CYP3A4 nor CYP3A7 mRNA were detected in A549 cells. Subsequent studies using a competitive antagonist of GR (ketoconazole) and siRNA selective for GR mRNA, demonstrated that inhibition of GR function prevented the induction of CYP3A5 mRNA by BDP and other glucocorticoids in A549 cells (Figure 4A–E and Figure 5B). It was also demonstrated that CAR mRNA was not expressed by lung cells, consistent with previous data15, and therefore could not be involved in the regulation of CYP3A5 expression by glucocorticoids as occurs in hepatocytes. It was concluded that CYP3A5 expression was directly regulated by GR (Figure 2B). Schuetz et al.24 previously described two “half sites” of GR (TGTTCT) separated by 160 bp in the promoter region of CYP3A5 in HepG2 cells and in human and rat hepatocytes. It was demonstrated that dexamethasone induced the expression of CYP3A5 by the GR homodimer binding to these two joined “half-sites” which could be blocked by RU-486, a GR antagonist. It is plausible these same sites are involved in the regulation of CYP3A5 in lung cells by BDP and other glucocorticoids.\n\nRegardless of the exact mechanism of regulation, the current results illustrate that glucocorticoids have the capacity to induce the expression of CYP3A5 in A549 cells. These data, in conjunction with prior metabolism studies of glucocorticoids10,11, support the hypothesis that treating patients with glucocorticoids could increase levels of CYP3A5 in the lung, and therefore increase pulmonary glucocorticoid metabolism, ultimately increasing clearance, and potentially decreasing the concentration of active drug in lung cells. Though most of the population expresses the inactive form of CYP3A5 (CYP3A5*3)13,28 those expressing CYP3A5*1, the active form of CYP3A513, would exhibit increased clearance of the drug, and therefore could account for at least some of the 30% of patients who do not respond to inhaled glucocorticoid therapy.\n\nIn order to further support the hypothetical scenario above, the induction of CYP3A enzymes by glucocorticoids in various lung cells was studied. CYP3A5 mRNA expression was quantified in primary lung cells, which presumably more closely model epithelial cells of the human respiratory tract and lung. NHBE, lobar, and cells recovered from tracheal washes of mechanically ventilated children were evaluated for CYP3A enzyme expression and induction by glucocorticoids. Results in Table 2 show that CYP3A mRNA was not expressed in cells of the conducting airways in response to glucocorticoid treatment, indicating that these epithelial cells likely do not play a role in CYP3A-dependent metabolism of glucocorticoids in the lung. In contrast, select donor samples of SAEC cells, representing cells of the distal bronchioles, alveolar ducts, and alveoli, did express CYP3A5 (Table 2). However, there was no change in CYP3A5 mRNA when these cells were treated with glucocorticoids. A thorough examination of potential confounding issues associated with cell culture revealed a high concentration of hydrocortisone (500 µM) in the growth media. Because cells were treated with only 10 µM BDP, it would stand to reason that no change in CYP3A5 mRNA would occur because CYP3A5 expression would already be maximized as a result of hydrocortisone activating the GR pathway.\n\nExperiments conducted in A549 cells showed that culturing cells in 500 µM hydrocortisone increased the basal expression of CYP3A5 mRNA by 2-fold, masking the induction routinely observed using 10 µM BDP for 24 h. When A549 cells were subsequently cultured in media without hydrocortisone for 48 h, providing sufficient time for a “wash out” of the hydrocortisone, the basal expression of CYP3A5 mRNA was reduced, and ~2-fold induction of CYP3A5 mRNA occurred with the 10 µM BDP, 24 h treatment. Therefore, hydrocortisone was omitted from the SAEC growth media. Subsequent experiments in SAEC cells showed no change in CYP3A5 mRNA in response to glucocorticoid treatment (Figure 6), albeit removal of hydrocortisone from the media caused a slight decrease in the basal level of CYP3A5 mRNA expression, suggesting that GR plays a role in the regulation of CYP3A5. It is feasible that because cells had been exposed to such high concentrations of hydrocortisone during their isolation and expansion, that 10 µM of BDP was not sufficient to induce CYP3A5 mRNA, even after culturing the cells in the absence of hydrocortisone for multiple division cycles. Therefore, the concentration of BDP was increased to 50 µM and an additional treatment group using 150 µM [M1] was added. Again no increases in CYP3A5 mRNA was observed. Heat-inactivated and charcoal-stripped FBS were also utilized to remove potential interfering compounds from FBS, and still no change was observed.\n\nTo our knowledge, no one has observed a change in CYP3A mRNA expression in any primary human lung cell cultures. However, Cyp3a11, 3a13, and 3a16 mRNA and protein induction have been documented in mouse lung following dexamethasone treatment29. As such, additional studies using animal models and relevant samples from human patients need to be evaluated in order to conclusively confirm or reject the hypothesis that CYP3A genes are regulated in human lung cells in response to glucocorticoid treatment since current in vitro models remain unexplainably limited in value for such studies.\n\nIn summary, the data presented herein demonstrate that, in A549 cells, glucocorticoid binding to the glucocorticoid receptor regulates the expression of CYP3A5, and therefore, corroborates the hypothesis that increased metabolism of glucocorticoids may occur in some patients. However, further research is needed to determine if changes in CYP3A5 expression occur in the human respiratory tissue similar to A549 cells, the precise mechanism by which this process occurs, and whether changes in the local metabolism of glucocorticoids by CYP3A5 ultimately impact glucocorticoid efficiency.",
"appendix": "Author contributions\n\n\n\nRoberts, Romero, Moore, Ward, Yost, and Reilly participated in research design. Roberts, Moore, and Romero conducted the experiments. Roberts, Moore, and Romero performed the data analysis. Roberts, Moore, Ward, Yost, and Reilly wrote or contributed to the writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe project described was supported by Grant Number R01HD060559 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health & Human Development or the National Institutes of Health. J.K. Roberts was also supported by the Howard Hughes Medical Institute under their HHMI Med to Grad initiative [Grant 56006777].\n\n\nAcknowledgments\n\nWe would like to thank and Dr. Roger Gaedigk for the CYP3A5*1 probe and primer sequences.\n\n\nReferences\n\nEPR-3. Guidelines for the Diagnosis and Management of Asthma-Summary Report 2007. J Allergy Clin Immunol. 2007; 120(5 Suppl): S94–138. PubMed Abstract | Publisher Full Text\n\nBateman ED, Hurd SS, Barnes PJ, et al.: Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J. 2008; 31(1): 143–178. PubMed Abstract | Publisher Full Text\n\nWilliams SG, Schmidt DK, Redd SC, et al.: Key clinical activities for quality asthma care. Recommendations of the National Asthma Education and Prevention Program. MMWR Recomm Rep. 2003; 52(RR-6): 1–8. PubMed Abstract\n\nJusko WJ: Corticosteroid pharmacodynamics: models for a broad array of receptor-mediated pharmacologic effects. J Clin Pharmacol. 1990; 30(4): 303–310. PubMed Abstract | Publisher Full Text\n\nLeung DY, Bloom JW: Update on Glucocorticoid Action and Resistance. J Allergy Clin Immunol. 2003; 111(1): 3–22. PubMed Abstract | Publisher Full Text\n\nMjaanes CM, Whelan GJ, Szefler SJ: Corticosteroid therapy in Asthma: Predictors of Responsiveness. Clin Chest Med. 2006; 27(1): 119–132. PubMed Abstract | Publisher Full Text\n\nWilcox JB, Avery GS: Beclomethasone dipropionate corticosteroid inhaler: a preliminary report of its pharmacological properties and therapeutic efficacy in asthma. Drugs. 1973; 6(2): 84–93. PubMed Abstract | Publisher Full Text\n\nJonsson G, Astrom A, Andersson P: Budesonide is metabolized by cytochrome P450 3A (CYP3A) enzymes in human liver. Drug Metab Dispos. 1995; 23(1): 137–142. PubMed Abstract\n\nPearce RE, Leeder JS, Kearns GL: Biotransformation of fluticasone: in vitro characterization. Drug Metab Dispos. 2006; 34(6): 1035–1040. PubMed Abstract | Publisher Full Text\n\nMoore CD, Roberts JK, Orton CR, et al.: Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes. Drug Metab Dispos. 2013; 41(2): 379–389. PubMed Abstract | Publisher Full Text\n\nRoberts JK, Moore CD, Ward RM, et al.: Metabolism of Beclomethasone Dipropionate by Cytochrome P450 3A Enzymes. J Pharmacol Exp Ther. 2013; 345(2): 308–316. PubMed Abstract | Publisher Full Text\n\nLeclerc J, Tournel G, Courcot-Ngoubo Ngangue E, et al.: Profiling gene expression of whole cytochrome P450 superfamily in human bronchial and peripheral lung tissues: Differential expression in non-small cell lung cancers. Biochimie. 2010; 92(3): 292–306. PubMed Abstract | Publisher Full Text\n\nWestlind-Johnsson A, Malmebo S, Johansson A, et al.: Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in drug metabolism. Drug Metab Dispos. 2003; 31(6): 755–761. PubMed Abstract | Publisher Full Text\n\nHukkanen J, Hakkola J, Anttila S, et al.: Detection of mRNA encoding xenobiotic-metabolizing cytochrome P450s in human bronchoalveolar macrophages and peripheral blood lymphocytes. Mol Carcinogen. 1997; 20(2): 224–230. PubMed Abstract | Publisher Full Text\n\nHukkanen J, Vaisanen T, Lassila A, et al.: Regulation of CYP3A5 by glucocorticoids and cigarette smoke in human lung-derived cells. J Pharmacol Exp Ther. 2003; 304(2): 745–752. PubMed Abstract | Publisher Full Text\n\nCourcot E, Leclerc J, Lafitte JJ, et al.: Xenobiotic metabolism and disposition in human lung cell models: comparison with in vivo expression profiles. Drug Metab Dispos. 2012; 40(10): 1953–1965. PubMed Abstract | Publisher Full Text\n\nLacroix D, Sonnier M, Moncion A, et al.: Expression of CYP3A in the human liver--evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur J Biochem. 1997; 247(2): 625–634. PubMed Abstract | Publisher Full Text\n\nSchuetz JD, Beach DL, Guzelian PS: Selective expression of cytochrome P450 CYP3A mRNAs in embryonic and adult human liver. Pharmacogenetics. 1994; 4(1): 11–20. PubMed Abstract\n\nGoodwin B, Redinbo MR, Kliewer SA: Regulation of cyp3a gene transcription by the pregnane x receptor. Annu Rev Pharmacol Toxicol. 2002; 42: 1–23. PubMed Abstract | Publisher Full Text\n\nKliewer SA, Goodwin B, Willson TM: The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. Endocr Rev. 2002; 23(5): 687–702. PubMed Abstract | Publisher Full Text\n\nRaunio H, Hakkola J, Pelkonen O: Regulation of CYP3A genes in the human respiratory tract. Chem Biol Interact. 2005; 151(2): 53–62. PubMed Abstract | Publisher Full Text\n\nDvorak Z, Pavek P: Regulation of drug-metabolizing cytochrome P450 enzymes by glucocorticoids. Drug Metab Rev. 2010; 42(4): 621–635. PubMed Abstract | Publisher Full Text\n\nUrquhart BL, Tirona RG, Kim RB: Nuclear receptors and the regulation of drug-metabolizing enzymes and drug transporters: implications for interindividual variability in response to drugs. J Clin Pharmacol. 2007; 47(5): 566–578. PubMed Abstract | Publisher Full Text\n\nSchuetz JD, Schuetz EG, Thottassery JV, et al.: Identification of a novel dexamethasone responsive enhancer in the human CYP3A5 gene and its activation in human and rat liver cells. Mol Pharmacol. 1996; 49(1): 63–72. PubMed Abstract\n\nDuret C, Daujat-Chavanieu M, Pascussi JM, et al.: Ketoconazole and miconazole are antagonists of the human glucocorticoid receptor: consequences on the expression and function of the constitutive androstane receptor and the pregnane X receptor. Mol Pharmacol. 2006; 70(1): 329–339. PubMed Abstract | Publisher Full Text\n\nKatome T, Obata T, Matsushima R, et al.: Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions. J Biol Chem. 2003; 278(30): 28312–28323. PubMed Abstract | Publisher Full Text\n\nDeKeyser JG, Laurenzana EM, Peterson EC, et al.: Selective phthalate activation of naturally occurring human constitutive androstane receptor splice variants and the pregnane X receptor. Toxicol Sci. 2011; 120(2): 381–391. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuehl P, Zhang J, Lin Y, et al.: Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet. 2001; 27(4): 383–391. PubMed Abstract | Publisher Full Text\n\nHaag M, Fautrel A, Guillouzo A, et al.: Expression of cytochromes P450 3A in mouse lung: effects of dexamethasone and pregnenolone 16alpha-carbonitrile. Arch Toxicol. 2003; 77(3): 145–149. PubMed Abstract"
}
|
[
{
"id": "1566",
"date": "21 Aug 2013",
"name": "Philip Ballard",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 addresses mechanisms of glucocorticoid metabolism in various cell lines and primary lung cells in vitro. In general, the experimental approaches are appropriate and the results are clearly presented. The following minor changes are suggested to improve clarity and data interpretation:In paragraph 1 of page 4, state explicitly whether hydrocortisone was present in the culture medium of the other cell lines and primary cultures.In paragraph 3 of page 8 and the final paragraph page 9; I suggest caution in the conclusion that the results, which were obtained in an adenocarcinoma cell line, support the proposal that glucocorticoids increase CYP3A5 and inhaled glucocorticoid metabolism in vivo. In fact, the negative data for primary cells argues against the hypothesis.",
"responses": []
},
{
"id": "1628",
"date": "18 Sep 2013",
"name": "Yassine Amrani",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 explores for the first time the expression of cytochrome P450 enzymes (CYP3A5) in lung cells. The merits of the present study are the investigation of CYP3A5 expression in different primary cell lines and the use of various corticosteroids in well-controlled experiments that have produced solid data. Main concern:Although the data in A549 cells are convincing, the hypothesis that CYP3A5 is expressed in “non-transformed” lung cells (tracheal/bronchial) potentially regulated by corticosteroid therapy is not apparent from the study. Unless the authors can show that CYP3A5 is indeed expressed in vivo in the epithelium in endobronchial biopsies from asthmatic patients, the physiological and/or clinical relevance of the present study in steroid insensitive patients is not apparent and this point should be revised in the discussion. Another concern relates to the data shown in figure 3A. The figure shows that the CYP-P450 suicide inhibitor (1-ABT) blocks the production of BDP active metabolite M1.This is a surprising finding as production of M1 is thought to result from the action of esterases in contrast to the production of inactive compounds (M5) occurs via CYP-P450 enzyme. How can the authors explain that M1 is sensitive to 1-ABT?",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-173
|
https://f1000research.com/articles/2-206/v1
|
07 Oct 13
|
{
"type": "Clinical Practice Article",
"title": "Laboratory tests and compliance of dermatologic outpatients",
"authors": [
"Won Ung Shin",
"Yoo Sang Baek",
"Tom Joonhwan Kim",
"Chil Hwan Oh",
"Jaehwan Kim",
"Won Ung Shin",
"Yoo Sang Baek",
"Tom Joonhwan Kim",
"Chil Hwan Oh"
],
"abstract": "Laboratory tests, including blood tests and urine analysis, are frequently performed in the dermatology outpatient clinic, but doctors often do not consider the cognitive or psychological effect of the examinations. Based on terror management theory, we hypothesized that performing laboratory tests increases the patient’s fear of mortality, and therefore has a positive effect on the patient’s attitude toward the doctor’s recommendations and willingness to accept them. The study employed a single factor between-subjects design, using a questionnaire completed by the patients. One group consisted of patients who had undergone laboratory tests 1 week before the survey, and the other group consisted of patients who had not undergone a laboratory test. Although the differences between two groups were not statistically significant, the patients who had laboratory tests had tendency to show even lower positive attitude toward the doctor’s recommendations and less intention to follow the recommendations. In contrast to our hypothesis, performing laboratory tests does not subliminally increase patients’ fears or anxieties about their disease or their compliance with doctors’ recommendations.",
"keywords": [
"Laboratory test",
"Compliance",
"Dermatologic Outpatients"
],
"content": "Introduction\n\nCompliance in health care has been defined as the extent to which a person’s behavior coincides with health-related advice1. Numerous factors have been suggested to influence patient compliance, including individual characteristics, illness type and severity, medication complexity, and emotional status2. Adherence to treatment of the patients with several chronic diseases such as diabetes, glaucoma, multiple sclerosis and psychological problems was studied in various trials that emphasized the influence of psychological components3–5. However, most surveys of patient compliance in dermatology have focused on dermatological diseases and types of treatment6,7.\n\nIn this study, we hypothesized that the examinations performed in dermatologic clinics may affect patient compliance. In a dermatologic clinic, laboratory tests such as blood and urine tests are one of the main examinations along with skin biopsy. Laboratory tests are usually performed to diagnose systemic disease associated with skin lesions or to monitor patient’s vital organ functions. Dermatologists usually focus on the results of laboratory tests without considering the psychological impact of the tests.\n\nOne of the studies on patient compliance in dermatology by Kim et al.8 examined the psychological influence of skin biopsy based on terror management theory (TMT). TMT proposes that awareness of death is a critical motivating force in human behavior9. The theory states that people cope with fear against death through sustaining faith in cultural worldviews, and get self-esteem by pursuing to the standards of values provided by the cultural worldviews. A cultural world-view means ‘humanly created and transmitted beliefs about the nature of the reality shared by groups of individuals’10. By following cultural values and engaging in culturally prescribed behaviors, one can pursue the meaning of existence and perpetuity10–12. The study by Kim et al.8 showed that receiving a skin biopsy led patients to have a greater tendency to follow a doctor’s recommendations.\n\nMost patients visiting dermatologic clinics are not concerned about systemic disease, vital organ dysfunction or mortality, unless they have chronic skin diseases or skin cancers. However, we hypothesized that performing laboratory tests subliminally reminds patients of systemic disease, vital organ dysfunction or mortality. Based on TMT, laboratory test could increase patients’ death-related thinking and might increase their willingness to accept the recommendations of doctors, whose authority is acknowledged by culture. We investigated the relationship between performing laboratory tests and acceptance of a doctor’s recommendations. In addition, the effects of the patient’s trust toward doctors, authoritarian personality, and mood on medical compliance were also studied.\n\n\nMaterial and methods\n\nAmong the patients that attended the dermatology clinic in Korea University Guro Hospital (Seoul, South Korea) between 1 July 2011 and 31 August 2011, a total of 100 first time visitors were requested to fill out the survey. Written informed consent was obtained from all participants in this study. A total of 45 male patients and 55 female patients were included in the study. A group of 50 patients underwent laboratory tests, while the other group of 50 patients did not have any laboratory tests. Laboratory tests included urine analysis and blood tests such as: complete blood count, liver function test, blood urea nitrogen analysis, and creatinine analysis. We included patients aged between 18 and 65 years old and excluded patients who had a history of psychological disorders, -had undergone other tests besides the above mentioned laboratory tests (such as a skin biopsy or a patch test), or who had undergone procedures such as laser treatment or cryotherapy.\n\nThe patients were asked to complete the survey within two weeks on their second visit to the clinic. The survey measured their trust towards doctors, authoritarian personality, current mood, attitude toward the doctor’s recommendation, and intention to follow the recommendations. Participants were asked to indicate the extent to which they agree with a given statement on the survey by using a seven-point scale (strongly disagree, disagree, disagree somewhat, undecided, agree somewhat, agree, strongly agree). The full questionnaire is available as supporting data.\n\nSeveral studies have measured patient’s trust in physicians using scales13,14, and we modified those scales so they included 5 statements to measure trust towards doctors: “I believe that doctors perform: 1. Perform only medically necessary tests and procedures, 2. keep personally sensitive medical information private, 3. perform necessary medical tests and procedures regardless of cost, 4. put my health and well-being above keeping down the health plan’s cost (reverse scale), and 5. I trust the doctors’ judgment when it comes to my medical care”. In case of fourth state reverse scale was used, and average of patients’ scale to these five questions was used.\n\nThe grades of authoritarianism are 1. adheres strongly to conventional moral values, 2. submissive to established authorities, and 3. willingness to be aggressive toward others if they are perceived as unconventional or threatening15. We hypothesized that the grade of authoritarian personality, together with the amount of trust towards doctors, might influences patients compliance with medical treatment. K. Min16 has developed standards for measuring Korean authoritarian personality grades, and the scale consist of 35 questions measuring conventionalism, authoritarian obedience, authoritarian aggressiveness, anti-introspectionism, stereotypical thinking, belief in power, cynicism, and sexism. Based on this scale, we randomly selected four items measuring authoritarian obedience, and average of patients’ scales to these questions was used.\n\nWe considered that the mood of the patients during the survey was a potential confounder affecting patient compliance, and patients who underwent laboratory tests might feel differently compared to patients who did not. To measure patient mood, they were asked to answer the twenty questions measuring their current mood (PANAS-X(17)). This measurement consists of 10 positive feelings (active, alert, attentive, determined, enthusiastic, excited, inspired, interested, proud, strong) and 10 negative feelings (afraid, scared, nervous, jittery, irritable, hostile, guilty, ashamed, upset, distressed). Average of patients’ scales to these twenty questions was used.\n\nIn the last part of the survey, patients read a scenario: “You have a severe and chronic skin disease, and your doctor recommends a newly developed treatment method for you. He tells you that this method can reduce treatment time, but it is more expensive than traditional treatment and safety is not fully guaranteed”. After reading the scenario, the attitude toward the scenario was measured using three 7 scale questions that were used in previous marketing studies18. Average of patients’ scales to these three questions was used.\n\nAs another dependent variable, the intention to accept recommendations was measured by answering the following two statements: “I will follow the recommendations of the doctor,” and “There is a possibility of following recommendations of the doctor”. The patients responded on a seven-point scale (strongly disagree, disagree, disagree somewhat, undecided, agree somewhat, agree, strongly agree). Average of patients’ scales to these two questions was used.\n\n\nStatistical analysis\n\nSPSS software (Version 15.00, SPSS Inc, Chicago, IL, USA) was used to conduct the statistical analyses and p-value less than 0.05 was considered to be statistically significant. Data analysis consisted of the unpaired t-test, two-way analysis of variance (ANOVA) test and correlation analysis.\n\n\nResults\n\nForty five (45%) male patients and fifty five (55%) female patients completed the survey. Each group (laboratory tests vs. no laboratory test) consisted of 50 patients, and the mean age of the groups was 37.42 years for the laboratory test group (range 19–65) and 39.56 years for the group not receiving laboratory tests (range 19–64). The dermatologic diagnoses were about evenly distributed over the two groups and were classified into cutaneous vascular disease, diseases of the skin appendages (hair and sebaceous gland), eczematous disease, erythema or urticaria, infectious disease, papulosquamous disease, pigment anomalies, pruritus, and tumors. Table 1 details the subject characteristics.\n\nCronbach’s α coefficients revealed that reliabilities of patients’ responses exceed 0.70, indicating an acceptable level of internal consistency.\n\nTo compare the two independent groups (laboratory tests vs. no laboratory test), an independent t-test was conducted. The laboratory test group had a lower positive attitude toward doctors and less intention to follow doctor's recommendations as compared to the no laboratory test groups. However this difference was not significant (Table 2). In addition, the statistical testing revealed no significant differences in the patient’s, ‘authoritarian personality’, ‘mood’, and ‘intention to accept recommendations’ between the two groups.\n\nNext, we investigated the effects of variables (laboratory tests vs. no laboratory test, high or low trust toward doctors, and high or low authoritarian personality) on patient attitude toward doctors and intention to accept their recommendations using two-way ANOVA. The groups (laboratory tests vs. no laboratory test) were divided into two groups based on the median value of two variables: trust toward doctors (cut-off value, 4.80) and authoritarian personality (cut-off value, 4.25).\n\nAccording to the analysis of effects of trust toward doctors, the patients who trust doctors expressed a more positive attitude (3.90 vs. 4.79, p<0.001) and an increased intention to follow the doctor’s recommendations (4.28 vs. 5.36, p<0.001) compared to patients that had less faith in their doctors. There was no relationship between trust in the doctor’s recommendations and the receipt of laboratory tests (Table 3).\n\nAnalysis of the effects of having an authoritarian personality showed a significant effect of authoritarian personality on both the attitude toward doctor’s recommendations, and the intention to follow the recommendations (Table 4). Patients with a more authoritarian personality were more positive toward the doctor’s recommendations (3.99 vs. 4.65, p=0.01) and had a more intention to follow the recommendation (4.35 vs. 5.24, p=0.001). In addition, there was a significant relationship between authoritarian personality and laboratory tests (Table 4). For patients with a more authoritarian personality, laboratory tests decreased the intention to follow the recommendation (5.74 vs. 4.77, p<0.05).\n\n†, ‡ Measure of the ‘Trust toward the doctors’ divided into two groups by median value.\n\n†, ‡ Measure of the ‘Authoritarian personality’ divided into two groups by median value.\n\nAdditionally, two-way ANOVA and correlation analysis were performed to evaluate variance by sex and age. Males had a more positive attitude toward the doctor’s recommendations (4.72 vs. 3.92, p<0.05) and more intention to follow the recommendations (5.06 vs. 4.47, p<0.05), as compared to females (Table 5). Also, measurements of authoritarian personality and trust in doctors were significantly higher in males (authority personality; 4.52 vs. 3.96, p<0.01, trust toward doctors; 5.04 vs. 4.64, p=0.05). There was no interaction between sex and two personal traits on compliance (p>0.1).\n\nBy correlation analysis, intention to follow the doctor’s recommendations and trust toward the recommendation had a tendency to increase in older patients (correlation coefficient was 0.374 (p<0.001) and 0.432 (p<0.001), respectively (Table 6).\n\n* Laboratory test group.\n\n** No laboratory test group.\n\nCompliance of a patient is ‘the extent to which the patient’s behavior matches agreed recommendation from the prescribers’19. It is critical element in determining the beneficial effect of medical care and decided by interaction of multiple factors20,21. Many psychological models including Becker’s health belief model have been introduced to explain compliance9. According to them, patients show increased compliance when they believe the threat of disease will be diminished by following health recommendations22. Also the doctor’s attitude towards the patient, doctor’s ability to bring out and respect the patient’s concerns, ability to provide appropriate information, and ability to show empathy are important factors of compliance23.\n\nAlthough the psychological aspects of patients and doctors have been emphasized in patient compliance, most of compliance studies in dermatology have focused on the diagnosis and treatment options as determining factors24–26. In 2010, Kim et al.8 first described the psychological impacts of skin biopsy, which is one of the most frequently performed diagnostic tools in dermatology. This study showed that skin biopsy subconsciously increase patients’ positive attitude toward doctor’s recommendations and potentially bias patients’ decision for the treatment. This phenomenon was explained by terror management theory (TMT), which is generally studied in psychology and marketing, but also has been applied to medicine27. TMT states that people behave to protect themselves from fear of death when they are reminded of their own deaths. Societies traditionally have built cultural worldview (such as laws, cultures, and religions), and individuals adhere to these cultural worldview when confronting fear of mortality10. In medicine, certain health behaviors such as breast self-exam can subconsciously activate and facilitate people to remind death, and death act as conditioning behavior towards a longer life27.\n\nIn this study, we hypothesized that the laboratory tests, which are frequently recommended in dermatologic outpatient clinic, could be a stimulant of mortality salience because it reminds patients of more severe diseases or vital organ dysfunctions. Based on TMT, we expected that patients who had laboratory tests adhere to doctor’s recommendations more than the patients who did not have laboratory tests.\n\nInterestingly, the result of this study was contrary to our expectation. Although the differences between two groups were not statistically significant, the patients who had laboratory tests had tendency to show even lower positive attitude toward the doctor’s recommendations and less intention to follow the recommendations. This suggests that laboratory tests do not subliminally increase the patient’s fear of their disease or increase compliance.\n\nDespite statistically insignificance in this study, no relationship between laboratory tests and adherence to doctors’ recommendation raises important implications for future research. For example, biomarkers for guiding therapy selection and disease monitoring have been developed with skin biopsy in the field of dermatology28. However, skin biopsy has a risk of subliminally increase the patient’s fear or anxiety and subconsciously bias patients’ decision for the treatment8. On the other hand, laboratory tests such as blood test and urine test do not influence patient compliance and supply a neutral circumstance for patients’ treatment decisions. Therefore, this study implicates that laboratory tests could be superior to skin biopsy as a source of biomarker development.\n\nThe limitation of this study is that we assessed patient compliance as a hypothetical construct and psychological concepts were examined using an indirect method. Also, the small sample size limited the statistical power of this study, and also the diagnosis of the participants and the types of treatments could not be randomized between groups.\n\nWe failed to prove our hypothesis based on TMT, but the results still have important implications in dermatologic outpatient clinics. The performance of laboratory tests does not seem to reinforce the patient’s fear of their own disease, and instead possibly decreases lower the patient’s trust in doctors. Doctors should not hesitate to perform laboratory tests when the situation requires this, but we suggest that careful consideration about possible psychological impacts is needed when ordering laboratory tests for patients. To enhance patient compliance with doctor’s recommendations, doctors should carefully consider not only their diagnosis or treatment plan, but also the psychological impact of commonly performed tests and the patient’s personality.",
"appendix": "Author contributions\n\n\n\nWon Ung Shin wrote the first draft of the article. Yoo Sang Baek edited the article, and reviewed the data. Tom Joonhwan Kim analyzed data, and interpreted results. Chil Hwan Oh interviewed the patients. Jaehwan Kim designed the study, edited the article, and has direct responsibility for the article.\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\nAcknowledgement\n\nWe thank Dr. Jung Woo Lee, Dr. Jae Woo Ahn, and Dr. Joo Ha Kim for critical comments on the manuscript.\n\n\nReferences\n\nRenzi C, Picardi A, Abeni D, et al.: Association of dissatisfaction with care and psychiatric morbidity with poor treatment compliance. Arch Dermatol. 2002; 138(3): 337–42. PubMed Abstract | Publisher Full Text\n\nInsel KC, Reminger SL, Hsiao CP: The negative association of independent personality and medication adherence. J Aging Health. 2006; 18(3): 407–18. PubMed Abstract | Publisher Full Text\n\nAxelsson M, Brink E, Lundgren J, et al.: The influence of personality traits on reported adherence to medication in individuals with chronic disease: an epidemiological study in West Sweden. PLoS One. 2011; 6(3): e18241. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBruce JM, Hancock LM, Arnett P, et al.: Treatment adherence in multiple sclerosis: association with emotional status, personality, and cognition. J Behav Med. 2010; 33(3): 219–27. PubMed Abstract | Publisher Full Text\n\nHollo G, Kothy P, Geczy A, et al.: Personality traits, depression, and objectively measured adherence to once-daily prostaglandin analog medication in glaucoma. J Glaucoma. 2009; 18(4): 288–92. PubMed Abstract | Publisher Full Text\n\nAugustin M, Holland B, Dartsch D, et al.: Adherence in the treatment of psoriasis: a systematic review. Dermatology. 2011; 222(4): 363–74. PubMed Abstract | Publisher Full Text\n\nMiyachi Y, Hayashi N, Furukawa F, et al.: Acne management in Japan: study of patient adherence. Dermatology. 2011; 223(2): 174–81. PubMed Abstract | Publisher Full Text\n\nKim JH, Kim J, Jung YH, et al.: Terror management theory in dermatology: skin biopsy influences patient compliance. Acta Derm Venereol. 2010; 90(3): 246–50. PubMed Abstract\n\nBecker MH, Maiman LA: Sociobehavioral determinants of compliance with health and medical care recommendations. Med Care. 1975; 13(1): 10–24. PubMed Abstract | Publisher Full Text\n\nHarmon-Jones E, Simon L, Greenberg J, et al.: Terror management theory and self-esteem: evidence that increased self-esteem reduces mortality salience effects. J Pers Soc Psychol. 1997; 72(1): 24–36. PubMed Abstract | Publisher Full Text\n\nLieberman EJ: Terror management theory. Am J Psychiatry. 2004; 161(8): 1508; author reply-9. PubMed Abstract | Publisher Full Text\n\nSimon L, Greenberg J, Harmon-Jones E, et al.: Terror management and cognitive-experiential self-theory: evidence that terror management occurs in the experiential system. J Pers Soc Psychol. 1997; 72(5): 1132–46. PubMed Abstract | Publisher Full Text\n\nAnderson LA, Dedrick RF: Development of the Trust in Physician scale: a measure to assess interpersonal trust in patient-physician relationships. Psychol Rep. 1990; 67(3 Pt 2): 1091–1100. PubMed Abstract | Publisher Full Text\n\nKao AC, Green DC, Zaslavsky AM, et al.: The relationship between method of physician payment and patient trust. JAMA 1998; 280(19): 1708–14. PubMed Abstract | Publisher Full Text\n\nHoffman J: The Authoritarian Personality-Adorno TW, Frenkelbrunswik E, Levinson D, Sanford RN. Contemp Psychol. 1965; 10(11): 500.\n\nMin K: Authoritarian personality and social prejudice among college students. Korean J Soc Psychol. 1989; 4: 146–68.\n\nWatson D, Clark LA, Tellegen A: Development and validation of brief measures of positive and negative affect: the PANAS scales. J Pers Soc Psychol. 1988; 54(6): 1063–70. PubMed Abstract | Publisher Full Text\n\nBobinski GS, Cox D, Cox A: Retail ''sale'' advertising, perceived retailer credibility, and price rationale. J Retailing. 1996; 72(3): 291–306.Publisher Full Text\n\nAronson JK: Compliance, concordance, adherence. Brit J Clin Pharmaco. 2007; 63(4): 383–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSherbourne CD, Hays RD, Ordway L, et al.: Antecedents of adherence to medical recommendations: results from the Medical Outcomes Study. J Behav Med. 1992; 15(5): 447–68. PubMed Abstract | Publisher Full Text\n\nKoberlein J, Kothe AC, Schaffert C: Determinants of patient compliance in allergic rhinoconjunctivitis. Curr Opin Allergy Clin Immunol. 2011; 11(3): 192–9. PubMed Abstract | Publisher Full Text\n\nVermeire E, Hearnshaw H, Van Royen P, et al.: Patient adherence to treatment: three decades of research. A comprehensive review. J Clin Pharm Ther. 2001; 26(5): 331–42. PubMed Abstract | Publisher Full Text\n\nRosenstock IM: Enhancing patient compliance with health recommendations. J Pediatr Health Care. 1988; 2(2): 67–72. PubMed Abstract | Publisher Full Text\n\nKatsambas AD: Why and when the treatment of acne fails - What to do. Dermatology. 1998; 196(1): 158–61. PubMed Abstract | Publisher Full Text\n\nRichards HL, Fortune DG, O'Sullivan TM, et al.: Patients with psoriasis and their compliance with medication. J Am Acad Dermatol. 1999; 41(4): 581–3. PubMed Abstract\n\nvan de Kerkhof PCM, de Hoop D, de Korte J, et al.: Patient compliance and disease management in the treatment of psoriasis in the Netherlands. Dermatology. 2000; 200(4): 292–8. PubMed Abstract | Publisher Full Text\n\nGoldenberg JL, Arndt J: The implications of death for health: a terror management health model for behavioral health promotion. Psychol Rev. 2008; 115(4): 1032–53. PubMed Abstract | Publisher Full Text\n\nZaba LC, Suárez-Fariñas M, Fuentes-Duculan J, et al.: Effective treatment of psoriasis with etanercept is linked to suppression of IL-17 signaling, not immediate response TNF genes. J Allergy Clin Immunol. 2009; 124(5): 1022–10.e1–395. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2020",
"date": "28 Oct 2013",
"name": "Mohamed Badawy Hassan Tawfik Abdel-Naser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written and informative. However, there some remarks in the following sections that could be be taken into consideration:Introduction:“In a dermatologic clinic, laboratory tests such as blood and urine tests are one of the main examinations” Is it better to use \"investigations\" or \"analyses\" instead of repeating the word \"tests\" likewise the second “the cultural worldviews” can be replaced by “them”“..blood urea nitrogen analysis, and creatinine analysis.” is it fine with “blood urea nitrogen and creatinine analyses” Survey:“towards” and “toward” are both used in the whole manuscript. You may adhere to one or same spellingTrust toward doctors:“..believe that doctors perform: 1. Perform only medically necessary…” you may delete the first “perform”.“In case of fourth state reverse scale was used, and average of patients’ scale to these five questions was used” please revise this statement.Authoritarian personality:“..amount of trust towards doctors,might influences” consider “might influence”“..the scale consist” consider “the scale consists”Reliability test of questionnaires:“Cronbach’s α coefficients” this statistical method has not been mentioned or referred to in the statistics sectionComparison of the groups:“..an independent t-test” is it equivalent to unpaired t test that has been mentioned in the statistics section? You should adhere to the same terminology. Moderating effects of patient’s personality:“The patients who trust doctors expressed a more positive attitude (3.90 vs. 4.79, p<0.001)” Please check these values with those in the table for errors. This is also applicable to all values mentioned in the text with their corresponding values in the tables. Also the arrangement of the values (higher values and then the lower).Discussion:“...skin biopsy subconsciously increase patients” consider “increases” also in other locations in the section.“ ..and death act as conditioning” consider “acts”\"Despite statistically insignificance in this study, no relationship between laboratory tests and adherence to doctors’ recommendation raises important implications for future research” Please revise this statement“..instead possibly decreases lower the patient’s trust in doctors” please revise the statement",
"responses": []
},
{
"id": "2291",
"date": "01 Nov 2013",
"name": "Roberto Arenas",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 interesting, practical and useful",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-206
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https://f1000research.com/articles/2-143/v1
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25 Jun 13
|
{
"type": "Research Article",
"title": "A fragmented alignment method detects a phosphorylation site and a new BRC repeat in the Drosophila melanogaster BRCA2 protein, and a new HAT repeat in Utp6 from yeast",
"authors": [
"Sandeep Chakraborty"
],
"abstract": "Mutations in the BRCA2 tumor suppressor protein leave individuals susceptible to breast, ovarian and other cancers. The BRCA2 protein is a critical component of the DNA repair pathways in eukaryotes, and also plays an integral role in fostering genomic variability through meiotic recombination. Although present in many eukaryotes, as a whole the BRCA2 gene is weakly conserved. Conserved fragments of 30 amino acids (BRC repeats), which mediate interactions with the recombinase RAD51, helped detect orthologs of this protein in other organisms. The carboxy-terminal of the human BRCA2 has been shown to be phosphorylated by checkpoint kinases (Chk1/Chk2) at T3387, which regulate the sequestration of RAD51 on DNA damage. However, apart from three BRC repeats, the Drosophila melanogaster gene has not been annotated and associated with other functionally relevant sequence fragments in human BRCA2. In the current work, the carboxy-terminal phosphorylation threonine site (E=9.1e-4) and a new BRC repeat (E=17e-4) in D. melanogaster has been identified, using a fragmented alignment methodology (FRAGAL). In a similar study, FRAGAL has also identified a novel half-a- tetratricopeptide (HAT) motif (E=11e-4), a helical repeat motif implicated in various aspects of RNA metabolism, in Utp6 from yeast. The characteristic three aromatic residues with conserved spacing are observed in this new HAT repeat, further strengthening my claim. The reference and target sequences are sliced into overlapping fragments of equal parameterized lengths. All pairs of fragments in the reference and target proteins are aligned, and the gap penalties are adjusted to discourage gaps in the middle of the alignment. The results of the best matches are sorted based on differing criteria to aid the detection of known and putative sequences. The source code for FRAGAL results on these sequences is available at https://github.com/sanchak/FragalCode, while the database can be accessed at www.sanchak.com/fragal.html.",
"keywords": [
"FRAGAL",
"alignment",
"BRCA2",
"HAT",
"Drosophila"
],
"content": "Introduction\n\nThe breast cancer susceptibility protein BRCA2, first identified in 19951, is a critical recombinase regulator2 that ensures genomic stability through high fidelity repair3,4 of double stranded breaks (DSB) and prevents stalled replication forks from replicating5 in the DNA. The primary recombinase in BRCA2 repair of DSB through homologous recombination is the RAD51 protein, belonging to the conserved RecA/RAD51 family6, that binds to the BRCA2 protein at various segments of ~30 amino acids (BRC repeats)7,8, and in the C-terminal region in most vertebrates9,10. Checkpoint kinases phosphorylate a serine9 and a threonine10 at the carboxy-terminal region of BRCA2, thereby regulating its interaction with RAD51. BRCA2 also plays a key role in fostering genomic variability through meiotic recombination11,12, although a different recombinase (DMC1) is implicated in this pathway in mammalian species13.\n\nThe BRC repeats have helped identify BRCA2 orthologs in various eukaryotic species14. Functional characterization of this gene in Drosophila melanogaster has demonstrated its interaction with RAD51, and a critical role in mitotic and meiotic DNA repair as well as homologous recombination11,15. The copy number of the BRC repeats differs considerably. The BRCA2 homolog in Ustilago maydis (a yeast like fungus) has a single BRC repeat16, the D. melanogaster homolog contains only three (known) repeats14, while there are eight repeats in the human BRCA2 gene7. Even among the Drosophila genus, the range of BRC repeat numbers is varied - the D. melanogaster species has only three repeats, while D. persimilis and D. pseudoobscura have up to eleven repeats17. RAD51 shows varying affinity for the different BRC motifs18,19. This difference in repeat numbers in Drosophila has raised doubts whether ‘this higher repeat number is real or a genome mis-assembly artifact’20, and also led to speculation on the evolution of these closely related organisms17,20. Any such hypothesis would need to be revisited if a new BRC motif were to be identified in D. melanogaster.\n\nIn the current work, the putative threonine phosphorylation site for checkpoint kinases (Chk1/Chk2) (E=9.1e-4) and a new BRC repeat (E=17e-4) in D. melanogaster has been identified, using a fragmented technique for the pairwise alignment of two sequences (FRAGAL). The reference and target sequences are sliced into fragments of equal parameterized length X, sliding along the sequence in intervals of length Y, such that Y is less than X. Thus, the slices have overlaps. An alignment of all pairs of slices in the reference and target proteins is done using the global alignment program ‘needle’21 from the EMBOSS suite22. The gap penalties are adjusted to discourage gaps in the middle of the alignment. The results of the best matches are sorted based on differing criteria to aid the detection of known and putative sequences. In order to establish the generic nature of the FRAGAL methodology, the detection of a new half-a-tetratricopeptide (HAT) repeat sequence (E=11e-4) in a nucleolar RNA-associated protein (Utp6) from Saccharomyces cerevisiae is also reported. HAT is a helical repeat motif implicated in various aspects of RNA metabolism23,24. The characteristic three aromatic residues with a conserved spacing are observed in this new HAT repeat, further strengthening my claim25.\n\nThe significant conservation of the DNA repair and checkpoint pathways in flies and higher organisms26, the advanced genetic tools available for Drosophila, and the viability of the Drosophila BRCA2 null mutants in contrast to mammalian mutants27 establishes Drosophila as a model organism for studying these pathways28. Significant divergence of key conserved sequences proves to be a serious hurdle for alignment techniques to annotate and associate the conserved sequences in the human BRCA2 to the Drosophila BRCA229. Thus, a generic methodology, applicable to distantly evolutionary related proteins like BRCA2 and nucleolar RNA-associated proteins is presented. The methodology has been validated by the identification of two novel functionally relevant sites in the BRCA2 protein from D. melanogaster, and a HAT repeat in Utp6 from S. cerevisiae.\n\n\nMaterials and methods\n\nThe FRAGAL methodology is shown in Supplementary Figure 1. The sequences are split into fragments of X amino acids, with the starting indices sliding across the sequence length in steps of Y amino acids (SI.A.fasta and SI.B.fasta in Data Files). The score for each match is either the %similarity or a score (FRscore) that is computed as shown in Equation 1 and Equation 2. FRscore is intended to give more weightage to identical residues in the alignment.\n\n%onlySimilarity = %similarity – %identity; (1)\n\nFRscore = 1/3 * %onlySimilarity + 2/3 * %identity; (2)\n\nOne sorting criteria is to rank the matches based on the best average score, while another takes the cumulative score of a stretch of fragment matches. Stretches of fragments are stitched while ensuring the slices in the sequences are in an increasing order and non-overlapping. The best average criteria will typically select single fragments, while the cumulative scoring criteria will bring forth longer conserved regions.\n\nThe threshold for sequence similarity for each fragment is parameterized, and set to 30% in the default mode. A large threshold will exclude more relevant matches, while a smaller threshold might include more false positives. The pairwise alignment for each fragment pair is done by a global alignment program ‘needle’ from the EMBOSS suite21,22. The parameters are set as follows - matrix=BLOSUM62, Gap penalty=25.0 and Extend penalty=0.5. The gap penalty is increased from the default value of 10 to ensure that gaps are discouraged in the middle of the alignment. Single deletions or insertions are rarely expected in conserved fragments.\n\nThe user is allowed to specify an annotation file for a given protein sequence using the uniprot accession syntax (Supplementary Figure 2). The results from FRAGAL can be filtered based on this annotation, and this provides a easier way to manually inspect and annotate corresponding segments in a query protein sequence.\n\nThe FRAGAL package is written in Perl on Ubuntu. Hardware requirements are modest - all results here are from a simple workstation (2GB RAM). The source code for FRAGAL results on these sequences is available at https://github.com/sanchak/FragalCode, while the database can be accessed at www.sanchak.com/fragal.html and in the Data Files below. The multiple sequence alignment was done using ClustalW30. PHYML has been used to generate phylogenetic trees from these alignments, which is based on the method of maximum likelihood31. The method searches for a tree with the highest probability or likelihood that, given a proposed model of evolution and the hypothesized history, would give rise to the observed data set. The alignment and cladograms images were generated using Seaview32. E-values and z-scores have been computed using the Protein Information Resource.\n\n\n\n\nResults\n\nThe D. melanogaster gene (CG30169)33 encodes a 971 amino acid protein (dmBRCA2, Uniprot Accession:Q9W157), and contains three BRC repeat units (conserved sequences of ~30 amino acids that binds to RAD51)8,14. In contrast, the human BRCA2 gene product (hBRCA2, Uniprot Accession:P51587) is 3418 amino acids long and contains eight BRC repeats7. Further, the hBRCA2 protein is annotated for several sites phosphorylated by checkpoint kinases, which regulate its interaction with RAD519,10. FRAGAL was run on the dmBRCA2 and hBRCA2 sequences. Table 1 shows the best matches obtained using two different sorting criteria - best average FRscore (see Methods) and best average %similarity - either when the match in hbrca2 is known to be conserved (Table 1A) based on an user defined input file (Supplementary Figure 2) or otherwise (Table 1B).\n\nThe scores are either FRscore (which gives more weightage to %identity, see Methods) or %similarity (%S). In A, the results are filtered out if the fragment in the hBRCA2 sequence is not marked as conserved (Supplementary Figure 2), whereas all results are included in B. The A91-B337 match corresponds to the phosphorylation site of checkpoint kinases in the carboxy-terminal of BRCA2, while the match A61-B151 (not shown in Table, FRscore=53), corresponds to the new BRC repeat identified in D. melanogaster. Note, that certain fragment alignments like A33-B176 are not characterized in either proteins, and are candidates for functional characterization due to their significant conservation in distant species.\n\nDetecting the threonine phosphorylation site in the carboxy-terminal region of dmBRCA2. Table 1 shows a significant match (E=9.1e-4, Z-score=100) between fragment 91 of dmBRCA2 to the fragment 337 in hBRCA2, which contains the T3387 that is phosphorylated by the checkpoint kinases Chk1 and Chk2. Z-scores above a value of 8 are considered to be significant34. The alignment shows that the T3387 corresponds to the T926 of dmBRCA2 (Figure 1a). The conservation of this region in the Drosophila and mammalian species is demonstrated by the multiple sequence alignment of three organisms from each species (Figure 1b). The highly conserved columns in the alignment are highlighted using an asterisk, and can be used to define a Prosite motif ([ST]-E-[ST][ST]-x-[ST]-x(6)-[ED]-x(4)-K-x(4)-[ST]-[ST]-[ST]-x(3)-[DE]-[DE])35. Either this motif or FRAGAL alignments failed to detect this site in other species distant from Drosophila or mammals (Ustilago maydis and Caenorhabditis elegans).\n\n(a) Putative phosphorylation site by checkpoint kinases in the carboxy-terminal of hBRCA2. The threonine that is phosphorylated is highlighted (T3387 in hBRCA2 and T926 in dmBRCA2) (E=0.00091, Zscore=100). (b) Conserved sequence in the carboxy-terminal of the BRCA2 protein sequence: Checkpoint kinases Chk1 and Chk2 phosphorylate threonine 3387 in hbrca2, and is seen to be conserved in the mammalian and Drosophila species (T926 in dmBRCA2). (c) Putative BRC repeat identified by the similarity of fragment 61 (634–664:LDTALKRSIESSEEMRSKASKLVVVDTTMR) in D. melanogaster to the BRC4 repeat in hBRCA2 (1517–1551) (E=0.0017, Zscore=95) (red for identity, green for similarity).\n\nDetecting an additional BRC repeat in Drosophila melanogaster. The correct identification of the three BRC repeats in D. melanogaster is seen by the significant scores of the FRscore matches of A67-B152 (64), A57-B100 (60) and A75-B152 (60) (Table 1). A significant alignment (E=17e-4, Z-score=95) between A61-B151 (35.8%similarity and 17%identity) (Figure 1c) was also observed. This sequence (634–664:LDTALKRSIESSEEMRSKASKLVVVDTTMR) is now added to the list of sequences previously studied in the Drosophila genus17. The multiple sequence alignment (obtained using ClustalW30) (Figure 2a) and phylogenetic trees (obtained using PHYML31) (Figure 2b) shows that this new BRC repeat is more related to D. willistoni than other organisms in the Drosophila genus. A detailed molecular phylogeny of Drosophilid species has noted that the subgenus Sophophora is ‘divided into D. willistoni and the clade of D. obscura and D. melanogaster groups’, possibly indicating the source of this BRC repeat that has been conserved between D. willistoni and D. melanogaster36. An iterative methodology, similar to PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool)37, can be automated to generate comprehensive motifs spanning distant species. The conservation of many key residues in this sequence fragment, as shown by comparing it to the sequence logo of the Prosite BRCA2 profile (PS50138) (Figure 2c) strongly suggests that this is a putative BRC repeat. However, it must be emphasized that such repeats are to be considered putative until verified experimentally38,39.\n\n(a) The multiple alignment for this new sequence (634–664:LDTALKRSIESSEEMRSKASKLVVVDTTMR) (using ClustalW) highlighted as melanogaster4 and other sequences compared previously in17. This putative sequence is more closely related to the sequences in D. willistoni than other members of the genus. (b) The phylogenetic tree (using PHYML) gives a graphical representation of the relation of the various repeats in the Drosophila genus, corroborating the closer relation of the new BRC repeat to D. willistoni. (c) Alignment of the new BRC repeat to the sequence logo of the Prosite BRCA2 repeat profile PS50138.\n\nHAT is a helical repeat motif implicated in various aspects of RNA metabolism and in protein-protein interactions23,24. These repeats are characterized by three aromatic residues with a conserved spacing25. A variable number of HAT repeats (9 to 12) are found in different proteins. Figure 3a shows a novel HAT repeat (E=11e-4, Z-score=116) detected in a nucleolar RNA-associated protein (Utp6) from Saccharomyces cerevisiae (Uniprot Accession:Q02354) by comparing it to HAT repeats from a human nucleolar RNA-associated protein (Uniprot Accession:Q9NYH9). Q9NYH9 has five annotated HAT repeats (121–153, 156–188, 304–335, 488–520 and 524–557), while Q02354 has three HAT repeats (87–119, 124–156 and 159–191). The new HAT sequence identified in Q02354 (SLIMKKRTDFEHRLNSRGSSINDYIKYINYESN) is from position 30 to 62. It can be seen from the multiple sequence alignment that this sequence has the desired aromatic residues at the proper spacing, a requisite for being considered a HAT repeat (Figure 3a and Figure 3b). Further, the MSA shows large variation amongst HAT sequences even within the same organism (Figure 3b). Finally, Figure 3b and Figure 3c shows that certain HAT repeats are more similar to HAT repeats from other organisms than to other HAT repeats in its own sequence.\n\n(a) Pairwise alignment of a previously unannotated HAT motif in S. cerevisiae (E=11e-4, Z-score=116) (red for identity, green for similarity). (b) The multiple alignment for this new sequence (using ClustalW) with other HAT motifs in S. cerevisiae and humans shows large variation amongst HAT sequences even within the same organism. The conserved spacing of the aromatic residues are also highlighted. (c) The phylogenetic tree (using PHYML) shows that certain HAT repeats are more similar to HAT repeats from other organisms than to other HAT repeats in its own sequence.\n\nA database (www.sanchak.com/fragal.html) which lists the results for the fragmented alignment of various proteins with BRC and HAT repeats sequences has been created. The results have been generated by varying two parameters - length of the fragments and the threshold %similarity value for a significant match in a fragment pair. As mentioned above, the results are presented in several formats - best cumulative score and best average score - where the score is either the %similarity or FRscore.\n\n\nDiscussion\n\nGenetic evolution over large time spans often leaves little trace of kinship in different organisms, even when the functional roles of the genes remains conserved. A relevant example is the BRCA2 gene which, although present in many eukaryotes, is weakly conserved40. The BRCA2 protein plays a major role in maintaining genomic stability, fostering genetic variability and also has other cellular functions2,41. Individuals with germline mutations in the BRCA2 gene are at significantly greater risk to a wide range of cancers42,43. This is supposed to be primarily due to the instability in chromosome structure and number induced by functional aberrations in BRCA244. Conserved fragments of ~30 amino acids (BRC repeats)7 that mediates the interaction of BRCA2 with the RAD51 recombinase45 have been instrumental in identifying BRCA2 orthologs in other species14,16. The BRCA2 protein in the Drosophila genus assumes significance in this context owing to the advanced tools available for Drosophila genetics28, and has been functionally characterized recently11,15.\n\nHowever, weak sequence conservation in this gene has proven to be an impediment in associating experimentally proven functionally relevant gene fragments in humans and Drosophila. The variability in the number of BRC repeats even within the Drosophila species has provided fodder for further speculation on the evolution of this gene17,20. The detection of a new BRC repeat would necessitate the reevaluation of such hypotheses.\n\nApart from the BRC repeats, RAD51 interacts with BRCA2 in the carboxy-terminal, and this interaction is modulated by checkpoint kinases9,10. Since the introduction of BRC repeats in the cell inhibits the formation of RAD51 nucleoprotein filaments8, a model has been suggested whereby RAD51 binds to both the BRC repeats and the carboxy-terminal in undamaged cells, and DNA damage triggers the release of the carboxy-terminal bound RAD51 via the phosphorylation of a threonine residue10.\n\nThus, it is noted that certain functionally significant domains are much more conserved compared to the complete protein40. In the current work, a methodology to annotate proteins in such ‘twilight’ zones29 by fragmenting and aligning two protein sequences (Figure 1) has been presented. The results are sorted based on differing criteria, and can be directed by a input file in case the sequences have already been annotated. This method helps in quickly honing onto conserved sites through visual inspection (Table 1 and Figure 1). The threonine phosphorylation site (E=9.1e-4) for checkpoint kinases (Chk1/Chk2) (Figure 1) and a new BRC repeat (E=17e-4) using FRAGAL (Figure 2) has been identified. Pruning out matches which do not have a corresponding conserved sequence in hBRCA2 helps us to select fragment 61 in dmBRCA2 as a new BRC repeat7,14, and fragment 91 in dmBRCA2 as the putative threonine site for phosphorylation by checkpoint kinase Chk1 and Chk210. It must be noted that the sites identified remain putative until verified by experimental data, in spite of the low E-values obtained.\n\nThe multiple alignments can be used to create (for the carboxy-terminal phosphorylation threonine site) or extend (for the new BRC repeat) Prosite motifs. However, the carboxy-terminal phosphorylation threonine site Prosite motif generated from the multiple alignment of sequences from Drosophila and mammals did not result in any matches in other organisms (Ustilago maydis and Caenorhabditis elegans).\n\nIn order to justify this method further, I concentrated on proteins that contain the Half-a-tetratricopeptide (HAT) repeat motifs. The HAT motif is much less ubiquitous than the related tetratricopeptide (TPR) repeat, and has been implicated in various aspects of RNA metabolism23,24. HAT motifs are also hypothesized to play a critical role in assembling RNA-processing complexes25. A recent study that combined bioinformatics, modeling and mutagenesis studies of the HAT domain used the three tandem HAT motifs in the Saccharomyces cerevisiae protein Utp6 to make inferences about the residues that confer structural and/or functional properties to the motif. In the current work, the detection of a new HAT repeat sequence (E=11e-4) in Utp6 from S. cerevisiae has been reported. This sequence has the desired aromatic residues at the proper spacing, a requisite for being considered a HAT repeat25. The above mentioned study would have gained further by the knowledge of this HAT repeat, a repeat that remained undetected by sequence analysis using other methods. The HAT repeats are much more varied, and thus not suitable for generating motifs (like Prosite35). For example, the consensus sequence has been derived from an alignment of 742 HAT motifs from Pfam46 and had to be manually edited since this alignment included gaps in greater than 90% of the sequences25. Moreover, FRAGAL detects that a particular HAT sequence in one protein is more related to HAT sequences from other species that other HAT repeats present in its own sequence. This raises interesting questions about their evolutionary history.\n\nExisting methods for detecting functional motifs in a given protein sequence have been unable to detect these putative sites. For example, meta servers (http://myhits.isb-sib.ch/cgi-bin/motif_scan, http://www.ebi.ac.uk/Tools/pfa/iprscan/, http://www.genome.jp/tools/motif/) for detecting motifs in a protein have been unable to detect the sites identified using the FRAGAL methodology. These meta servers use one or more motif databases35,46–49. It is fair to mention that the FRAGAL method is much more computationally intensive than the above mentioned methods. At the same time, FRAGAL makes no assumption of any knowledge of the conserved regions (either the sequence or their position). The choice of the fragment length in FRAGAL depends on the length of repeats that is expected to be present in the protein. Since both repeats (BRC and HAT) discussed in this manuscript are around ~30 amino acid long, I have chosen a fragment length of 50. A larger fragment length might mask the similarity in the core region due to variations in the non-critical regions, whereas a smaller fragment would match irrelevant portions and thus increase false positives.\n\nIn some of the significant matches in Table 1 the fragment in hBRCA2 is not annotated to be functionally relevant - for example fragments 33 and 87 of dmBRCA2 and fragments 176 and 194 in hBRCA2, respectively. These fragments might suggest an important, yet unknown, functional relevance of that stretch of the human gene as well, since it is conserved across distant species. An excellent database for Drosophila related information is available at http://flybase.org/50. A database (www.sanchak.com/fragal.html) for BRCA2 and nucleolar RNA-associated proteins from different organisms, and will be updating this on a regular basis to include more organisms and different repeats has been created. The increasing importance of Drosophila as a model system for cancer research51 in the search for human therapeutics52–54 can be exploited to the hilt once the conserved mechanism is fully understood. FRAGAL presents the first step by annotating putative conserved sequence fragments in Drosophila and humans.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the Tata Institute of Fundamental Research (Department of Atomic Energy), and the Department of Science and Technology (JC Bose Award Grant). 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 gratefully acknowledge Chaitali Khan for introducing me to the lack of annotation of the BRCA2 gene in Drosophila melanogaster, and for simulating technical discussions. I am also indebted to B. J. Rao for technical inputs. I would also like to thank Ishita Mehta for helping me in preparing the manuscript.\n\n\nSupplementary materials\n\nThe BRCA2 protein sequences from Drosophila melanogaster and humans are split into fragments of parameterized length (50 in this case), at a parameterized interval (10 in this case). All pairs of fragments are aligned, and the results stitched such that there are no overlap in any given match and the order of the match is not interspersed. The alignment is done using the global alignment program ‘needle’ from the EMBOSS suite, and the gap penalties are set to 25 to discourage gaps in the middle of the alignment.\n\nThe syntax is similar to the one used in the UNIPROT accession site.\n\n\nReferences\n\nWooster R, Bignell G, Lancaster J, et al.: Identification of the breast cancer susceptibility gene BRCA2. Nature. 1995; 378(6559): 789–792. PubMed Abstract | Publisher Full Text\n\nThorslund T, West SC: BRCA2: a universal recombinase regulator. Oncogene. 2007; 26(56): 7720–7730. PubMed Abstract | Publisher Full Text\n\nJasin M: Homologous repair of DNA damage and tumorigenesis: the BRCA connection. Oncogene. 2002; 21(58): 8981–8993. PubMed Abstract | Publisher Full Text\n\nLiu Y, West SC: Distinct functions of BRCA1 and BRCA2 in double-strand break repair. Breast Cancer Res. 2002; 4(1): 9–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchlacher K, Christ N, Siaud N, et al.: Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell. 2011; 145(4): 529–542. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolthausen JT, Wyman C, Kanaar R: Regulation of DNA strand exchange in homologous recombination. DNA Repair (Amst). 2010; 9(12): 1264–1272. PubMed Abstract | Publisher Full Text\n\nBork P, Blomberg N, Nilges M: Internal repeats in the BRCA2 protein sequence. Nat Genet. 1996; 13(1): 22–23. PubMed Abstract | Publisher Full Text\n\nPellegrini L, Yu DS, Lo T, et al.: Insights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature. 2002; 420(6913): 287–293. PubMed Abstract | Publisher Full Text\n\nEsashi F, Christ N, Gannon J, et al.: CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair. Nature. 2005; 434(7033): 598–604. PubMed Abstract | Publisher Full Text\n\nBahassi EM, Ovesen JL, Riesenberg AL, et al.: The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage. Oncogene. 2008; 27(28): 3977–3985. PubMed Abstract | Publisher Full Text\n\nKlovstad M, Abdu U, Schupbach T: Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint. PLoS Genet. 2008; 4(2): e31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharan SK, Pyle A, Coppola V, et al.: BRCA2 deficiency in mice leads to meiotic impairment and infertility. Development. 2004; 131(1): 131–142. PubMed Abstract | Publisher Full Text\n\nThorslund T, Esashi F, West SC: Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1. EMBO J. 2007; 26(12): 2915–2922. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLo T, Pellegrini L, Venkitaraman AR, et al.: Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer. DNA Repair (Amst). 2003; 2(9): 1015–1028. PubMed Abstract | Publisher Full Text\n\nBrough R, Wei D, Leulier S, et al.: Functional analysis of Drosophila melanogaster BRCA2 in DNA repair. DNA Repair (Amst). 2008; 7(1): 10–19. PubMed Abstract | Publisher Full Text\n\nKojic M, Kostrub CF, Buchman AR, et al.: BRCA2 homolog required for proficiency in DNA repair, recombination, and genome stability in Ustilago maydis. Mol Cell. 2002; 10(3): 683–691. PubMed Abstract | Publisher Full Text\n\nBennett SM, Noor MA: Molecular evolution of a Drosophila homolog of human BRCA2. Genetica. 2009; 137(2): 213–219. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong AK, Pero R, Ormonde PA, et al.: RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene brca2. J Biol Chem. 1997; 272(51): 31941–31944. PubMed Abstract | Publisher Full Text\n\nChen CF, Chen PL, Zhong Q, et al.: Expression of BRC repeats in breast cancer cells disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and loss of G(2)/M checkpoint control. J Biol Chem. 1999; 274(46): 32931–32935. PubMed Abstract | Publisher Full Text\n\nBennett SM, Mercer JM, Noor MA: Slip-sliding away: serial changes and homoplasy in repeat number in the Drosophila yakuba homolog of human cancer susceptibility gene BRCA2. PLoS One. 2010; 5(6): e11006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeedleman SB, Wunsch CD: A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970; 48(3): 443–453. PubMed Abstract | Publisher Full Text\n\nRice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000; 16(6): 276–277. PubMed Abstract | Publisher Full Text\n\nPreker PJ, Keller W: The HAT helix, a repetitive motif implicated in RNA processing. Trends Biochem Sci. 1998; 23(1): 15–16. PubMed Abstract | Publisher Full Text\n\nHammani K, Cook WB, Barkan A: RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression. Proc Natl Acad Sci U S A. 2012; 109(15): 5651–5656. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChampion EA, Kundrat L, Regan L, et al.: A structural model for the HAT domain of Utp6 incorporating bioinformatics and genetics. Protein Eng Des Sel. 2009; 22(7): 431–439. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSekelsky JJ, Brodsky MH, Burtis KC: DNA repair in Drosophila: insights from the Drosophila genome sequence. J Cell Biol. 2000; 150(2): F31–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLudwig T, Chapman DL, Papaioannou VE, et al.: Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos. Genes Dev. 1997; 11(10): 1226–1241. PubMed Abstract | Publisher Full Text\n\nBier E: Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev Genet. 2005; 6(1): 9–23. PubMed Abstract | Publisher Full Text\n\nRost B: Twilight zone of protein sequence alignments. Protein Eng. 1999; 12(2): 85–94. PubMed Abstract | Publisher Full Text\n\nLarkin MA, Blackshields G, Brown NP, et al.: Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23(21): 2947–2948. PubMed Abstract | Publisher Full Text\n\nGuindon S, Lethiec F, Duroux P, et al.: PHYML Online-a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 2005; 33(Web Server issue): W557–559. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGouy M, Guindon S, Gascuel O: SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 2010; 27(2): 221–224. PubMed Abstract | Publisher Full Text\n\nClark AG, Eisen MB, Smith DR, et al.: Evolution of genes and genomes on the Drosophila phylogeny. Nature. 2007; 450(7167): 203–218. PubMed Abstract | Publisher Full Text\n\nComet JP, Aude JC, Glemet E, et al.: Significance of Z-value statistics of Smith-Waterman scores for protein alignments. Comput Chem. 1999; 23(3–4): 317–331. PubMed Abstract | Publisher Full Text\n\nSigrist CJ, Cerutti L, de Castro E, et al.: PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res. 2010; 38(Database issue): D161–166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRusso CA, Takezaki N, Nei M: Molecular phylogeny and divergence times of drosophilid species. Mol Biol Evol. 1995; 12(3): 391–404. PubMed Abstract\n\nAltschul SF, Madden TL, Schaffer AA, et al.: Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–3402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShivji MK, Davies OR, Savill JM, et al.: A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange. Nucleic Acids Res. 2006; 34(14): 4000–4011. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShivji MK, Mukund SR, Rajendra E, et al.: The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange. Proc Natl Acad Sci U S A. 2009; 106(32): 13254–13259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWarren M, Smith A, Partridge N, et al.: Structural analysis of the chicken BRCA2 gene facilitates identification of functional domains and disease causing mutations. Hum Mol Genet. 2002; 11(7): 841–851. PubMed Abstract | Publisher Full Text\n\nAyoub N, Rajendra E, Su X, et al.: The carboxyl terminus of Brca2 links the disassembly of Rad51 complexes to mitotic entry. Curr Biol. 2009; 19(13): 1075–1085. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStratton MR, Wooster R: Hereditary predisposition to breast cancer. Curr Opin Genet Dev. 1996; 6(1): 93–97. PubMed Abstract\n\nMeyer P, Landgraf K, Hogel B, et al.: BRCA2 mutations and triple-negative breast cancer. PLoS One. 2012; 7(5): e38361. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVenkitaraman AR: Linking the cellular functions of BRCA genes to cancer pathogenesis and treatment. Annu Rev Pathol. 2009; 4: 461–487. PubMed Abstract | Publisher Full Text\n\nRajendra E, Venkitaraman AR: Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase. Nucleic Acids Res. 2010; 38(1): 82–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFinn RD, Mistry J, Tate J, et al.: The Pfam protein families database. Nucleic Acids Res. 2008; 36(Database issue): D281–288. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPedruzzi I, Rivoire C, Auchincloss AH, et al.: HAMAP in 2013, new developments in the protein family classification and annotation system. Nucleic Acids Res. 2013; 41(Database issue): D584–589. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoua D, Cerutti L, Falquet L, et al.: PeroxiBase: a database with new tools for peroxidase family classification. Nucleic Acids Res. 2009; 37(Database issue): D261–266. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAttwood TK, Coletta A, Muirhead G, et al.: The PRINTS database: a fine-grained protein sequence annotation and analysis resource-its status in 2012. Database (Oxford). 2012; 2012: bas019. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcQuilton P, St Pierre SE, Thurmond J, et al.: FlyBase 101-the basics of navigating FlyBase. Nucleic Acids Res. 2012; 40(Database issue): D706–714. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVidal M, Cagan RL: Drosophila models for cancer research. Curr Opin Genet Dev. 2006; 16(1): 10–16. PubMed Abstract | Publisher Full Text\n\nChelouah S, Monod-Wissler C, Bailly C, et al.: An integrated Drosophila model system reveals unique properties for F14512, a novel polyamine-containing anticancer drug that targets topoisomerase II. PLoS One. 2011; 6(8): e23597. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGladstone M, Su TT: Chemical genetics and drug screening in Drosophila cancer models. J Genet Genomics. 2011; 38(10): 497–504. PubMed Abstract | Publisher Full Text\n\nDas T, Cagan R: Drosophila as a novel therapeutic discovery tool for thyroid cancer. Thyroid. 2010; 20(7): 689–695. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1147",
"date": "12 Aug 2013",
"name": "Himanshu Sinha",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA well written paper that proposes a new method, FRAGAL for identifying functional putative motifs within protein sequences which have been hidden from previous analyses. By splitting the sequences into overlapping fragments, this method is able to discover additional motifs. The author has tested his technique on BRC repeats in Drosophila dmBRCA2 and HAT repeats in budding yeast Utp6, by comparing them to corresponding human protein sequences. The BRC repeat has been well analysed with comparisons across several Drosophila species. However the author does not provide extensive comparison of HAT repeats in Saccharomyces species. Since the sequences of several Saccharomyces sibling species and closely related fungi such as Aspergillus, Candida, etc. are known, it would be interesting to see how conserved this new HAT repeat is within the overall conservation of Utp6. While the author establishes the advantage of FRAGAL technique, it is too early to say that this is a useful generic tool to identify known and novel motifs in protein sequences. I would request the author to run his FRAGAL code on several protein sequences with small motifs to estimate success rates and false discovery rates of his method. A supplementary table should be provided describing several sequences analysed by this method and these rates. A minor comment, Table 1 should be simplified with the two BRCA2 protein sequences presented in two sub-tables. Please explain why certain ranks are missing in FRscore and %S.",
"responses": [
{
"c_id": "534",
"date": "23 Aug 2013",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Sinha,I greatly appreciate your comments on my manuscript. I will incorporate your suggested changes and update the manuscript. Your suggestion of including more of such repeats (I plan to do BIR and TPR) is something that will take some computational time and thus the delay.Best regards,Sandeep"
},
{
"c_id": "561",
"date": "17 Sep 2013",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "I am grateful for the encouraging comments on the work. ...author does not provide extensive comparison of HAT repeats in Saccharomyces species. ... it would be interesting to see how conserved this new HAT repeat is within the overall conservation of Utp6.I have implemented this interesting idea using proteins which have the HAT repeat from Aspergillus nidulans and Candida glabrata. This is now a Supplementary figure. However, these do not provide any further insights into the evolution of the HAT repeat, and would require sophisticated analyses beyond my expertise. I would request the author to run his FRAGAL code on several protein sequences with small motifs to estimate success rates and false discovery rates of his method. A supplementary table should be provided describing several sequences analyzed by this method and these rates. In accordance with this suggestion, I have run FRAGAL on two more motifs (BIR and TPR). However, I failed to detect any new repeats using these two motifs. These are now part of the database - http://sanchak.com/fragal.html. A minor comment, Table 1 should be simplified with the two BRCA2 protein sequences presented in two sub-tables. Please explain why certain ranks are missing in FRscore...I have simplified the table considerably based on the comments of another reviewer (please see below). The naming of the sub tables as A and B was confusing given that the query and target sequences were named A and B. Further; the columns for the similarity scoring has been removed. We did not ever use the similarity only score, and this was adding to the confusion. I have now clearly stated the reason for some missing ranks. I apologize for the confusing aspects of this table."
}
]
},
{
"id": "1106",
"date": "22 Aug 2013",
"name": "Satish Chikkagoudar",
"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 presents an interesting technique for detecting new BRC repeats. The paper is generally well written, but needs some additional material to bolster its case. The introduction section needs more discussion of the 'state-of-the-art' in the alignment and motif detection area (especially with respect to detecting BRC repeats). The paper’s argument can be made stronger by explicitly mentioning the advantages of fragmented alignment over any other recursively applied local alignment method or homology search method. Some discussion of existing methods exists in the discussion/results section, but that needs to be made available in the introduction in order to justify the need for creating a new method An explanation of the choice of parameter values needs to be given. For example, why does FRscore have weights of 1/3 for only Similarity (equation 2)? Also, the reasoning for choosing particular values for gap open and gap extend penalties needs to be mentioned along with whether parameter tuning/search was done to arrive at those values. Otherwise, those numbers seem arbitrary. The author needs to discuss whether he tried any other alignment algorithms apart from Clustal-W. Some other algorithms such as MAFFT, ProbCons or CONTRAlign may yield better results. The author may want to discuss the data in a separate section under methods/materials. A figure describing the FRAGAL pipeline will be useful to visually describe the pipeline/algorithm.",
"responses": [
{
"c_id": "533",
"date": "23 Aug 2013",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "Dear Dr Chikkagoudar,I would like to thank you for your insightful suggestions which will help improve the manuscript. I will make the suggested changes, and incorporate them in a new version shortly.A small clarification - you have asked for a \"figure describing the FRAGAL pipeline\". There is a supplementary figure S1 doing this. Do you think that this figure is insufficient, or were you suggesting that I move this to the main manuscript?Best regards,Sandeep"
},
{
"c_id": "562",
"date": "17 Sep 2013",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "I appreciate the positive comments, and hope to have addressed the concerns detailed below. The introduction section needs more discussion of the 'state-of-the-art' in the alignment and motif detection area (especially with respect to detecting BRC repeats) ... Some discussion of existing methods exists in the discussion/results section, but that needs to be made available in the introduction in order to justify the need for creating a new method. I have moved this section to the introduction. In response to the comments of another reviewer (Dr Saurabh Sinha), I have also mentioned the possible reasons why existing tools have failed to detect these repeats. Further, I have cited methods that use spliced alignment methods for genome assembly, noting that these methods use graph algorithms to solve the computationally difficult problem of exon chaining. FRAGAL does the converse by finding best matches in known exon chains (i.e. protein sequences). An explanation of the choice of parameter values needs to be given. For example, why does FRscore have weights of 1/3 for only Similarity (equation 2)? The extra weightage given to % identity in the score is due to the fact that one expects more sequence conservation in repeats. ...the reasoning for choosing particular values for gap open and gap extend penalties needs to be mentioned along with whether parameter tuning/search was done to arrive at those values. The gap penalties are set to discourage gaps, but not gap extensions. The gap opening has been set to two values - 10 and 25. Results using both values have been uploaded in the database http://sanchak.com/fragal/BRCA2.html . The results using 25 have been observed to be better. However, a complete statistical analysis of these values is beyond the scope of this work. The author needs to discuss whether he tried any other alignment algorithms apart from Clustal-W. Some other algorithms such as MAFFT, ProbCons or CONTRAlign may yield better results. I have used another tool (MAFFT) to generate the multiple sequence alignment. The results from the new alignment tool, mirrors the inference drawn from Clustal-W. This is now a supplementary figure. A figure describing the FRAGAL pipeline will be useful to visually describe the pipeline/algorithm. I have added a pseudo code of the FRAGAL program in the main manuscript."
}
]
},
{
"id": "1731",
"date": "06 Sep 2013",
"name": "Saurabh Sinha",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well-written manuscript, Chakraborty presents a tool for local alignment of two protein sequences that includes a fragment-chaining step. He then uses this tool to identify important putatively functional fragments in two different Drosophila proteins by comparison to the respective human ortholog. A database containing results of many more similar applications is also presented and is a nice aspect of the work. I have the following specific comments, mostly related to the presentation, that might help the author improve the clarity of the manuscript.The current title is rather long. The contribution of this work is mainly in the form of the FRAGAL tool, and the title could be trimmed to emphasize only that. The two example applications to finding the phosphorylation site, BRC repeat etc. are not experimentally substantiated biological claims, and may be better off being left out of the title. Clear discussion should be provided regarding other previous work where pairs of aligned fragments are stitched together (e.g. exon chaining, see Jones & Pevzner 2004, and the chain/net approach to whole genome alignments). Since the FRScore does not include gap penalties, I am assuming that each pair of fragments is subjected to two distinct similarity-scoring approaches; the gap-based approach when aligning that pair of fragments using 'needle' and the match/mismatch based approach when ranking the aligned pairs. This should be stated clearly, to avoid confusion. Is there a reason why the needle score was not used in place of the FRScore?It appears that the FR score is the unweighted sum of %similarity and %identity. This should be stated explicitly.I did not quite understand the formatting of Table 1. I think there should be a line separating ‘A’ from ‘B’ (which I think comes after a row with rank 14 in the second column). It took me some time to see that there are two sub-tables being shown here. Also, the fact that the same row is used to show different entities was confusing; usually a table is constructed so that a row shows different pieces of information about one entity. I assume something like A91-B337 refers to the starting positions of a matching fragment between sequences A and B, and the length of that fragment is not indicated in the row. Is this correct? (On reading further I realize that this interpretation is incorrect, and the numbers in a match are arbitrary indices and not coordinates. This was not clear from the legend.)I found that presenting sub-tables ‘A’ and ‘B’ (which I finally realized does not relate to ‘A’ and ‘B’ sequences) leads to more confusion than it helps. If both sub-tables present the same ranked list and the only difference is that ‘A’ filters for “conserved” fragments, then it might be better to show only sub-table ‘B’ and add a column indicating if this is a fragment marked conserved.Where is the E-value of an FRScore coming from? (I am not familiar with what the 'Protein Information Resource' provides.) Perhaps this E-value correspond to the global alignment score reported by needle?The results of Table 1 do not aid ones understanding as to how the fragmented alignment, i.e. stitching together of fragments, helps in this case. As shown, this appears similar in form to a ranked list of matches from a standard local-aligner. Similarly, with respect to Figure 2, the author may wish to discuss why FRAGAL finds the 'melanogaster4' fragment as a BRC repeat where previous annotations (that found three repeats) failed. Was this a matter of previous methods ‘missing the threshold’? (This seems unlikely given the strong E-value reported for this.) Similar clarifications for the HAT repeat finding exercise will also be helpful.",
"responses": [
{
"c_id": "563",
"date": "17 Sep 2013",
"name": "Sandeep Chakraborty",
"role": "Author Response",
"response": "I greatly appreciate the positive comments.The current title is rather long. The contribution of this work is mainly in the form of the FRAGAL tool, and the title could be trimmed to emphasize only that. The two example applications to finding the phosphorylation site, BRC repeat etc. Are not experimentally substantiated biological claims, and may be better off being left out of the title. Since this work began in search for unannotated fragments of the dmBRCA2 sequence, and is being followed in the lab actively, I think the reference to an application of FRAGAL is warranted. However, I have removed the reference to the HAT repeat and stated the fact that the phosphorylation site and the BRC repeat is putative in the title.Clear discussion should be provided regarding other previous work where pairs of aligned fragments are stitched together... I have discussed the spliced alignment approach to genome assembly in the discussion. However, I have noted that while these methods use graph algorithms to solve the computationally difficult problem of exon chaining, FRAGAL does the converse by finding best matches in known exon chains (i.e. protein sequences).Since the FRScore does not include gap penalties, I am assuming that each pair of fragments is subjected to two distinct similarity-scoring approaches; the gap-based approach when aligning that pair of fragments using 'needle' and the match/mismatch based approach when ranking the aligned pairs. This should be stated clearly, to avoid confusion. Is there a reason why the needle score was not used in place of the FRScore? The needle score includes gap penalties, which is something that is not intended for use in FRScore, as you have correctly pointed out. The idea is to direct the alignment to discourage gaps – but once the alignment is done a gap should not have a penalty. It is ‘real' and therefore only the identity or similarity that matters.It appears that the FR score is the unweighted sum of %similarity and %identity. This should be stated explicitly.I have empirically assigned more weightage to the %identity based on the fact that we are searching for repeats, and expect higher conservation. This conservation is magnified a bit more by assigning higher weightage.I did not quite understand the formatting of Table 1. ... I found that presenting sub-tables A and B (which I finally realized does not relate to A and B sequences) leads to more confusion than it helps. I apologize for this confusion. The line demarcating subtables A and B was lost in the typesetting - and I missed out on detecting this error. Further, naming the subtables A and B was a poor choice of names, since the sequences were also named A and B. Finally, I agree that the second column was unnecessary, as was two subtables. I have simplified this table.I assume something like A91-B337 refers to the starting positions of a matching fragment between sequences A and B, and the length of that fragment is not indicated in the row. Is this correct? (On reading further I realize that this interpretation is incorrect, and thenumbers in a match are arbitrary indices and not coordinates. This was not clear from the legend.)I apologize for this oversight. This is mentioned in the web pages - http://sanchak.com/fragal/ALLRUNS.BRCA2/Caenorhabditiselegans.G5EG86.Homosapiens.P51587.gap.25.size30/cumu.results.thresh30.size30.doiden1.onlyanno1.html as `Multiply index with 10 to get sequence starting position in original sequence'. Thus A91 refers to the sequence starting at 910 in `A' and going till 959, since the fragmenting length is 50. I have now mentioned this at the beginning of the Results section, and in the legend.Where is the E-value of an FRScore coming from? ... Perhaps this E-value corresponds to the global alignment score reported by needle?I have specified the website (http://pir.georgetown.edu/pirwww/search/pairwise.shtm ), and cited the paper by Wu C et al. (2003) The Protein Information Resource. Nucleic Acids Res 31: 345347. ... with respect to Figure 2, the author may wish to discuss why FRAGAL finds the 'melanogaster4' fragment as a BRC repeat where previous annotations (that found three repeats) failed. ... Similar clarifications for the HAT repeat finding exercise will also be helpful.I could only make an educated guess as to why other tools failed to detect these repeats. I believe that the tools used had a ‘sequential' methodology and therefore one match fixed the order of the next searches. Not all known BRC repeats have a low E-value when aligned with the new BRC repeat. For example, the first BRC repeat in hBRCA2 when aligned to the new dmBRCA2 repeat has an E=0.04, much more than the E=17e-4 observed for the fourth repeat (which is the one I report here). Ideally, if one took all the BRC repeats and did a search in the dmBRCA2 sequence, this new repeat would be reported. Essentially, this is what FRAGAL does, albeit implicitly, by automatically fragmenting the sequence. The same logic applies to the HAT repeat, where the sequences are more varied and thus the choice of the repeat would affect the detection of new motifs."
}
]
}
] | 1
|
https://f1000research.com/articles/2-143
|
https://f1000research.com/articles/2-205/v1
|
04 Oct 13
|
{
"type": "Research Article",
"title": "Seizure evoked regulation of LIM-HD genes and co-factors in the postnatal and adult hippocampus",
"authors": [
"Vanisha Lakhina",
"Lakshmi Subramanian",
"Dhananjay Huilgol",
"Ashwin S Shetty",
"Vidita A. Vaidya",
"Shubha Tole",
"Vanisha Lakhina",
"Lakshmi Subramanian",
"Dhananjay Huilgol",
"Ashwin S Shetty"
],
"abstract": "The LIM-homeodomain (LIM-HD) family of transcription factors is well known for its functions during several developmental processes including cell fate specification, cell migration and axon guidance, and its members play fundamental roles in hippocampal development. The hippocampus is a structure that displays striking activity dependent plasticity. We examined whether LIM-HD genes and their co-factors are regulated during kainic acid induced seizure in the adult rat hippocampus as well as in early postnatal rats, when the hippocampal circuitry is not fully developed. We report a distinct and field-specific regulation of LIM-HD genes Lhx1, Lhx2, and Lhx9, LIM-only gene Lmo4, and cofactor Clim1a in the adult hippocampus after seizure induction. In contrast none of these genes displayed altered levels upon induction of seizure in postnatal animals. Our results provide evidence of temporal and spatial seizure mediated regulation of LIM-HD family members and suggest that LIM-HD gene function may be involved in activity dependent plasticity in the adult hippocampus",
"keywords": [
"Transcription factors regulate gene expression in the mammalian brain",
"playing a critical role in both neurodevelopment and in neuronal plasticity during its lifespan. During development",
"transcription factor mediated regulation is essential for appropriate cell fate specification",
"cell migration and connectivity1–3. Transcription factors also regulate plasticity including activity-dependent process of dendritic pruning",
"axonal sprouting and cell proliferation and survival4–6."
],
"content": "Introduction\n\nTranscription factors regulate gene expression in the mammalian brain, playing a critical role in both neurodevelopment and in neuronal plasticity during its lifespan. During development, transcription factor mediated regulation is essential for appropriate cell fate specification, cell migration and connectivity1–3. Transcription factors also regulate plasticity including activity-dependent process of dendritic pruning, axonal sprouting and cell proliferation and survival4–6.\n\nOne family of transcription factors, the LIM-homeodomain (LIM-HD) family, is known to play critical roles in regulating cell proliferation, axon outgrowth and pathfinding across several systems7–11. The LIM-HD proteins have a C-terminal homeodomain which binds to DNA and two zinc finger “LIM” domains that bind co-factors encoded by the Clim genes. The transcriptionally active complex is a tetramer comprising two LIM-HD molecules bridged by a dimer of two Clim molecules12,13. LIM-only (Lmo) proteins lack the homeodomain but can bind Clim molecules, and function as dominant-negative regulators of LIM-HD function12,14,15. At least thirteen LIM-HD (Lhx) genes four Lmo genes and two Clim genes have been identified in the mouse. A subset of genes is expressed in the embryonic and mature hippocampus and of these, Lhx2 and Lhx5 are critical to hippocampal development16. Lhx2 plays a fundamental role in early telencephalic development as a cortical selector gene11. The neocortex and hippocampus do not form in the absence of Lhx211,17. At later stages, Lhx2 plays a new role in the developing hippocampus, as a necessary and sufficient repressor of astrogliogenesis18. Lhx2 continues to be expressed in the mature hippocampus. Lhx5 is critical for hippocampal development at early stages, but is not expressed in the embryonic hippocampus once it is specified19. Lhx1, Lhx9, Clim1a, Clim2, Lmo3 and Lmo4 are all expressed in the hippocampus at embryonic and adult stages16, but no loss of function phenotypes have been reported in the hippocampus.\n\nWhile several studies have implicated the LIM-HD family as a key modulator of important neurodevelopmental events, the understanding of the role of this transcription factor family in the postnatal and adult brain remains relatively unexplored. These transcription factors are known to regulate cell proliferation8,20, axon pathfinding21,22 and neurite outgrowth23,24. These phenomena have parallels in the structural plasticity that occurs in postnatal and adult life. It is now well established that the same molecules that bring about the early development of the hippocampus are often reutilized in adult reorganization and structural plasticity25–27. Several LIM-HD family members continue to be expressed in the adult hippocampus16 (this study). Therefore, we explored whether these genes display activity-dependent regulation in the adult hippocampus, to provide a basis for studies that may uncover new functions for these genes in maturity.\n\nActivity dependent neuronal plasticity has been suggested to reutilize key developmental pathways to evoke plasticity in the mature nervous system. In particular, seizure models have been shown to induce dramatic changes in progenitor proliferation, axonal sprouting, dendritic reorganization, changes in neuronal cell survival and progenitor differentiation within the hippocampus28–31. Intriguingly, the nature of neuroplastic changes evoked by seizures differs quite dramatically in the postnatal versus the adult brain32–34. Regulation at the level of signaling and transcription factors has been shown to be important for structural plasticity in the hippocampus35. While neuronal activity and seizures are likely to recruit major developmental signalling pathways in the hippocampus, thus far the role of key developmental transcription factor families as targets is relatively unexplored.\n\nAn earlier study reported that LIM-only genes Lmo1, 2 and 3 are differentially regulated in a field-specific manner in the adult rat hippocampus in response to kainic acid-induced seizure36. We examined a broader set of Lmo and LIM-HD genes as well as their co-factors in a similar paradigm, not only in the adult rat hippocampus, but also in early postnatal stages when hippocampal circuitry is not fully developed37–39. Our study provides evidence that LIM-HD, LIM-only, and Clim gene mRNA displays selective field-specific regulation in the hippocampus in response to kainate induced seizures. This provides a basis to explore potential new functions of these genes in activity-dependent synaptic plasticity.\n\n\nResults\n\nIn this study we focused on LIM-HD genes that are expressed in the adult hippocampus, Lhx1, Lhx2 and Lhx9 and their co-factors, Clim1a and Clim2. Among the LIM-only genes, Lmo1, Lmo2, and Lmo3 have been previously reported to display differential regulation in kainate-induced seizure36. In our study, we included Lmo3 as a control to allow comparison with the earlier study36, and also Lmo4 which was not examined previously. We examined the mRNA expression of these genes at postnatal day P7 when the hippocampal circuitry is not yet fully developed, and also in adult rats (2–3 months old) with mature hippocampal neurocircuitry.\n\nWe used non-radioactive in-situ hybridization to examine gene expression in the CA1 and CA3 fields of the Ammon’s horn as well as the dentate gyrus (DG) of control animals (Figure 1a). Lhx1 transcripts were not detectable in the hippocampus at P7, and only weakly expressed in the adult DG (Figure 1b). In contrast, Lhx2 and Lhx9 are expressed intensely in the DG and CA3, with weaker expression in CA1 at P7. In the adult, expression was strong in the DG, but weak in CA1 and CA3 (Figure 1c, d). Lmo3 and Clim2 are strongly expressed in CA1 and DG, with weaker expression in the CA3 region at both stages (Figure 1e, h). Lmo4 shows strong expression in CA1 but is weakly expressed in CA3 and DG at both stages (Figure 1f). Clim1a displays expression in all fields at P7, but is weak to undetectable in CA3 in the adult (Figure 1g).\n\n(a) A schematic illustrating hippocampal subfields; dentate gyrus (DG) (yellow), CA3 (red) and CA1 (green) fields of Ammon’s horns. (b–d) Non-radioactive in-situ hybridization of LIM-homeodomain genes at postnatal day (P)7 and in adult control animals showing differential expression of LIM-homeodomain genes; Lhx1 (b), Lhx2 (c) and Lhx9 (d), Lmo3 (e), Lmo4 (f) Clim1a (g), Clim2 (h) across the hippocampal subfields. Scale bars = 200µm\n\nActivity is known to regulate structural plasticity and neurogenesis in the adult hippocampus40,41. We administered kainate intraperitoneally to both early postnatal and adult rats to induce seizures as a model of activity and analysed whether there is differential regulation of LIM genes in response to kainate-evoked seizures 6 hours later. All animals administered kainate exhibited classical hallmarks of seizure. Using radioactive in-situ hybridization and optical densitometry we assessed the expression of Lhx1, Lhx2, Lhx9, Lmo3, Lmo4, Clim1a and Clim2 in the postnatal and adult hippocampal subfields (see Materials and methods). Radioactive in-situ hybridization has an important advantage over quantitative PCR since it provides spatial resolution. The hippocampal CA1 and CA3 fields are molecularly distinct, and the dentate gyrus contains a distinct cell population from the Ammon’s horn42. Therefore it is necessary to quantitate the gene expression in each region individually.\n\nThe DG displays robust structural changes in response to seizure. Increase in dentate granule cell neurogenesis43 and extensive mossy fiber sprouting41,44 are hallmarks of kainate induced seizure. Upon kainate treatment, the expression of Lhx1 showed a striking increase (25%; p = 0.019) in the adult DG. This is in contrast with the adult Lhx2 and Lhx9 expression, the mRNA levels of which show a drastic reduction (60% for Lhx2, p = 0.0004 and 36% for Lhx9, p = 0.003; Figure 2, Figure 3a). Interestingly, the LIM-only genes Lmo3 and Lmo4 also showed opposite changes: whereas Lmo3 levels decreased significantly (53%, p = 0.002), Lmo4 mRNA levels showed a remarkable increase (55%, p = 0.009) in kainate-treated animals. The decrease in Lmo3 levels was consistent with that reported previously36. The mRNA levels of the cofactor Clim1a decreased slightly in treated animals (15%, p = 0.048) whereas no significant difference was observed with Clim2 (Figure 2, Figure 3b).\n\nRepresentative images of sections of brains from control and kainate-administered animals processed for radioactive in-situ hybridization of LIM-homeodomain genes in the hippocampus. 1 section from each condition is shown for adult (a) and P7 (b) animals. Colored lines mark the areas for quantification of expression in different hippocampal subfields: DG (yellow); CA3 (red); CA1 (green). Scale bars = 200µm.\n\nQuantitative densitometric analysis of the adult DG region following kainate administration in adult (a,b) and P7 (c,d) rats. Grey bars are controls, white bars are kainate treated animals. Results are expressed as mean ± SEM percentage of control for mRNA expression (*p < 0.05, unpaired Student’s t test).\n\nThe CA3 subfield has pyramidal neurons, which receive input from the dentate granule cells. They display profound alterations in dendritic structure and branching in response to seizure. In our experiments using kainate-induced seizure, Lhx1 mRNA increased (20%, p = 0.014) in the adult CA3. In contrast, Lhx2 and Lhx9 levels decreased (30%, p = 0.028; 35%, p = 0.044 respectively; Figure 2, Figure 4a). Levels of both Lmo3 and Lmo4 were reduced (40%, p = 0.007; 25%, p = 0.002 respectively). The levels of the cofactor Clim1a also decreased (15%, p = 0.047) whereas Clim2 levels remained unaltered in the adult CA3 (Figure 2, Figure 4b).\n\nQuantitative densitometric analysis of the adult CA3 region following kainate administration in adult (a,b) and P7 (c,d) rats. Grey bars are controls, white bars are kainate treated animals. Results are expressed as mean ± SEM percentage of control for mRNA expression (*p < 0.05, unpaired Student’s t test).\n\nThe CA1 pyramidal neurons receive input from the CA3 neurons. They displayed altered dendritic shape and density and also axon sprouting as a result of seizure45. In the CA1 field, Lhx1 mRNA increased (20%, p = 0.02), whereas Lhx2 levels decreased (19%, p = 0.024) but, there was no change in Lhx9 mRNA levels in the adult CA1 upon kainate-induced seizure (Figure 2, Figure 5a). Lmo3 mRNA levels decreased (41%, p = 0.0018) whereas Lmo4, Clim1a, and Clim2 levels remained unchanged (Figure 2, Figure 5b).\n\nQuantitative densitometric analysis of the adult CA1 region following kainate administration in adult (a,b) and P7 (c,d) rats. Grey bars are controls, white bars are kainate treated animals. Results are expressed as mean ± SEM percentage of control for mRNA expression (*p < 0.05, unpaired Student’s t test).\n\nTable 1 summarizes the data such that seizure-induced regulation can be compared within a particular field as well as for a particular gene across all fields. For example, upon kainate induced seizure, Lhx1 mRNA shows a significant increase over very low baseline expression in all the hippocampal fields in response to seizure. In contrast, Lhx2 and Lmo3 show a significant decrease in all hippocampal fields. Interestingly, Lhx9 and Clim1a show a significant decrease in CA3 and DG, but not in CA1. Lmo4 transcript levels increase in the DG, decrease in the CA3 and show no change in the CA1. This correlates with the fact that the DG and CA3 undergo a more drastic structural reorganization in response to seizure46,47. Clim2 shows no alteration suggesting it may not have any additional roles in kainate-induced plasticity, but continues to be available to LIM-HD transcription factors at the same levels.\n\nSeizure evoked structural plasticity differs between the postnatal and adult hippocampus in its extent as well as the type of changes seen. Although postnatal kainate treatment evokes powerful seizures, the immature brain is relatively resistant to seizure-evoked structural remodeling. For example, mossy fiber sprouting is absent or delayed48–50, and DG neurogenesis is unaltered or biphasically regulated with an initial decline and a delayed increase51–53 in response to seizure in the postnatal hippocampus. We asked whether the postnatal hippocampus differs from the adult hippocampus in kainic acid induced regulation of LIM genes and co-factors. We administered kainic acid to rat pups on postnatal day P7 and analyzed changes in transcript levels of several LIM genes 6 hours later. In striking contrast to the changes observed in the adult brain, the postnatal hippocampus appears refractory to regulation of the LIM-HD family following kainate evoked seizures (Figure 2, Figure 3c, d, Figure 4c, d, Figure 5c, d).\n\nIn summary, the LIM gene family and its co-factors display distinct and highly field-specific regulation in response to kainate induced seizure in the adult, but not in the postnatal hippocampus.\n\n\n\n\nDiscussion\n\nSeizures can lead to different forms of hippocampal plasticity, which include axonal/dendritic remodeling and neurogenesis. Chemical-induced seizures like the kainic acid (kainate) treatment are used as models for epilepsy and have been shown to increase neurogenesis in the adult DG28 and extensive mossy fiber sprouting where mossy fibers aberrantly synapse onto dentate granule cells instead of CA3 pyramidal neurons41,44. Kainic acid administration causes animals to display motor signs including convulsions. In our experiments, we observed changes in the transcript levels 6 hours post kainic acid administration, after the animals displayed all the characteristic physical stages of seizures. In future experiments it would be interesting to examine whether any LIM gene transcript regulation occurs in a shorter time window post kainic acid administration, prior to the physical manifestation of seizure by the animal.\n\nTranscription factors important for brain development are also known to regulate structural changes and reorganization in the adult brain, one example being members of the basic Helix-Loop-Helix (bHLH) family25,26. Members of the LIM-HD family of transcription factors are necessary for different aspects of the development of the hippocampus11,18,19, a structure that is vulnerable to changes in response to activity. LIM genes are differentially expressed in both the postnatal and adult hippocampus, suggesting that there might be a role for these genes in postnatal circuit development and adult reorganization16. We therefore hypothesized that the LIM-HD family members are differentially regulated in response to activity. Indeed, from our analysis of radioactive in-situ hybridization, we find that each hippocampal field displays differential expression and post-seizure regulation of different LIM genes. LIM-only genes Lmo1, 2 and 3 were previously shown to be regulated in response to kainate-induced seizures in the adult hippocampus36. We report that Lmo4 is also regulated by kainate-induced seizures throughout the hippocampus. We also discovered that LIM-HD genes Lhx1, Lhx2, Lhx9 and cofactor Clim1a are differentially regulated in response to seizures in a field-specific manner. Furthermore, we show that this differential regulation of LIM genes is restricted to adult animals and when we administered kainic acid to postnatal pups, no such regulation was observed. This is intriguing because these results highlight that a developing system such as the hippocampal circuitry in the early postnatal brain is relatively resistant to seizure-induced structural remodelling and plasticity32. For example, in the adult, seizure induces an increase in DG neurogenesis whereas in early postnatal stages, it is either decreased or unchanged34. Our results raise the intriguing possibility that such differences in molecular regulation of transcription factors may underlie the differing nature of cellular changes evoked by seizures in the postnatal versus adult brain.\n\nSeizure leads to an increase in neuronal activity thereby inducing the transcription of several immediate early genes (IEGs). The IEGs are hypothesized to be involved in seizure-induced structural remodelling54. The LIM family of transcription factors could be part of effector cascades downstream of these IEGs, which may eventually lead to the structural changes seen in different hippocampal subfields. CREB, a well-known activity regulated transcription factor, has been shown to interact in the same transcriptional complex as Lmo4 in response to activity55. It is also interesting to note that well known seizure-responsive IEGs in the adult hippocampus, such as the AP-1 complex, are not regulated by postnatal seizures56. This further supports the idea that distinct molecular changes evoked by postnatal versus adult seizures may contribute to the age-dependent differences in seizure-evoked plasticity.\n\nDistinct structural changes occur in response to seizure in different subfields of the hippocampus. On seizure induction, DG shows an increase in the granule cell neurogenesis46, enhanced integration of granule cells into the neurocircuitry, a profound increase in mossy fiber sprouting by these neurons and formation of recurrent synapses57–59. The CA3 and CA1 pyramidal neurons show a loss of dendritic spine and dendritic branches47 post seizure. Some axon sprouting is also seen in CA1 neurons45,60. LIM genes may bring about activity induced structural changes in the hippocampus. They are known to regulate neurite outgrowth24. Some LIM-HD genes also control key axon guidance molecules such as Eph/ephrins61, which affect mossy fiber sprouting in the DG62. Lhx1 is known to regulate the transcription of Eph/ephrins in a subset of motor neurons61. Our results show increased Lhx1 mRNA levels in the DG in response to seizure that could lead to increased Eph/ephrin levels therefore contributing to mossy fiber sprouting. Lhx2 represses Robo1 and 2 expression in the thalamus during thalamocortical pathfinding22 and so down regulation of Lhx2 mRNA in response to seizure could be important for mossy fiber sprouting via upregulation of the Robo receptors. Lmo4 has been shown to confer a neuroprotective role in response to hypoxia63. Interestingly, we find an increase in the Lmo4 mRNA after kainate treatment, which could lead to neuronal survival in response to seizure.\n\nOur study provides new evidence of seizure mediated regulation of LIM-HD transcription factors. We show that this regulation is age-dependent and field specific. Future experiments will aim at testing whether LIM genes are necessary for mediating seizure induced structural alterations. Examining the effect of kainic acid treatment on structural changes such as DG neurogenesis in LIM gene loss-of-function mutants will begin to address this issue. In addition, determining the interactions of LIM gene family proteins with other factors known to mediate structural changes such as the bHLH family members25,26 will open avenues for the mechanistic understanding of this process. These results therefore provide impetus for future studies to explore the role of the LIM-HD transcription factors, LIM only genes, and their cofactors in activity-dependent reorganization and plasticity in the mature nervous system.\n\n\nMaterials and methods\n\nSprague-Dawley rats were bred in the Tata Institute of Fundamental Research (TIFR) Animal house, maintained under normal 12-hour light/dark cycle and were provided with food and water ad libitum. A total of 84 adults and 101 pups (P7) were used. Adults were between 2–3 months old and weighed between 200–250 grams. All animal procedures were performed in accordance with the NIH guidelines for use and maintenance of animals and were approved by the TIFR Institutional Animal Ethics committee. The male rats were sexed at P21 and were used for experiments when they reached adulthood. Postnatal pups of both sexes were used for experiments at P7. All animals were grouped based on their treatment with either saline (control group; n = 44 adults; n = 47 P7 pups) or with 10mg/kg kainic acid (Sigma, USA; n = 40 adults; n = 54 P7 pups) administered intraperitoneally and were housed isolated for 6 hours after the treatment. The kainic acid treated group was observed every 30 minutes across the 6 hours and displayed all the characteristic stages of seizures. The animals displayed facial clonus (Racine Stage 1) to front and hindlimb clonus and continuous falling down (Racine Stage 5).\n\nAnimals were decapitated using a guillotine 6 hours after treatment and the brains were immediately frozen on dry ice and stored at -70°C. Coronal sections (14µm) were generated on the cryostat and mounted onto Probe-plus RNase free slides (Electron Microscopy Sciences, USA). Slides were then treated with 4% paraformaldehyde (PFA; Merck Chemicals), washed in 1X phosphate-buffered saline, acetylated with acetic acid (Qualigens Fine Chemicals) in 0.1M triethanolamine (Sigma-Aldrich), rinsed in 2X sodium saline citrate (SSC), pH 4.5 and then dehydrated through grades (30%, 70% and 100% in double distilled water) of ethanol (Commercial Alcohols, Ontario, Canada) prior to storage at -70°C.\n\nThe in-situ hybridization for DIG-labeled probes was carried out as described previously (Bulchand et al., 2003)16. Plasmid DNAs encoding different LIM genes and co-factors were linearized by restriction digestion to provide template for making DIG-labeled RNA probe16. Briefly, the slides were incubated in hybridization buffer (50% formamide, 5X SSC and 1% SDS) containing DIG-labeled riboprobes (Roche) for 16 hours at 70°C followed by post-hybridization washes using Solution X (50% formamide, 2X SSC and 1% SDS), 2X SSC and 0.2X SSC.\n\nRadioactive in-situ hybridization was carried out as described previously64. Briefly, the slides were incubated in the hybridization buffer (50% formamide, 0.6M sodium chloride, 10mM Tris pH 7.4, 1X Denhardts solution, 10mM dithiotheritol (DTT), 250µg/ml yeast tRNA, 50µg/ml Salmon sperm DNA, 10% Dextran sulphate) containing35 S-UTP labeled riboprobes (Amersham, Buckinghamshire, UK) at a concentration of 106cpm/250µl for 20–24 hours at 60°C. Post-hybridization, the slides were washed with 2XSSC, treated with RNase A (20µg/ml for 30 minutes at 37°C; USB Corporation, Cleveland, Ohio), 0.5X SSC for 30 minutes at 60°C, 0.1X SSC for 20 minutes and then rinsed in double distilled water. Slides were air dried and exposed to Biomax film (Kodak) for 3–6 weeks. To confirm the specificity of the signal observed with antisense riboprobes, controls used were sense riboprobes or RNase treatment (40µg/ml at 37°C for 30 minutes) prior to hybridization.\n\nDensitometric analysis of LIM gene transcript levels was performed using the Macintosh-based Scion Imaging software (Scion, Frederick, Maryland, USA). Sections were observed directly on the monitor using a Sony 3 CCD color video camera (Model DXC-390P).14C standards were used for calibration to correct for non-linearity. An equivalent area was outlined for each of the hippocampal subfields and optical density measurements from both hemispheres of 3–4 individual sections from each animal were analysed to calculate the mean value. Results were subjected to statistical Student’s t-test. Significance was determined at p < 0.05 using GraphPad inSTAT (version 3.05, LaJolla, California, USA). The following numbers of animals were used for each condition: Control adults, n = 7 (Clim1a), 9 (Clim2), 5 (Lhx1), 5 (Lhx2), 5 (Lhx9), 4 (Lmo3), 9 (Lmo4). Kainate treated adults, n = 8 (Clim1a), 8 (Clim2), 5 (Lhx1), 4 (Lhx2), 4 (Lhx9), 3 (Lmo3), 8 (Lmo4). Control pups, n = 6 (Clim1a), 8 (Clim2), 6 (Lhx1), 8 (Lhx2), 5 (Lhx9), 7 (Lmo3), 7 (Lmo4). Kainate treated pups, n = 8 (Clim1a), 8 (Clim2), 8 (Lhx1), 8 (Lhx2), 7 (Lhx9), 8 (Lmo3), 7 (Lmo4).",
"appendix": "Author contributions\n\n\n\nVL, LS, VV, ST conceived the project. VL, LS, DH, AS performed the experiments, analyzed the data, and helped to critically revise the paper. DH, VV, ST analyzed the data and wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a Wellcome trust Senior Fellowship (056684/Z/99/Z), a Swarnajayanti Fellowship (Dept. of Science and Technology, Govt. of India) and a Lady Tata Memorial Trust to ST; a Wellcome Trust International Senior Research Fellowship (04082003114133) to VV, and a Kanwal Rekhi Career Development Award (Tata Institute of Fundamental Research Endowment Fund) to LS.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank B. Anderson, F. Porter, Y. Nakagawa, I. Bach and E. A. Grove for gifts of plasmid DNA to make cRNA probes for in situ hybridization; SB Banerjee for assistance with the radioactive in-situ hybridization experiments. We thank Dr. Yashwantrao Mane and the TIFR Animal House staff for excellent support.\n\n\nReferences\n\nGuillemot F: Cell fate specification in the mammalian telencephalon. Prog Neurobiol. 2007; 83(1): 37–52. PubMed Abstract | Publisher Full Text\n\nPolleux F, Ince-Dunn G, Ghosh A: Transcriptional regulation of vertebrate axon guidance and synapse formation. Nat Rev Neurosci. 2007; 8(5): 331–40. PubMed Abstract | Publisher Full Text\n\nNóbrega-Pereira S, Marín O: Transcriptional control of neuronal migration in the developing mouse brain. Cereb Cortex. 2009; 19(Suppl 1): i107–13. PubMed Abstract | Publisher Full Text\n\nLonze BE, Ginty DD: Function and regulation of CREB family transcription factors in the nervous system. Neuron. 2002; 35(4): 605–23. PubMed Abstract | Publisher Full Text\n\nParrish JZ, Emoto K, Kim MD, et al.: Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields. Annu Rev Neurosci. 2007; 30: 399–423. PubMed Abstract | Publisher Full Text\n\nWest AE, Greenberg ME: Neuronal activity-regulated gene transcription in synapse development and cognitive function. Cold Spring Harb Perspect Biol. 2011; 3(6): a005744. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLundgren SE, Callahan CA, Thor S, et al.: Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous. Development. 1995; 121(6): 1769–73. PubMed Abstract\n\nPorter FD, Drago J, Xu Y, et al.: Lhx2, a LIM homeobox gene, is required for eye forebrain, and definitive erythrocyte development. Development. 1997; 124(15): 2935–4. PubMed Abstract\n\nThor S, Andersson SG, Tomlinson A, et al.: A LIM-homeodomain combinatorial code for motor-neuron pathway selection. Nature. 1999; 397(6714): 76–80. PubMed Abstract | Publisher Full Text\n\nAndo H, Kobayashi M, Tsubokawa T, et al.: Lhx2 mediates the activity of Six3 in zebrafish forebrain growth. Dev Biol. 2005; 287(2): 456–468. PubMed Abstract | Publisher Full Text\n\nMangale VS, Hirokawa KE, Satyaki PR, et al.: Lhx2 selector activity specifies cortical identity and suppresses hippocampal organizer fate. Science. 2008; 319(5861): 304–309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilan M, Cohen SM: Regulation of LIM homeodomain activity in vivo: a tetramer of dLDB and apterous confers activity and capacity for regulation by dLMO. Mol Cell. 1999; 4(2): 267–273. PubMed Abstract | Publisher Full Text\n\nThaler JP, Lee SK, Jurata LW, et al.: LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. Cell. 2002; 110(2): 237–249. PubMed Abstract | Publisher Full Text\n\nMilan M, Diaz-Benjumea FJ, Cohen SM: Beadex encodes an LMO protein that regulates Apterous LIM-homeodomain activity in Drosophila wing development: a model for LMO oncogene function. Genes Dev. 1998; 12(18): 2912–2920. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeihe U, Milan M, Cohen SM: Regulation of Apterous activity in Drosophila wing development. Development. 2001; 128(22): 4615–4622. PubMed Abstract\n\nBulchand S, Subramanian L, Tole S: Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex. Dev Dyn. 2003; 226(3): 460–469. PubMed Abstract | Publisher Full Text\n\nBulchand S, Grove EA, Porter FD, et al.: LIM-homeodomain gene Lhx2 regulates the formation of the cortical hem. Mech Dev. 2001; 100(2): 165–175. PubMed Abstract | Publisher Full Text\n\nSubramanian L, Sarkar A, Shetty AS, et al.: Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus. Proc Natl Acad Sci U S A. 2011; 108(27): E265–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao Y, Sheng HZ, Amini R, et al.: Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5. Science. 1999; 284(5417): 1155–8. PubMed Abstract | Publisher Full Text\n\nYan CH, Levesque M, Claxton S, et al.: Lmx1a and lmx1b function cooperatively to regulate proliferation, specification, and differentiation of midbrain dopaminergic progenitors. J Neurosci. 2011; 31(35): 12413–25. PubMed Abstract | Publisher Full Text\n\nLakhina V, Falnikar A, Bhatnagar L, et al.: Early thalamocortical tract guidance and topographic sorting of thalamic projections requires LIM-homeodomain gene Lhx2. Dev Biol. 2007; 306(2): 703–13. PubMed Abstract | Publisher Full Text\n\nMarcos-Mondéjar P, Peregrín S, Li JY, et al.: The lhx2 transcription factor controls thalamocortical axonal guidance by specific regulation of robo1 and robo2 receptors. J Neurosci. 2012; 32(13): 4372–85. PubMed Abstract | Publisher Full Text\n\nHobert O, Tessmar K, Ruvkun G: The Caenorhabditis elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons. Development. 1999; 126(7): 1547–1562. PubMed Abstract\n\nManetopoulos C, Hansson A, Karlsson J, et al.: The LIM-only protein LMO4 modulates the transcriptional activity of HEN1. Biochem Biophys Res Commun. 2003; 307(4): 891–9. PubMed Abstract | Publisher Full Text\n\nElliott RC, Khademi S, Pleasure SJ, et al.: Differential regulation of basic helix-loop-helix mRNAs in the dentate gyrus following status epilepticus. Neuroscience. 2001; 106(1): 79–88. PubMed Abstract | Publisher Full Text\n\nElliott RC, Miles MF, Lowenstein DH: Overlapping microarray profiles of dentate gyrus gene expression during development- and epilepsy-associated neurogenesis and axon outgrowth. J Neurosci. 2003; 23(6): 2218–27. PubMed Abstract\n\nTakasu MA, Dalva MB, Zigmond RE, et al.: Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors. Science. 2002; 295(5554): 491–5. PubMed Abstract | Publisher Full Text\n\nParent JM, Lowenstein DH: Seizure-induced neurogenesis: are more new neurons good for an adult brain? Prog Brain Res. 2002; 135: 121–131. PubMed Abstract | Publisher Full Text\n\nNadler JV: The recurrent mossy fiber pathway of the epileptic brain. Neurochem Res. 2003; 28(11): 1649–58. PubMed Abstract | Publisher Full Text\n\nOverstreet-Wadiche LS, Bromberg DA, Bensen AL, et al.: Seizures accelerate functional integration of adult-generated granule cells. J Neurosci. 2006; 26(15): 4095–103. PubMed Abstract | Publisher Full Text\n\nScharfman HE, McCloskey DP: Postnatal neurogenesis as a therapeutic target in temporal lobe epilepsy. Epilepsy Res. 2009; 85(2–3): 150–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSperber EF, Haas KZ, Stanton PK, et al.: Resistance of the immature hippocampus to seizure-induced synaptic reorganization. Brain Res Dev Brain Res. 1991; 60(1): 88–93. PubMed Abstract | Publisher Full Text\n\nLynch M, Sayin U, Bownds J, et al.: Long-term consequences of early postnatal seizures on hippocampal learning and plasticity. Eur J Neurosci. 2000; 12(7): 2252–64. PubMed Abstract | Publisher Full Text\n\nPorter BE: Neurogenesis and epilepsy in the developing brain. Epilepsia. 2008; 49(Suppl 5): 50–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlberini CM: Transcription factors in long-term memory and synaptic plasticity. Physiol Rev. 2009; 89(1): 121–45. PubMed Abstract | Publisher Full Text\n\nHinks GL, Shah B, French SJ, et al.: Expression of LIM protein genes Lmo1, Lmo2, and Lmo3 in adult mouse hippocampus and other forebrain regions: differential regulation by seizure activity. J Neurosci. 1997; 17(14): 5549–59. PubMed Abstract\n\nAltman J, Das GD: Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol. 1965; 124(3): 319–35. PubMed Abstract | Publisher Full Text\n\nGuéneau G, Privat A, Drouet J, et al.: Subgranular zone of the dentate gyrus of young rabbits as a secondary matrix. A high-resolution autoradiographic study. Dev Neurosci. 1982; 5(4): 345–58. PubMed Abstract | Publisher Full Text\n\nEckenhoff MF, Rakic P: Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey. J Neurosci. 1988; 8(8): 2729–47. PubMed Abstract\n\nGray WP, Sundstrom LE: Kainic acid increases the proliferation of granule cell progenitors in the dentate gyrus of the adult rat. Brain Res. 1998; 790(1–2): 52–9. PubMed Abstract | Publisher Full Text\n\nWenzel HJ, Woolley CS, Robbins CA, et al.: Kainic acid-induced mossy fiber sprouting and synapse formation in the dentate gyrus of rats. Hippocampus. 2000; 10(3): 244–60. PubMed Abstract | Publisher Full Text\n\nLein ES, Zhao X, Gage FH: Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization. J Neurosci. 2004; 24(15): 3879–89. PubMed Abstract | Publisher Full Text\n\nDong H, Csernansky CA, Goico B, et al.: Hippocampal neurogenesis follows kainic acid-induced apoptosis in neonatal rats. J Neurosci. 2003; 23(5): 1742–9. PubMed Abstract\n\nOkazaki MM, Evenson DA, Nadler JV: Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats: visualization after retrograde transport of biocytin. J Comp Neurol. 1995; 352(4): 515–34. PubMed Abstract | Publisher Full Text\n\nSmith BN, Dudek FE: Short- and long-term changes in CA1 network excitability after kainate treatment in rats. J Neurophysiol. 2001; 85(1): 1–9. PubMed Abstract\n\nParent JM, Yu TW, Leibowitz RT, et al.: Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci. 1997; 17(10): 3727–38. PubMed Abstract\n\nJiang M, Lee CL, Smith KL, et al.: Spine loss and other persistent alterations of hippocampal pyramidal cell dendrites in a model of early-onset epilepsy. J Neurosci. 1998; 18(20): 8356–68. PubMed Abstract\n\nRibak CE, Navetta MS: An immature mossy fiber innervation of hilar neurons may explain their resistance to kainate-induced cell death in 15–day-old rats. Brain Res Dev Brain Res. 1994; 79(1): 47–62. PubMed Abstract | Publisher Full Text\n\nCornejo BJ, Mesches MH, Coultrap S, et al.: A single episode of neonatal seizures permanently alters glutamatergic synapses. Ann Neurol. 2007; 61(5): 411–26. PubMed Abstract | Publisher Full Text\n\nCross DJ, Cavazos JE: Synaptic reorganization in subiculum and CA3 after early-life status epilepticus in the kainic acid rat model. Epilepsy Res. 2007; 73(2): 156–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGray WP, May K, Sundstrom LE: Seizure induced dentate neurogenesis does not diminish with age in rats. Neurosci Lett. 2002; 330(3): 235–8. PubMed Abstract | Publisher Full Text\n\nBender RA, Dubé C, Gonzalez-Vega R, et al.: Mossy fiber plasticity and enhanced hippocampal excitability, without hippocampal cell loss or altered neurogenesis, in an animal model of prolonged febrile seizures. Hippocampus. 2003; 13(3): 399–412. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu H, Kaur J, Dashtipour K, et al.: Suppression of hippocampal neurogenesis is associated with developmental stage, number of perinatal seizure episodes, and glucocorticosteroid level. Exp Neurol. 2003; 184(1):196–213. PubMed Abstract | Publisher Full Text\n\nWatanabe Y, Johnson RS, Butler LS, et al.: Null mutation of c-fos impairs structural and functional plasticities in the kindling model of epilepsy. J Neurosci. 1996; 16(12): 3827–36. PubMed Abstract\n\nKashani AH, Qiu Z, Jurata L, et al.: Calcium activation of the LMO4 transcription complex and its role in the patterning of thalamocortical connections. J Neurosci. 2006; 26(32): 8398–408. PubMed Abstract | Publisher Full Text\n\nPennypacker KR, McMillian MK, Douglass J, et al.: Ontogeny of kainate-induced gene expression in rat hippocampus. J Neurochem. 1994; 62(2): 438–44. PubMed Abstract | Publisher Full Text\n\nTauck DL, Nadler JV: Evidence of functional mossy fiber sprouting in hippocampal formation of kainic acid-treated rats. J Neurosci. 1985; 5(4): 1016–22. PubMed Abstract\n\nRepresa A, Jorquera I, Le Gal La Salle G, et al.: Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites? Hippocampus. 1993; 3(3): 257–68. PubMed Abstract | Publisher Full Text\n\nLynch M, Sutula T: Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats. J Neurophysiol. 2000; 83(2): 693–704. PubMed Abstract\n\nPerez Y, Morin F, Beaulieu C, et al.: Axonal sprouting of CA1 pyramidal cells in hyperexcitable hippocampal slices of kainate-treated rats. Eur J Neurosci. 1996; 8(4): 736–748. PubMed Abstract | Publisher Full Text\n\nLee SK, Pfaff SL: Synchronization of neurogenesis and motor neuron specification by direct coupling of bHLH and homeodomain transcription factors. Neuron. 2003; 38(5): 731–45. PubMed Abstract | Publisher Full Text\n\nXu B, Li S, Brown A, et al.: EphA/ephrin-A interactions regulate epileptogenesis and activity-dependent axonal sprouting in adult rats. Mol Cell Neurosci. 2003; 24(4): 984–99. PubMed Abstract | Publisher Full Text\n\nChen HH, Schock SC, Xu J, et al.: Extracellular ATP-dependent upregulation of the transcription cofactor LMO4 promotes neuron survival from hypoxia. Exp Cell Res. 2007; 313(14): 3106–16. PubMed Abstract | Publisher Full Text\n\nNair A, Vadodaria KC, Banerjee SB, et al.: Stressor-specific regulation of distinct brain-derived neurotrophic factor transcripts and cyclic AMP response element-binding protein expression in the postnatal and adult rat hippocampus. Neuropsychopharmacology. 2007; 32(7): 1504–19. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2010",
"date": "11 Oct 2013",
"name": "Bin Chen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLakhina et al. examine the effect of chemically induced seizure on the expression of Lim family members and their cofactors, specially within the rodent hippocampus. Using in situ hybridization to measure mRNA levels they demonstrate that transcription of Lim, Lmo and Clim family members is altered in a field-specific manner specifically in adult but not early postnatal (P7) rats. These interesting observations extend previous work on seizure induced changes in Lmo1-3 expression (Hinks et al.) and raise several important questions for future study:How rapid and persistent are the changes in gene expression following kainic acid administration? Do alterations in gene expression drive physical remodeling within hippocampal neurons or vise verse? Are there field specific differences in the timing of transcriptional changes? What are the functional consequences of these transcriptional changes? It is interesting that changes in transcription were observed in adult but not juvenile animals, whose circuits generally believed to be more plastic. Might these transcriptional changes be a means by which to counteract decreasing circuit plasticity in adults?",
"responses": []
},
{
"id": "4163",
"date": "28 Mar 2014",
"name": "Stan Leung",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 showed field-specific changes in expression of LIM-HD genes Lhx1, Lhx2, and Lhx9, LIM-only gene Lmo4, and cofactor Clim1a in the adult hippocampus at 6 hours after seizure induction by kainic acid. No significant changes in expression were observed after seizure induction with the same dose of kainic acid in rat pups of postnatal day 7 (P7). The subfield-specific changes in expression after kainic acid seizures were clearly presented for different members of the LIM-HD/ LIM-only family of transcription factors. This work extends previous studies that showed seizure-induced changes in expression of LIM-only genes in adult rats. The lack of expression change in P7 rat pups may be related to the lack of seizure-induced structural plasticity in immature animals, i.e., lack of cell death, mossy fiber sprouting and neurogenesis. Members of the LIM-HD family of transcription factors are necessary for different aspects of the development of the hippocampus. Thus, the changes in these transcription factors after seizures in adult rats may be related to seizure-induced neural plasticity, such as axon sprouting, progenitor proliferation, and changes in neuronal cell survival. The central question of whether LIM-HD/ LIM-only transcription factors are causally related to the neural plasticity induced by kainic acid seizures remains unanswered. Are transient changes in these transcription factors, at ~6 hours after seizures, sufficient or necessary for the structural changes induced by kainic acid seizures? The design of the present study does not allow the consequences of kainic acid seizures to be studied, and not all adult rats induced with kainic acid seizures showed plasticity to the same extent. In fact, whether seizures are essential for the expression changes is not totally clear. In other studies, a single dose of kainic acid (intraperitoneal 10 mg/kg) may not induce seizures in all adult rats, and the 10 mg/kg i.p. given to P7 rats was >5 times the dose needed for seizures as reported by Lynch et al. (Eur J Neurosci, 2000). The authors should provide some justification of the kainic acid dose used, in terms of seizure induction in their adult and P7 rats. “Regulation” in the title apparently means “changes in expression” rather than better control or lack of control of the genes and cofactors.The results are presented in an organized manner.I would prefer to see the sample sizes (Ns) in the figure legend rather than in the “Quantitation and data analysis” section.The issue of whether seizures are necessary and sufficient for the expression changes can be further discussed. Perhaps other molecules implicated for seizure-induced plasticity, e.g., neurotrophins, should be mentioned.Overall, the experimental study appears to be well done.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-205
|
https://f1000research.com/articles/2-204/v1
|
04 Oct 13
|
{
"type": "Research Article",
"title": "Isoform-specific anti-MeCP2 antibodies confirm that expression of the e1 isoform strongly predominates in the brain",
"authors": [
"Lara Kaddoum",
"Nicolas Panayotis",
"Honoré Mazarguil",
"Giuseppina Giglia-Mari",
"Jean Christophe Roux",
"Etienne Joly",
"Nicolas Panayotis",
"Honoré Mazarguil",
"Giuseppina Giglia-Mari",
"Jean Christophe Roux"
],
"abstract": "Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene. MeCP2 transcripts are alternatively spliced to generate two protein isoforms (MeCP2_e1 and MeCP2_e2) that differ at their N-termini. Whilst mRNAs for both forms are expressed ubiquitously, the one for MeCP2_e1 is more abundant than for MeCP2_e2 in the central nervous system. In transfected cells, both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci. With a view to understanding the physiological contribution of each isoform, and their respective roles in the pathogenesis of Rett syndrome, we set out to generate isoform-specific anti-MeCP2 antibodies. To this end, we immunized rabbits against the peptides corresponding to the short amino-terminal portions that are different between the two isoforms. The polyclonal antibodies thus obtained specifically detected their respective isoforms of MeCP2 in Neuro2a (N2A) cells transfected to express either form. Both antisera showed comparable sensitivities when used for Western blot or immunofluorescence, and were highly specific for their respective isoform. When those antibodies were used on mouse tissues, specific signals were easily detected for Mecp2_e1, whilst Mecp2_e2 was very difficult to detect by Western blot, and even more so by immunofluorescence. Our results thus suggest that brain cells express low amounts of the Mecp2-e2 isoform. Our findings are compatible with recent reports showing that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.",
"keywords": [
"Rett syndrome (RTT) is a dominant X-linked neurological disorder that affects girls. It is a progressive disease with symptoms appearing around 6 to 18 months after birth. After a normal developmental period",
"girls show growth retardation",
"microcephaly",
"stereotypic hand movements",
"motor abnormalities",
"mental retardation and communication dysfunction1. Most RTT cases are sporadic",
"but using information from rare familial cases",
"Amir et al. did manage to identify mutations in the MECP2 gene as the origin of 95% of classic RTT cases2."
],
"content": "Introduction\n\nRett syndrome (RTT) is a dominant X-linked neurological disorder that affects girls. It is a progressive disease with symptoms appearing around 6 to 18 months after birth. After a normal developmental period, girls show growth retardation, microcephaly, stereotypic hand movements, motor abnormalities, mental retardation and communication dysfunction1. Most RTT cases are sporadic, but using information from rare familial cases, Amir et al. did manage to identify mutations in the MECP2 gene as the origin of 95% of classic RTT cases2.\n\nThe MECP2 gene encodes for the methyl-CpG binding protein 2, an abundant nuclear protein identified in 1992 for its capacity to bind methylated DNA3. MeCP2 is particularly abundant in mature neurons and favors brain development and maturation4–6. Although MeCP2 was initially thought to be mostly a transcription factor, it has become apparent, over the past few years, that MeCP2 is expressed at extremely high levels in mature neurons, and that one of its central functions is to influence chromatin architecture by assuming a histone H1-like role7.\n\nThe MECP2 gene consists of four exons giving rise to two different isoforms of the protein due to an alternative splicing of the mRNA. In addition, the MECP2 mRNA has a long highly conserved 3′-UTR with three sites of polyadenylation generating three different transcripts for each isoform. The first isoform to be described was MeCP2_e2, which contains all four exons with the initiation site in exon 2 giving rise to a protein of 486 amino acids in humans and 484 in mice8. MeCP2_e2 is also sometimes referred to as MeCP2A, mostly in humans, or MeCP2β, mostly in mice. The MeCP2_e1 isoform (also called MeCP2B or MeCP2α was identified eleven years later, both in human9 and mouse10. It lacks exon 2, and thus consists of exons 1, 3 and 4, with the starting codon in exon 1, giving rise to a protein of 498 amino acids in humans, and 501 in mice (see Bienvenu and Chelly8 and Figure 1A).\n\nAdaptation from Mnatzakanian et al.9 and Kriaucionis and Bird10, showing alternative splicing for the e1 (or B, or α) isoform above the gene and for the e2 (or A or β) isoform below the gene. B). Alignment of mouse (m) and human (h) MeCP2_e1 and MeCP2_e2 proteins sequences. Boxes delineate the peptides’ sequences chosen for immunization: in the N-terminal region peptides correspond to regions that differ between the two isoforms, whereas in the C-terminal region, the amino acid sequences are identical to both isoforms. The sequence of the 15-mer peptide used to produce the Abcam2828 antibody is indicated by the red line.\n\nRT-PCR analyses have revealed the presence of two transcripts in all tissues in human and mouse, and also demonstrated that the MeCP2_e1 mRNA isoform is more abundant than MeCP2_e2 in the brain, thymus and lung5,9–11. Although mRNAs for MeCP2 containing exon 2 have been identified in many placental mammals, including primates, carnivores and herbivores, many arguments suggest that the MeCP2_e1 protein may be the dominant form expressed in the brain, and the one which is more relevant to the physiopathology of RTT.\n\nFirstly, in the MeCP2_e2 mRNA, the ATG start codon present in exon 1 is followed by a very short open reading frame that terminates after 55 nucleotides (in mouse) before the starting codon in exon 2. Kriaucionis and Bird actually demonstrated that the presence of this first ATG results in very inefficient translation of the MeCP2_e2 protein10.\n\nSecond, the ancestral form of MeCP2 inferred from sequence comparisons with non-mammalian vertebrates corresponds to MeCP2_e110.\n\nThird, until now, in the hundreds of sequences for MeCP2 genes obtained from patients affected by RTT, no mutation has yet been found in exon 2. On the other hand, work carried out between 2005 and 2009 has already revealed the presence of more than 10 different mutations (deletions and missense) in exon 1 in patients with classical or atypical (mild and severe form) RTT12.\n\nFourth, in two patients showing classical phenotypes of RTT, but without seizures or microcephaly, Saunders et al. identified mutations affecting the initiation codon of MeCP2_e112, which would result in the lack of translation of the MeCP2_e1 protein, but would be expected to somewhat ‘restore’ higher expression of MeCP2_e2, similarly to what has been reported for the mouse cDNA10. Furthermore, at the meeting of the American Society for Human Genetics held in San Francisco in November 2012, the group of J. LaSalle reported that transgenic KO mice carrying the equivalent single point mutation in exon 1, invalidating the initiation codon for MeCP2-e1, recapitulated a typical RTT-like phenotype13.\n\nLastly, a recent report documented that, in four classical RTT patients harbouring mutations in exon 1, there were normal levels of MeCP2_e2 mRNA14.\n\nOn the other hand, even if the mRNA coding for the MeCP2_e2 protein isoform does not suffice to prevent a RTT phenotype15, this isoform must be able to fulfill most functions of MeCP2_e1 since, in MeCP2 KO mice, the pathologic phenotype could be rescued by a tau-driven transgene based on the MeCP2_e2 cDNA16. It is also worthy of note that for historical reasons, many of the studies based on the expression of recombinant MeCP2 were using cDNA constructs coding for the MeCP2_e2 isoform. A recent study has, however, established that expression of either isoform could prevent the RTT-like phenotype in MeCP2-/y mice, although the rescue of the clasping and motor phenotypes was significantly higher with MeCP2_e117.\n\nTo explore the expression of each protein isoform in vivo, and understand their respective contributions to Mecp2-related pathologies and especially RTT, we thought that it would be very valuable to have access to antibodies that could detect them specifically and separately. In this regard, however, all the anti-MeCP2 antibodies that we had at our disposal had been raised against portions common to the two isoforms, and would thus recognize them both. With a view to obtain truly isoform-specific antibodies, and since the commonly accepted minimal size for a peptide epitope is five amino acids, we chose to immunize rabbits with shorter peptides that overlapped by only three amino acids between the two MeCP2 isoforms. Since the initiation of our study, we have been made aware of the existence of commercially available polyclonal rabbit antibodies generated against the first 15 amino acids of MeCP2_e2, namely Abcam2828, and Thermo PA1-881. For our purpose, however, a concern with these antibodies is that the 15 amino acid sequence of the peptides used for immunizations overlaps with the MeCP2_e1 sequence by six amino acids, and the possibility thus remains that this could be sufficient to lead to some cross-reaction against e1.\n\nHere, we describe the generation of these two isoform-specific antibodies, and their characterization in comparison to Abcam2828 by staining of cells transfected to express either one or the other of the two isoforms, both by immunofluorescence and Western blotting. Those antibodies were then used to compare expression of the two isoforms in the brain, which revealed a prominent expression of the e1 isoform, whilst e2 remained barely detectable.\n\n\nMaterial and methods\n\nAll experimental procedures involving animals were carried out in keeping with the European guidelines for the care and use of laboratory animals (EU directive 2010/63/EU). No specific approval was required for this study since animals were bred and sacrificed under approved conditions, and not submitted to any experimental procedures. Both Jean Christophe Roux in Marseille and Etienne Joly hold licenses to experiment on animals, and to oversee other people doing experiments under their supervision and responsibility. Their respective numbers are 13–405 for Dr Roux, and 31–40 for Etienne Joly.\n\nFor each antibody, two New Zealand white rabbits were immunized with a synthetic peptide corresponding either to amino acids 1–12 of human MeCP2_e2 (MVAGMLGLREEK-C), or to amino acids 14–24 of human MeCP2_e1 (C-GGGEEERLEEK) (Figure 1). The cysteine residues were added for conjugation to carrier proteins for immunizations, or to resins for affinity purification. For immunization, we chose thyroglobulin, to which each peptide was coupled using maleimide. Rabbit immunizations were carried out by Eurogentec, Angers, France. Four injections with the thyroglobulin-conjugated peptides were performed on day 0, 21, 49, and 77 (two rabbits per peptide). With the e2 peptide, the sera obtained from both rabbits gave indistinguishable results in all immunofluorescence and Western blot experiments where they were initially compared, and most subsequent experiments were thus carried out with only one of the two. For the e1 peptide, however, only one rabbit responded satisfactorily. The sera from the three rabbits having responded satisfactorily were affinity-purified by MilleGen (Labège, France) on columns coupled to the appropriate peptides. We will refer to those antibodies as αe1 and αe2 respectively.\n\nThe third antibody, αe1+e2, recognizes both MeCP2 isoforms, and was raised against (C-PRPNREEPVDSRTP) as described previously by others18 and ourselves19. This polyclonal antibody was produced for us by MilleGen (Labège, France). Rabbits received five injections on day 0, 12, 23, 44 and 57 with the KLH-conjugated peptide we provided. One of the two rabbits responded satisfactorily, and the serum obtained from that rabbit gave sufficient titers and specificity to be used without further purification.\n\nAll three peptides used were synthesized by FMOC chemistry, purified by HPLC and coupled to carrier proteins by the IPBS core facility. Thyroglobulin, KLH and maleimide were all purchased from Sigma.\n\nThis antibody was purchased from Abcam (Cambridge, UK), kept as frozen aliquots as recommended by the manufacturer, and used in parallel to the three antibodies we generated ourselves.\n\nMice used in this study were male B6.129P2(C)-Mecp2tm1.1Bird/J obtained from the Jackson Laboratory (USA), carrying either the inactivated Mecp2 allele, or their wild type (WT) littermates. For the mouse experiments, hemizygous mutant males (Mecp2-/y) were generated by crossing heterozygous females (Mecp2+/-) with C57BL/6 males. Genotyping was performed by routine PCR technique according to the Jackson Laboratory protocols as previously described20. A total number of 10 Mecp2−/y and 10 WT mice were used in the study. For histology, the Mecp2−/y and WT mice were sacrificed at postnatal day 24 (P24; n=2 WT; n=2 Mecp2−/y) and day 55 (P55; n=4 WT; n=4 Mecp2−/y). For Western blotting, a total of eight mice (four pairs of WT and Mecp2−/y littermates) were sacrificed over the course of the study, at ages ranging from 3 to 6 weeks.\n\npcDNA3.1(A) vectors expressing Myc-tagged human MeCP2_e1 and MeCP2_e2 were provided by Dr. Berge A. Minassian of the Hospital for Sick Children, Toronto, Ontario, Canada9.\n\nNeuro2a (N2A) cells, a mouse neuroblastoma cell line, were originally obtained from the ATCC (USA), and G418-resistant clones of stably transfected N2A cells expressing Myc-His-tagged human MeCP2_e1 or MeCP2_e2 were maintained in DMEM 10% fetal calf serum supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate (+ 0.5 mg/ml G418 for the transfectants). All tissue culture reagents were from Gibco Life Technologies.\n\n2.5×105 N2A cells were plated overnight on glass coverslips in 24-well plates. Cells were washed twice with PBS and fixed with paraformaldehyde 3.7% in PBS for 30 min at room temperature (RT). After two washes with PBS, cells were permeabilized in 0.3% Triton-X100 for 10 min at RT. After 2×15 min washes with PBS/FCS 2%, cells were incubated with Rabbit anti-MeCP2 serum (1:300 dilution in PBS/FCS 10%) for 1 h at RT. Cells were again washed 3×5 min before incubation with Alexa Fluor 633-labelled polyclonal goat anti-rabbit, (Invitrogen, cat n° A-21070) diluted 1:300 in PBS/FCS 10% for 1 h at RT. Cells were washed 3×5 min with PBS/FCS 2% and after a final wash with PBS, coverslips were mounted in Vectashield Mounting medium with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride; Vector Labs, Peterborough, UK). We used a Zeiss LSM710 confocal laser scanning microscope, with a 40× oil immersion objective to visualize the stained cells. Images were acquired and analyzed with the ImageJ software (Version 1.47, http://rsb.info.nih.gov/ij/).\n\nCellular extracts from N2A cells or mouse brain tissues were prepared as described previously21. After evaluation of the protein contents using a Bradford assay (BioRad), adjusted amounts of proteins from these extracts were loaded onto acrylamide gels. After PAGE separation, proteins were transferred to nitrocellulose membrane (0.45 μm Whatman) (VWR, France). Membranes were blocked overnight at 4°C with TBS (Tris 10 mM, NaCl 0.15 M, pH 7.4 containing 3% skimmed powder milk (non-fat Régilait, France) and 0.1% Tween-20). TBS blocking buffer was always centrifuged (100000 g for 45 min) and filtered with 0.2 mm filters. Membranes were then incubated with one of the four rabbit anti-MeCP2 antibodies diluted in blocking buffer for 1 h at RT (αe1 was diluted to 1/4000, αe2 to 1/4000, Abcam2828 to 1/1000 and αe1+e2 to 1/4000). After 4×10 min washes in PBS/0.1% Tween buffer, membranes were incubated with goat anti-rabbit conjugated to horseradish peroxidase (HRP) (BioRad, 1:10000 dilution in blocking buffer) for 1 h at RT. Finally, the blots were washed 4×10 min in PBS/0.1% Tween buffer and revealed with an ECL kit (Pierce, Thermo Fisher Scientific, Brebières, France) and using Amersham hyperfilm ECL (high performance chemiluminescence, GE Healthcare).\n\nMice were sacrificed with a lethal pentobarbitone injection (100 mg kg−1 i.p.) and transcardially perfused with NaCl for 1 min, followed by PBS 0.1 M containing 4% paraformaldehyde for 10 min. Dissected brains were then postfixed for 5 h in the same solution, placed overnight in PBS 0.1 M containing 20% sucrose and frozen at −80°C.\n\nBrain sections (20 µm) were cut using a cryostat (Microm Microtech, France), permeabilized for 15 min in PBS Triton 0.1%, blocked for 45 min with 7% normal goat serum (NGS, cat n° 005–000_121, Jackson ImmunoResearch Laboratories, Newmarket, England), and incubated overnight at RT with primary antibodies (αe1, αe2 or Abcam2828) diluted 1/300 in PBS containing 3.5% NGS. The following day, sections were washed, incubated 2 h at RT with goat polyclonal anti-rabbit alexa 546 (Life technologies, cat. n° 11010 diluted 1:400 in PBS containing 3.5% NGS and re-washed. After 5 min incubation with DAPI, slides were mounted in Shandon Immu-Mount (ThermoFisher). Immunostained slices were analyzed using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP camera (Princeton Instruments, Trenton, NJ, USA). Pictures were then analyzed with ImageJ software.\n\nImmunoquantification of the staining levels was performed using the same protocol previously described in detail22. In order to compare the intensity of staining in Mecp2-deficient mice and their respective controls, great care was taken at all stages to perform all steps of fixation, freezing, cutting, staining and scanning of the images under the same conditions, using the same solutions and timing, and by alternating tissues coming from the two genotypes. The fluorescence intensity was carefully selected in order to avoid reaching saturation. Densitometric analysis of the staining level was performed on 8-bit images using ImageJ software. The integrated density was calculated as the sum of the values of the pixels region of interest, i.e. in the nuclei as defined by DAPI staining. We analyzed pictures at high magnification (63×) and for each section stained by either αe1, αe2 or Abcam 2828, we measured the Mecp2 staining level of at least 50 DAPI-positive cells. Immunoquantifications were always performed by successively alternating the wild-type and the Mecp2-deficient samples.\n\n\nResults and discussion\n\nMeCP2_e1 and MeCP2_e2 differ only by short stretches in their N-terminal regions, which makes it challenging to define isoform-specific peptides that will be long enough to serve as useful immunogens (Figure 1). In addition, to obtain reagents that would be useful both in mouse and human samples, we had to design and synthesize two peptides that matched both the human and mouse sequences. The first sequence finally chosen corresponded to amino acids 14–24 of human MeCP2_e1 (C-GGGEEERLEEK) and the second to amino acids 1–12 of human MeCP2_e2 (MVAGMLGLREEK-C). Although these peptides do carry the same C-terminal sequence of the three amino acids EEK, we felt that this was very unlikely to lead to cross reactivity since it is commonly accepted that the minimal size for a peptide as an antibody epitope is five amino acids. Cysteine residues were added to the N-terminus of the e1 peptide and to the C-terminus of the e2 peptide for coupling to carrier proteins for immunizations or resins for purification. Rabbits were immunized as described in materials and methods and sera were harvested by terminal exsanguination 9 days after the final injection. Antibodies were affinity purified on columns coupled to their respective peptides, and yielded a total of 2 mg for the αe1 antibody and 3 and 5 mg for the two αe2 antibodies respectively.\n\nIn addition, we also generated αe1+e2, a polyclonal antibody reacting against a peptide present in both isoforms of human and mouse MeCP2, as described previously by others18 and ourselves19. This peptide corresponds to amino acids 467–480 of human MeCP2_e1, and 479–492 of human MeCP2_e2.\n\nFinally, we also purchased the commercial antibody Abcam2828, which has been raised against the first 15 amino acids of MeCP2_e2. Since the immunogenic peptide used overlaps only by 6 amino acids with the MeCP2_e1, this antibody would be expected to recognize MeCP2_e2 preferentially, but we feared that it may show some cross reactivity against MeCP2_e1.\n\nTo test the specificity of the antibodies, we used transfected clones of the mouse neuroblastoma cell line N2A which stably express either MeCP2_e1 or MeCP2_e2 (see materials and methods). Despite the neuronal lineage of the N2A cells, it is worth emphasizing that undifferentiated N2A cells do not express any detectable level of endogenous MeCP223. As can be seen in Figure 2, after staining with the αe1+e2 antibody, both isoforms were expressed at similar levels in their respective clones, and, as expected, distributed similarly in nuclei, leading to labeling that co-localized closely with heterochromatin, as revealed by DAPI staining.\n\nN2A cells (left columns), or stable N2A transfectants expressing either hMeCP2_e1-myc-his (middle columns) or hMeCP2_e2-myc-his (right columns) were plated on coverslips and labeled with the following antibodies: αe1 (first line), αe2 (second line), Abcam2828 (third line) and αe1+e2 (bottom line), followed by anti-rabbit Alexa633 secondary antibody. Slides were mounted with DAPI-containing medium, and observed with a 40x oil immersion objective. Similar results were obtained in at least five other experiments.\n\nFor the αe1 antibody, we found that staining was highly specific of the MeCP2_e1 isoform, with no staining detectable either in the MeCP2_e2 transfectants, or in untransfected N2A cells. Conversely, with the αe2 and Abcam2828 antibodies, we found that the staining obtained was highly specific of the MeCP2_e2 isoform and absolutely no staining was detectable at the level of the heterochromatin in the MeCP2_e1 transfectants. On the other hand, both antibodies gave some background staining on untransfected N2A, but with different and remarkably reproducible patterns: the αe2 antibody led to cytoplasmic background signals, whilst the Abcam2828 antibody gave weak signals that were intra-nuclear, but that were more nucleoplasmic rather than overlapping with heterochromatin (Figure 2 and Figure S1). It is clear, however, that none of these signals were due to MeCP2 since untransfected N2A cells did not express any detectable levels of MeCP2 with the αe1+e2 antibody (Figure 2). Quite remarkably, we noticed that the levels of cytoplasmic staining with the αe2 antibody appeared much more intense when there were no cells expressing MeCP2_e2. This suggests that, in the αe2 polyclonal mix, some antibodies that bind with high affinity to the N-terminal portion of MeCP2 may actually cross react with some other cytoplasmic component(s), but with lower affinity. Blast searches of mammalian protein sequences with the sequence of the peptide used for immunizing the rabbits identified proteins of the plectin family as prime candidates since those have cytoplasmic distributions, and many start with MVAGML, the same six amino acids as found in the MeCP2_e2 peptide used for immunizations (Figure 1B).\n\nTo characterize the specificity of these antibodies further, we next used Western blot analysis of nuclear and total extracts prepared from the same transfected N2A cells, as well as extracts from either WT or MeCP2 KO mouse brains (Figure 3).\n\nAs described in materials and methods, protein extracts were prepared from N2A cells or from stable N2A transfectants expressing either hMeCP2_e1-myc-his or hMeCP2_e2-myc-his, as well as from brains of two C57 male MeCP2 KO mice, and from their two respective WT littermates. Four nitrocellulose membranes were prepared in parallel from the same extracts, stained respectively with the four antibodies αe1+e2, αe1, αe2 and Abcam2828, and revealed with chemiluminescence. All blots were exposed on the same piece of film for 5 minutes. Similar results were obtained in at least three other experiments.\n\nStaining with the αe1+e2 antibody confirmed the observation made by immunofluorescence that untransfected N2A cells expressed absolutely no detectable endogenous MeCP2, and that both sets of transfectants were expressing similar levels of either MeCP2_e1 or MeCP2_e2. In all experiments, and in agreement with previous reports5,10,18,25,26, the apparent electrophoretic mobility of the MeCP2 proteins was around 70 KDa, slightly slower than would be expected from the predicted 52 KDa molecular weight. As expected, the e1 isoform, which is 11 amino acids longer, was also found to migrate slightly more slowly than e2. Although the mouse MeCP2_e1 is predicted to be longer than the human form by three amino acids, the slightly slower electrophoretic mobility seen for the human MeCP2 proteins expressed by transfected N2A cells compared to the ones expressed in mouse brains can be explained by the myc-his C-terminal tag carried by this protein, which amounts to slightly more than 20 amino acids9.\n\nStaining with the αe1 and αe2 antibodies confirmed their specificity for their respective isoform, with each revealing only the band corresponding to the MeCP2 isoform it was raised against. For the Abcam2828 antibody, although the signals obtained were clearly almost exclusively detected in the lane containing MeCP2_e2, we could not rule out that this antibody may cross react weakly on MeCP2_e1. Indeed, this antibody shows nonspecific cross-reactivity against two bands which migrate just above and just below the level where MeCP2 is expected, but, in the case shown here, there also was a slightly stronger signal in between these two bands in the N2A-MeCP2_e1 lane than in the N2A control lane. Having repeated this experiment at least four times, we have, however, found it very difficult to document this cross-reactivity reproducibly, with the signal being sometimes undetectable, and sometimes even more prominent than the example shown here.\n\nIf we now consider the results obtained with cellular brain extracts of MeCP2-KO and WT mice, we can first see that both αe1+e2 and αe1 efficiently detected a specific signal for MeCP2 in the WT and not in the KO brain extracts. For αe2, although there was a specific signal of the expected size, it was much weaker than the signal obtained with αe1. In fact, in a first set of experiments, we had initially failed to detect this band, and it is only by using very optimized protocols and very sensitive photographic films that we have managed to detect this band in brain extracts reliably.\n\nAnother important factor for the detection of this faint band was to use electrophoretic runs that led to good resolution around 50 kDa to separate the MeCP2 signal from the non specific bands running just below. Those two prominent bands, which were not present in the N2A extracts, are clearly non-specific since they are equally present in WT and KO extracts. Since they are also weaker in nuclear than in total cell extracts, they probably correspond to a cytoplasmic protein, possibly of the plectin family alluded to earlier.\n\nWith the Abcam2828 antibody, the specific signals corresponding to MeCP2 were much easier to detect than with the αe2 antibody. Since the Abcam2828 antibody may cross-react weakly on MeCP2_e1, the possibility remains that it is not our αe2 antibody which works less well than Abcam2828, but that the stronger signal obtained with the latter may be due to the sum of the detection of the two isoforms.\n\nAltogether, these observations support the already established notion that the e1 isoform is expressed at higher levels in the brain than the e2 isoform.\n\nNext, we prepared tissue sections from different regions of the brains of normal C57BL/6 and MeCP2 KO mice. Figure 4 shows examples of the results obtained on lateral posterior thalamic nucleus regions of 55 days old mice. Similar results were obtained with several sections, and on sections of other brain areas such as the cortex and the Substantia Nigra reticulata, and on the cortex of 24 days old mice (see Figure S2–Figure S4). Using the αe1 antibody, we confirmed the absence of labeling on KO tissues while the staining of WT samples revealed the expected nuclear punctuated labeling which co-localized perfectly with the DAPI signals. By contrast, with the αe2 or the Abcam2828 antibodies, we only obtained very faint levels of nuclear staining, which appeared slightly stronger for the Abcam2828 antibody, but were in both cases too weak to be able to discern whether they co-localized with heterochromatin. Although it only provides indicative values, it is possible to use the ImageJ software to quantify the signals obtained by immunofluorescence (see materials and methods). By doing this specifically in nuclei, we found WT/KO signal ratios of about 30 for the αe1 antibody, but only 1,3 for αe2 and 1,8 for Abcam2828, thus bringing further support to the notion that it is mostly the e1 isoform which is expressed in the brain, whilst e2 is only present in trace amounts.\n\n20 μm brain sections from a C57 male MeCP2 KO mouse, and from a WT littermate, were prepared as described in materials and methods. These sections were stained with αe1 (top section), αe2 (middle section) and Abcam2828 (bottom section), followed by anti-rabbit Alexa 546 secondary antibody, and mounted in DAPI-containing mounting medium.\n\n\nDiscussion\n\nWe have succeeded in generating rabbit polyclonal antibodies directed against each of the two MeCP2 isoforms, MeCP2_e1 and MeCP2_e2, and also compared the latter to the commercially available antibody Abcam2828. Whilst each of the two antibodies we have generated were found to be clearly specific of their respective isoform, and suitable for using by Western blot and immunofluorescence, we could not rule out that Abcam2828 did not cross react slightly on MeCP2_e1. Hence, the stronger signals obtained with Abcam2828 on immunostaining brain sections as well as on Western blots with brain extracts could either be due to a better reactivity of this antibody on MeCP2_e2 when it is expressed in vivo, or to a weak cross reaction on MeCP2_e1. At any rate, even if the stronger signals obtained with Abcam2828 were due to a better reactivity with MeCP2_e2, our data strongly support the already established views that the MeCP2_e1 isoform is not only the most abundant isoform in the organism, but also the one involved in the physiopathology of RTT15.\n\nThis raises the question of why the e2 exon should be conserved in so many, if not all, placental mammals. One possibility is that e2 could be predominantly expressed in other tissues outside the brain. In preliminary experiments, however, we have found a similar predominance of the e1 isoform in lung, heart, liver, spleen and thymus, without managing to detect any significant levels of the e2 protein by Western blot in any of those tissues (unpublished observation). It may be, however, that we did not look in the right place or at the right time. Indeed, very recently published works have suggested that the e2 isoform may play important roles for embryo viability and embryogenesis15, as well as in regulating apoptosis in post-mitotic neurons27. In future experiments, it will thus be interesting to use the αe2 antibody to investigate whether the expression of the e2 isoform can be detected during embryogenesis, or in the brain parenchyma under conditions where post-mitotic neurons undergo apoptosis.\n\nAnother intriguing observation in our results concerns the differences seen between the reactivities of the αe2 and Abcam2828 antibodies in immunofluorescence on cells in culture and on brain sections. Indeed, while both antibodies stain N2A cells expressing the e2 isoform with high efficiency, Abcam2828 resulted in noticeably stronger signals on brain sections than the αe2 antibody, albeit still much lower than those obtained with the e1-specific or the pan-reactive reagents. Whilst this may be due to better reactivity of the Abcam2828 antibody on the e2 protein when it is expressed in vivo, the possibility remains that this stronger signal may be due to a cross reactivity of that antibody on the e1 isoform since it is expressed at very high levels in the CNS. Although Abcam2828 showed no detectable cross reactivity on the e1 isoform expressed in N2A cells by immunofluorescence, this may be due to the conformation of the e1 isoform not being exactly the same in transfected N2A cells and in mature neurons. It could thus be that, when it is expressed in mature neurons, the e1 isoform can adopt a conformation which is more prone to cross react with Abcam2828, as may well be the case in the context of Western blots. An alternative explanation would be that the e2 isoform is found in a different conformation when expressed in transfected N2A cells or in vivo, and that the latter is recognized less efficiently by our αe2 antibody than by Abcam2828. In future experiments, it should be possible to use transgenic mice in which either one or the other of the MeCP2 isoforms has been specifically knocked out13,15 to discriminate between these two possibilities.\n\nOf note, a very recent report by the group of M. Rastegar has described the generation of an e1-specific chicken polyclonal antibody raised against the very same e1 peptide which we used to immunize rabbits28. Comfortingly, the results we obtained with αe1 are in complete agreement with those in that report, i.e. absolute specificity for the e1 isoform. On tissue slices, both antibodies revealed the same patterns of expression, i.e. strongest in cortical neurons, and overlapping perfectly with the patterns obtained with antibodies that do not discriminate between the two isoforms. While it is somewhat more practical to have reagents raised in the same species to compare the levels of expression of each of the isoforms, having access to reagents raised in different species, such as rabbits and chickens in this case, could prove very useful to combine the two types of reagents for double staining experiments, to monitor expression of the two isoforms in parallel in the same tissue sections.",
"appendix": "Author contributions\n\n\n\nLK, NP, JCR, EJ: conceived, designed and performed the experiments, analysed the data and wrote the manuscript. GGM supervised some of LK’s work. HM prepared the peptides and conjugated them to the carrier proteins.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Association Française du Syndrome de Rett (AFSR), the Fondation pour la Recherche Medicale (FRM) and the E-RARE EuroRETT network. JCR and EJ were supported by INSERM (Institut National pour la Santé et la Recherche Médicale). NP was supported by an INSERM-Région Provence-Alpes-Côte d’Azur fellowship, HM was supported by CNRS (Centre National pour la Recherche Scientifique).\n\n\nAcknowledgements\n\nWe are grateful to Laurent Villard for helpful discussions and suggestions, as well as to the anonymous referees who, when this manuscript was submitted to a different journal, made useful comments and suggestions, and pointed out the existence of commercial antibodies that may have been e2-specific.\n\n\nSupplementary figures\n\nFor αe2, the signals are mostly cytoplasmic, and for Abcam2828, they are intra-nuclear, but overlap with regions that are less intensely stained with DAPI, and thus more likely correspond to nucleoli than to heterochromatin.\n\n20 μm brain sections from a C57 male MeCP2 WT mouse were prepared as described in materials and methods. These sections were stained with αe1 (top section), αe2 (middle section) and Abcam2828 (bottom section), followed by anti-rabbit Alexa 546 secondary antibody, and mounted in DAPI-containing mounting medium.\n\n20 μm brain sections from a C57 male MeCP2 WT mouse were prepared as described in materials and methods. These sections were stained with αe1 (top section), αe2 (middle section) and Abcam2828 (bottom section), followed by anti-rabbit Alexa 546 secondary antibody, and mounted in DAPI-containing mounting medium.\n\n20 μm brain sections from a C57 male MeCP2 WT mouse were prepared as described in materials and methods. These sections were stained with αe1 (top section), αe2 (middle section) and Abcam2828 (bottom section), followed by anti-rabbit Alexa 546 secondary antibody, and mounted in DAPI-containing mounting medium.\n\n\nReferences\n\nChahrour M, Zoghbi HY: The story of Rett syndrome: from clinic to neurobiology. Neuron. 2007; 56(3): 422–437. PubMed Abstract | Publisher Full Text\n\nAmir RE, Van den Veyver IB, Wan M, et al.: Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 1999; 23(2): 185–188. PubMed Abstract | Publisher Full Text\n\nLewis JD, Meehan RR, Henzel WJ, et al.: Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell. 1992; 69(6): 905–914. PubMed Abstract | Publisher Full Text\n\nKaufmann WE, Johnston MV, Blue ME: MeCP2 expression and function during brain development: implications for Rett syndrome's pathogenesis and clinical evolution. Brain Dev. 2005; 27(Suppl 1): S77–S87. PubMed Abstract | Publisher Full Text\n\nShahbazian MD, Antalffy B, Armstrong DL, et al.: Insight into Rett syndrome: MeCP2 levels display tissue- and cell-specific differences and correlate with neuronal maturation. Hum Mol Genet. 2002; 11(2): 115–124. PubMed Abstract | Publisher Full Text\n\nKishi N, Macklis JD: MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Mol Cell Neurosci. 2004; 27(3): 306–321. PubMed Abstract | Publisher Full Text\n\nSkene PJ, Illingworth RS, Webb S, et al.: Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state. Mol Cell. 2010; 37(4): 457–468. PubMed Abstract | Publisher Full Text\n\nBienvenu T, Chelly J: Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized. Nat Rev Genet. 2006; 7(6): 415–426. PubMed Abstract | Publisher Full Text\n\nMnatzakanian GN, Lohi H, Munteanu I, et al.: A previously unidentified MECP2 open reading frame defines a new protein isoform relevant to Rett syndrome. Nat Genet. 2004; 36(4): 339–341. PubMed Abstract | Publisher Full Text\n\nKriaucionis S, Bird A: The major form of MeCP2 has a novel N-terminus generated by alternative splicing. Nucleic Acids Res. 2004; 32(5): 1818–1823. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDragich JM, Kim YH, Arnold AP, et al.: Differential distribution of the MeCP2 splice variants in the postnatal mouse brain. J Comp Neurol. 2007; 501(4): 526–542. PubMed Abstract | Publisher Full Text\n\nSaunders CJ, Minassian BE, Chow EW, et al.: Novel exon 1 mutations in MECP2 implicate isoform MeCP2_e1 in classical Rett syndrome. Am J Med Genet A. 2009; 149A(5): 1019–1023. PubMed Abstract | Publisher Full Text\n\nYasui DH, Dunaway KW, Golub MS, et al.: Isoform-selective MeCP2-e1 deficient mice recapitulate the neurologic defecits of Rett syndrome. Meeting Abstract, ASHG San Francisco. 2012. Reference Source\n\nGianakopoulos PJ, Zhang Y, Pencea N, et al.: Mutations in MECP2 exon 1 in classical Rett patients disrupt MECP2_e1 transcription, but not transcription of MECP2_e2. Am J Med Genet B Neuropsychiatr Genet. 2012; 159B(2): 210–216. PubMed Abstract | Publisher Full Text\n\nItoh M, Tahimic CG, Ide S, et al.: Methyl CpG-binding protein isoform MeCP2_e2 is dispensable for Rett syndrome phenotypes but essential for embryo viability and placenta development. J Biol Chem. 2012; 287(17): 13859–13867. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuikenhuis S, Giacometti E, Beard CF, et al.: Expression of MeCP2 in postmitotic neurons rescues Rett syndrome in mice. Proc Natl Acad Sci U S A. 2004; 101(16): 6033–6038. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKerr B, Soto CJ, Saez M, et al.: Transgenic complementation of MeCP2 deficiency: phenotypic rescue of Mecp2-null mice by isoform-specific transgenes. Eur J Hum Genet. 2012; 20(1): 69–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou Z, Hong EJ, Cohen S, et al.: Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation. Neuron. 2006; 52(2): 255–269. PubMed Abstract | Publisher Full Text\n\nKaddoum L, Magdeleine E, Waldo G, et al.: One-step split GFP staining for sensitive protein detection and localization in mammalian cells. Biotechniques. 2010; 49(4): 727–8, 730, 732 passim. PubMed Abstract | Publisher Full Text\n\nMiralves J, Magdeleine E, Joly E: Design of an improved set of oligonucleotide primers for genotyping MeCP2tm1.1Bird KO mice by PCR. Mol Neurodegener. 2007; 2: 16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews NC, Faller DV: A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991; 19(9): 2499. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoux JC, Dura E, Villard L: Tyrosine hydroxylase deficit in the chemoafferent and the sympathoadrenergic pathways of the Mecp2 deficient mouse. Neurosci Lett. 2008; 447(1): 82–86. PubMed Abstract | Publisher Full Text\n\nYoung JI, Hong EP, Castle JC, et al.: Regulation of RNA splicing by the methylation-dependent transcriptional repressor methyl-CpG binding protein 2. Proc Natl Acad Sci U S A. 2005; 102(49): 17551–17558. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShi SR, Key ME, Kalra KL: Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991; 39(6): 741–748. PubMed Abstract | Publisher Full Text\n\nChen RZ, Akbarian S, Tudor M, et al.: Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nat Genet. 2001; 27(3): 327–331. PubMed Abstract | Publisher Full Text\n\nTao J, Hu K, Chang Q, et al.: Phosphorylation of MeCP2 at Serine 80 regulates its chromatin association and neurological function. Proc Natl Acad Sci U S A. 2009; 106(12): 4882–4887. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDastidar SG, Bardai FH, Ma C, et al.: Isoform-specific toxicity of Mecp2 in postmitotic neurons: suppression of neurotoxicity by FoxG1. J Neurosci. 2012; 32(8): 2846–2855. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZachariah RM, Olson CO, Ezeonwuka C, et al.: Novel MeCP2 isoform-specific antibody reveals the endogenous MeCP2E1 expression in murine brain, primary neurons and astrocytes. PLoS One. 2012; 7(11): e49763. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "2161",
"date": "23 Oct 2013",
"name": "Mario Costa",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 small but interesting paper in which the authors describe the generation of two new and more specific anti-MeCP2 antibodies recognizing the Mecp2 isoforms (Mecp2_e1, and Mecp2_e2). In addition the authors, by using these new tools, are able to revisit and confirm the recent data indicating that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.The data and the ideas herein presented are clear and, as also discussed by the authors, there is a growing interest in understanding the functional relationship between the two isoforms of Mecp2. The approach in the antibody preparation is classical, the techniques are well accepted, the data analysis are rigorous and the results are convincing.Intriguingly, ERK 1/2 (Extracellular Regulated Kinase 1 and 2) like MeCP2 (e1 and e2) presents two different isoforms (in this case originated from two different genes) and the ERK1 isoform posses an N terminal basically identical to that of MeCP2. This data, together with the highly conserved sequence in MeCP2s and ERKs reinforce the idea that this kind of N-terminal could be involved in important biological functions. I think that further speculation on this topic could add additional value to the manuscript.",
"responses": []
},
{
"id": "2346",
"date": "13 Nov 2013",
"name": "Jean-François Brunet",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, backed by very convincing and carefully discussed images of immunofluorescence stains and Western blots, describes novel antibodies specific for each of the two MeCP2 isoforms, useful tools no doubt, for the Rett syndrome community. These antibodies allow the authors to conclude, in line with many other clearly summarized previous findings, that one isoform (e1) massively dominates in the adult brain. The text could be shortened a bit.",
"responses": []
},
{
"id": "2439",
"date": "14 Nov 2013",
"name": "Mike Johnston",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting paper provides new evidence that the e2 isoform is absent in brain and it is consistent with the earlier paper by Itoh et al., 2012 demonstrating that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-204
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https://f1000research.com/articles/2-202/v1
|
03 Oct 13
|
{
"type": "Case Report",
"title": "Epidermal growth factor receptor inhibitor therapy for recurrent respiratory papillomatosis",
"authors": [
"Matthew M. Moldan",
"Bruce C Bostrom",
"Robert J Tibesar",
"Timothy A. Lander",
"James D. Sidman",
"Matthew M. Moldan",
"Bruce C Bostrom",
"Robert J Tibesar",
"Timothy A. Lander"
],
"abstract": "The epidermal growth factor pathway has been implicated in various tumors, including human papillomavirus (HPV) lesions such as recurrent respiratory papillomatosis (RRP). Due to the presence of epidermal growth factor receptors in RRP, epidermal growth factor receptor (EGFR) inhibitors have been utilized as adjuvant therapy. This case series examines the response to EGFR inhibitors in RRP. Four patients with life-threatening RRP were treated with EGFR inhibitors. Operative frequency and anatomical Derkay scores were calculated prior to, and following EGFR inhibitor treatment via retrospective chart review. The anatomical Derkay score decreased for all four patients after initiation of EGFR inhibitor therapy. In one patient, the operative frequency increased after switching to an intravenous inhibitor after loss of control with an oral inhibitor. In the other patients there was a greater than 20% decrease in operative frequency in one and a more than doubling in the time between procedures in two. This study suggests that EGFR inhibitors are a potential adjuvant therapy in RRP and deserve further study in a larger number of patients.",
"keywords": [
"Recurrent respiratory papillomatosis (RRP) occurs with human papillomavirus (HPV) infection of the respiratory tract epithelium",
"typically by HPV types 6 and 111",
"2. It is the most common benign neoplastic laryngeal disorder in children3. RRP has been associated with an increased risk of airway obstruction4. Juvenile onset RRP is more severe and results in more intensive therapy2. The mainstay of RRP management remains surgical debulking",
"however",
"adjuvant therapies are offered in up to a fifth of cases of RRP5."
],
"content": "Introduction\n\nRecurrent respiratory papillomatosis (RRP) occurs with human papillomavirus (HPV) infection of the respiratory tract epithelium, typically by HPV types 6 and 111,2. It is the most common benign neoplastic laryngeal disorder in children3. RRP has been associated with an increased risk of airway obstruction4. Juvenile onset RRP is more severe and results in more intensive therapy2. The mainstay of RRP management remains surgical debulking; however, adjuvant therapies are offered in up to a fifth of cases of RRP5.\n\nAs a result of the low prevalence of RRP, large controlled trials of adjuvant therapies have been limited. Epidermal growth factor receptor (EGFR) inhibitors have been used as an adjuvant therapy due to the presence of EGFR in papillomas6. This case series examines the use of EGFR inhibitors in four patients with life-threatening RRP.\n\n\nMaterials and methods\n\nThis was an institutional review board (IRB) approved study to examine the response to EGFR inhibitors in RRP. A waiver of informed consent and an IRB waiver of HIPPA authorization were approved by the IRB prior to the advent of the study as the data was anonymised. Those patients with severe RRP, defined as requiring more than four surgical procedures per year with rapid regrowth of papilloma leading to airway compromise, who had been treated with EGFR inhibitors, were identified and their medical charts reviewed. The interval between operations was based upon the physicians’ intraoperative determination of disease burden and was not determined by a specific protocol. Surgical procedures used included carbon dioxide (CO2) laser and microdebrider or a combination thereof. The operative notes were reviewed to determine a modified Derkay Severity Score, utilizing the anatomical portion of the scoring system, at the time of each surgical debulking7. The EGFR inhibitors used, all of which are FDA approved for EGFR expressing malignancies, included erlotinib (Tarceva®), gefitinib (Iressa®), and panitumumab (Vectibix®). Erlotinib (starting dose of 85 mg/m2 PO rounded to nearest 12.5 mg) or gefitinib (starting dose of 325 mg/m2 PO rounded to nearest 50 mg) were administered daily while panitumumab (starting dose of 150 mg/m2 IV) was given immediately following each operation with a minimum of every two weeks. An oral medication was initiated first (gefitinib if available or erlotinib if gefitinib was not available). If there was a concern for oral bioavailability or inadequate response to the oral EGFR inhibitor, patients were then transitioned to IV panitumumab. Informed consent was obtained for all patients.\n\n\nResults\n\nFour patients from 2003 through 2012 met the criteria listed above. Table 1 includes patient demographic and disease-specific information. EGFR expression and associated grade was determined by immunohistochemical analysis prior to initiation of EGFR inhibitor therapy and is also reported in Table 18. The outcome measures for each patient following adjuvant therapy were compared to their own measures prior to therapy; therefore the diversity in regard to age, viral type, and number of operations within the patient group had no effect on outcome.\n\nM=Male, F=Female, EGFR=Epidermal Growth Factor Receptor.\n\nThe outcome data is included in Table 2. Prior to start of adjuvant therapy the Derkay scores of three of the patients were increasing. Following initiation of the EGFR inhibitor therapy, the Derkay score decreased for all four patients. In one patient, the operative frequency actually increased. She was previously well controlled on oral gefitinib but her condition acutely worsened. This prompted a switch to intravenous panitumumab. Contrastingly, in another patient there was a greater than 20% improvement (decrease) in the operative frequency. In the remaining two patients the time interval between procedures more than doubled.\n\n* Data are reported as median.\n\nPatient 1. A 3 year old female was diagnosed at 6 months of age and was solely managed surgically until 14 months of age. Prior to panitumumab treatment her Derkay score was increasing and she required weekly operations. She had previously received a trial of erlotinib and celecoxib with minimal effect. She had 23 operations prior to starting IV panitumumab at a dose of 200 mg/m2.\n\nFollowing the switch, her operation frequency decreased, with an operation every 26 days and modified Derkay score of 6 on average (Table 2). The patient has undergone 15 IV treatments with some minimal side effects. These have included a skin rash that decreased with ongoing therapy and resolved off therapy, diarrhea limited by probiotic use, an MRSA leg abscess and untreated iron deficiency without anemia that resolved off therapy. During the most recent operation, her airway was free from papillomas and panitumumab treatment was discontinued and the patient has remained free of recurrence off therapy for 12 months.\n\nPatient 2. A 6 year old male was diagnosed with RRP at the age of 2. He received nine microdebrider procedures at an outside hospital prior to referral to us for adjuvant therapy. He was first started on erlotinib and celecoxib, which failed to control his disease. The patient had received 30 surgical procedures prior to initiation of IV panitumumab at a dose of 130 mg/m2, later increased to 200 mg/m2 to improve response.\n\nFollowing panitumumab treatment, his operative frequency and Derkay score have decreased (Table 2). Side effects have included increased thirst, nocturnal enuresis, an acneform rash and untreated iron deficiency without anemia. His height has fallen from the 50th to 25th percentile over two years while on panitumumab. The patient began the HPV vaccination series a few months after starting panitumumab. He has also received diindolylmethane (DIM, Bioresponse®) 150 mg 2–3 times daily. Due to a recent improvement in his condition, his surgical interval has been lengthened to approximately twelve weeks.\n\nPatient 3. A 7 year old female was diagnosed at 9 months. Shortly thereafter she received a tracheostomy and G-tube at an outside institution due to the extent of the RRP. Her condition continued to worsen and her care was transferred to our institution for adjuvant therapy.\n\nAfter failure of a short trial of erlotinib (60 mg/m2 daily for 1 month followed by 85 mg/m2 daily for 1 month) she was managed briefly with interferon alpha 1.5 million units by subcutaneous injection daily prior to initiation of gefitinib at an initial dose of 325 mg. She was managed with gefitinib and surgical debulking for nearly five years. The patient was discovered to be non-compliant once in the past with worsening of her modified Derkay score after which her mother closely monitored gefitinib administration. More recently, her RRP acutely worsened after which it was decided to switch to IV panitumumab at a dose of approximately 150 mg/m2 (later increased to 200 mg/m2). The patient had received 69 laser procedures and four requiring combined mechanical and laser debridement prior to panitumumab.\n\nSide effects have included a rash, increased urinary frequency and nocturnal enuresis, and occasional mild fevers with no known source. Her supraglottic disease was so severe that she was only being treated, and thus scored, for tracheal disease. She was also noted to have progressive iron deficiency without anemia that did not respond to oral iron, though it did improve with IV iron sucrose. She has been less than 5th percentile for height but continues to grow. A recent bone age study showed delay. After another period of worsening RRP, panitumumab was increased to 265 mg/m2 and DIM was added. Subsequently oral propranolol was added at 3 mg/kg/day divided into three doses per day (TID) without effect after more than a month. Subsequent to this regime, the panitumumab dose was decreased to 200 mg/m2 and daily gefitinib (270 mg/m2/day) restarted with no significant change in operative frequency.\n\nPatient 4. A 23 year old male was diagnosed at the age of 3 months. His case has been reported in a previous study6. The patient’s comorbidities include interferon-induced nephrotic syndrome with stage III acute renal failure from focal segmental glomerulosclerosis, hypertension and developmental delay. He was born at 28 weeks gestation. He required a tracheostomy as a result of extensive laryngeal stenosis secondary to RRP. The patient was managed with interferon alfa-2a until approximately 8 years of age.\n\nDue to life-threatening airway obstruction 10 years ago, he was started on gefitinib at an initial dose of approximately 400 mg/m2/day divided twice daily because it was thought the patient would not survive. His Derkay score was based upon the trachea and bronchi only as these were the areas being treated due to complete stenosis of the larynx.\n\nAdverse effects have included acne-form rash, dry skin, and minimal diarrhea. He also developed iron deficiency anemia two years ago requiring iron infusions and occasional blood transfusion. An extensive workup has not found a source of blood loss. He continued to receive gefitinib with satisfactory control of his RRP until June 2013 when a trial period off gefitinib was started in conjunction with increased surgical debridement at the family’s request.\n\n\nDiscussion\n\nOur interest in EGFR inhibitors stemmed from the benefit observed with the use of gefitinib in a patient with life-threatening RRP (patient 4 above)6. Three EGFR inhibitors—panitumumab, gefitinib, and erlotinib—have been utilized in these four patients. In our hands, decrease in papilloma severity has been observed only with panitumumab and gefitinib. The lack of response to erlotinib is surprising given that it targets EGFR and there is a prior report of response to erlotinib and celecoxib9. Interestingly there is one prior report of the use of intravenous cetuximab, another EGFR inhibitor, in RRP without effect10. The use of EGFR inhibitors in the treatment of RRP is off-label. However, due to the EGFR expression in these patients’ papillomas it was hypothesized that EGFR antagonists could offer benefit.\n\nThis case series is justifiably limited in scope; it nevertheless suggests that EGFR inhibitors are a potential adjuvant therapy for the treatment of RRP. Beneficial results were observed in all four patients as evidenced by improvement in the Derkay score. In three of the four patients the operative frequency decreased, with the time interval between operations lengthening to twice what was required prior to EGFR inhibitor therapy in two subjects.\n\nNone of the patients experienced serious adverse effects from the medications with minimal tolerable adverse events noted. Common side effects of EGFR inhibitors include skin rash, hypomagnesemia, paronychia, fatigue, abdominal pain, nausea and diarrhea. Two patients (patients 2 and 3) developed nocturnal enuresis on panitumumab one of which (patient 3) did not have this side effect while on gefitinib. This has not been previously described with panitumumab or other EGFR inhibitors. All patients developed iron deficiency and one severe anemia which has also not been described previously for the use of EGFR inhibitors. A possible mechanism is EGFR inhibition leading to an increase in hepcidin preventing absorption of oral iron11.\n\nVarious factors could play a role in the response to adjuvant therapy. These may include: the extent of surgical excision, the method used for surgical debulking, the natural course of the disease and the aggressiveness of the virus. None of the patients have a systemic immune deficiency as an explanation for this variability. For these reasons the study of adjuvant therapies in RRP is problematic. Additionally, as with other adjuvant treatments studied for RRP, the number of patients included in this series is very limited. Larger trials comparing the efficacy of EGFR inhibitors with other promising therapies, such as intralesional cidofovir or vorinostat, are needed in order to determine the relative efficacy of these treatments12,13. EGFR inhibitors appear to improve papilloma disease control as an adjunct to surgical therapy—at least in some patients. They do not appear to eradicate papillomas and at the current time the optimal duration of therapy is unknown. The major side effect to date has been skin rash, which has been successfully managed with emollients, sunscreen for sun exposure and selective use of antibiotics for acneform lesions. Although there may be unknown risks from long-term therapy with EGFR inhibitors, our opinion is that the benefits described outweigh the possibility of future unknown risks.\n\n\nConclusion\n\nThis case series suggests that EGFR inhibitors are a potential adjuvant therapy for the treatment of RRP as evidenced by the improvement in the modified Derkay scores and the general decrease in operative frequency.\n\n\nConsent\n\nA waiver of informed consent and an IRB waiver of HIPPA authorization were approved by the IRB prior to advent of the study.",
"appendix": "Author contributions\n\n\n\nJS and BB conceived the study. All authors assisted with the design of the study. MM carried out the research. All authors contributed to the analysis and interpretation of the data. MM prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\nMatthew Moldan had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\n\nCompeting interests\n\n\n\nJS has served as a consultant to Medtronic. All other authors have no competing interests to disclose.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nSara Froyen-Gernbacher, RN for outstanding nurse case management and infusion therapy for patients in this study.\n\nMeixia Liu for assistance with analysis of and interpretation of data.\n\n\nReferences\n\nWiatrak B, Derkay CS: Recurrent respiratory papillomatosis: a review. Laryngoscope. 2008; 118(7): 1236–1247. PubMed Abstract | Publisher Full Text\n\nReeves WC, Ruparelia SS, Swanson KI, et al.: National registry for juvenile-onset recurrent respiratory papillomatosis. Arch Otolaryngol Head Neck Surg. 2003; 129(9): 976–982. PubMed Abstract | Publisher Full Text\n\nMorgan AH, Zitch RP: Recurrent respiratory papillomatosis in children: a retrospective study of management and complications. Ear Nose Throat J. 1986; 65(9): 19–28. PubMed Abstract\n\nLeung R, Hawkes M, Campisi P: Severity of juvenile onset recurrent respiratory papillomatosis is not associated with socioeconomic status in a setting of universal health care. Int J Pediatr Otorhinolaryngol. 2007; 71(6): 965–972. PubMed Abstract | Publisher Full Text\n\nSchraff S, Derkay CS, Burke B, et al.: American Society of Pediatric Otolaryngology members’ experience with recurrent respiratory papillomatosis and the use of adjuvant therapy. Arch Otolaryngol Head Neck Surg. 2004; 130(9): 1039–1042. PubMed Abstract | Publisher Full Text\n\nBostrom B, Sidman J, Marker S, et al.: Gefitinib therapy for Life-threatening Laryngeal Papillomatosis. Arch Otolaryngol Head Neck Surg. 2005; 131(1): 64–67. PubMed Abstract | Publisher Full Text\n\nDerkay CS, Malis DJ, Zalzal G, et al.: A Staging System for Assessing Severity of Disease and Response to Therapy in Recurrent Respiratory Papillomatosis. Laryngoscope. 1998; 108(6): 935–937. PubMed Abstract | Publisher Full Text\n\nLyford-Pike S, Westra WH, Loyo M, et al.: Differential expression of epidermal growth factor receptor in juvenile and adult-onset recurrent respiratory papillomatosis. Histopathology. 2010; 57(5): 768–770. PubMed Abstract | Publisher Full Text\n\nLimsukon A, Susanto I, Soo Hoo GW, et al.: Regression of Recurrent Respiratory Papillomatosis with Celecoxib and Erlotinib Combination Therapy. Chest. 2009; 136(3): 924–926. PubMed Abstract | Publisher Full Text\n\nLoyo M, Pai SI, Netto GJ, et al.: Aggressive recurrent respiratory papillomatosis in a neonate. Int J Pediatr Otorhinolaryngol. 2008; 72(6): 917–920. PubMed Abstract | Publisher Full Text\n\nGoodnough JB, Ramos E, Nemeth E, et al.: Inhibition of hepcidin transcription by growth factors. Hepatology. 2012; 56(1): 291–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDerkay CS, Volsky PG, Rosen CA, et al.: Current use of intralesional cidofovir for recurrent respiratory papillomatosis. Larygoscope. 2012; 123(3): 705–712. PubMed Abstract | Publisher Full Text\n\nYuan H, Myers S, Wang J, et al.: Use of Reprogrammed Cells to Identify Therapy for Respiratory Papillomatosis. N Engl J Med. 2012; 367(13): 1220–1227. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2261",
"date": "03 Dec 2013",
"name": "Julie Bauman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nM. Moldan and colleagues report an intriguing, retrospective case series of four patients with severe recurrent respiratory papillomatosis (RRP) treated with epidermal growth factor receptor (EGFR) inhibitors. Two descriptive features, including a numerical increase in the intersurgical interval for three of four cases, and a numerical decrease in anatomic Derkay score in all four, prompted the authors’ conclusion that EGFR inhibitors have a potential adjuvant role in the management of RRP. Inclusion in this report required both severe disease (defined by the authors as requiring at least four debridements per year) and treatment with an EGFR inhibitor. Authors describe neither a systematic process for case identification, nor how many cases were excluded – raising the question of how selective reporting was avoided. Particular caution is warranted in clinical interpretation of the results, due to the variable natural history of RRP. The average pediatric patient requires approximately 20 lifetime surgical procedures, including airway debridement. However, 19% of patients demonstrate a more aggressive course requiring more than 40 lifetime procedures, and a small percentage of patients will spontaneously remit1-3. Patterns of surgical frequency for individuals with RRP indicate significant fluctuation in intersurgical intervals, independent of adjuvant use4. Moreover, the anatomic Derkay score improved in both the cidofovir and placebo groups in a randomized, controlled trial in juvenile RRP5. Thus, descriptive improvements in intersurgical interval or Derkay score as noted in this selective, retrospective report must be treated with caution. This case series, along with other anecdotal reports in the literature 6-8, may justify a prospective, randomized trial but do not establish EGFR inhibitor therapy as an adjuvant for RRP.The use of EGFR inhibitor therapy in RRP, including this report, has been justified by the presence of EGFR over-expression in laryngeal papillomas relative to normal laryngeal epithelium9. Of note, EGFR expression by immunohistochemistry has been a disappointing biomarker of response to EGFR inhibitor therapy in cancers of the lung, colon and head and neck – areas where EGFR inhibitors are currently FDA approved10-12. EGFR expression is unlikely to be an appropriate selection factor for patients with RRP to be treated with EGFR inhibitor therapy. Of particular relevance to this question, in head and neck cancer, EGFR expression is lower in cancers driven by human papillomavirus (HPV) than those driven by tobacco and other environmental carcinogens13,14. Nonetheless, HPV appears to deregulate EGFR independent of expression level: in preclinical models, the E6 HPV oncoprotein increases phosphorylation of EGFR, downstream signaling, and internalization of activated receptor forms15. The importance of EGFR inhibitor therapy, if confirmed in prospective, randomized trials in RRP or other HPV-related neoplasms, will likely relate to HPV biology rather than EGFR expression level per se.",
"responses": []
},
{
"id": "4085",
"date": "31 Mar 2014",
"name": "Lluís Nisa",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nJuvenile-onset respiratory papillomatosis is often a severe disease, with an extraordinary burden on the quality of life of children and their families. Surgical approaches, especially debulking and, when not avoidable, tracheotomy, remain the mainstay of therapy for this devastating disease. Therefore, the need for adjuvant medical therapies that may allow long-term remission is evident1-3. In this sense, the current case series by Moldan and collaborators is worthy of attention. The series also stresses the fact that several medical adjuvant treatments are available but, unfortunately, none has shown conclusive evidence in terms of efficacy. The authors treated four patients with an off-label indication for EGFR inhibitors, often combining several molecules (antibodies and tyrosine kinase inhibitors) in the same patients. Furthermore, most patients had in the past received other forms of medical therapy (e.g. celecoxib, ribavirin, INF alpha-2A). It is interesting that patients initially treated with erlotininb (patients 1, 2, and 3), when switched to panitumumab responded better in two cases. This is worthy of attention, as the same holds true for the improved results of cetuximab versus erlotinib in the treatment for head and neck cancer4. As the authors and Dr. Bauman state, the major difficulty in the choice of a given adjuvant treatment, including EGFR inhibitors, is the lack of readouts for response stratification. As for oropharyngeal cancer, immunohistochemical evidence of EGFR overexpression is not an accurate predictor of treatment responses. The same holds true for every medical adjuvant therapy. The relatively low incidence of severe RRP makes it difficult to set up proper randomized clinical trials, a problem which may be palliated by multicenter efforts.As the authors correctly state, their findings must be considered as preliminary data which need further in-depth evaluation before any specific recommendations may be formulated. As an outlook, it is likely that further understanding of the molecular pathogenesis of HPV-related tumorigenesis is going to lead to the development of new approaches, for example PI3K inhibitors (given the association between HPV and PI3K mutations in cancer)5.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-202
|
https://f1000research.com/articles/2-200/v1
|
02 Oct 13
|
{
"type": "Correspondence",
"title": "Issues with data transformation in genome-wide association studies for phenotypic variability",
"authors": [
"Xia Shen",
"Lars Rönnegård",
"Lars Rönnegård"
],
"abstract": "The purpose of this correspondence is to discuss and clarify a few points about data transformation used in genome-wide association studies, especially for phenotypic variability. By commenting on the recent publication by Sun et al. in the American Journal of Human Genetics, we emphasize the importance of statistical power in detecting functional loci and the real meaning of the scale of the phenotype in practice.",
"keywords": [
"Recently",
"Sun et al.1 raised an interesting suggestion concerning the use of variance-stabilization transformations in genome-wide association studies (GWAS) for phenotypic variability. Specifically",
"Sun et al. revisited Yang et al.’s2 results on the variability-controlling locus FTO for human body mass index (BMI) and claimed that the underlying variability across genotypes might not be as large as Yang et al. had seen. Although it was an important point that Sun et al. discussed",
"especially when quantitatively studying phenotypic variability has become such a hot topic",
"it is our opinion that there are some issues with the transformation approach that Sun et al. proposed."
],
"content": "Correspondence\n\nRecently, Sun et al.1 raised an interesting suggestion concerning the use of variance-stabilization transformations in genome-wide association studies (GWAS) for phenotypic variability. Specifically, Sun et al. revisited Yang et al.’s2 results on the variability-controlling locus FTO for human body mass index (BMI) and claimed that the underlying variability across genotypes might not be as large as Yang et al. had seen. Although it was an important point that Sun et al. discussed, especially when quantitatively studying phenotypic variability has become such a hot topic, it is our opinion that there are some issues with the transformation approach that Sun et al. proposed.\n\nFirst of all, if we take Sun et al.’s transformation according to Yang et al.’s phenotypic mean and variance per FTO genotype class, i.e. a one-to-one map through an inverse hyperbolic sine function, the BMI scale will become rather different compared with the ordinary measurement that we normally use (Figure 1). On the transformed scale of BMI, the difference between two persons who have a BMI of 24 and 25 kg/m2 is much larger than that between two BMIs of 20 and 21 kg/m2, which is strange in reality since the original BMI scale is what we commonly use and also what we care about. Sun et al.’s main argument here is that nearly all the measurement units are manmade. However, considering one of the traits of most interest, e.g. height, why should we regard the difference between 160cm and 170cm different from 170cm and 180cm? Although the definitions of most units can be arbitrary, some measurement scales do have meaning in real life.\n\nThe transformation was determined by the phenotypic distribution across FTO genotypes reported by Yang et al.2.\n\nSecondly, a key problem with Sun et al.’s transformation in practice is that such a transformation is marker-specific. Namely, when performing a GWAS, one needs to transform the phenotypic records differently for different markers, according to the phenotypic distribution across the genotypes per marker. This does not make much sense in practical analyses, because if there is a \"best\" scale of the phenotype, it should be used for all the markers across the genome, before testing the association between the phenotype and the markers. Using the tested marker to determine the transformation of the phenotype is strange. If a marker-specific transformation can be estimated, one should estimate a genome-specific transformation for GWAS, instead of doing different transformations marker-by-marker.\n\nThirdly, if the transformation of the phenotype is determined by one marker showing a significant effect on the phenotypic variability before testing the other markers, another significant effect on the phenotypic variability might be created due to such a transformation. In such a situation, it is problematic to decide which phenotypic scale we should choose.\n\nFourthly, several recent studies discussed that gene-gene or gene-environment interactions could cause significant variance heterogeneity across genotypes3–6, which makes testing variance-controlling loci a powerful tool to reveal potential interaction effects. Reducing the difference in variance across genotypes using a marker-specific variance-stabilization transformation would dramatically reduce such power. Regarding the biological sense of genetically regulated variance heterogeneity, empirical evidence has shown that a single causal locus could show a much higher significant effect on variance compared with the mean6. In a particular population, such a locus may only be mappable through testing the variability rather than the magnitude of the phenotype.\n\nThe above issues cause us to question Sun et al.’s transformation in practice. The scale of the phenotype is certainly an important concern when interpreting an effect on phenotypic variability7. However, one needs to be careful for the points above before applying any transformation on the data. In particular, the statistical power in detecting functional loci and the real meaning of the scale used should be emphasized.",
"appendix": "Author contributions\n\n\n\nXS and LR initiated the study. XS performed the analysis. Both authors contributed to writing the report.\n\n\nCompeting interests\n\n\n\nThe authors declare no competing interest.\n\n\nGrant information\n\nXS is funded by a Future Research Leaders grant from Swedish Foundation for Strategic Research (SSF) to Örjan Carlborg. LR is funded by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS).\n\n\nReferences\n\nSun X, Elston R, Morris N, et al.: What Is the Significance of Difference in Phenotypic Variability across SNP Genotypes? Am J Hum Genet. 2013; 93(2): 390–397. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYang J, Loos RJ, Powell JE, et al.: FTO genotype is associated with phenotypic variability of body mass index. Nature. 2012; 490(7419): 267–272. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRönnegård L, Valdar W: Detecting major genetic loci controlling phenotypic variability in experimental crosses. Genetics. 2011; 188(2): 435–447. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStruchalin MV, Dehghan A, Witteman JC, et al.: Variance heterogeneity analysis for detection of potentially interacting genetic loci: method and its limitations. BMC Genet. 2010; 11: 92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParé G, Cook NR, Ridker PM, et al.: On the use of variance per genotype as a tool to identify quantitative trait interaction effects: a report from the Women's Genome Health Study. PLoS Genet. 2010; 6(6): e1000981. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen X, Pettersson M, Rönnegård L, et al.: Inheritance Beyond Plain Heritability: Variance-Controlling Genes in Arabidopsis thaliana. PLoS Genet. 2012; 8(8): e1002839. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRönnegård L, Valdar W: Recent developments in statistical methods for detecting genetic loci affecting phenotypic variability. BMC Genet. 2012; 13: 63. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1949",
"date": "07 Oct 2013",
"name": "William G. Hill",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nShen and Rönnegård (SR) comment critically and succinctly on the paper by Sun et al. published in AJHG which advocates that, before any claim of differences in variance among genotypes in a GWAS or similar study, a check should first be made whether these can be removed by a monotonic transformation. Each of SR’s four criticisms seems well justified. As 105 or more SNPs may be fitted in a GWAS study, what biological interpretation could be given to that number of different transformations or even on a limited subset of loci showing possible variance differences? If some loci give signals of mean but not variance difference, should these then be transformed to eliminate the scale effect on mean and perhaps reveal variance differences? Any concept of an original scale of measurement is lost, as SR point out. It is not obvious why the mere existence of a transformation designed to minimise differences in variance should prevent discussion of variance heterogeneity on the chosen scale. Equivalently, if we considered means of the three genotypes at the locus rather than just average effects, would our ability to transform the data at each locus such that heterozygotes were intermediate imply there was no dominance, or only that it was on a particular scale?On a further point. Sun et al. (p395) comment: ‘In the absence of genotypic mean differences, we can hardly infer that differences in variances are per se of biological interest.’ That is to take too narrow a view: the mean and phenotypic variance (or CV) of a quantitative trait in any species take typical values, e.g. the CV for adult human height is ca. 4% and for BMI ca. 16% . There is direct evidence of genetic differences within species in environmental variance, with GWAS and other single gene studies, that cannot be removed by scale, so the level of the environmental variance is subject to evolutionary forces (e.g. Hill & Mulder 2010 Genet. Res. 92:381). To view variance as a biological phenomenon which is just some adjunct to the mean seems simplistic, as SR argue. Indeed one has to ask whether scale transformations have value unless there is a biological basis, such as a log transformation to account for multiplicative genetic effects; but that must then apply across all loci.",
"responses": []
},
{
"id": "1948",
"date": "01 Nov 2013",
"name": "Yurii Aulchenko",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 agree with criticism raised by Shen and Ronnegard in their points 2 and 3 concerning the application of the transformation of Sun et al. in the context of whole-genome scans. Indeed, applying this transformation in SNP-specific manner is difficult to adopt conceptually. Sun et al. rightly suggest that “the scales on which we measure interval-scale quantitative traits are man-made and have little intrinsic biological relevance”, but the underlying intrinsic scale, and the function reflecting this scale into the observed, is likely to be unique and does not change with SNP. In that, the transformation applied to a trait should not change through the markers studied. Practically, this is not very difficult to implement, and as a simplest option one could think of the estimation of Sun’s transformation parameters from upper, middle and lower tertiles of the total phenotypic distribution. A more general approach (without restricting the data into three groups, but modelling the variance as a function of the mean) should be straightforward to implement.We also understand the reasoning behind the Shen and Ronnegard’s points 1 and 4, but here we are less certain that the problem raised could be easily addressed. Specifically, one could argue with point 1 (“why should we regard the difference between 160cm and 170cm different from 170cm and 180cm?”): it is not that hard to imagine a biologically relevant model in which same changes of an “intrinsic scale” lead to different changes on the observed scale as the mean advances (an example would be Michaelis–Menten kinetics). Also both points 1 and 4 (losing power after transformation) relate not only to Sun et al.’s transformation, but to almost any transformation in wide use (e.g. Log, Box-Cox, Gaussenization/inverse-normal). While it is true that analysis of transformed trait may lead to reduced power (and specifically in case of Sun’s transformation applied in marker-specific manner to the analysis of variance heterogeneity it should), we have a feeling that one still would like to check whether the variance heterogeneity found can be modeled as a function of the mean (in which case any SNP affecting the mean is likely to show “control” of the variance as well).Finally, we fully agree with comment of William Hill and Ian White who criticize Sun et al.'s statement that “‘In the absence of genotypic mean differences, we can hardly infer that differences in variances are per se of biological interest”. We think that the differences in variance per se are biologically and genetically plausible and interesting.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-200
|
https://f1000research.com/articles/2-199/v1
|
30 Sep 13
|
{
"type": "Commentary",
"title": "Advancing the activity cliff concept",
"authors": [
"Ye Hu",
"Dagmar Stumpfe",
"Jürgen Bajorath",
"Ye Hu",
"Dagmar Stumpfe"
],
"abstract": "The activity cliff concept has experienced increasing interest in medicinal chemistry and chemoinformatics. Activity cliffs have originally been defined as pairs of structurally similar compounds that are active against the same target but have a large difference in potency. Activity cliffs are relevant for structure-activity relationship (SAR) analysis and compound optimization because small chemical modifications can be deduced from cliffs that result in large-magnitude changes in potency. In addition to studying activity cliffs on the basis of individual compounds series, they can be systematically identified through mining of compound activity data. This commentary aims to provide a concise yet detailed picture of our current understanding of activity cliffs. It is also meant to introduce the further refined activity cliff concept to a general audience in drug development.",
"keywords": [
"Activity cliffs have been discussed in the medicinal and computational chemistry literature since the early 1990s1–4. In the practice of medicinal chemistry",
"structurally similar compounds with large potency differences are often encountered",
"mostly during the chemical optimization of individual compound series. Moreover",
"activity cliffs have also been systematically identified by computational compound data mining3",
"4",
"which has sparked further interest in the activity cliff concept. Hence",
"in addition to the study of activity cliffs on a case-by-case basis in medicinal chemistry",
"a large knowledge base of activity cliff information is emerging. In addition",
"in recent years",
"the activity landscape concept has also become popular5. Activity landscapes are generally defined as graphical representations that integrate similarity and potency relationships of compounds sharing the same biological activity5",
"and activity cliffs are their most prominent features3",
"5. As compound data sets rapidly grow in size",
"activity landscape representations are increasingly used as tools for SAR visualization6",
"which further emphasizes the notion of activity cliffs. Hence",
"the activity landscape and cliff concepts go hand in hand."
],
"content": "Introduction\n\nActivity cliffs have been discussed in the medicinal and computational chemistry literature since the early 1990s1–4. In the practice of medicinal chemistry, structurally similar compounds with large potency differences are often encountered, mostly during the chemical optimization of individual compound series. Moreover, activity cliffs have also been systematically identified by computational compound data mining3,4, which has sparked further interest in the activity cliff concept. Hence, in addition to the study of activity cliffs on a case-by-case basis in medicinal chemistry, a large knowledge base of activity cliff information is emerging. In addition, in recent years, the activity landscape concept has also become popular5. Activity landscapes are generally defined as graphical representations that integrate similarity and potency relationships of compounds sharing the same biological activity5, and activity cliffs are their most prominent features3,5. As compound data sets rapidly grow in size, activity landscape representations are increasingly used as tools for SAR visualization6, which further emphasizes the notion of activity cliffs. Hence, the activity landscape and cliff concepts go hand in hand.\n\nTwo recent perspective articles have provided detailed accounts of activity cliff research and new developments3,4. For an in-depth review of the activity cliff research area, the interested reader is referred to these publications and the references therein. This commentary does not aim to present a full account of activity cliffs and their utility in drug discovery. Rather, it aims to distill out the information that is most relevant to provide a differentiated and critical, yet easy-to-understand view of activity cliffs. In addition, some new findings are reported concerning the target distribution of activity cliffs and coordination of cliffs, which further complement the picture. As mentioned above, computational approaches have substantially influenced our current understanding of activity cliffs. Since we strive for a widely accessible presentation of the activity cliff concept, the discussion of computational details is kept herein to an essential minimum. Furthermore, given that a number of recent activity cliff investigations have originated from our laboratory, some of the views and recommendations presented herein are at least partly subjective. However, it is hoped that they might, nevertheless, stimulate further exploration and discussion of the activity cliff concept. Several recommendations made should also aid in practical applications.\n\n\nDefinition-related key aspects\n\nAn activity cliff has originally been defined as a pair of structurally similar compounds with a large difference in potency2,3. This general definition has four key aspects, which require further consideration and specification:\n\n(i) Only a pair of compounds is considered.\n\n(ii) Both compounds are active (against the same target).\n\n(iii) A structural similarity criterion must be specified (i.e., how is similarity assessed and how similar must compounds be?).\n\n(iv) A potency difference criterion must be established (i.e., when is a potency difference considered to be large?).\n\nIn the following, these key points will be further evaluated (in reverse order).\n\nTo clearly establish the potency difference criterion, it must not only be decided how large a potency difference between two compounds should be but also considered which type of potency measurements should be utilized. We emphasize that activity cliff information is only useful if the description of cliffs is accurate and interpretable (vide infra). This also relates to potency comparisons. Different types of potency measurements should not be combined, e.g., assay-dependent IC50 measurements and (in theory) assay-independent equilibrium constants (Ki values) should be separately considered. Moreover, the use of approximate potency annotations (such as \"% inhibition\") should be avoided to ensure that SAR information encoded by activity cliffs is accurate. As our understanding of activity cliffs has evolved over the years, we have become increasingly conservative in the assessment of cliffs (\"conservatism\" will indeed be a recurrent theme in our discussion). Therefore, we generally prefer Ki values (vide infra), which are in principle the most accurate measurements.\n\nConcerning the magnitude of potency differences, there is no generally applicable rule for the definition of activity cliffs. We have found that statistical significance assessment typically yields data set-dependent results. On the basis of large-scale SAR exploration of many different data sets, we have also concluded that the presence of an at least 100-fold difference in potency as a cliff criterion generally leads to the identification of \"significant\" activity cliffs in compound data sets from which SAR information can often be deduced. Clearly, this represents a heuristic and not a rule written in stone.\n\nWithout doubt, the assessment of compound similarity is the most difficult task for activity cliff definition and analysis, for several reasons. The quantification of compound similarity is strongly dependent on chosen molecular representations (descriptors). In addition, there are no generally accepted similarity measures. For activity cliff definition, the calculation of Tanimoto similarity7 on the basis of different fingerprint representations7–9 has thus far been most popular3. Fingerprints are generally defined as bit representations of molecular structure and/or properties. As such, they are fairly abstract descriptions of compounds. Two fingerprints of different design that have often been used for the description of activity cliffs are the \"molecular access system (MACCS) structural keys\" (http://accelrys.com)8, one of the \"classical\" fingerprints, and the \"extended connectivity fingerprint with bond diameter 4 (ECFP4)\"9, a more recent design. MACCS consists of a set of 166 defined structural fragments whose presence or absence in a compound is monitored and ECFP4 is a topological fingerprint that generates varying numbers of atom environments for test compounds. These fingerprints are calculated from molecular graphs and are thus 2D representations. Furthermore, for the purpose of our discussion, it is sufficient to appreciate that the Tanimoto coefficient (Tc) is a similarity measure that ranges from 0 to 1 and quantifies fingerprint overlap as a measure of molecular similarity (i.e., a Tc value of 0 is produced by fingerprints that share no features and a value of 1 by identical fingerprints). A MACCS Tc value of 0.85 (corresponding to Tanimoto similarity of 85%) has often been applied as a similarity criterion for activity cliff formation3. This value approx. corresponds to an ECFP4 Tc value of 0.56 because the same percentage of compound pairs reaches or exceeds these MACCS- and ECFP4-dependent values in systematic compound comparisons4. Because computational similarity methods are strongly representation- and compound class-dependent, the assessment of activity cliffs suffers from the same dependencies. Consequently, activity cliff distributions often vary significantly dependent on the representations and similarity measures used10,11. Furthermore, calculated fingerprint Tc values are often difficult to interpret from a medicinal chemistry point of view3–5, which further complicates matters.\n\nIn light of these difficulties, attempts have been made to replace calculated similarity values for activity cliff assessment by structurally more conservative and intuitive similarity criteria. For example, a substructure-based similarity criterion has been introduced on the basis of the matched molecular pair (MMP) formalism12,13. An MMP is defined as a pair of compounds that are only distinguished by a structural change at a single site12, i.e., the exchange of a substructure, which is termed a chemical transformation13. Importantly, the presence of a defined substructure relationship such as the formation of an MMP can also be applied as a similarity criterion. For the definition of activity cliffs, transformation size-restricted MMPs have been introduced in which transformations are limited to relatively small and chemically meaningful replacements14. Figure 1A and 1B show exemplary Tanimoto similarity- and MMP-based activity cliffs, respectively. The latter activity cliff category has been termed MMP cliff14. The similarity criterion underlying MMP cliffs is simple and intuitive. MMP cliffs are often found to further improve the chemical interpretability of activity cliffs compared to cliffs defined on the basis of calculated similarity values4,14.\n\nIn (A)–(D), four categories of activity cliffs are shown. Structural differences between activity cliff compounds are highlighted (red). For ECFP4 (A) and 3D cliffs (D), calculated similarity values are reported, i.e., 0.65 and 0.90 refer to the value of the Tanimoto and property density function similarity coefficient17, respectively (both of which range from 0 to 1). Compound potency (pKi) values are given in blue.\n\nAnother intuitive categorization of activity cliffs has been introduced on the basis of consistently defined scaffolds (obtained from compounds by removal of R-groups)15 and the presence of different scaffold/R-group relationships, as illustrated in Figure 1C16. This categorization makes it possible, for example, to distinguish activity cliffs that are caused by R-group replacements, small chemical changes in core structures, or chiral centers16.\n\nMoreover, activity cliffs can also be defined by comparing compound binding modes in complex ligand/target X-ray structures and calculating their 3D similarity17,18, as shown in Figure 1D. These \"3D cliffs\" enable the interpretation of activity cliff formation on the basis of experimentally observed ligand-target interactions and substantially add to the ligand-centric view of 2D activity cliffs. Although the Protein Data Bank19 provides a steadily growing source of public domain 3D structures of therapeutically relevant proteins, only small numbers of activity cliffs can be rationalized in three dimensions and compared to 2D cliffs. In addition, 3D cliffs also rely on the calculation of similarity values. The quantification of the 3D similarity of compound binding modes is a fairly complex task because positional and conformational changes need to be taken into account.\n\nTaken together, the examples in Figure 1 illustrate that activity cliffs can be defined in rather different -and more or less intuitive- ways, depending on the applied similarity criteria.\n\nBased on the original definition of activity cliffs, both compounds are required to be active against a specific target (vide supra). Several extensions of the activity cliff concept have been introduced that depart from this theme (for example, by considering selectivity against a pair of targets instead of single-target activity)3. In principle, there is no requirement to exclusively consider active compounds for activity cliff assessment. Rather, active and inactive compounds might also be compared, provided confirmed inactive compounds are available for a given target20. For SAR analysis in medicinal chemistry, the identification of small structural changes that render compounds active or inactive is of high interest, and the inclusion of confirmed inactive compounds further increases the frequency of activity cliff formation and hence our knowledge base20. However, if inactive compounds are taken into consideration, a potency difference criterion is no longer applicable to define activity cliffs. Instead, a potency threshold must be set for active compounds as a cliff criterion. For example, one might require an active compound to have a potency of at least 100 nM to qualify for the formation of an activity cliff with an inactive one. The choice of this threshold is essentially subjective and it might be adjusted, depending on the application. It should also be noted that only small numbers of confirmed inactive compounds are typically available from compound optimization projects. Rather, confirmed inactive compounds mostly result from biological screening campaigns. Thus, care must be taken to obtain high-confidence activity data. For example, confirmatory bioassays from PubChem21 presently provide a source of confirmed inactive compounds for more than 100 different targets.\n\nThe definition of activity cliffs on the basis of compound pairs might imply that cliffs are mostly formed in an \"isolated\" manner. This means that cliff partners are only involved in a single activity cliff and have no structural neighbors with large potency differences. However, this is clearly not the case. For example, series of highly and lowly potent structural analogs have been identified in a variety of compound data sets that form multiple and overlapping activity cliffs22, giving rise to the notion of \"coordinated\" activity cliffs23. Figure 2 shows an example of a compound set in which highly coordinated activity cliffs are formed. Higher-order activity cliff configurations involving multiple compounds are of particular interest for medicinal chemistry, given their high SAR information content. Such activity cliff arrangements can be systematically explored through data mining22,23. On the basis of our most recent survey (vide infra), only small proportions of activity cliffs are formed in isolation.\n\nMMP cliffs involving inhibitors of mitogen-activated protein kinase 14 are shown in a network representation. Nodes represent compounds that are connected by edges if they form an MMP cliff. Furthermore, nodes are color-coded according to pKi values of all inhibitors applying a continuous color spectrum from red (lowest potency) via yellow (medium) to green (highest). Only one isolated activity cliff was detected in the inhibitor data set (indicated by a dashed red box). All other activity cliffs were formed in a coordinated manner involving multiple active compounds. Structures of the inhibitors forming the isolated activity cliff are shown.\n\n\nPreferred definition\n\nAs discussed above, there are many different ways to represent activity cliffs. Is it, then, possible to formulate a generally preferred detailed definition? The answer is: in principle, no; in practice, yes. From first principles, one is unable to determine (at least at present) how compound similarity should best be accounted for. In addition, setting the potency difference criterion for meaningful activity cliff and SAR analysis is subject to heuristic approximations. However, on the basis of our experience with a variety of data analyses and practical applications, we generally prefer the following activity cliff definition10:\n\n(a) Similarity criterion: Formation of a size-restricted MMP14.\n\n(b) Potency difference criterion: At least two orders of magnitude.\n\n(c) Activity measurements: Equilibrium constants.\n\nThis MMP cliff definition is conservative both from a compound similarity and activity data perspective and favors chemical interpretability of activity cliffs.\n\n\nFrequency of occurrence\n\nHow often are activity cliffs found in bioactive compounds? What is the proportion of active compounds that participate in the formation of cliffs? Up-to-date results providing answers to these and other questions are reported in Table 1. These results were obtained from a large-scale analysis of compound data sets extracted from ChEMBL (https://www.ebi.ac.uk/chembl/)24. Further details are provided in the legend of Table 1. For this survey, equilibrium constants were exclusively used and a potency difference of at least two orders of magnitude was required. Thus, the MMP cliffs reported in Table 1 correspond to our preferred activity cliff definition (vide supra). It should be noted that 3D cliffs are statistically underrepresented compared to 2D activity cliffs and that their frequencies of occurrence should not be directly compared. Depending on the molecular representations used, between 5.2% and 6.8% of all qualifying pairs of similar compounds form activity cliffs. The molecular representation dependence of activity cliff assessment is also reflected by the percentage of compounds that participate in activity cliffs, which ranges from 27.6% for MMP- over 35.3% for ECFP4- to 41% for MACCS-based cliffs. MMP cliffs occur with slightly lower frequency than fingerprint-based cliffs and involve a smaller proportion of active compounds. Nevertheless, on average, MMP cliffs are formed by on average every fourth active compound across many different data sets. Hence, even on the basis of this conservative assessment, activity cliffs frequently occur and provide direct access to SAR information. We also note that ~96.5%–98.6% of all 2D cliffs are not formed in isolation but in a coordinated manner involving more than two compounds. The example in Figure 2 illustrates that the degree of coordination is often high. For public domain 3D cliffs, the rate of isolated activity cliffs is much higher (20.4%) than for 2D cliffs. This is likely the case because structurally distinct ligands are often chosen for crystallization in order to explore different compound binding modes.\n\nThe average frequency of occurrence of activity cliffs and cliff-forming compounds is reported for 129 target sets4 and different molecular representations including the MACCS and ECFP4 fingerprints and MMPs. The proportion of activity cliffs was calculated on a per-target basis relative to the total number of compound pairs meeting similarity criteria and the proportion of cliff-forming compounds relative to all active compounds. Also reported is the propensity of 3D activity cliffs based on comparison of ligand binding modes in complex X-ray structures. In each case, isolated and coordinated activity cliffs are distinguished. Potency difference criterion: at least 100-fold on the basis of equilibrium constants. Similarity criteria: For MACCS and ECFP4, Tc values of at least 0.85 and 0.56, respectively4; for 3D cliffs, a binding mode similarity coefficient of at least 0.8017; for MMP cliffs, formation of a transformation size-restricted MMP14. Target sets: A target set is defined as a set of compounds with activity against the same target. Target sets were extracted from ChEMBL24 on the basis of two selection criteria: Each set had to contain at least 100 compounds and for all compounds equilibrium constants had to be available. The 129 target sets included more than 35,000 unique compounds4.\n\n\nUtilization\n\nGiven the considerable frequency with which activity cliffs are formed in different compound sets, a key question is to what extent activity cliff information might currently be utilized in the practice of medicinal chemistry? This question is very difficult to answer since it is hardly possible to systematically track this information with medicinal chemists on a per-project basis. However, data mining studies can provide at least some evidence for the potential utilization of activity cliffs. In a recent study, activity cliffs were systematically identified in compound data sets evolving over time25. For each highly potent activity cliff partner, it was determined whether structural analogs of this compound were reported after the activity cliff became available. If structural analogs of a highly potent cliff partner were detected in subsequent years, the possibility existed that activity cliff information provided a starting point for further compound optimization. Alternatively, if no such analogs were identified, no evidence existed for activity cliff progression, as outlined in Figure 3. On the basis of this analysis, evidence for the utilization of activity cliffs was only available for 25% of all available cliffs. By contrast, no evidence for cliff progression was detected for the remaining 75% of activity cliffs25. Thus, in light of these findings, we would conclude that existing activity cliff information is currently under-utilized in the practice of medicinal chemistry. It is apparently difficult to bridge between data mining investigations and practical medicinal chemistry applications and consider information from the public domain early in the course of compound optimization projects. This would recommend striving for much closer links between chemoinformatics and practical medicinal chemistry.\n\nProgression of activity cliffs over time is evaluated by searching for analogs of highly potent activity cliff partners. One of the compound pathways originating from the activity cliff on the left is leading over a sequence of analogs to one of the most potent data set compounds. For the activity cliff on the right, no analogs of the highly potent cliff partner are detected. Thus, in this case, there is no evidence for the potential utilization of activity cliff information.\n\nAnother related question should also be of interest. If activity cliff information is utilized, is there an \"SAR evolution advantage\" detectable compared to other optimization efforts not involving activity cliff compounds? To answer this question, the computational compound pathway model schematically shown in Figure 3 was applied to monitor the progression of activity cliffs towards the most potent compounds in a data set and compare activity cliff-dependent and -independent pathways26. Therefore, for each active compound, a search for series of pairwise similar compounds (MMP sequences) was carried out that ultimately reached one of the 10% most potent compounds in a data set, thus delineating putative compound optimization paths in accord with the pathway model in Figure 3. Although the mean potency of activity cliff compounds and other active compounds was overall very similar, compound pathways originating from 54% of all activity cliffs successfully reached highly potent compounds, compared to only 28% of pathways originating from compounds not involved in cliff formation26. Hence, activity cliff-dependent pathways reached highly potent compounds with higher frequency than cliff-independent pathways, indicating the presence of activity cliff-associated SAR advantages.\n\n\nTarget distribution\n\nAre activity cliffs differently distributed in compounds active against different targets? This is another question of considerable interest for medicinal chemistry, which has only recently been addressed4. Figure 4 reports the distribution of the frequency of occurrence of MMP cliffs and, in addition, compounds participating in cliff formation for more than 200 different target sets of increasing size (each target set consists of compounds active against a specific target). The proportion of MMP cliffs relative to all MMPs and the percentage of activity cliff compounds among all active compounds were monitored. In small compound sets, significant frequency fluctuations were observed, as one would expect (for statistical reasons). By contrast, the distribution of activity cliffs and cliff-forming compounds was relatively stable for target sets containing 200 or more compounds. The box plot representations in Figure 5 indicate that there is surprisingly little variation in the frequency of activity cliffs across many different targets for data sets of moderate to large size (despite the presence of many different specific ligand-target interactions and binding constraints).\n\nIn (A) and (B), the size of target sets is compared to the proportion of MMP cliffs and cliff-forming compounds they contain, respectively. Dots represent individual target sets. Dashed vertical lines mark a target set size of 200 compounds. The analysis was based on 218 target sets with available equilibrium constants extracted from ChEMBL24.\n\nThe distributions of MMP cliffs (red) and cliff-forming compounds (blue) over target sets are reported as box plots. Each box plot provides the smallest value (bottom line), lower quartile (lower boundary of the box), median value (thick line), upper quartile (upper boundary of the box), and the largest value (top line). The dashed line indicates the value range.\n\n\nConclusions\n\nHerein, we have focused on the activity cliff concept and provided a further refined view of activity cliffs. For medicinal chemistry, activity cliffs are of particular interest because they are associated with high SAR information content. For a meaningful assessment of activity cliffs, similarity and potency difference criteria need to be clearly defined. Care must be taken to utilize high-confidence activity data for activity cliff analysis. However, similarity assessment is the most critical step in activity cliff analysis. Although calculated similarity values can be conveniently used to describe activity cliffs, they often limit the interpretability of activity cliffs in medicinal chemistry. Therefore, substructure-based activity cliff definitions have been introduced such as MMP cliffs that further support chemical interpretation. Activity cliffs are formed with relatively high frequency among active compounds, indicating that they provide a substantial source of SAR information. In fact, if activity cliff information is utilized, as assessed on the basis of pathway modeling, compound paths originating from activity cliffs more frequently yield highly potent compounds than optimization paths originating from other active compounds. However, there currently is no evidence for utilization of about three quarters of activity cliffs in compound data sets evolving over time, which indicates that available activity cliff information is under-utilized in the practice of medicinal chemistry. Thus, there should be significant potential for further improvement of compound optimization efforts by taking activity cliff information from data mining into consideration. It has also been determined that activity cliffs are relatively evenly distributed across compounds active against a variety of targets, perhaps surprisingly so. In summary, the activity cliff concept provides an intuitive access to SAR information and can be evaluated from different perspectives. Recent analyses have yielded in part unexpected results that further differentiate our current view of activity cliffs and associated SAR features.",
"appendix": "Author contributions\n\n\n\nJB designed the study, YH and DS collected and organized the data, YH, DS, and JB wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors thank Dilyana Dimova for help with data sets.\n\n\nReferences\n\nLajiness M: Evaluation of the Performance of Dissimilarity Selection Methodology. In QSAR: Rational Approaches to the Design of Bioactive Compounds; Silipo, C., Vittoria, A., Eds.; Elsevier: Amsterdam, Netherlands, 1991; pp 201–204.\n\nMaggiora GM: On Outliers and Activity Cliffs – Why QSAR often Disappoints. J Chem Inf Model. 2006; 46(4): 1535. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Exploring Activity Cliffs in Medicinal Chemistry. J Med Chem. 2012; 55(7): 2932–2942. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Hu Y, Dimova D, et al.: Recent Progress in Understanding Activity Cliffs and their Utility in Medicinal Chemistry. J Med Chem. 2013; in press. PubMed Abstract | Publisher Full Text\n\nWassermann AM, Wawer M, Bajorath J: Activity Landscape Representations for Structure-Activity Relationship Analysis. J Med Chem. 2010; 53(23): 8209–8223. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Methods for SAR Visualization. RSC Adv. 2012; 2(2): 369–378. Publisher Full Text\n\nWillett P, Barnard JM, Downs GM: Chemical Similarity Searching. J Chem Inf Comput Sci. 1998; 38(6): 983–996. Publisher Full Text\n\nMACCS Structural Keys. Accelrys: San Diego, CA.\n\nRogers D, Hahn M: Extended-Connectivity Fingerprints. J Chem Inf Model. 2010; 50(5): 742–754. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Frequency of Occurrence and Potency Range Distribution of Activity Cliffs in Bioactive Compounds. J Chem Inf Model. 2012; 52(9): 2348–2353. PubMed Abstract | Publisher Full Text\n\nMedina-Franco JL, Martínez-Mayorga K, Bender A, et al.: Characterization of Activity Landscapes using 2D and 3D Similarity Methods: Consensus Activity Cliffs. J Chem Inf Model. 2009; 49(2): 477–491. PubMed Abstract | Publisher Full Text\n\nKenny PW, Sadowski J: Structure Modification in Chemical Databases. In Chemoinformatics in Drug Discovery; Oprea, T. I., Ed.; Wiley-VCH: Weinheim, Germany, 2005; pp 271–285. Publisher Full Text\n\nHussain J, Rea C: Computationally Efficient Algorithm to Identify Matched Molecular Pairs (MMPs) in Large Data Sets. J Chem Inf Model. 2010; 50(3): 339–348. PubMed Abstract | Publisher Full Text\n\nHu X, Hu Y, Vogt M, et al.: MMP-Cliffs: Systematic Identification of Activity Cliffs on the Basis of Matched Molecular Pairs. J Chem Inf Model. 2012; 52(5): 1138–1145. PubMed Abstract | Publisher Full Text\n\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, Bajorath J: Extending the Activity Cliff Concept: Structural Categorization of Activity Cliffs and Systematic Identification of Different Types of Cliffs in the ChEMBL Database. J Chem Inf Model. 2012; 52(7): 1806–1811. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Exploration of 3D Activity Cliffs on the Basis of Compound Binding Modes and Comparison of 2D and 3D cliffs. J Chem Inf Model. 2012; 52(3): 670–677. PubMed Abstract | Publisher Full Text\n\nHu Y, Furtmann N, Gütschow M, et al.: Systematic Identification and Classification of Three-dimensional Activity Cliffs. J Chem Inf Model. 2012; 52(6): 1490–1498. PubMed Abstract | Publisher Full Text\n\nBerman HM, Westbrook J, Feng Z, et al.: The Protein Data Bank. Nucleic Acids Res. 2000; 28(1): 235–242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu Y, Maggiora GM, Bajorath J: Activity cliffs in PubChem confirmatory bioassays taking inactive compounds into account. J Comput Aided Mol Des. 2013; 27(2): 115–124. PubMed Abstract | Publisher Full Text\n\nWang Y, Xiao J, Suzek TO, et al.: PubChem’s BioAssay Database. Nucleic Acids Res. 2012; 40(Database issue): D400−D412. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVogt M, Huang Y, Bajorath J: From Activity Cliffs to Activity Ridges: Informative Data Structures for SAR Analysis. J Chem Inf Model. 2011; 51(8): 1848–1856. PubMed Abstract | Publisher Full Text\n\nNamasivayam V, Bajorath J: Searching for Coordinated Activity Cliffs Using Particle Swarm Optimization. J Chem Inf Model. 2012; 52(4): 927–934. PubMed Abstract | Publisher Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: A Large-scale Bioactivity Database for Drug Discovery. Nucleic Acids Res. 2012; 40(Database issue): D1100–D1107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDimova D, Heikamp K, Stumpfe D, et al.: Do Medicinal Chemists Learn from Activity Cliffs? A Systematic Evaluation of Cliff Progression in Evolving Compound Data Sets. J Med Chem. 2013; 56(8): 3339–3345. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Dimova D, Heikamp K, et al.: Compound Pathway Model to Capture SAR progression: Comparison of Activity Cliff-Dependent and -Independent Pathways. J Chem Inf Model. 2013; 53(5): 1067–1072. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1940",
"date": "07 Oct 2013",
"name": "Gerhard Müller",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nYe Hu, Dagmar Stumpfe, and Jürgen Bajorath elaborate in their commentary entitled 'Advancing the activity cliff concept' on the application of an abstract chemoinformatics-based analysis of differentiated structure-activity relationships within given compound sets. Given the high level of complexity and the expert computational chemistry knowledge often required to comprehend and to apply those latest developments in the field of chemoinformatics, I consider this commentary as extremely helpful in bridging exactly that obvious gap between the practitioners of bioactive compound data mining on the one hand, and the laboratory-based practitioners of medicinal chemistry on the other hand, respectively.Within the context of today’s practice of medicinal chemistry, the sound and conclusive interpretation of structure-activity data is one of the most relevant, though challenging assignments in any small molecule lead finding and optimization campaign. In the course of those projects, involved teams are permanently required to make high-impact decisions on e.g. further prosecution of specific chemotypes based on systems that are, from an experimental point of view, notoriously under determined. Hence, it is of great value to account for all publicly available information on the compound-centric aspects in a given drug discovery project in order to make the most educated decision, specifically on the 'tractability' of a given chemotype.More specifically, compound classes that after a certain chemical elaboration fail to exhibit a differentiated activity profile, i.e. do not show activity cliffs, should not be prosecuted any further since all too often those 'flat SAR' or 'rolling-hill' analogues were repeatedly identified as cul-de-sac series in many lead finding campaigns. In that context, the authors provide very helpful guidelines for the medicinal chemistry community on how to access the concept of activity cliffs by sharing expert insights they have gained over many systematic studies on practical aspects such as choosing the right potency difference and molecular similarity criteria to reliably define activity cliff patterns. Different molecular similarity metrics are introduced and comparatively ranked on their applicability and interpretability within a potential medicinal chemistry project. We all might have heard about Tanimoto, MACCS and ECFP4 fingerprints; but I assume that not too many of us are acquainted to the MMP (matched molecular pair) concept and utilize that concept pro-actively in pursued lead finding and optimization campaigns.The group around Jürgen Bajorath actively de-mystifies those similarity concepts for us by providing quantitative measures on the inter-relation, thereby sharing their experience on the practicability of all those computational-based approaches. And for a medicinal chemist it is inspiring to see how e.g. scaffold-based cliffs unfold in a comprehensible way. I find it especially useful that the authors share their preferred definitions and settings in terms of applied similarity criteria, potency difference criteria, as well as activity measurements, thus highlighting scope and limitations of the underlying methodology. The more interested reader who wants to get really deeply involved into algorithmic details or further applications of the activity cliff concept is referred to the most recent and relevant research references.Within the framework of a baseline study on 35000 bioactive compounds acting at 129 targets in the ChEMBL database, the overall occurrence of activity cliffs is analysed underlining the finding that activity cliffs occur quite frequently and contain valuable intrinsic SAR information that is not applied systematically. It becomes apparent that activity cliff-dependent optimization pathways seem to have a cliff-associated SAR advantage over other optimization strategies. Based on those findings, I can only support the claim that is made by the authors, notably to establish much closer links between chemoinformatics and practising medicinal chemists for a better and earlier integration of existing activity cliff information into pursued projects.Despite the multiple and rather different ways towards activity cliffs, the concept as such becomes a tangible and intuitive asset that should be applied more frequently in the today’s pre-clinical discovery arena.I see especially the attempt of reaching highest user-friendliness for a putative end-user that renders this commentary as very helpful to get the medicinal chemistry community alerted for this useful but still under-appreciated concept. The group around Jürgen Bajorath undoubtedly qualifies as an advocate in that sense, and the community of practising medicinal chemists should start to move in their direction accordingly.",
"responses": []
},
{
"id": "1944",
"date": "09 Oct 2013",
"name": "Patrick Walters",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides an overview of the concept of activity cliffs in structure-activity relationships and provides a number of examples of how the technique can be applied in a drug discovery setting. Activity cliffs can provide a means of better understanding the relationships between chemical structure and biological activity, as well as identifying regions of a molecule that are critical to activity. The review is directed toward the non-expert and should provide an adequate background for those unfamiliar with the topic. Those familiar with activity cliffs may want to consult more specialized reviews written by the same authors. This review is well-written, easy to follow, and provides many key references for those who would like to learn more about the topic.The authors’ breakdown of the activity cliff concept into components will be helpful for those unfamiliar with the topic. It may be useful to point out that utilizing log differences in activity can make activity cliff scores more intuitive to chemists and biologists. Those with a limited knowledge of chemoinformatics will appreciate the discussion of fingerprint similarity. However, a figure might make these concepts even more clear. The authors' point out that \"calculated fingerprint Tc values are often difficult to interpret from a medicinal chemistry point of view\". In my experience, Tanimoto similarity typically makes sense, this sentence might be better as \"calculated fingerprint Tc values are *sometimes* difficult to interpret\". The authors mention matched molecular pairs (MMP) as a means of expressing molecular similarity.In addition to references 12 and 13, it would be useful to include what I consider to be the seminal paper on matched molecular pairs.Leach, A. G.; Jones, H. D.; Cosgrove, D. A.; Kenny, P. W.; Ruston,L.; MacFaul, P.; Wood, M.; Colclough, N.; Law, L. Matched Molecular Pairs as a Guide in the Optimization of Pharmaceutical Properties; A Study of Aqueous Solubility, Plasma Protein Binding and Oral Exposure. J. Med. Chem. 2006, 49, 6672–6682.As chemists and biologists pursue more complex targets, it is necessary to increase the sophistication of our SAR analysis. I appreciate the fact that the authors point out how activity cliffs can also be used to examine multi-target activity relationships and links between sometimes-distant functional groups.I agree with the authors' assessment that the activity cliff concept is underutilized in medicinal chemistry. Hopefully with the introduction of robust commercial software tools for the identification and exploitation of activity cliffs, the technique will be more widely used.This review provides and excellent introduction to activity cliffs for those unfamiliar with the method. The paper is well referenced and will hopefully inspire a new group of users to continue to advance the method.",
"responses": []
},
{
"id": "2111",
"date": "16 Oct 2013",
"name": "Meir Glick",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe commentary provides an up to date view of activity cliffs and assess their prevalence in ChEMBL. In addition, it discusses how activity cliffs can be defined using potency and similarity criteria. The paper is well written. My only suggestion is to make table 1 more detailed; that is to include the activity cliff frequency around target families or even around the 129 individual target sets.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-199
|
https://f1000research.com/articles/2-178/v1
|
21 Aug 13
|
{
"type": "Research Article",
"title": "TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation",
"authors": [
"Devavani Chatterjea",
"Luisa Paredes",
"Tijana Martinov",
"Evelyn Balsells",
"Juliann Allen",
"Akilah Sykes",
"Alyssa Ashbaugh",
"Luisa Paredes",
"Tijana Martinov",
"Evelyn Balsells",
"Juliann Allen",
"Akilah Sykes",
"Alyssa Ashbaugh"
],
"abstract": "Background: Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-α neutralizing antibody on compound 48/80-provoked thermal hyperalgesia. Methods: We treated ND4 Swiss male mice with intravenous anti-TNF-α antibody or vehicle 30 minutes prior to bilateral, intra-plantar compound 48/80 administration and measured changes in the timing of hind paw withdrawal observed subsequent to mice being placed on a 51oC hotplate. We also assessed changes in tissue swelling, TNF-α gene expression and protein abundance, mast cell degranulation, and neutrophil influx in the hind paw tissue. Findings: We found that TNF-α neutralization significantly blocked thermal hyperalgesia, and reduced early tissue swelling. TNF-α neutralization had no significant effect on mast cell degranulation or neutrophil influx into the tissue, however. Moreover, no changes in TNF-α protein or mRNA levels were detected within 3 hours of administration of compound 48/80. Interpretation: The neutralizing antibodies likely target pre-formed TNF-α including that stored in the granules of tissue-resident mast cells. Pre-formed TNF-α, released upon degranulation, has immediate effects on nociceptive signaling prior to the induction of neutrophil influx. These direct effects on nociceptors are abrogated by TNF-α blockade resulting in compromised nociceptive withdrawal responses to acute, harmful environmental stimuli.",
"keywords": [
"Mast cell degranulation",
"Thermal sensitivity",
"TNF-alpha"
],
"content": "Introduction\n\nPain is one of the cardinal components of inflammation and tissue-resident immune cells are important players in the regulation of protective nociceptive responses1. Compound 48/80 (c48/80)-provoked thermal hyperalgesic responses in the hind paw tissue of male ND4 Swiss mice are mediated by the degranulation of tissue-resident mast cells2. The increased sensitivity to thermal stimulus is dependent, in part, on neutrophil influx into the affected tissue, and on histamine signaling; the response is completely abrogated in mast cell-deficient mice, and is substantially reduced by blocking neutrophil influx or treatment with histamine receptor antagonists2. The importance of neutrophil influx in nociceptive cascades has been shown in different rodent models3–5 and the important regulatory roles of inflammatory cytokines including IL-1β, TNF-α, and IL-6 in pain signaling have also been demonstrated6,7. Here we show that c48/80-induced early thermal sensitivity in ND4 Swiss mice is markedly reduced by pre-treatment with a neutralizing antibody against TNF-α. Anti-TNF-α antibody administration does not significantly affect levels of mast cell degranulation or neutrophil influx into the affected tissue. We also found no increase in protein and mRNA levels of TNF-α in the tissue within the first 3 hours following c48/80 administration suggesting that this blockade targets pre-formed TNF-α including that stored in the granules of tissue-resident mast cells. Our data suggest that pre-formed TNF-α may act rapidly and directly on nociceptors that are known to reside in close proximity to mast cells8 and modulate nociceptor sensitization thresholds9 and function10. TNF-α blockade therefore compromises the early, protective nociceptive withdrawal responses that normally act to protect tissues from further exposure to acute, injurious environmental stimuli11.\n\n\nMethods\n\nThree-six month old male ND4 Swiss mice (Harlan Laboratories, Indianapolis, IN) were housed in Macalester College’s research animal facility with a 12-hour light/dark cycle and free access to food and water. A total of 73 mice were used for the experiments shown here (21 controls; 52 experimental) to ensure that appropriate statistical analysis could be performed on data acquired from these experiments. Macalester College’s Institutional Animal Care and Use Committee approved all experimental procedures (Protocol B08Su1).\n\nAll drugs were administered using 0.9% saline (VWR, Radnor, PA) vehicle or phosphate buffered saline (EMD Millipore, Billerica, MA). Mice received bilateral intra-plantar (i.pl.) treatments with c48/80 (1.5μg/paw; 10μl; (Sigma-Aldrich, St. Louis, MO)) or saline alone as previously described2. Either 200μg/kg of anti-TNF-α neutralizing antibody (R&D Systems, Minneapolis, MN, Polyclonal Goat IgG, Catalog #: AB-410-NA) or 200μl vehicle was injected intravenously 30 minutes prior to c48/80 injection in a protocol adapted from Rocha et al.12. Rocha et al. administered anti-TNF-α antibodies i.v. 5 mins prior to carrageenan treatment in a mouse model of mechanical hyperalgesia12.\n\nTo assess thermal sensitivity, single mice treated with i.pl. c48/80 or vehicle were placed in a Plexiglas cylinder on a hotplate analgesia meter (Harvard Laboratories, Edenbridge, KY) maintained at 51.0 ± 0.5°C and removed when prolonged retraction, flipping/licking of the hind paw, or jumping with both hind paws off the hotplate were observed, but no later than 40 seconds, as previously described2. Two baseline hotplate latencies were taken 24 and 48 hours before the experiment. Mice with >10 second differences between baselines or <15 second averages were excluded from the experiment. Nociceptive thermal sensitivity was quantified by subtracting the mean baseline thermal latency from the experimental thermal latency at each time point for each mouse.\n\nChange in hind paw width measured using digital calipers (±0.1mm; VWR, Radnor, PA) was calculated as an average of the left and right paw widths. Baseline paw widths for each mouse were taken pre-treatment and subtracted from post-treatment paw widths to calculate tissue edema as previously described2.\n\nFootpads were extracted from hind paws of mice euthanized by CO2 inhalation, weighed, frozen at -80°C in 5.6μl/mg tissue weight of 50mM K2HPO4 buffer (pH 6.0) (Sigma-Aldrich, St. Louis, MO) containing 0.05% hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich, St. Louis, MO), thawed, homogenized in 5x the storage volume of HTAB buffer, sonicated with a 550 Sonic Dismembrator (Fisher Scientific, Waltham, MA), freeze-thawed, and centrifuged (AllegraX-15R; Beckman Coulter, Inc., Pasadena, CA) as previously described2,13. Absorbance was recorded using a BioTek PowerWave XS plate reader (BioTek, Winooski, VT) at 450nm after a 20-minute incubation in 50mM phosphate buffer (pH 6.0) with 0.025% hydrogen peroxide (Sigma Aldrich, St. Louis, MO) and 0.167mg/ml o-dianisidine-dihydrochloride (Sigma Aldrich, St. Louis, MO) at room temperature in the dark. Myeloperoxidase (MPO) levels were normalized to tissue weight and presented as OD/g of wet tissue.\n\nFor protein and gene expression studies, hind paws were excised from mice euthanized by CO2 inhalation, flash-frozen in liquid nitrogen and stored at -80°C.\n\nFor protein studies, flash-frozen paws were homogenized in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA) supplemented with protease inhibitor (Cocktail Set IV; EMD Biosciences, Billerica, MA) using a Tissue-Tearor (BioSpec; Model 985370). Homogenates were incubated on ice for 20 minutes, centrifuged at 2000 rpm for 10 minutes at 4°C, and lysate supernatants stored at -80°C. We quantified TNF-α cytokine levels by ELISA according to the manufacturer’s instructions (eBioscience, San Diego, CA).\n\nTo measure cytokine gene expression, total RNA was extracted from flash-frozen plantar tissue (Total RNA Mini Kit, Midwest Scientific, St. Louis, MO), quantified with Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE), and reverse-transcribed in a 2720 Thermal Cycler (Life Technologies, Grand Island, NY) using the Superscript III First-Strand Synthesis System (Life Technologies, Grand Island, NY) with 100ng of RNA per reaction. Relative transcript abundance was determined by quantitative reverse transcriptase PCR (qRT-PCR) using TaqMan Gene Expression Assay Primer/Probe Sets and TaqMan MasterMix (Life Technologies, Grand Island, NY) in a Bio-Rad iCycler (Bio-Rad, Hercules, CA) using β-2-microglobulin (β2m; Mm00437762_m1, Life Technologies, Grand Island, NY) and TNF-α (Mm00443260_g1, Life Technologies, Grand Island, NY) primer sets. Fold expression was normalized to β-2-microglobulin levels and calculated as previously described14.\n\nExcised hind paws (from mice euthanized by CO2 inhalation) were fixed in 10% buffered formalin (VWR, Radnor, PA) for 24 hours, transferred to 70% ethanol (Sigma-Aldrich, St. Louis, MO), decalcified for 1–2 weeks in 15% EDTA, hydrated, and embedded in paraffin. 4μm sagittal sections were stained with toluidine blue (Tb) (Sigma-Aldrich, St. Louis, MO) or hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO) for the detection of mast cells or neutrophils, respectively, at 400x magnification with ≥3 biological replicates per treatment. Tb-stained mast cells were counted in 10 fields/section and degranulation was scored based on the number of granules observed outside the boundary of the cell: intact (0), mild (1–10), moderate (10–20), and extensive (20+) as previously described2. The same investigator performed all counts and was blinded to treatment groups. Neutrophils were imaged between the heel and the toes on the plantar side of the hind paw section using an Olympus CX21LED light microscope and camera (Olympus Corporation of the Americas, Center Valley, PA).\n\nData were processed using Microsoft Excel (Redmond, WA) and graphed with PRISM 5.0 (GraphPad, San Diego, CA). All statistical analyses were performed using JMP 9.0 (SAS, Cary, NC). All data are presented as the mean ±SEM. Data were analyzed using one-way ANOVA, and the Tukey-Kramer HSD post hoc test, at each time point. Statistical significance was defined as p<0.05. Thermal sensitivity and hind paw edema measurements included 15–18 mice per treatment group. Other analyses used 3–9 mice per treatment group. All data represent 2–3 independent experiments.\n\n\nResults\n\nMale ND4 Swiss mice bilaterally injected with 1500ng of c48/80 in the hind paws showed hyperalgesic withdrawal responses ~10 seconds sooner than their baseline withdrawal 30 minutes after treatment; these responses were resolved by 2.5 hours (Figure 1A). Pre-treatment with an intravenous injection of 200μl/kg TNF-α neutralizing antibody 30 minutes prior to c48/80 injection significantly abrogated these responses; pre-treated mice showed hyperalgesic withdrawal responses <5 seconds sooner than their baseline responses 30 minutes after treatment (Figure 1A). Withdrawal responses of control and anti-TNF-treated groups were significantly different at 1.5 hours after treatment and indistinguishable at 2.5 hours when the c48/80-provoked hyperalgesic responses were resolved. Anti-TNF-α pretreatment also significantly reduced hind paw edema compared to mice without pre-treatment at 30 minutes but not at 1.5 and 2.5 hours (Figure 1B). Saline-treated control mice had no increase in thermal sensitivity and showed no tissue swelling after c48/80 treatment (Figure 1A–B). Overall, thermal sensitivity was reduced in a more sustained manner compared to tissue edema.\n\nMice were pre-treated with 200μl/kg anti-TNF-α neutralizing antibody or vehicle (200μl, i.v.) 30 minutes before bilateral intra-plantar c48/80 or 0.9% saline injections (1.5μg/paw; 10μl). The bars represent the mean change in thermal paw withdrawal latency (A) and the change in paw width (B) from baseline and error bars represent ±SEM. Anti-TNF-α administration abrogated thermal hyperalgesia at 0.5 and 1.5h after c48/80 injection (A) and reduced paw edema significantly at 0.5h (B). Significances are compared to Sal/Sal (# = p<0.05; ### = p<0.001) and Sal/c48/80 (* = p<0.05; *** = p<0.001). n = 12 in Sal/Sal; n = 18 for Sal/c48/80; n = 19 for anti-TNF/c48/80 treatment groups; data are pooled from 2 independent experiments.\n\nTo confirm that the antibody pre-treatment did not have an effect on the extent of mast cell degranulation caused by c48/80 that would have consequently affected mast cell-mediated hyperalgesia, we evaluated the levels of degranulation in toluidine blue-stained 4μm paraffin sections as previously described2 and confirmed that the levels of mild, moderate and extensive degranulation were not altered in anti-TNF-α treated mice (Figure 2).\n\nMice were pre-treated with 200μl/kg anti-TNF-α neutralizing antibody or vehicle (200μl, i.v.) 30 minutes before bilateral intra-plantar c48/80 injection (1.5μg/paw; 10μl). Mice were euthanized 2.5h after c48/80 administration; their hind paws were harvested, preserved in 10% buffered formalin and ethanol, and 4μm paraffin sections stained with toluidine blue for mast cell visualization. Mast cells were counted at 400x and their degranulation status was assessed as described previously2. Bars represent mean percentages of mast cells examined that are assigned to intact or mild, moderate and extensive degranulation status respectively and the error bars represent ±SEM. n = 3 mice per treatment group; data are representative of 2 experiments.\n\nTaken together, pre-treatment with anti-TNF-α neutralizing antibody did not change the extent of local mast cell degranulation in the mouse hind paw tissue but markedly reduced resulting thermal sensitivity and tissue swelling provoked by injection of the basic mast cell secretagogue c48/80.\n\nMast cells contain pre-formed TNF-α in their granules15 that is released early upon degranulation15. We analyzed TNF-α protein and mRNA levels in c48/80-treated and untreated hind paw tissue to determine whether there was new synthesis of TNF-α immediately following treatment with the basic mast cell secretagogue. We found that neither protein nor mRNA levels of the cytokine detectably increased in the tissue within 3 hours following intra-plantar c48/80 injection (Figure 3A–B). Thus, it is most likely that the anti-TNF-α neutralizing antibody treatment targets pre-formed, rather than newly synthesized, TNF-α molecules and this blockade contributes to the resulting decrease in thermal sensitivity in the hind paw tissue.\n\nMice were pretreated with 200μl/kg anti-TNF-α neutralizing antibody or vehicle (200μl, i.v.) 30 minutes before bilateral intra-plantar c48/80 injection (1.5μg/paw; 10μl). 2.5h after c48/80 treatment, their hind paws were excised and preserved for TNF-α protein quantification by ELISA (A). Another set of mice was euthanized at 3h after c48/80 treatment; their footpads were excised for TNF-α mRNA abundance quantification by qRT-PCR (B). Bars represent average pg/ml TNF-α protein (A) and fold-expression of TNF-α transcripts compared to β2m (B) and the error bars represent ±SEM. n = 6 (Sal/Sal); 9 (Sal/c48/80) for ELISA assays and n = 3 mice per treatment group for qRT-PCR; data are representative of 3 (ELISA) and 2 (qPCR) experiments.\n\nNeutrophil influx into the affected tissue contributes to c48/80-provoked hind paw thermal sensitivity2 and TNF-α is a known neutrophil attractant3,16. We have previously shown that the blockade of neutrophil influx can abrogate mast cell-dependent thermal sensitivity in the mouse hind paw2. Therefore, we investigated whether pre-treatment with the TNF-α neutralizing antibody had an effect on neutrophil influx. We found that levels of myeloperoxidase (an enzyme indicating the presence of activated neutrophils) in the hind paw tissue of mice injected bilaterally with 1500ng of c48/80 were not significantly reduced with anti TNF-α pre-treatment (Figure 4A). We further confirmed this by examining the presence of neutrophils in 4μm paraffin embedded hind paw tissue sections stained with H&E and found that infiltrating neutrophils were present in the hind paws of mice that received TNF-neutralizing antibody (Figure 4B). Neutrophil numbers in the hind paw tissue of mice following c48/80 injection with and without anti-TNF-α pre-treatment were approximately a total of ~150 neutrophils in 10 randomly chosen visualized sections per paw (see Data File). Therefore, pre-treatment with anti-TNF-α neutralizing antibody had little to no inhibitory effect on the levels of tissue infiltrating neutrophils and myeloperoxidase enzyme activity in the hind paw tissue of c48/80-treated mice.\n\nMice that received c48/80 intra-plantar injections following anti-TNF-α pretreatment did not have significantly different levels of myeloperoxidase enzyme compared to mice that received vehicle pretreatment (A). Hind paws were harvested from euthanized mice and assayed for MPO activity; bars represent average MPO activity as OD/g wet tissue and error bars represent ±SEM (A). Mice were euthanized 2.5h after c48/80 administration, their hind paws were harvested, preserved in 10% buffered formalin and ethanol, and 4μm paraffin sections stained with hematoxylin & eosin for neutrophil visualization at 1000x. Similar to c48/80-treated mice (B), mice pre-treated with anti-TNF-α neutralizing antibody showed clear evidence of neutrophil influx (indicated by black arrowheads) into the affected hind paw tissue (C). n = 3 mice per treatment group for MPO assay; data are representative of 2 experiments.\n\n\n\n\nDiscussion\n\nPain is a complex physiological and pathological phenomenon with intricate underlying neuro-immune circuitry. Pain can be either protective or maladaptive1,11 and in the former instance, serves as an early warning system that prompts an organism to retreat from noxious environmental stimuli.\n\nWe have previously shown that mast cells are required for enhanced sensitivity to heat stimulus following c48/80 treatment in the hind paw tissue of mice and blockade of neutrophil influx or histamine signaling can abrogate these responses2. Here we show that pre-treatment with TNF-α neutralizing antibody just prior to c48/80 administration significantly reduces early thermal hyperalgesic responses. Mast cells contain pre-formed TNF-α in their cytoplasmic granules and this pro-inflammatory cytokine is one of the earliest mediators released by these cells upon activation15. We looked for evidence of changes in TNF-α protein and mRNA levels shortly after c48/80 treatment and found none, suggesting that the pre-formed TNF released by mast cells may be the main source of this cytokine in these experiments. As TNF-α is a known attractant of neutrophils16, we expected TNF-α blockade to result in a reduction of infiltrating neutrophils. We have previously shown that blockade of neutrophil influx can abrogate c48/80-provoked thermal sensitivity2. However, we found here that in the first 2.5 hours following c48/80 administration, TNF-α neutralizing antibody pre-treatment had little to no effect on recruitment of tissue neutrophils. Our observations are in keeping with earlier studies that show that mast cell mediators other than TNF-α can contribute to the induction of neutrophil influx17. However, pain was significantly abrogated as early as 30 minutes after c48/80 treatment despite the presence of neutrophils.\n\nTNF-α is one of the early inflammatory cytokines with a known role in various nociceptive cascades6,7. Direct injection of TNF-α in the hind paw tissue produces thermal and mechanical hyperalgesia in rodents6,18; anti-TNF-α antibody administration shortly before challenge reduces the hyperalgesic responses to carrageenan12 in mice and blockade of TNF-α induced neutrophil influx impairs thermal carrageenan pain in rats3.\n\nOur findings here add nuance to the prevailing model of mast cell and neutrophil modulation of peripheral nociception with evidence that TNF-α blockade can also abrogate very early thermal pain responses without a proportional reduction in neutrophil influx. Hind paw thermal pain provoked by c48/80 is markedly abrogated despite the presence of infiltrating neutrophils in the tissue. In a model of carrageenan-induced pain in rats, Cunha and colleagues have shown that levels of TNF-α in the tissue are not affected by the absence of infiltrating neutrophils i.e. these cells are not the primary sources of TNF-α3 but rather the release of TNF-α and other cytokines such as IL-1β in the tissue following an inflammatory insult serves to attract infiltrating neutrophils that continue to fuel the nociceptive cascade3. In preliminary experiments, we have found that pre-treatment with antibodies against IL-1β reduces neutrophil influx into the tissue but does not abrogate c48/80-provoked thermal pain at early time points (data not shown). Therefore, the rapid anti-hyperalgesic effect that we observe here may be due to the blockade of earlier, rapid nociceptive effects of TNF-α in pain potentiation mediated by direct modulation of activation thresholds of nociceptive neurons9,19 or effects of TNF-α on nociceptor ion channels10. Our observations here suggest that pre-formed TNF-α, e.g. from mast cell granules in the skin mast cells, is the likely target of the TNF-α neutralizing antibody that produces an early reduction of thermal sensitivity. This is underscored by the documented proximity of nerves and mast cells in the tissue8 and the known effects of mast cell-derived TNF-α on nerve physiology - elongation of cutaneous nerves in oxazolone contact hypersensitivity20, and susceptibility of sensory neurons to agents such as capsaicin, in a model of tracheal vascular hyper-permeability21. These known interactions further support the idea that nociceptive neurons could be early downstream targets of the first release of pre-formed TNF-α immediately after mast cell degranulation. In this study, we show that early, protective, nociceptive withdrawal responses to heat stimulus are compromised by the blockade of TNF-α signaling. It has been documented that individuals who use TNF-targeted biological therapeutics to manage chronic autoimmune and inflammatory conditions can experience a higher risk of bacterial and viral infections22. Our findings here suggest that the use of TNF-α blockade therapies may also compromise the protective, nociceptive responses of such individuals leaving them more vulnerable to tissue damage from injurious environmental stimuli.",
"appendix": "Author contributions\n\n\n\nLP contributed to study design, conducted hyperalgesia, edema and histology experiments and analyzed the data. TM contributed to study design and data analyses, conducted protein analyses and co-wrote the manuscript. EB and LP conducted enzyme activity assays and data analyses. JA and AS performed histological analysis and imaging. AA performed gene expression studies. DC designed the study, supervised experiments, analyzed data and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nDC is supported by Macalester College and NIH Academic Research Enhancement Award (Grant R15 NS067536-01A).\n\n\nAcknowledgements\n\nThe Comparative Pathology Shared Resource at the University of Minnesota provided technical support. In the Chatterjea laboratory, we thank Grace Linder and Emily Ewan for assistance with protein and mRNA quantification studies, Kidist Zewdie and Elena Tonc for logistical assistance, and all other lab members for their support.\n\n\nReferences\n\nRen K, Dubner R: Interactions between the immune and nervous systems in pain. Nat Med. 2010; 16(11): 1267–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChatterjea D, Wetzel A, Mack M, et al.: Mast cell degranulation mediates compound 48/80-induced hyperalgesia in mice. Biochem Biophys Res Commun. 2012; 425(2): 237–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCunha TM, Verri WA Jr, Schivo IR, et al.: Crucial role of neutrophils in the development of mechanical inflammatory hypernociception. J Leukoc Biol. 2008; 83(4): 824–32. PubMed Abstract | Publisher Full Text\n\nGuerrero AT, Verri WA Jr, Cunha TM, et al.: Involvement of LTB4 in zymosan-induced joint nociception in mice: participation of neutrophils and PGE2. J Leukoc Biol. 2008; 83(1): 122–30. PubMed Abstract | Publisher Full Text\n\nLavich TR, Siqueira Rde A, Farias-Filho FA, et al.: Neutrophil infiltration is implicated in the sustained thermal hyperalgesic response evoked by allergen provocation in actively sensitized rats. Pain. 2006; 125(1-2): 180–7. PubMed Abstract | Publisher Full Text\n\nCunha FQ, Poole S, Lorenzetti BB, et al.: The pivotal role of tumour necrosis factor alpha in the development of inflammatory hyperalgesia. Br J Pharmacol. 1992; 107(3): 660–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVerri WA Jr, Cunha TM, Parada CA, et al.: Hypernociceptive role of cytokines and chemokines: targets for analgesic drug development? Pharmacol Ther. 2006; 112(1): 116–38. PubMed Abstract | Publisher Full Text\n\nForsythe P, Bienenstock J: The mast cell-nerve functional unit: a key component of physiologic and pathophysiologic responses. Chem Immunol Allergy. 2012; 98: 196–221. PubMed Abstract | Publisher Full Text\n\nZhang XC, Kainz V, Burstein R, et al.: Tumor necrosis factor-α induces sensitization of meningeal nociceptors mediated via local COX and p38 MAP kinase actions. Pain. 2011; 152(1): 140–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCzeschik JC, Hagenacker T, Schäfers M, et al.: TNF-alpha differentially modulates ion channels of nociceptive neurons. Neurosci Lett. 2008; 434(3): 293–8. PubMed Abstract | Publisher Full Text\n\nWoolf CJ: What is this thing called pain? J Clin Invest. 2010; 120(11): 3742–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRocha AC, Fernandes ES, Quintão NL, et al.: Relevance of tumour necrosis factor-alpha for the inflammatory and nociceptive responses evoked by carrageenan in the mouse paw. Br J Pharmacol. 2006; 148(5): 688–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBradley PP, Priebat DA, Christensen RD, et al.: Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol. 1982; 78(3): 206–9. PubMed Abstract | Publisher Full Text\n\nLivak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method. Methods. 2001; 25(4): 402–8. PubMed Abstract | Publisher Full Text\n\nGalli SJ, Kalesnikoff J, Grimbaldeston MA, et al.: Mast cells as \"tunable\" effector and immunoregulatory cells: recent advances. Annu Rev Immunol. 2005; 23: 749–86. Review. PubMed Abstract | Publisher Full Text\n\nMalaviya R, Ikeda T, Ross E, et al.: Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature. 1996; 381(6577): 77–80. PubMed Abstract | Publisher Full Text\n\nMalaviya R, Abraham SN: Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis. J Leukoc Biol. 2000; 67(6): 841–6. PubMed Abstract\n\nCunha TM, Verri WA Jr, Silva JS, et al.: A cascade of cytokines mediates mechanical inflammatory hypernociception in mice. Proc Natl Acad Sci U S A. 2005; 102(5): 1755–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJunger H, Sorkin LS: Nociceptive and inflammatory effects of subcutaneous TNFalpha. Pain. 2000; 85(1-2): 145–51. PubMed Abstract | Publisher Full Text\n\nKakurai M, Monteforte R, Suto H, et al.: Mast cell-derived tumor necrosis factor can promote nerve fiber elongation in the skin during contact hypersensitivity in mice. Am J Pathol. 2006; 169(5): 1713–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Houwelingen AH, Kool M, de Jager SC, et al.: Mast cell-derived TNF-alpha primes sensory nerve endings in a pulmonary hypersensitivity reaction. J Immunol. 2002; 168(10): 5297–302. PubMed Abstract\n\nXie X, Li F, Chen JW, et al.: Risk of tuberculosis infection in anti-TNF-α biological therapy: From bench to bedside. J Microbiol Immunol Infect. 2013. pii: S1684-1182(13)00039-X. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1561",
"date": "29 Aug 2013",
"name": "Peter D. Drummond",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper makes a strong case for the early involvement of mast cell-derived TNF-alpha in thermal hyperalgesia and oedema during inflammatory reactions. The only contentious issue is the suggestion that TNF-alpha acts directly on nociceptors, rather than via the cascade of inflammatory mediators triggered by TNF release. The references cited in support of this view either did not directly address this point, or did not provide the intended support. For example, Zhang et al. (2011) (reference 9 in the paper) proposed that TNF-alpha sensitizes meningeal nociceptors indirectly via local COX activity and the p38 MAPK signalling cascade, rather than by direct actions on nociceptors. Wang et al. (J Mol Neurosci 2007;32:207-216) reported that TNF receptors in the sciatic nerve were expressed primarily on Schwann cells rather than neurons, leaving open the possibility that Schwann cells are involved in the inflammatory cascade triggered by TNF-alpha.One other minor point: I think this sentence in the first paragraph of the Results \"Saline-treated control mice had no increase in thermal sensitivity and showed no tissue swelling after c48/80 treatment\" should be \"Saline-treated control mice had no increase in thermal sensitivity and showed no tissue swelling\".",
"responses": [
{
"c_id": "568",
"date": "24 Sep 2013",
"name": "Devavani Chatterjea",
"role": "Author Response",
"response": "Dear Dr. Drummond,Thank you for reviewing our paper. Following your suggestion, we have rephrased the sentence describing the effects of TNF-a on nociceptors as possibly mediated by inflammatory intermediates. We have also corrected the sentence in the first paragraph of the results section."
}
]
},
{
"id": "1562",
"date": "29 Aug 2013",
"name": "Adrian Piliponsky",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study by Chatterjea D et al. is well-designed; its conclusions are balanced and justified based on the results shown. I think that it could be relevant to know whether TNF-alpha injections can mimic the effects of c48/80 injections in the model used: hyperalgesic withdrawal responses.Figure 4a leads to the conclusion that neutrophil influx is not affected by treatment with anti-TNFalpha antibodies and c48/80. Data is based on n=3 according to figure legend, though there is a trend for neutrophil numbers to be reduced by treatment with anti-TNFalpha antibodies. I am wondering whether the authors have considered increasing the number of mice for this experiment. If it is definitely not the case that neutrophils numbers are reduced, it remains unexplained why there is a discrepancy with a previous study, in which the authors demonstrated that neutrophils are an important component of the hyperalgesic response induced by c48/80.",
"responses": [
{
"c_id": "567",
"date": "24 Sep 2013",
"name": "Devavani Chatterjea",
"role": "Author Response",
"response": "Dear Dr. Piliponsky,Thank you for reviewing our paper. In our discussion of the finding in Figure 4A, we have now added some clarifying language indicating that these findings are based on a relatively small sample size but that multiple lines of evidence indicate that at least in these experiments, treatment with anti-TNF neutralizing antibodies did not completely abrogate neutrophil influx into the c48/80-treated hind paw tissue."
}
]
},
{
"id": "1559",
"date": "02 Sep 2013",
"name": "Rosa Torres",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nChatterjea et al. show that TNF-alpha blockade reduced early tissue swelling in a mast cell-dependent thermal hyperalgesia model. The paper is well constructed and clear, and experiments have been well designed and conducted. There are minor questions that I mention below. Anti-TNF-a neutralizing antibody dose is mainly expressed in the article as ul/Kg, please write it as ug/Kg.Since toluidine blue staining is performed differently in different tissues please indicate pH and the time of incubation.In the Methods for TNF-a expression measurements it is said “relative transcript abundance was determined by quantitative RT-PCR” but the technique used is semi-quantitative.When explaining Figure 1A does not mention that at 30 minutes it is also significantly different when comparing the control and TNF treated groups.Please comment more on the mast cells degranulation status of figure 2. Is 60% mild degranulation for this model an expected value? Have you seen or would you expect different percentages using different C4880 doses?Pre-treatment with TNF-a antibody seems to have a small effect on MPO activity but not a significant one, probably because of the low n number (n = 3). This could be discussed as a possibility.",
"responses": [
{
"c_id": "566",
"date": "24 Sep 2013",
"name": "Devavani Chatterjea",
"role": "Author Response",
"response": "Dear Dr. Torres,Thank you for reviewing our paper. Our responses to your comments follow: 1. We have now corrected this throughout the manuscript.2. We have added this information to the relevant methods section.3. We have clarified that our approach uses comparative quantitative PCR analysis and added an additional reference for this nomenclature. 4. We have added this to the description of the report of the results from Figure 1A.5. We have added a sentence to the discussion indicating that the levels of degranulation seen here are similar to those we have previously observed.6. We have added some clarifying language indicating that these findings are based on a relatively small sample size but that multiple lines of evidence indicate that at least in these experiments, treatment with anti-TNF neutralizing antibodies did not completely abrogate neutrophil influx into the c48/80-treated hind paw tissue."
}
]
}
] | 1
|
https://f1000research.com/articles/2-178
|
https://f1000research.com/articles/2-197/v1
|
24 Sep 13
|
{
"type": "Short Research Article",
"title": "Novel targets of miR-30, a microRNA required for biliary development",
"authors": [
"Claire L. Le Guen",
"Joshua R. Friedman",
"Nicholas J. Hand",
"Claire L. Le Guen"
],
"abstract": "MicroRNAs have been found to play a profound role in embryonic and post-natal development through their regulation of processes such as cell proliferation, differentiation, and morphogenesis. The microRNA-30 (miR-30) family is necessary for vertebrate hepatobiliary development; however, the mechanism through which miR-30 regulates these processes is not fully understood. Here, we identify genes directly regulated by miR-30 that have been characterized as key developmental factors. The targets were confirmed via a luciferase reporter assay, following exogenous over-expression of miR-30a and miR-30c2 in cultured cells. Five novel miR-30ac2 targets were identified using this approach, all of which play crucial roles in hepatobiliary development or are involved in hepatocellular carcinoma and cholangiocarcinoma.",
"keywords": [
"MicroRNAs are short non-coding RNAs that regulate cell function",
"differentiation",
"organ development",
"and disease states (reviewed in1–4). MiRNAs regulate gene expression via the RNA-induced silencing complex (RISC)",
"leading to translational repression and/or transcript degradation5. The identification of gene targets for individual miRNAs is key to understanding their function",
"thus necessitating accurate methods to identify significant mRNA targets. The prevailing methods of miRNA target identification begin with computational algorithms based on free energy change calculations of complementary binding",
"other sequence features",
"and evolutionary conservation6",
"7. However",
"false positive rates are high",
"as interactions may be predicted in physiological settings in which targeting does not naturally occur8. The data gathered from these computational methods are typically validated by luciferase reporter assays in cultured cells",
"in which the activity of the relevant miRNA is altered (i.e.",
"by miRNA over-expression or repression)."
],
"content": "Background\n\nMicroRNAs are short non-coding RNAs that regulate cell function, differentiation, organ development, and disease states (reviewed in1–4). MiRNAs regulate gene expression via the RNA-induced silencing complex (RISC), leading to translational repression and/or transcript degradation5. The identification of gene targets for individual miRNAs is key to understanding their function, thus necessitating accurate methods to identify significant mRNA targets. The prevailing methods of miRNA target identification begin with computational algorithms based on free energy change calculations of complementary binding, other sequence features, and evolutionary conservation6,7. However, false positive rates are high, as interactions may be predicted in physiological settings in which targeting does not naturally occur8. The data gathered from these computational methods are typically validated by luciferase reporter assays in cultured cells, in which the activity of the relevant miRNA is altered (i.e., by miRNA over-expression or repression).\n\nMiR-30a, expressed as part of an intronic cluster with miR-30c-2, is required for hepatobiliary development in zebrafish9. In our previous study9, we found that within the fetal mouse liver and infant human liver, members of the miR-30 family are predominantly expressed within the biliary primordium, post-natal bile ducts, and hepatocytes. Using a zebrafish model of miR-30a deficiency, we confirmed that miR-30a plays a critical role in vertebrate bile duct development9. The next step in understanding the function of the miR-30 family in biliary development is to identify the genes that it targets.\n\nUsing gene expression profiling in cultured hepatoblasts9, we previously identified a set of mRNAs whose levels are increased following antisense oligonucleotide (ASO)-mediated inhibition of miR30a9. From this dataset, we have selected 7 novel candidate targets (Ccne2, Celsr3, Mdm2, Mtdh, Smad1, Twf, and Timp3) on the basis of biological relevance and two bioinformatic prediction methods: Targetscan and PITA6,7. In addition, we evaluated four candidate targets previously tested via ASO-mediated miR-30a inhibition in cultured cells (Ak1, Tnrc6a, Egfr, and Inhba)9 (Table 1). All 11 candidates were tested using luciferase reporter assays coupled with over-expression of miR-30a. Here we show that several of these candidate target genes - selected on the basis of their known roles in growth and morphogenesis - are direct miR-30a targets.\n\nSee text for references.\n\n\nMethods\n\nThe 3’ untranslated regions (UTRs) of predicted target genes were amplified from C57B/6 mouse genomic DNA by nested PCR and cloned into pMirCheck2 plasmids10, a modified derivative of pSiCheck™-2 (Promega, Madison, WI) (Data File 1).\n\nThe miR-30ac2 expression plasmid was subcloned into the pSLIK lentiviral vector platform11 as follows: the murine miR-30ac2 cluster was amplified via nested PCR from C57B/6 genomic DNA and cloned into the ScaI, MfeI sites of the pSLIK tet-inducible, GFP-expressing entry vector pEN-TTGmiRc2. This entry vector was recombined into the pSLIK vector using Gateway® LR Clonase® II Enzyme mix (Life Technologies, Grand Island, NY).\n\nOne male C57B/6 mouse was used as the source of PCR template DNA in plasmid construction. It was kept in standard caging in a controlled environment (12 hour/12 hour light/dark cycle; temperature 22°±2°C, in a cage with clear plastic walls (17 cm x 19 cm base, 16 cm high). It was fed standard mouse chow and water ad libitum. It was euthanized at 12 weeks of age by anesthesia with ketamine (200 mg/kg) and xylazine (15 mg/kg) (Sigma-Aldrich) followed by decapitation. All procedures were in accordance with federal and institutional guidelines under the supervision of the Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee.\n\nThe human embryonic kidney cell line HEK293FT (Life Technologies, Carlsbad, CA) was maintained at 37ºC and 5% CO2 in Dulbecco’s modified Eagle’s Minimum Essential Medium High Glucose GlutaMAX™ (Life Technologies) supplemented with 10% Tet-system approved fetal bovine serum (Clontech, Mountain View, CA). For luciferase reporter experiments, 5×104 HEK293FT cells were seeded into 24-well plates. After 24 hours, cells were transfected with 900ng expression plasmid and 100ng of reporter plasmid per well using FuGENE® HD transfection reagent (Promega, Madison, WI). Twenty-four hours post-transfection, cells were induced with 1μg/ml doxycycline (Sigma-Aldrich, St. Louis, MO) for an additional 24 hours of outgrowth. For the over-expression validation experiment, 6×104 HEK293FT cells transfected with 1μg of miR-30ac2 over-expression plasmid, using 3μl of FuGENE® transfection reagent (Promega). After 16 hours of outgrowth, cells were induced with 1μg/ml doxycycline and outgrown for an additional 24 hours. Total RNA was isolated using the miRVana™ miRNA Isolation Kit (Life Technologies) according to manufacturer’s instructions.\n\nCells were washed with 1×PBS and lysed in 150μl 1×Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System (Promega) on a GloMax Multi luminometer (Promega) according to manufacturer’s instructions. Relative light units were calculated as the ratio of Renilla to firefly luciferase activity, and the reporters were normalized to the control expression plasmid and to empty pMirCheck2 reporters to correct for nonspecific effects. Three biological replicates were performed for each condition.\n\nMature miRNAs were reverse transcribed from 10ng total RNA using the TaqMan® MicroRNA Reverse Transcription Kit and TaqMan® MicroRNA Assays (Life Technologies). qRT-PCR was performed in duplicate for 4 biological replicates. Relative expression was calculated using comparative CT method and normalized to RNU44.\n\nStatistical significance was determined between groups using an unpaired Student’s t-test in Microsoft Excel, using a p-value ≤ 0.05 to determine significance.\n\n\nResults\n\nTo determine the regulatory effect of the miR-30a and miR-30c cluster (“mir-30ac2”) on the candidate target genes, we induced the over-expression of mir-30ac2 in cultured cells. We transfected a human embryonic kidney cell line transformed with the SV40 large T antigen (HEK293FT) with a doxycycline-inducible green fluorescent protein (GFP) and miR-30ac2 expression plasmid. We observed GFP-positive cells 24 hours post-induction. Mir-30a transcript levels were quantified using qRT-PCR, and revealed a 28-fold increase of miR-30a in cells transfected with the miR-30ac2 expression plasmid versus a control encoding a scrambled sequence (Figure 1). The unrelated control miRNA let-7c was also assayed and expression levels were not significantly altered between the two treatment groups, confirming that miR-30ac2 over-expression did not have an effect on miRNA production in general.\n\nA plasmid encoding miR-30a cluster miRNAs (miR-30a and miR-30c2) or a scrambled miRNA sequence was transfected into HEK293T cells. The resulting levels of miR-30a and let-7a were measured by qRT-PCR. Error bars indicate the standard error of the mean. * p < 0.05. The data represent three separate transfections, each with three technical replicate wells.\n\nWe selected nine potential miR-30 target genes for further investigation and included two confirmed miR-30 target genes in our study to serve as positive controls. We constructed luciferase reporter plasmids for each of the genes of interest by subcloning each 3′-UTR immediately downstream of the Renilla (hRluc) luciferase cDNA (Data File 1). Following co-transfection of HEK293FT cells with the miR-30ac2 over-expression plasmid and each reporter plasmid, we observed that reporters containing the 3′-UTRs of all the candidate genes were significantly down-regulated relative to the control reporter, with the exception of Inhba (Figure 2). The degree of down-regulation ranged from 28–88% of basal activity.\n\nHEK293T cells we transfected with the miR-30ac2 expression plasmid and the indicated reporter plasmids. The ratio of firefly to Renilla luciferase activity for each reporter was normalized to that of an empty reporter. Error bars indicate the standard error of the mean. * p < 0.05. The data represent three separate transfections, each with three technical replicate wells.\n\nTo determine if the degree of down-regulation correlated to predicted miRNA target affinity, we correlated their PITA and TargetScan prediction scores with the reporter assay results. A slight positive correlation (R2 = 0.68) was observed between the PITA target score and the reporter assay (Figure 3). No correlation was observed between the Targetscan score and the degree of repression.\n\nThe PITA (left) and Targetscan (right) scores are plotted on the x-axis, with the relative level of luciferase activity following miR-30ac2 over-expression on the y-axis.\n\n\n\n\nDiscussion\n\nThe regulation of gene expression plays a critical role in establishing the complex protein patterning that is a hallmark of development and growth. While transcriptional control determines which genes have the potential to be expressed, translational control presents an additional mechanism to fine tune protein level output spatially and temporally. In the fetal liver, miR-30a and miR-30c2 are expressed in ductal plate cholangiocytes and decrease dramatically in the postnatal bile duct9.\n\nTo investigate what role differential miR-30ac2 expression may be having on biliary development, we wanted to determine if miR-30ac2 directly targeted the 3′-UTR of genes that have an important function in cell growth and morphogenesis. The panel of candidate miR-30 target genes selected for validation was based on the results of gene expression profiling by microarray following miR-30a knockdown in miR-30a ASO-treated mouse embryonic liver (BMEL) cells9. From this data, nine genes were selected for further validation due to the potential role they play in hepato-biliary development. Of the nine experimental targets, eight were identified as direct miR-30ac2 targets, with three of these eight targets (Mtdh12, Smad113, and Twf14) validated as direct miR-30ac2 targets in separate studies during the preparation of this manuscript.\n\nThe novel genes identified as directly regulated by miR-30ac2 are Ccne2, Celsr3, Egfr, Mdm2, and Timp3. Additionally, in our study, Mtdh, Smad1, and Twf, were identified as miR-30ac2 targets, corroborating the results of the previous studies and validating our method. Inhba was the only gene included in our study that was not identified as a direct target. The targeting of these genes by miR-30ac2 support the hypothesis that the miR-30 family is required for biliary development, as the targeted genes all also have roles in hepatocyte proliferation. CcnE2 (Cyclin E2) plays a role in promoting S-phase entry from quiescence, and has also been implicated in regulatory pathways promoting liver regeneration and fibrogenesis15,16. Celsr3 (cadherin, EGF LAG seven-pass G-type receptor 3) is a cadherin important for the maintenance of planar cell polarity17, an essential feature of hepatic epithelial cells18. Egfr (Epidermal growth factor receptor) is an essential activator of liver regeneration19, and its over-expression has also been associated with cholangiocarcoma20. Mdm2 (E3 ubiquitin protein ligase) regulates tumor suppressor proteins, such as p53, by marking them for proteasomal degradation, and its aberrant expression is linked to hepatocellular carcinoma, indicating a role in liver cell proliferation21. Timp3 (tissue inhibitor of metallopeptidase 3) inhibits matrix metalloproteinases which function to degrade extracellular matrix, and has also been implicated in hepatocellular carcinoma22 .\n\nDuring the preparation of this manuscript, three of our confirmed miR-30ac2 targets were independently verified as miR-30 targets and described in other publications. Mtdh12, Smad113, and Twf14. Tnrc6a and Ak1 were included in this study as verified positive controls and have been previously described9. Inhba (Inhibin, beta A) was not targeted by miR-30ac2 in our assay suggesting that it is not a direct target, but does not rule out the possibility of indirect regulation.\n\nThe degree of target repression as a result of miR-30ac2 over-expression is loosely correlated to the predicted PITA score which calculates aggregate ∆∆G of all 3′-UTR binding sites. Those targets with the greatest PITA score demonstrated a high level of repression, in the order of 28–42% expression relative to our controls. The Targetscan scores did not correlate with the reporter assay results. This difference between the two methods most likely reflects their distinct algorithms; PITA relies on thermodynamic prediction of miRNA:mRNA binding affinity, whereas Targetscan places more emphasis on evolutionary conservation6,7. Overall, our results highlight the necessity of confirming predicted miRNA target genes by experimental approaches.\n\nIn conclusion, this study presents five new novel targets of miR-30a and miR-30c, all of which play roles in biliary and liver development, and additionally have implications in cancers of the liver. Given the crucial role of miR-30 in biliary development, we speculate that aberrant expression of miR-30 may be a factor in the development of human biliary diseases, and can therefore be considered as a potential target for therapeutic manipulation.",
"appendix": "Author contributions\n\n\n\nJRF and NJH conceived and designed the study. NJH provided direct supervision of CLL, who performed all of the experiments and wrote the first draft of the manuscript. All authors contributed to manuscript editing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe studies were supported by NIH R01DK079881 (JRF) and the Fred and Suzanne Biesecker Pediatric Liver Center.\n\n\nReferences\n\nIorio MV, Croce CM: MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med. 2012; 4(3): 143–159. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMendell JT, Olson EN: MicroRNAs in Stress Signaling and Human Disease. Cell. 2012; 148(6): 1172–1187. PubMed Abstract | Publisher Full Text | Free Full Text\n\nD’Alessandra Y, Pompilio G, Capogrossi MC: MicroRNAs and myocardial infarction. Curr Opin Cardiol. 2012; 27(3): 228–235. PubMed Abstract | Publisher Full Text\n\nRottiers V, Näär AM: MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol. 2012; 13(4): 239–250. PubMed Abstract | Publisher Full Text\n\nHuntzinger E, Izaurralde E: Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet. 2011; 12(2): 99–110. PubMed Abstract | Publisher Full Text\n\nLewis BP, Burge CB, Bartel DP: Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005; 120(1): 15–20. PubMed Abstract | Publisher Full Text\n\nKertesz M, Iovino N, Unnerstall U, et al.: The role of site accessibility in microRNA target recognition. Nat Genet. 2007; 39(10): 1278–1284. PubMed Abstract | Publisher Full Text\n\nReyes-Herrera PH, Ficarra E: One decade of development and evolution of microRNA target prediction algorithms. Genomics Proteomics Bioinformatics. 2012; 10(5): 254–263. PubMed Abstract | Publisher Full Text\n\nHand NJ, Master ZR, Eauclaire SF, et al.: The microRNA-30 family is required for vertebrate hepatobiliary development. [Internet]. Gastroenterology. 2009; 136(3): 1081–1090. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHand NJ, Horner AM, Master ZR, et al.: MicroRNA Profiling Identifies miR-29 as a Regulator of Disease-Associated Pathways in Experimental Biliary Atresia. [Internet]. J Pediatr Gastroenterol Nutr. 2011; 54(2): 186–192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShin K-J, Wall EA, Zavzavadjian JR, et al.: A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci USA. 2006; 103(37): 13759–13764. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang N, Wang X, Huo Q, et al.: MicroRNA-30a suppresses breast tumor growth and metastasis by targeting metadherin. Oncogene. [published online ahead of print: July 15, 2013]. PubMed Abstract | Publisher Full Text\n\nWu T, Zhou H, Hong Y, et al.: miR-30 family members negatively regulate osteoblast differentiation. J Biol Chem. 2012; 287(10): 7503–7511. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBockhorn J, Dalton R, Nwachukwu C, et al.: MicroRNA-30c inhibits human breast tumour chemotherapy resistance by regulating TWF1 and IL-11. Nat Commun. 2013; 4: 1393. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNevzorova YA, Tschaharganeh D, Gassler N, et al.: Aberrant cell cycle progression and endoreplication in regenerating livers of mice that lack a single E-type cyclin. Gastroenterology. 2009; 137(2): 691–703703.e1–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNevzorova YA, Bangen JM, Hu W, et al.: Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice. Hepatology. 2012; 56(3): 1140–1149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y: Wnt/Planar cell polarity signaling: a new paradigm for cancer therapy. Mol Cancer Ther. 2009; 8(8): 2103–2109. PubMed Abstract | Publisher Full Text\n\nCui S, Capecci LM, Matthews RP: Disruption of planar cell polarity activity leads to developmental biliary defects. Dev Biol. 2011; 351(2): 229–241. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParanjpe S, Bowen WC, Tseng GC, et al.: RNA interference against hepatic epidermal growth factor receptor has suppressive effects on liver regeneration in rats. Am J Pathol. 2010; 176(6): 2669–2681. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoshikawa D, Ojima H, Iwasaki M, et al.: Clinicopathological and prognostic significance of EGFR, VEGF, and HER2 expression in cholangiocarcinoma. Br J Cancer. 2008; 98(2): 418–425. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang J, Zheng T, Chen X, et al.: MDM2 antagonist can inhibit tumor growth in hepatocellular carcinoma with different types of p53 in vitro. J Gastroenterol Hepatol. 2011; 26(2): 371–377. PubMed Abstract | Publisher Full Text\n\nWang B, Hsu SH, Majumder S, et al.: TGFbeta-mediated upregulation of hepatic miR-181b promotes hepatocarcinogenesis by targeting TIMP3. Oncogene. 2010; 29(12): 1787–1797. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1945",
"date": "01 Oct 2013",
"name": "Tushar Patel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reports the identification and subsequent validation of several targets of miR-30a/mir-30c cluster using luciferase-based assays in a human embryonic kidney cell line. The use of computational algorithms to identify downstream targets may not always identify targets with biological significance. Thus, studies such as these are necessary to identify relevant downstream miRNA targets. The studies are well described, with adequate detail, and the conclusions are justified. Future studies to explore these targets could consider analyses using gene-specific 3-UTR luciferase constructs in which putative miRNA binding sites have been mutated, as well as an evaluation of their effects in the specific target cells of interest, such as hepatoblasts.",
"responses": []
},
{
"id": "2015",
"date": "07 Oct 2013",
"name": "Kalpana Ghoshal",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFive different miR-30 variants (a-e), all sharing the same seed sequence, are expressed in mammals. Previously, Hand et al. have shown that miR-30 family members are necessary for development of the hepatobiliary track in vertebrates [PMID: 19185580]. However, the underlying molecular mechanism through which miR-30 regulates bile duct development has not been completely understood.In this manuscript Le Guen et al. report identification and validation of novel targets of two biliary-specific hepatic miRNAs, namely miR-30a and miR-30c2. To do this, authors have cloned 3’-UTRs of candidate targets into psiCHEK2 vector downstream of renilla luciferase coding sequence, and measured the ratio of renilla to firefly (internal control) luciferase activities in H293T cells co-transfected with the recombinant psiCHECK2 and an expression vector harboring miR-30ac2 gene. Using this approach, they have identified and validated 5 novel miR-30ac2 targets, namely Ccne2, Celsr3, Egfr, Mdm2, and Timp3, that are known to play an important role in hepatobiliary development, or are involved in hepatocarcinogenesis and cholangiocarcinoma development. Additionally, they confirmed Mtdh, Smad1, and Twf as miR-30ac2 targets, reported recently by others. The extent of target repression by ectopic miR-30ac2 varied among these genes and is correlated, albeit loosely, to the predicted PITA score but not the TargetScan score. Overall, this is a very well written manuscript and the data is well presented. The targets of miR-30ac2, two bile duct enriched miRNA, identified in this study could explain the key function of these two miRs in liver development, and their potential causal role in hepato- and cholangio-carcinogenesis.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-197
|
https://f1000research.com/articles/2-196/v1
|
24 Sep 13
|
{
"type": "Case Report",
"title": "A rare aggravation of severe mucositis post chemotherapy in a child with acute lymphoblastic leukemia",
"authors": [
"Adlette Inati",
"Grace Akouri",
"Hussein A Abbas",
"Grace Akouri",
"Hussein A Abbas"
],
"abstract": "Oral mucositis is a debilitating manifestation in children undergoing chemotherapy and radiotherapy. Children with mucositis should be properly managed in order to prevent further exacerbation and adverse complications. We hereby present the first report of a severe chemotherapy-induced mucositis, plausibly aggravated by improper dental hygiene leading to shedding of the ventral part of the tongue in a child with pre-B acute lymphoblastic leukemia (ALL). The patient steadily and gradually recovered her oral maneuvers and ability to speak several months later. Her tongue underwent hypertrophy as a compensatory mechanism. We recommend that critical and regular assessment of the oral mucosa and proper dental care and oral hygiene be emphasized in all pediatric patients receiving chemotherapy. Families of affected children need to be educated about the benefits and modes of optimal oral hygiene for their children and the need to seek immediate care for mouth pain and or lesions. Optimal treatment for mucositis needs to be instituted without delay in this high risk pediatric population. Such a preventive and therapeutic approach may prevent associated life threatening oral and systemic complications, promote rapid and complete mucosal healing, alleviate pain and improve quality of life in children with cancer.",
"keywords": [
"mucositis",
"chemotherapy",
"pre-B ALL",
"pediatric"
],
"content": "Case report\n\nWe report a 5-year old female with average risk (AR1) pre-B acute lymphoblastic leukemia (ALL) who presented with severe oral mucosal pain and fever of 38.7oC on day 22 induction (EORTC Children’s Leukemia Group protocol AR1 shown in Table 1).\n\nOn physical exam, the patient was sick-looking, lethargic and moderately pale. Her vital signs were: temperature = 38.7oC, HR = 120/min, RR = 24/min and BP = 90/60 mm Hg. Her weight was 12.2 kg (<3rd % for her age) and her height was 110 cm (75% for her age). Her conjunctivae were mildly injected and her sclerae were non icteric. The oral cavity showed diffuse dental caries, severe mucosal erythema with whitish membranes and multiple necrotic and hemorrhagic lesions in the tongue, buccal mucosa and lower lip (Figure 1). She was unable to drink fluids or eat solid food (WHO mucositis stage IV)1. Her heart sounds were regular; a GII/VI systolic hemic murmur was heard all over the precordium. Lungs were clear and resonant. Abdomen was soft, non-tender with no hepatosplenomegaly or masses or ascites. There was no pathologic lymphadenopathy. Femoral pulses were symmetrical bilaterally and there was no peripheral edema. Neurologic exam, fundoscopy and skeletal exams were all normal. Laboratory tests upon admission showed severe neutropenia with moderate anemia and thrombocytopenia, increased prothrombin time (PT), partial thromboplastin time (PTT) and low fibrinogen (Table 2).\n\nINR = International normalized ratio\n\nSGOT = serum glutamic oxaloacetic transaminase\n\nSGPT = serum glutamic-pyruvic transaminase\n\nGGT = gamma-glutamyl transpeptidase\n\nThe patient was started on intravenous cefepime (150 mg/Kg/d), vancomycin (60 mg/Kg/d), fluconazole (12 mg/Kg/d on day 1 followed by 6 mgs/Kg/d afterwards), topical mycostatin, 0.12% chlorhexidine mouthwash, adequate hydration and IV analgesia (perfelgan 15 mg/Kg given q6 hours). She also received vitamin K, platelet transfusion and fresh frozen plasma. On hospitalization day 3, the patient was still febrile with progressive mucositis and no oral intake. Micafungin 2.5 mg/Kg/day was started and fluconazole was discontinued. Total parenteral nutrition (TPN) was also initiated and packed red blood cell and platelet transfusions were continued as needed. On day 4, admission cultures of the lesions of the tongue and buccal mucosa grew Candida albicans and coagulase-negative staphylococci while blood culture was negative.\n\nThe patient was steadily but slowly improving when on hospitalization day 6, she had an unexpected spontaneous shedding of the ventral part of her tongue (Figure 2). Pathological analysis of the detached tongue confirmed necrosis and bacterial and fungal cultures were negative. Her speech and feeding were further impeded by this trauma. The patient was continued on IV imipenem (100 mg/Kg/d), vancomycin (60 mg/Kg/d), amikacin (20 mg/Kg/d) and fluconazole (12 mg/Kg/day day 1 followed by 6 mgs/Kg/day afterwards) antimicrobials and TPN. A psychology consultation was offered to the parents and the patient. On hospitalization day 13, her mucositis resolved and her oral food and fluid intake gradually recovered. Chemotherapy was initiated on hospitalization day 17 with no complications and the patient was discharged on day 22 in good condition. The family was instructed to adhere to the recommended daily oral care regimen consisting of topical mycostatin (100,000 unit/g applied BID), 0.12% chlorhexidine mouthwash, gentle tooth brushing and adequate oral hydration.\n\nEight months after the incident, the patient recovered her ability to undergo oral maneuvers and her articulation steadily and progressively improved. Her tongue underwent compensatory hypertrophy (Figure 3). She continues, though, to have slightly unintelligible speech at times. Currently, the patient is in complete remission and receiving her maintenance chemotherapy.\n\n\nDiscussion\n\nLeukemia and its treatment can adversely affect oral health. Leukemic cells are capable of infiltrating the gingiva and the deeper periodontal tissues resulting in local ulceration and infection. Mucosal cells are susceptible to chemotherapy due to their high mitotic rate2. Oral mucositis is the most frequent and debilitating complication of chemotherapy in children with cancer and can be associated with serious morbidities and increased risk of infection2–4.\n\nThe decreased salivary flow rate, salivary pH and total salivary antioxidant levels in leukemic children compared to controls can lead to further deterioration in their oral health status, gingival status and increased dental caries3,4. Our patient suffered from poor dental hygiene and severe dental caries when diagnosed with pre-B ALL (Figure 4). Oxidative stress per se may result in the onset of inflammatory oral pathologies. Saliva constitutes the first line of defense against free radical-mediated oxidative stress4. The biological manifestations of mucositis include a surge in lipopolysaccharides or endotoxins which activate macrophages and other mononuclear cells leading to production of nitric oxide, tumor necrosis factor (TNF), interleukin-6 (IL-6) and other cytokines5,6.\n\nTo our knowledge, this is the first report of very severe chemotherapy-induced mucositis leading to shedding of a part of the tongue. We hypothesize other factors in this patient may have led to this fulminant and unique clinical presentation. The poor oral hygiene and the advanced dental caries the patient had at the time of ALL diagnosis could have contributed to the flare up of chemotherapy induced mucositis and to the shedding of part of the tongue. This hypothesis conforms to the previously reported association of exacerbated oral mucositis with trauma from the teeth4. The degree of severity leading to tongue shedding in this patient was, however, remarkable. Notably, the tongue recovery and hypertrophic sublingual tissue seen in this patient 8 months after the incident are not unexpected and are a result of intrinsic tissue compensatory mechanisms which aided the patient in using her tongue for eating and speaking.\n\nChildren receiving chemotherapy should have critical and regular assessment of their oral mucosa before, during and after treatment. There are several oral assessment rating scales that can be used in cancer patients receiving chemotherapy such as the Oral Assessment Guide (OAG), Oral Mucositis Index (OMI) and Oral Mucositis Assessment Scale (OMAS)7. While OAG addresses the oral functionality and tissue keratinization, the OMAS and OMI focus on the degree of tissue abrogation7.\n\nCareful oral management of children with cancer is critical and aims at preventing and treating mucositis and secondary infections as well as relieving pain and improving quality of life. An oral care protocol consisting of daily mouth rinsing with 0.12% chlorhexidine and tooth-brushing has been found to significantly decrease the incidence of ulcerative lesions, severity of oral mucositis and the related pain intensity compared to controls in pediatric cancer patients8. In infants and very young children who are unable to rinse, care-givers need to use the chlorhexidine or its equivalent as an oral swab.\n\n\nConclusion\n\nThis case represents a very severe chemotherapy-induced mucositis leading to a unique and previously unreported complication in a child with leukemia. It illustrates the fact that critical and regular assessment of the oral mucosa and proper dental care and oral hygiene are mandatory in all pediatric patients receiving chemotherapy. Physicians caring for children with cancer need to be aware of this rare complication and educate families about the benefits and modalities of optimal oral hygiene and the need to immediately report to their child’s health provider for mouth pain and or lesions. Additionally, vigilant treatment of mucositis needs to be instituted without delay in this high risk patient population. Such a preventive and therapeutic approach may prevent life threatening oral and systemic complications, promote mucosal healing and potentiate the hypertrophic compensatory action of linguinal cells thereby ensuring rapid and complete recovery.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient’s legal guardian.",
"appendix": "Author contributions\n\n\n\nDr. Inati and Dr. Akouri diagnosed, followed up the patient and reviewed the manuscript. Dr. Abbas followed up on the case and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nWHO: Handbook for reporting results of cancer treatment. Geneva, Switzerland: World Health Organization. 1979; 45. Reference Source\n\nSchwab M, Zanger UM, Marx C, et al.: Role of genetic and nongenetic factors for fluorouracil treatment-related severe toxicity: a prospective clinical trial by the German 5–FU Toxicity Study Group. J Clin Oncol. 2008; 26(13): 2131–2138. PubMed Abstract | Publisher Full Text\n\nHegde AM, Joshi S, Rai K, et al.: Evaluation of oral hygiene status, salivary characteristics and dental caries experience in acute lymphoblastic leukemic (ALL) children. J Clin Pediatr Dent. 2011; 35(3): 319–323. PubMed Abstract\n\nRaber-Durlacher JE, Elad S, Barasch A: Oral mucositis. Oral Oncol. 2010; 46(6): 452–456. PubMed Abstract | Publisher Full Text\n\nGifford GE, Lohmann-Matthes ML: Gamma interferon priming of mouse and human macrophages for induction of tumor necrosis factor production by bacterial lipopolysaccharide. J Natl Cancer Inst. 1987; 78(1): 121–124. PubMed Abstract\n\nSonis ST: Mucositis as a biological process: a new hypothesis for the development of chemotherapy-induced stomatotoxicity. Oral Oncol. 1998; 34(1): 39–43. PubMed Abstract | Publisher Full Text\n\nEilers J, Million R: Prevention and management of oral mucositis in patients with cancer. Semin Oncol Nurs. 2007; 23(3): 201–212. PubMed Abstract | Publisher Full Text\n\nCheng KK, Molassiotis A, Chang AM, et al.: Evaluation of an oral care protocol intervention in the prevention of chemotherapy-induced oral mucositis in paediatric cancer patients. Eur J Cancer. 2001; 37(16): 2056–2063. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1889",
"date": "24 Sep 2013",
"name": "Bassem Razzouk",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case report about severe oral mucositis in a patient with ALL receiving induction therapy. The existing severe dental caries present at the diagnosis of ALL most likely contributed to the severity of the condition, which was managed appropriately and aggressively.",
"responses": []
},
{
"id": "1892",
"date": "25 Sep 2013",
"name": "Jan Stary",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present the unusually severe complication of mucositis developed during the induction treatment of acute lymphoblastic leukemia. They stress the need for education of family and patients about the importance of oral hygiene during the intensive chemotherapy. The paper is well written and documented.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-196
|
https://f1000research.com/articles/2-190/v1
|
17 Sep 13
|
{
"type": "Research Article",
"title": "On the intrinsic disorder status of the major players in programmed cell death pathways",
"authors": [
"Alexey V Uversky",
"Bin Xue",
"Zhenling Peng",
"Lukasz Kurgan",
"Vladimir N Uversky",
"Alexey V Uversky",
"Bin Xue",
"Zhenling Peng",
"Lukasz Kurgan"
],
"abstract": "Earlier computational and bioinformatics analysis of several large protein datasets across 28 species showed that proteins involved in regulation and execution of programmed cell death (PCD) possess substantial amounts of intrinsic disorder. Based on the comprehensive analysis of these datasets by a wide array of modern bioinformatics tools it was concluded that disordered regions of PCD-related proteins are involved in a multitude of biological functions and interactions with various partners, possess numerous posttranslational modification sites, and have specific evolutionary patterns (Peng et al. 2013). This study extends our previous work by providing information on the intrinsic disorder status of some of the major players of the three major PCD pathways: apoptosis, autophagy, and necroptosis. We also present a detailed description of the disorder status and interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathways.",
"keywords": [
"Many biologically active proteins do not have a unique 3-D structure as a whole or in part1–5",
"and are as such described as intrinsically disordered proteins (IDPs) and hybrid proteins containing ordered domains and IDP regions (IDPRs). These proteins and regions possess highly flexible structures and exist as conformational dynamic ensembles characterized by different degree and depth of disorderedness2",
"4",
"6–10. Globally",
"the structure of IDPs/IDPRs can be described as collapsed-disordered (molten globule-like)",
"partially collapsed-disordered (pre-molten globule-like)",
"or extended-disordered (coil-like)8",
"11. IDPs/IDPRs are highly abundant in virtually any given proteome1",
"3",
"5",
"12. As they are abundant constituents of all cells",
"tissues",
"and organs",
"and are responsible for crucial controling and regulating functions",
"IDPs are commonly involved in the pathogenesis of various human diseases13. This conclusion is based on numerous case studies in which a particular IDP was shown to be associated with a particular disease (including many cancer-14–19 and neurodegeneration-related proteins20–27)",
"as well as on the results of systematic bioinformatics studies12",
"28–36."
],
"content": "Introduction\n\nMany biologically active proteins do not have a unique 3-D structure as a whole or in part1–5, and are as such described as intrinsically disordered proteins (IDPs) and hybrid proteins containing ordered domains and IDP regions (IDPRs). These proteins and regions possess highly flexible structures and exist as conformational dynamic ensembles characterized by different degree and depth of disorderedness2,4,6–10. Globally, the structure of IDPs/IDPRs can be described as collapsed-disordered (molten globule-like), partially collapsed-disordered (pre-molten globule-like), or extended-disordered (coil-like)8,11. IDPs/IDPRs are highly abundant in virtually any given proteome1,3,5,12. As they are abundant constituents of all cells, tissues, and organs, and are responsible for crucial controling and regulating functions, IDPs are commonly involved in the pathogenesis of various human diseases13. This conclusion is based on numerous case studies in which a particular IDP was shown to be associated with a particular disease (including many cancer-14–19 and neurodegeneration-related proteins20–27), as well as on the results of systematic bioinformatics studies12,28–36.\n\nThe phenomenon of intrinsic disorder in proteins is spreading through modern protein science, and is implemented in more and more aspects of protein functionality. The biological functions of IDPs/IDPRs include envolvement in regulation, signaling, and controlling pathways29,37,38. These disorder-based functions represent a crucial complementation to the functional repertoire of ordered proteins30,39–41. Conformational plasticity, pliability, and adjustability combined with functional versatility define the overall natural abundance of IDP/IDPRs. As a result, many (if not the vast majority) of the non-catalytic functions of proteins frequently rely on the advantages provided by the lack of a fixed unique structure30,39–41.\n\nIn addition to being crucial for the functionality of many individual proteins, intrinsic disorder plays a vital role in the control of almost all cellular processes. For example, in our recent study, we analyzed the abundance and roles of intrinsic disorder in proteins involved in the various pathways related to programmed cell death (PCD)42. PCD represents a suicidal cellular response to the exposure to a set of environmental factors that trigger a chain of specific intracellular biochemical events leading to characteristic morphological cellular changes, and ultimately to cell death.\n\nThe three PCD routes (or PCD types I, II and III, which correspond to apoptosis, autophagy, and necroptosis, respectively) have very different biological roles. Apoptosis is important for the development, immune regulation, and homeostasis of a multi-cellular organism. Necroptosis plays a role in the modulation of the inflammatory response in the skin and intestine, serves as a backup mechanism to clear pathogens, and is involved in the immunologically silent maintenance of the T cell homeostasis43. Autophagy controls a wide range of physiological processes such as starvation, cell differentiation, cell survival, and death44–46. It regulates the turnover of long-lived proteins, the disposal of damaged organelles and misfolded proteins, and the turnover of cellular building blocks following nutrient deprivation. As a result, autophagy typically has crucial pro-survival roles in cellular homeostasis and during stress. However, under some circumstances, it can initiate characteristic cell death44,47. Therefore, although apoptosis and necroptosis both invariably contribute to cell death, autophagy might play either pro-survival or pro-death roles47–49. As a result, the fate of the cells and the fine balance between cell death and survival of healthy cells is jointly decided by the interplay between these three PCD pathways49.\n\nSince these three major PCD mechanisms involve different signaling pathways, they can be easily distinguished from each other based on their specific morphological features and on the unique biochemical changes developing in dying cells47,50. Among the characteristic morphological features of the three PCD types are the apoptosis-specific fragmentation and condensation of chromatin, combined with the fragmentation of chromosomal DNA, characteristic fragmentation of nucleous, shrinkage of cells, and production of cell fragments known as apoptotyc bodies49; the autophagy-linked formation of autophagosomes, which are the double- or multimembrane-bound structures around the recycling-destined cytoplasmic macromolecules and organelles49,51–55; and the necroptosis-related dysfunction of organnelles accompanied by the swelling and lysis of cells56.\n\nAlthough a wide range of cell signals of either extracellular or intracellular origin can lead to the activation of various pathways eventually resulting in the initiation of apoptosis, the major cause of the cell death is related to the organized degradation of cellular organelles by activated members of the caspase family of cysteine proteases57. Depending on the origin of the triggering signal, apoptotic pathways are classified as extrinsic and intrinsic, and each of these pathways can be regulated at multiple levels. Furthermore, depending on the nature of the cell signals, both apoptotic pathways can be either initiated or repressed. Among the extracellular (or extrinsic) inducers of apoptosis are various cytokines, nitric oxide58–60, hormones, growth factors, and toxins61, which somehow cross the plasma membrane or transduce to affect a response. Among the major players of the extrinsic apoptosis pathway are cell surface receptors known as death receptors, decoy receptors serving as inhibitory counterparts of these death receptors, are a set of related cytoplasmic proteins62. Furthermore, this pathway is regulated by changes in the transcription levels of the death inhibitory proteins (e.g., FLIP) and by the variations in the expression levels of the specific cytoplasmic adapter proteins, such as FADD and other apoptosis-activating ligands, leading to the procaspase activation62,63. Among the intracellular triggers of apoptosis are specific signals released by a damaged cell in response to different types of stress, such as heat, radiation, nutrient deprivation, increased intracellular calcium concentration64, hypoxia, or viral infection. The intrinsic pathway centers on the mitochondria that contains several key apoptogenic factors such as cytochrome c, AIF, SMAC/DIABLO, Htra2/Omi65,66, and endoG67–69; the release of these factors from mitochondria is regulated by the pro- and anti-death members of the BCL-2 family70. Also, members of the inhibitor of apoptosis protein (IAP) family of functionally and structurally related proteins control both intrinsic and extrinsic apoptosis pathways, serving as endogeneous apoptosis inhibitors62. Finally, a unique regulatory role is played by a transcription factor p53 via its modulation of many key control points in both the extrinsic and intrinsic pathways62.\n\nAmong the major molecular players of autophagy are the mammalian target of rapamycin kinase (mTOR), the ULK1 kinase complex32, the class III PI(3)-kinase VPS34, Beclin-131, and several members of the ATG family that possess various biological functions47. Regulators of necroptosis are specific to cell types being different for the apoptosis-competent and the apoptosis-incompetent cells. In fact, RIP1 kinase and death receptors play a role in activation and control of the necroptosis in the apoptosis-incompetent cells71, whereas the necroptosis of the apoptosis-competent cells is regulated by reactive oxygen species, apoptosis inducing factor (AIF), death-associated protein kinase (DAPK), and c-Jun N-terminal kinase (JNK)72–75.\n\nEarlier we reported the results of a comprehensive computational analysis of 1138 human apoptosis-related proteins, 137 human proteins associated with and autophagy, and 35 human necroptosis-related proteins, and also studied 3,458 proteins from DeathBase76 (http://deathbase.org/) that included proteins from five manually curated species: human, mouse, zebrafish, D. melanogaster, and C. elegans, and 23 reference species42. This previous analysis revealed that proteins involved in the regulation and execution of PCD possess substantial amounts of intrinsic disorder; that disorder has numerous functional roles across and within apoptosis, autophagy, and necroptosis processes; and that IDPRs of the PCD-related proteins are engaged in protein-protein interactions, interactions with other partners including nucleic acids and other ligands, and are also enriched with posttranslational modification sites42. In the current study, we extend our previous work and provide information on the intrinsic disorder status of some of the major players of the three major PCD pathways: apoptosis, autophagy, and necroptosis. The extention is related to the more focused consideration of the disorder status of several specific human proteins related to the PCD pathways and consideration of the roles of disorder in functions of these proteins;, aspects not covered in the previous article. We also present a detailed description of the disorder status and interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathways, which was not covered in the previous article. Finally, we provide analysis of available structural information for human proteins related to the p53-controlled apoptotic pathways.\n\n\nMethods\n\nIn this study we extend the previous analysis of the abundance and roles of intrinsic disorder in the control and execution of PCD pathways42. We report the results of the focused evaluation of intrinsic disorder in specific sub-sets of human proteins in three major modules of the programmed cell death, namely apoptosis, necroptosis, and autophagy. The analyzed proteins are shown in Figure 1 which represents an interlinked network of protein-protein interactions related to PCD. We also paid special attention to 29 human proteins related to the p53-mediated apoptotic pathways (see Figure 2). All human proteins considered in this study were reviewed entries in the UniProtKB Protein Knowledgebase (http://www.uniprot.org/uniprot/), and the corresponding FASTA files were downloaded from UniProt.\n\nDiagram shows a map of the regulators and molecular components of the apoptosis, autophagy, and necroptosis death pathways, constituting the three known modules of the programmed cell death (PCD) network. The proteins are color coded according to their intrinsic disorder content evaluated by PONDR-FIT, with highly ordered ([IDP score]<10%), moderately disordered (10%≤ [IDP score]<30%), and highly disordered ([IDP score]≥30%) being shown as blue, pink, and red boxes, respectively. This diagram is based on the PCD maps published in47,150,151 and is reproduced from42.\n\nDiagram shows a map of the regulators and molecular components of this pathway. The proteins are color coded according to their intrinsic disorder content evaluated by PONDR-FIT, with highly ordered ([IDP score]<10%), moderately disordered (10%≤ [IDP score]<30%), and highly disordered ([IDP score]≥30%) being shown as blue, pink, and red boxes, respectively. This diagram is based on the maps of the p53-mediated apoptotic signaling pathways available at (http://www.ebioscience.com/resources/pathways/p53-mediated-apoptosis-pathway.htm) and (http://www.qiagen.com/Products/Genes and Pathways/Pathway Details/?pwid=338) and on related information62,109,151,152 and many other papers.\n\nThe intrinsic disorder status in these proteins was evaluated by PONDR FIT (http://www.disprot.org/pondr-fit.php)77, which is a meta-predictor that combines six individual predictors: PONDR® VLXT78, PONDR® VSL279, PONDR® VL380, FoldIndex81, IUPred82, TopIDP83 and by PONDR® VLXT78. PONDR FIT is somewhat more accurate than the individual predictors used for its development77. We also analyzed the disorder status of human protein related to the p53-mediated apoptosis signaling pathways by PONDR® VLXT. The use of PONDR® VLXT is determined by the ability of this computational tool to visualize potential functional sites important for molecular recognition, signaling, and regulation. These potential binding sites, which are now recognized as Molecular Recognition Features (MoRFs)84,85, occur as dips on the plot of disorder score, and correspond to segments with an increased propensity towards order that are flanked by disordered regions.\n\nMoRFs are short order-prone motifs flanked by disordered regions that are involved in molecular recognition and are able to undergo disorder-to-order transition during binding to a specific partner. The recognition capability of MoRFs and their ability to undergo induced folding at binding are defined by their specific amino acid biases, since these regions usually have a much higher content of aliphatic and aromatic amino acids than disordered regions in general and therefore are computationally identifiable84,85. We used α-MoRF-Pred and α-MoRF-Pred II tools to find α-helix-forming MoRFs84,85.\n\nThe ANCHOR algorithm represents an alternative approach for finding potential binding sites in IDPs and IDPRs86,87 (http://anchor.enzim.hu/). At the foundation of this approach and its parental tool, the general disorder predictor IUPred82,88, is the estimation of the pairwise energy. Here, the potential binding sites are found as protein segments that cannot form enough favorable intra-chain interactions to fold on their own, but are likely to gain stabilizing energy via specific interactions with globular protein partners86,87. In line with our earlier studies89–91, these potential binding regions are termed here as ANCHOR-indicated binding site (AIBS).\n\nFinally, we looked at the interactions of the functional analysis of these proteins using the STRING database92. The STRING database (http://string-db.org), which covers more than 1100 completely sequenced organisms, including Homo sapiens, represents the online database resource search tool for the retrieval of interacting genes providing both experimental and predicted interaction information92. For each protein, STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins. The edges represent the functional associations evaluated based on experiments, search of databases, and text mining. The thickness of edges is proportional to the confidence level92. We used two sets of parameters in this analysis. To show the breadth of the p53-centered interactome, the number of interactors was set to 500 and the STRING confidence level was set to 0.900. In the analysis of other human proteins involved in the p53-controlled apoptotic signaling pathways, the number of interactors was set to 20 and the medium confidence level of 0.4 was used.\n\n\nResults and discussion\n\nFigure 1 (which is adopted from Peng et al.42) schematically represents the interlinked nature of the three PCD-related modules by showing some major events taking place within a cell undergoing apoptosis, autophagy, or necroptosis47. Figure 1 clearly shows that the various programmed cell death processes are under tight control and that the involved proteins are strongly interconnected. In fact, it is clear that inside the challenged cell, there is a common programmed cell death network that integrates three PCD modules, which include many pathways that are intertwined and interconnected, and many death regulatory proteins that are used by more than one module47. Our previous study suggested that one of the common structural features of these PCD-related proteins is their strong propensity for being disordered or to possess long IDPRs42. We believe that this disordered nature of PCD-controlling proteins allows them to be uniquely and effectively modulated via multiple specific interaction with various partners effectively, and to control the regulation and execution of different PCD modules42.\n\nThis conclusion is supported by a simple visualization technique used in Figure 1, namely, coding the involved proteins according to their intrinsic disorder content evaluated by PONDR-FIT. We used two arbitrary cutoffs for the levels of intrinsic disorder to classify proteins as highly ordered ([IDP score]<0.1, blue boxes), moderately disordered (0.1≤ [IDP score]<0.3, pink boxes) and highly disordered ([IDP score]≥0.3, red boxes)36. According to this classification, only 11 human proteins related to the controlled cell death pathways shown in Figure 1 are characterized by low disorder scores, whereas the absolute majority of the PCD-related proteins are moderately or highly disordered. Figure 1 also shows that the highly connected PCD-related proteins (i.e., proteins involved in several functional interactions) are typically more disordered than proteins with a lesser number of interaction partners. Interestingly, Figure 1 also illustrates that although many of the human PCD-related proteins are enzymes (kinases, ribonucleases, deoxyribonuclease, proteases, protein and ubiquitin ligases, polymerases, oxidureductase, GTPases, etc.), they possess significant disorder levels. The discussion below provides a very brief overview of some major players in three PCD modules and their disorder status.\n\nThe paragraphs below provide a more focused description of some major functions ascribed to the proteins in the three PCD modules shown in Figure 1. This description is further enhanced by our analysis of the disorder status of these proteins, where all mean PONDR-FIT scores were determined in this study.\n\nApoptosis is a type I programmed cell death that is essential for the elimination of unwanted cells during normal development, and for the maintenance of tissue homeostasis. Apoptosis starts with the formation of multiprotein complexes. One of these complexes is the extrinsic pathway-specific death-inducing signaling complex (DISC) triggered by binding of extracellular death ligands to death receptors. Typical DISC consists of the death receptor FAS (mean PONDR FIT score of 0.281), death ligand (mean PONDR FIT score of 0.394), adaptor proteins such as FADD (mean PONDR FIT score of 0.274) and TRADD (mean PONDR FIT score of 0.298)93. Another important complex formed at these early apoptosis stages is the intrinsic pathway-specific apoptosome, consisting of APAF1 (mean PONDR FIT score of 0.154) and cytochrome c (mean PONDR FIT score of 0.327), formation of which is triggered by the cytochrome c release from the mitochondria94,95. At the next stage, initiator caspases (such as caspase-2 (mean PONDR FIT score of 0.122), caspase-8 (mean PONDR FIT score of 0.178), and caspase-9 (mean PONDR FIT score of 0.271)) are recruited and activated by these initial complexes. These secondary complexes then cleave and activate effector caspases, including caspase-3 (mean PONDR FIT score of 0.303) and caspase-7 (mean PONDR FIT score of 0.221), that target specific cellular substrates for proteolysis57. Caspases are directly inhibited by members of the IAP family (such as HIAP1 (mean PONDR FIT score of 0.212), HIAP2 (mean PONDR FIT score of 0.215), XIAP (mean PONDR FIT score of 0.177)), as well as by Bruce (mean PONDR FIT score of 0.223) and Survivin (mean PONDR FIT score of 0.373). Anti-apoptotic and pro-apoptotic members of the BCL-2 family (such as BCL-xL (mean PONDR FIT score of 0.330), BCL-2 (mean PONDR FIT score of 0.347), and BCL-W (mean PONDR FIT score of 0.093)) regulate the release of apoptogenic factors from the mitochondria, including cytochrome c and an IAP inhibitor SMAC (mean PONDR FIT score of 0.234)94,95. Caspase-8 mediated cleavage of BID (mean PONDR FIT score of 0.374), a BH3-only member of the BCL-2 family, links the extrinsic and intrinsic pathways96.\n\nAutophagy is induced via the suppression of mTOR (mean PONDR FIT score of 0.089), a sensor of growth factors and nutrient availability, leading to the release of its inhibitory effects on the ULK1 kinase complex (mean PONDR FIT score of 0.546)32. Membrane nucleation requires the class III PI(3)-kinase VPS34 (mean PONDR FIT score of 0.209) and its associated proteins, including Beclin-1 (mean PONDR FIT score of 0.400)31. Two ubiquitin-like conjugation schemes (the ATG5–ATG12 (mean PONDR FIT scores of 0.091 and 0.464, respectively) and LC3-phosphatidylethanolamine (PE) systems (mean PONDR FIT score of 0.306)) mediate the elongation of the autophagosome membrane. At the molecular level, autophagy is mediated by several members of the ATG family that possess various biological functions47. For example, ATG1 (mean PONDR FIT score of 0.546) is a serine/threonine-protein kinase that acts upstream of phosphatidylinositol 3-kinase PIK3C3 to regulate the formation of autophagophores, the precursors of autophagosomes. ATG10 (mean PONDR FIT score of 0.114) and ATG3 (mean PONDR FIT score of 0.452) act as E2-like enzymes responsible for conjugation of ubiquitin-like ATG12 (mean PONDR FIT score of 0.464) to ATG5 (mean PONDR FIT score of 0.091) and ATG8-like proteins (mean PONDR FIT score of 0.306) to PE, respectively, whereas ATG7 (mean PONDR FIT score of 0.064) serves as an E1-like enzyme that facilitates the reaction of conjugation of ATG8-like proteins to PE by forming an E1-E2 complex with ATG3.\n\nFinally, a few words about the major players in necroptosis are given below. One of the better understood necroptosis models is where this pathway is initiated by the ligand-bound tumor necrosis factor receptor 1 (TNFR1) trimers (mean PONDR FIT score of 0.167)48, which recruit multiple proteins such as TNFR-associated death domain (TRADD, mean PONDR FIT score of 0.298), receptor-interacting protein kinase 1 (RIPK1, better known as RIP1, mean PONDR FIT score of 0.276), TRAF2 (mean PONDR FIT score of 0.036), and TRAF5 (mean PONDR FIT score of 0.079). Polyubiquitination of RIP1 triggers the canonical pathway of activation of the transcription factor NF-κB (mean PONDR FIT score of 0.202) which conveys protection against cell death97. However, TNFR1 internalization and RIP1 deubiquitination may lead to the formation of the DISC complex that includes RIP1 (mean PONDR FIT score of 0.276), receptor interacting protein kinase 3 (RIPK3, better known as RIP3, mean PONDR FIT score of 0.431), TRADD (mean PONDR FIT score of 0.298), FAS-associated protein with a death domain (FADD, mean PONDR FIT score of 0.274), and caspase-8 (mean PONDR FIT score of 0.178), which, when activated by the DISC complex, typically initiates apoptosis48. Caspase-8 can be activated not only by TNFR1, but also by other death receptors (such as FAS (mean PONDR FIT score of 0.281)98, TNF-related apoptosis-inducing ligand receptors 1 and 2 (TRAIL-R1, mean PONDR FIT score of 0.323 and TRAIL R2, mean PONDR FIT score of 0.495)48), ultimately leading to the initiation of apoptosis. However, when caspase-8 cannot be activated (i.e., when it is inhibited or depleted), the cell undergoes necroptosis, which is induced by the formation of a necrosis-inducing complex, necrosome, comprised of RIP1 and RIP399. This RIP1-RIP3 necrosome generates several pronecroptotic signals, including activation of c-JUN N-terminal kinases 1 and 2 (JNK1, mean PONDR FIT score of 0.155, and JNK2, mean PONDR FIT score of 0.184) that eventually mediates a signaling cascade affecting the iron storage compartment99,100. Another necroptosis activating pathway is related to the overactivation of poly-ADP-ribose polymerase 1 (PARP1, mean PONDR FIT score of 0.194) leading to the depletion of ATP and NAD, accumulation of poly-ADP-ribose (PAR), and the cytosolic release of apoptosis-inducing factor (AIF, mean PONDR FIT score of 0.162), a protein that is normally secured within the mitochondrial intermembrane space, but upon release from the mitochondria rapidly translocates to the nucleus where it initiates large-scale, caspase-independent DNA fragmentation, leading to further PARP1 activation and subsequent cell death48. Finally, necroptosis is accompanied by the release of high-mobility group box 1 protein (HMGB1, mean PONDR FIT score of 0.774), which is both a nuclear factor (which serves as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets) and a secreted protein serving as a potent mediator of inflammation101. This HMGB1 release is a diffusible signal of necroptosis, which can be used as a cue to nearby cells102.\n\nFigure 2 zooms into the bottom left corner of Figure 1 and shows the p53-mediated apoptotic signaling pathways in more detail. At the center of this signaling pathways is the tumor-related protein p53 (mean PONDR FIT score of 0.519). p53 is a crucial transcription factor, its activation in response to genotoxic or cellular stresses is known to induce or inhibit more than 150 genes103,104. Some of the p53 targets are important players in apoptosis, growth arrest, or senescence pathways62,105–107. Since p53 is an important regulator of various cellular processes (including apoptosis), it has a short life-time and is normally maintained at low levels in unstressed mammalian cells via continuous ubiquitination and subsequent proteasomal degradation. Normally, ubiquitination of non-phosphorylated p53 is driven by the mouse double minute-2 ubiquitin ligase (Mdm2, mean PONDR FIT score of 0.550), which is capable of targeting p53 for degradation108. This process is regulated via negative feedback between the p53 and Akt pathways109. Here, Akt (mean PONDR FIT score of 0.173) is activated in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components110.\n\nIn humans, there is a myriad of growth factors that are able to stimulate cellular growth, proliferation, and cellular differentiation, and some of them can also be involved in apoptosis111. Since growth factor proteins typically occur as members of rather large families of related proteins, the real number of the representatives of this protein class is noticeably larger. For example, there are 23 members in the fibroblast growth factor family (the comprehensive analysis of this very interesting class of proteins is outside the scope of this study). One of the characteristic members of this family of proteins is fibroblast growth factor 1 (FGF1, mean PONDR FIT score of 0.291) that is involved in the regulation of apoptosis. The binding of growth factors to growth factor receptors (e.g., to the fibroblast growth factor receptor 2, FGFR2, mean PONDR FIT score of 0.248) leads to their dimerization and activates their tyrosine kinase activity, causing autophosphorylation of tyrosine residues on the intracellular domain of the receptor. This recruits the p85 regulatory subunit of the phosphoinositol 3-kinase (PI3K p85, mean PONDR FIT score of 0.413) via its SH2 domain, mediating the association of the PI3K p110 catalytic unit (mean PONDR FIT score of 0.164) to the plasma membrane109,110. Activated PI3K phosphorylates membrane-bound PIP2 (phosphatidylinositol-3,4-bisphosphate) to generate PIP3 (phosphatidylinositol-3,4,5-triphosphate) which then binds to Akt via its PH domain, eventually leading to Akt activation109,110. Activated Akt then specifically phosphorylates Mdm2 at position Ser166 which promotes cell survival via Mdm2-mediated inhibition and destruction of p53109. This cell survival pathway is blocked under stress conditions where Akt is degraded and PI3K is inhibited via phosphatase PTEN (phosphatase and tensin homolog, mean PONDR FIT score of 0.280). The multifarious roles of intrinsic disorder on the functions of PTEN and its interactomes were covered in a recent study112. Particularly, it was shown that PTEN possesses an IDPR at its C-terminus. This disordered C-tail, with its set of MoRFs, conserved eukaryotic linear motifs, and numerous sites of posttranslational modifications, plays a crucial role in a multitude of PTEN-based protein-protein interactions112. Since many proteins in the primary and secondary interactomes of PTEN possessed significant amount of intrinsic disorder, and since many of these PTEN-interacting IDPs were cancer-related, it has been concluded that PTEN represents a pliable, intrinsically disordered, cancer-related hub located within a flexible network of cancer-related IDPs112.\n\nOne of the most important mechanisms defining the ability of p53 to possess various regulatory functions consists of the precise control of its posttranslational modifications (PTMs), such as phosphorylation, acetylation, sumoylation, and ubiquitination. For example, in response to DNA damage, the cell cycle checkpoint kinases 1 and 2 (Chk1 and Chk2, mean PONDR FIT scores of 0.252 and 0.343, respectively) become activated by ataxia-telangiectasia and Rad3 related kinase (ATR, mean PONDR FIT score of 0.170) and ataxia-telangiectasia mutated kinase (ATM, mean PONDR FIT score of 0.194), respectively. Activated Chk1 and Chk2 phosphorylate p53 at multiple positions, with Chk1 phosphorylating multiple sites within the intrinsically disordered C-terminus of p53113 and Chk2 primarily targeting the intrinsically disordered N-terminal region of p53114. It is important to note that the Chk2-induced p53 phosphorylation at Ser20 and ATM-driven p53 phosphorylation at position Ser15 stabilize p53 and promote accumulation of activated p53 in the nucleus114. Activated p53 binds to the consensus p53 response elements of several genes, particularly those encoding proteins from both the extrinsic and intrinsic apoptotic pathways103,104.\n\nIn the intrinsic apoptotic pathway, p53 induces the expression of several members of the BCL-2 family (whose family members are classified on the basis of their structural similarity to the BH (BCL-2 homology) domains (BH1, BH2, BH3, and BH4) and a transmembrane domain), such as multidomain BCL-2 family member BAX (mean PONDR FIT score of 0.310)115, together with the BH3-only BCL-2 family members PUMA (p53-upregulated modulator of apoptosis, mean PONDR FIT score of 0.896)116, NOXA (mean PONDR FIT score of 0.578)117, and BID (BH3 interacting domain death agonist, mean PONDR FIT score of 0.374)118.\n\nUnder stress conditions, homodimeric BAX undergoes a conformation change that causes translocation to the mitochondrial membrane and formation of higher BAX oligomers. Mulitmerization of BAX and subsequent translocation to mitochondria are promoted by a family of PUMA proteins produced via the alternative splicing of a p53 transactivation target gene, PUMA116. Another p53 target gene, Noxa encodes a BH3-only protein, which contributes to the p53-controlled apoptosis in a similar manner to PUMA and BAX. Also, there is another p53 target gene encoding for the p53-regulated apoptosis-inducing protein 1 (p53AIP1, mean PONDR FIT score of 0.492), which is located in the mitochondrial membrane and is directly involved in the p53-dependent mitochondrial apoptosis119. Translocation of BAX to the mitochondrion membrane initiates the release of cytochrome c (mean PONDR FIT score of 0.327). At the next stage, the apoptosome complex comprising of cytochrome c, the oligomeric form of the apoptotic protease-activating factor-1 (APAF1, mean PONDR FIT score of 0.153), and procaspase-9 (mean PONDR FIT score of 0.271) is formed. This complex then activates caspase-9, which promotes the activation of caspase-3 (mean PONDR FIT score of 0.303), caspase-6 (mean PONDR FIT score of 0.233), and caspase-7 (mean PONDR FIT score of 0.221), leading to the subsequent cleavage of vital death substrates and culminating in cell death.\n\nThe p53-stimulated extrinsic apoptotic pathway relies on the engagement of the specific cell-surface death receptors from the TNDR family needed for the activation of specific caspases, such as caspase-8 (mean PONDR FIT score of 0.178) and caspase-3 (mean PONDR FIT score of 0.303), leading to the eventual induction of apoptosis. Among the most common death receptors involved in extrinsic apoptosis are TNDR superfamily member 6 or apoptosis-mediating surface antigen FAS (FAS, CD95, or Apo-1, mean PONDR FIT score of 0.281), death receptor-5 (DR5, mean PONDR FIT score of 0.437), and p53 apoptosis effector related to PMP-22 (PERP, mean PONDR FIT score of 0.215). FAS is activated by binding its ligand, FasL (mean PONDR FIT score of 0.394), which is a homotrimeric protein expressed predominantly by T-cells that causes FAS oligomerization on binding. FAS oligomerization is accompanied by the clustering of their death domains and the formation of the death-inducing signaling complex (or DISC) comprising of FAS (mean PONDR FIT score of 0.281), FADD (mean PONDR FIT score of 0.274), death ligand (mean PONDR FIT score of 0.448), and procaspase-8 (mean PONDR FIT score of 0.301) that binds to the death effector domain of FADD via a homologous motif120,121.\n\nIn relation to the p53-mediated extrinsic apoptotic pathway, p53 plays a dual role in FAS-induced apoptosis. Firstly, in response to the gamma-irradiation of specific tissues, FAS mRNA expression is induced by p53 binding to the specific elements within the promoter and first intron of the FAS gene. Secondly, overexpressed p53 can rapidly sensitize cells to FAS-induced apoptosis before the transcription-dependent effect operates, by increasing FAS levels at the cell surface via the promotion of the FAS receptor trafficking from the Golgi apparatus120,121.\n\nThe second member of the TNDR family induced by p53 is the death-domain-containing receptor for TNF-related apoptosis-inducing ligand (TRAIL, mean PONDR FIT score of 0.349), killer or death receptor-5 (DR5, mean PONDR FIT score of 0.437). In response to DNA damage, DR5 is induced by p53 in a cell type specific manner, initiating apoptosis through caspase-8. Finally, the p53 apoptosis effector related to PMP-22 (PERP, mean PONDR FIT score of 0.215) is assumed to serve as a direct p53 target, since the PERP promoter contains a p53-responsive element and since PERP production is induced in response to DNA damage. PERP is a member of the PMP-22/Gas family of tetraspan transmembrane proteins implicated in the regulation of cell growth. Although the precise role of PERP in p53-mediated apoptosis is yet to be understood, this protein can be involved in at least three potential mechanisms. First, PERP could serve as a cell death receptor that receives specific apoptosis-initiating signals. Second, due to its sequence similarity to the calcium channel, PERP could possess channel or pore activity and play a role in apoptosis activation by passing through some crucial molecules. Third, PERP might directly affect some regulators of apoptotic machinery, such as BAX (mean PONDR FIT score of 0.310) or BCL-2 (mean PONDR FIT score of 0.347), or even directly act on some crucial apoptotic effectors, such as the caspases122,123.\n\nTo further delve into the abundance and functional roles of intrinsic disorder in the proteins discussed above from the intrinsic and extrinsic apoptotic pathways mediated by p53, Figure 3–Figure 8 represent disorder profiles plotted using the results of the analysis of these proteins by PONDR-FIT and PONDR® VLXT disorder predictors, and also shows the results of the functional analysis of these proteins using the STRING database92. Figure 3–Figure 8 show that all the members of the p53-controlled intrinsic and extrinsic apoptosis signaling pathways possess noticeable amounts of intrinsic disorder and are involved in multiple interactions with other apoptosis-related proteins (and also with many other proteins not directly related to the apoptosis), which are often predicted to be disordered. In protein-protein interaction networks, the highly connected nodes (proteins) with many edges (interactions) are known as hubs. Earlier analysis revealed that protein intrinsic disorder is crucial for the binding promiscuity of hub proteins37,124. In fact, it has been shown that although hub proteins can be entirely disordered or contain long IDPRs or be highly structured, they utilize intrinsic disorder for protein-protein interactions via at least two mechanisms: one disordered region binding to many partners or many disordered region binding to one partner37,124–132. Therefore, in agreement with these previous studies, our analysis revealed that the major players of the p53-modulated apoptosis can be considered as disordered hub proteins.\n\nA. and a. Growth/survival factor (Fibroblast growth factor 1, UniProt ID: P05230); B. and b. TRAIL (UniProt ID: P50591); C. and c. FasL (UniProt ID: P48023); D. and d. Growth factor receptor (Fibroblast growth factor receptor 2, UniProt ID: P21802); E. and e. PERP (UniProt ID: Q96FX8). Intrinsic disorder propensity was evaluated by PONDR-FIT (red curves and pink shadow) and PONDR® VLXT (blue curves). The shadow around PONDR-FIT curves represents distribution of statistical errors. STRING database is the online database resource search tool for the Retrieval of Interacting Genes, which provides both experimental and predicted interaction information92. For each protein, STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins. The edges represent the functional associations evaluated based on experiments, search of databases, and text mining. The thickness of edges is proportional to the confidence level92.\n\nA. and a. DR5 (UniProt ID: O14763); B. and b. FAS (UniProt ID: P25445); C. and c. PI3K regulatory subunit (UniProt ID: O00459); D. and d. PI3K catalytic subunit (UniProt ID: P42336); E. and e. PTEN (UniProt ID: P60484). Intrinsic disorder propensity was evaluated by PONDR-FIT (red curves and pink shadow) and PONDR® VLXT (blue curves). The shadow around PONDR-FIT curves represents distribution of statistical errors. STRING database is the online database resource search tool for the Retrieval of Interacting Genes, which provides both experimental and predicted interaction information92. For each protein, STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins. The edges represent the functional associations evaluated based on experiments, search of databases, and text mining. The thickness of edges is proportional to the confidence level92.\n\nA. and a. AKT1 (UniProt ID: P31749); B. and b. Procaspase-8 (UniProt ID: Q14790); C. and c. Caspase-8 (residues 217–374 and 385–479; ID: Q14790); D. and d. Mdm2 (UniProt ID: Q00987); E. and e. ATM (UniProt ID: Q13315). Intrinsic disorder propensity was evaluated by PONDR-FIT (red curves and pink shadow) and PONDR® VLXT (blue curves). The shadow around PONDR-FIT curves represents distribution of statistical errors. STRING database is the online database resource search tool for the Retrieval of Interacting Genes, which provides both experimental and predicted interaction information92. For each protein, STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins. The edges represent the functional associations evaluated based on experiments, search of databases, and text mining. The thickness of edges is proportional to the confidence level92.\n\nA. and a. ATR (UniProt ID: Q13535); B. and b. p53 (UniProt ID: P04637); C. and c. CHK2 (UniProt ID: O96017); D. and d. CHK1 (UniProt ID: O14757); E. and e. Caspase-3 (UniProt ID: P42574). Intrinsic disorder propensity was evaluated by PONDR-FIT (red curves and pink shadow) and PONDR® VLXT (blue curves). The shadow around PONDR-FIT curves represents distribution of statistical errors. STRING database is the online database resource search tool for the Retrieval of Interacting Genes, which provides both experimental and predicted interaction information92. For each protein, STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins. The edges represent the functional associations evaluated based on experiments, search of databases, and text mining. The thickness of edges is proportional to the confidence level92.\n\nA. and a. Caspase-6 (UniProt ID: P55212); B. and b. Caspase-9 (UniProt ID: P55211); C. and c. BID (UniProt ID: P55957); D. and d. p53PAI-1 (UniProt ID: Q9HCN2); E. and e. NOXA (UniProt ID: Q13794). Intrinsic disorder propensity was evaluated by PONDR-FIT (red curves and pink shadow) and PONDR® VLXT (blue curves). The shadow around PONDR-FIT curves represents distribution of statistical errors. STRING database is the online database resource search tool for the Retrieval of Interacting Genes, which provides both experimental and predicted interaction information92. For each protein, STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins. The edges represent the functional associations evaluated based on experiments, search of databases, and text mining. The thickness of edges is proportional to the confidence level92.\n\nA. and a. BAX (UniProt ID: Q07812); B. and b. BCL-2 (UniProt ID: P10415); C. and c. PUMA (UniProt ID: Q96PG8); D. and d. Cytochrome c (UniProt ID: P99999); E. and e. APAF-1 (UniProt ID: O14727). Intrinsic disorder propensity was evaluated by PONDR-FIT (red curves and pink shadow) and PONDR® VLXT (blue curves). The shadow around PONDR-FIT curves represents distribution of statistical errors. STRING database is the online database resource search tool for the Retrieval of Interacting Genes, which provides both experimental and predicted interaction information92. For each protein, STRING produces the network of predicted associations for a particular group of proteins. The network nodes are proteins. The edges represent the functional associations evaluated based on experiments, search of databases, and text mining. The thickness of edges is proportional to the confidence level92.\n\nOne should remember, however, that the interactomes of various proteins involved in the regulation and execution of p53-mesdiated apoptosis are in fact essentially larger than those shown in Figure 3–Figure 8. In fact, to generate the corresponding plots, the standard set of parameters was used in the STRING database, namely, Active Prediction Methods: Neighborhood, gene fusion, co-occurrence, co-expression, experiments, databases, and textmining; Required confidence (score): medium confidence (0.400); Interactors shown: no more than 20 interactors. Figure 9 illustrates how the predicted interactome of p53 changes when the number of interactors is increased to 500 (which is the highest allowed value) and the STRING confidence level is boosted to the value of 0.900. Figure 9 gives further support to the notion that, being an intrinsically disordered hub, p53 is located at the middle of a large and well-developed network of protein-protein interactions, serving as a crucial linker that connects multiple important pathways. Analysis of the disorder status in the 500 proteins known to interact with p53 is outside the scope of this study and will be reported elsewhere.\n\nThis network was built by setting the number of interactors to 500 and the STRING confidence level of 0.900. Therefore, this network includes only partners whose interactions are predicted with the highest confidence level.\n\nFigure 10–Figure 14 represent structural information available in protein databank (PDB)133 for many of the human proteins associated with the p53-mediated apoptotic signaling pathways. In fact, at least partial structural information is available for 23 of the 29 human proteins discussed above. The lack of complete structural information for these proteins can be explained, at least in part, by the abundance of intrinsic disorder which hinders crystallization. In fact, two of the structurally uncharacterized proteins, p53PAI-1 and PUMA (see Figure 8), are among the most disordered members of the analyzed group, possessing average disorder scores of 0.492 and 0.896, respectively. Figure 3C shows that a significant part of FasL is predicted to be highly disordered. Although ATM and ATR are mostly ordered (see Figure 5 and Figure 6), these are large kinases that possess a noticeable amount of relatively long disordered regions. Finally, it is expected that crystallization of PERP (Figure 3E) would be a challenging task since this p53 apoptosis effector is a typical transmembrane protein with four transmembrane domains, and transmembrane proteins are known to be rather tough targets for structural characterization.\n\nA. Fibroblast growth factor 1 (PDB ID: 2RQ9); B. TRAIL (PDB ID: 1DG6); C. Fibroblast growth factor receptor 2 (residues 153–362; PDB ID: 1DJS); D. Fibroblast growth factor receptor 2 (residues 465–768; PDB ID: 1GJO); E. DR5 (PDB ID: 1D4V); F. FAS (Residues 17–172; PDB ID: 3THM); G. FAS (Residues 218–335; PDB ID: 1DDF); H. PI3K (regulatory subunit, 1–80; PDB ID: 2KT1); I. PI3K (regulatory 108–298; PDB ID: 2XS6); J. PI3K (regulatory subunit, 433–610; PDB ID: 3MTT); K. PI3K (catalytic subunit; PDB ID: 2RD0).\n\nA. PTEN (PDB ID: 1D5R); B. AKT1 (PDB ID: 3O96); C. Caspase-8 (PDB ID: 2K7Z); D. Caspase-3 (PDB ID: 1CP3); E. Caspase-6 (PDB ID: 2WDP); F. Caspase-9 (Residues 1–96; PDB ID: 3YGS); G. Caspase-9 (Residues 140–416; PDB ID: 1JXQ).\n\nA. CHK2 (PDB ID: 3I6W); B. CHK1 (PDB ID: 1IA8); C. BID (PDB ID: 2BID); D. NOXA (PDB ID: 3MPQ); E. BAX (PDB ID: 1F16); F. BCL-2 (PDB ID: 1GM5).\n\nA. Cytochrome c (PDB ID: 1J3S); B. APAF-1 (Residues 1–591; PDB ID: 1Z6T); C. APAF-1 (PDB ID: 3J2T).\n\nA. Residues 1–93 of p53 (red ribbon) in a complex with the bromodomain of CREB-binding protein (blue surface) (PDB ID: 2LY4); B. Residues 94–293 of p53 in a complex with DNA (red bonds) (PDB ID: 3IGK); C. Residues 319–360 of p53 in a homotetrameric complex (PDB ID: 1OLH); D. Residues 367–386 of p53 (red ribbon) in a complex with the bromodomain of CREB-binding protein (blue surface) (PDB ID: 1JSP); E. Residues 1–117 of MDM2 (PDB ID: 1Z1M); F. Residues 17–125 of MDM2 (blue surface) in a complex with the transactivation domain of p53 (residues 15–29, red ribbon) (PDB ID: 1YCR); G. Residues 147–150 of Mdm2 (red ribbon) in a complex with the N-terminal domain of HAUSP/USP7 (blue surface) (PDB ID: 2FOP); H. Residues 290–335 of Mdm2 (PDB ID: 2C6A).\n\nAnalysis of structures shown in Figure 10–Figure 14 and obtained by protein crystallization and NMR studies suggests that many of the human proteins associated with the p53-mediated apoptotic signaling pathways possess noticeable conformational flexibility. This is evidenced by the following facts:\n\na) Crystallization of a full length protein was often impossible and many structures shown in Figure 10–Figure 14 correspond to the protein domains and short fragments;\n\nb) Proteins of interest and their fragments were frequently co-crystallized in the complexes with specific binding partners;\n\nc) Proteins or protein fragments often do not possess globular shape suggesting that the resulting structures are induced by binding to specific partners (e.g., Figure 10E, 10F, 10J, Figure 14A, 14C, 14D, 14F, and 14G);\n\nd) The NMR structures are characterized by the \"fuzzy\" appearance typical of the highly dynamic structures (e.g., see Figure 10A, 10H, Figure 11C, Figure 12E, Figure 13A, Figure 14E, and 14H);\n\ne) Many proteins contain long structureless tails and loops (e.g., see Figure 10B, 10G, Figure 11A, 11E, 11G, Figure 12B, 12C, 12D, and 12F);\n\nf) Even the determined structures of many proteins contain structurally undefined regions known as regions of missing electron density (see Table 1). The flexibility or disorder of a protein region determines the failure of this region to scatter X-ray coherently and therefore explains the invisibility of this region in a crystal structure134.\n\nAll these observations provide strong support to the notions that intrinsic disorder is abundant in human proteins associated with the p53-mediated apoptotic signaling pathways and that disordered regions are frequently used for protein-protein interactions needed for the efficient control and regulation of the mentioned pathways.\n\nIDPs and IDPRs are commonly engaged in protein-protein interactions and play crucial roles in molecular recognition2,8,10,30,84,135–143. In the process of their function and interaction with their binding partners, many IDPs and IDPRs are known to undergo at least partial disorder-to-order transitions, and these conformational transitions are crucial for recognition, regulation, and signaling4,8,84,143–149. Based on the specific features and patterns in the per-residue disorder propensity curves calculated by the PONDR® VLXT tool combined with the analysis of several characteristic sequence-based parameters, short disordered regions capable of undergoing the disorder-to-order transitions on binding can be found by specific predictors84,85. These disorder-based binding regions are known as α-helix forming MoRFs (α-MoRFs). Table 2 shows that α-MoRFs are common in human proteins involved in p53-mediated apoptotic pathways, and that some long disordered proteins have multiple α-MoRFs, which likely serve as promiscuous binders interacting with multiple partners.\n\na ANCHOR-indicated binding site. Numbers shown in italic correspond to regions (longer than 5 residues) that were predicted as potential binding site by ANCHOR, but filtered due to low IUPred disorder scores (below 0.1)86,87.\n\nb NLS, Nuclear localization signal.\n\nMoRF, Molecular recognition feature.\n\nAnother way to find potential disorder-based binding sites is based on looking for residues in disordered regions that cannot form enough favorable intra-chain interactions to fold on their own, but which are likely to gain stabilizing energy by interacting with a globular protein partner86,87. This approach is based on using the ANCHOR algorithm86,87 to find disordered but foldable binding regions, ANCHOR-identified binding sites (AIBSs). ANCHOR relies on the pairwise energy estimation approach that is the basis for IUPred, a general disorder prediction method82, whereas MoRF identifiers rely on specific patterns in the per-residue disorder propensity curves calculated by the PONDR® VLXT78. Since methodologically and logistically ANCHOR and MoRF identifiers are very different, the application of both tools provides complementary information. Table 2 shows that AIBSs are very common among the human proteins involved in the p53-mediated apoptotic pathways. In fact, the majority of these proteins possess more than one AIBS, and many of them have multiple AIBSs. Furthermore, there is typically a reasonable agreement between the outputs of these two tools.\n\nFinally, Table 2 lists some of the experimentally validated binding sites found in the human proteins involved in the p53-mediated apoptotic pathways. These potential binding sites were typically found based on the analysis of the reported x-ray crystallography or NMR data on structural characterization of a fragment of a target protein bound to its partner(s). Table 2 shows that the majority of the experimentally found binding sites are correctly predicted by at least one algorithm. This observation provides indirect support to the validity of the results of the computational analysis and to the conclusion that the major function of many IDPRs in the human proteins involved in the p53-mediated apoptotic pathways is related to regulation and control via the protein-protein interactions.\n\n\nConcluding remarks\n\nIn conclusion, the results presented in this paper clearly show that many of the human proteins involved in regulation and execution of three major programmed cell death pathways: apoptosis, autophagy, and necroptosis, as well as human proteins that are involved in the p53-mediated apoptotic signaling pathways possess substantial amounts of intrinsic disorder. Since functional repertoires of ordered and disordered proteins are very different, finding high prevalence of disorder in PCD-related proteins clearly indicates that careful consideration of this important feature is absolutely critical for better understanding the structure and conformational behavior of these proteins, their promiscuity, and molecular mechanisms of their functions, regulation, and control. In agreement with this idea, we show here that most major players in three major PCD pathways and in particular proteins related to the p53-mediated apoptosis have relative high levels of intrinsic disorder and that there is a positive relationship between protein disorder and the number of interactions a protein has. Furthermore, many of these proteins contain multiple disorder-based protein interaction sites. These observations suggest that intrinsic disorder might be intimately related to all the aspects of activity of these proteins and plays indispensible roles in their functional interactions.\n\n\nList of abbreviations\n\nAIBS, ANCHOR-indicated binding site\n\nAIF, apoptosis inducing factor\n\nAPAF1, apoptotic protease-activating factor 1\n\nATM, ataxia-telangiectasia mutated kinase\n\nATR, ataxia-telangiectasia and Rad3 related kinase\n\nBID, BH3-interacting domain death agonist\n\nChk1, cell cycle checkpoint kinase 1\n\nChk2, cell cycle checkpoint kinase 2\n\nDAPK, death-associated protein kinase\n\nDISC, death-inducing signaling complex\n\nDR5, death receptor-5\n\nFADD, FAS-associated death domain protein\n\nFasL, FAS ligand\n\nFGFR2, fibroblast growth factor receptor 2\n\nGFG1, fibroblast growth factor 1\n\nHMGN1, high-mobility group box 1\n\nIAP, inhibitor of apoptosis protein\n\nIDP, intrinsically disordered protein\n\nIDPR, intrinsically disordered protein region\n\nJNK, c-Jun N-terminal kinase\n\nMdm2, mouse double minute-2 ubiquitin ligase\n\nMoRF, molecular recognition feature\n\nmTOR, mammalian target of rapamycin kinase\n\np53AIP1, p53-regulated apoptosis-inducing protein 1\n\nPAR, polyADP-ribose\n\nPARP1, poly-ADP-ribose polymerase 1\n\nPE, phosphatidylethanolamine\n\nPERP, p53 apoptosis effector related to PMP-22\n\nPCD, programmed cell death\n\nPDB, protein databank\n\nPI3K, phosphoinositol 3-kinase\n\nPIP2, phosphatidylinositol-3,4-bisphosphate\n\nPTEN, Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN\n\nPTM, posttranslational modification\n\nRIPK1, receptor-interacting protein kinase 1\n\nSMAC, second mitochondria-derived activator of caspase\n\nTNDR, tumor necrosis factor receptor\n\nTNFR1, tumor necrosis factor receptor 1\n\nTRADD, tumor necrosis factor receptor type 1-associated DEATH domain protein\n\nTRAF, TNF receptor-associated factor\n\nTRAIL, TNF-related apoptosis-inducing ligand\n\nTRAIL-R, TNF-related apoptosis-inducing ligand receptor",
"appendix": "Author contributions\n\n\n\nAVU implemented experiments, performed analysis, generated figures, and helped with manuscript drafting. BX and ZP implemented experiments and performed analysis. LK helped in experiment design and participated in the manuscript drafting. VNU proposed the idea of the study, designed and implemented experiments, carried out the analysis, drafted the manuscript, and coordinated study.\n\n\nCompeting interests\n\n\n\nAuthors have no competing interests.\n\n\nGrant information\n\nThis work was supported in part by University of South Florida (V.N.U. and B.X.), Temple University (A.V.U.), the Programs of the Russian Academy of Sciences for the “Molecular and Cellular Biology” (to V.N.U.), the Alberta Innovates Graduate Scholarship in Omics (to Z.P.), and the Natural Sciences and Engineering Research Council (NSERC) Discovery grant (to L.K.).\n\n\nReferences\n\nDunker AK, Obradovic Z, Romero P, et al.: Intrinsic protein disorder in complete genomes. Genome Inform Ser Workshop Genome Inform. 2000; 11: 161–171. PubMed Abstract\n\nUversky VN, Dunker AK: Understanding protein non-folding. Biochim Biophys Acta. 2010; 1804(6): 1231–1264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWard JJ, Sodhi JS, McGuffin LJ, et al.: Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J Mol Biol. 2004; 337(3): 635–645. PubMed Abstract | Publisher Full Text\n\nUversky VN, Gillespie JR, Fink AL: Why are \"natively unfolded\" proteins unstructured under physiologic conditions? Proteins. 2000; 41(3): 415–427. PubMed Abstract | Publisher Full Text\n\nXue B, Dunker AK, Uversky VN: Orderly order in protein intrinsic disorder distribution: disorder in 3500 proteomes from viruses and the three domains of life. J Biomol Struct Dyn. 2012; 30(2): 137–149. PubMed Abstract | Publisher Full Text\n\nDunker AK, Garner E, Guilliot S, et al.: Protein disorder and the evolution of molecular recognition: theory, predictions and observations. Pac Symp Biocomput. 1998; 473–484. PubMed Abstract\n\nWright PE, Dyson HJ: Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol. 1999; 293(2): 321–331. PubMed Abstract | Publisher Full Text\n\nDunker AK, Lawson JD, Brown CJ, et al.: Intrinsically disordered protein. J Mol Graph Model. 2001; 19(1): 26–59. PubMed Abstract | Publisher Full Text\n\nTompa P: Intrinsically unstructured proteins. Trends Biochem Sci. 2002; 27(10): 527–533. PubMed Abstract | Publisher Full Text\n\nDaughdrill GW, Pielak GJ, Uversky VN, et al.: Natively disordered proteins. In: Buchner J Kiefhaber T(eds) Handbook of Protein Folding. Wiley-VCH, Verlag GmbH & Co., Weinheim, Germany, 2005; pp 271–353. Publisher Full Text\n\nUversky VN: Natively unfolded proteins: a point where biology waits for physics. Protein Sci. 2002; 11(4): 739–756. PubMed Abstract | Free Full Text\n\nUversky VN: The mysterious unfoldome: structureless, underappreciated, yet vital part of any given proteome. J Biomed Biotechnol. 2010; 2010: 568068. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUversky VN, Oldfield CJ, Dunker AK: Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu Rev Biophys. 2008; 37: 215–246. PubMed Abstract | Publisher Full Text\n\nLee H, Mok KH, Muhandiram R, et al.: Local structural elements in the mostly unstructured transcriptional activation domain of human p53. J Biol Chem. 2000; 275(38): 29426–29432. PubMed Abstract | Publisher Full Text\n\nAdkins JN, Lumb KJ: Intrinsic structural disorder and sequence features of the cell cycle inhibitor p57Kip2. Proteins. 2002; 46(1): 1–7. PubMed Abstract | Publisher Full Text\n\nChang BS, Minn AJ, Muchmore SW, et al.: Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2. EMBO J. 1997; 16(5): 968–977. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCampbell KM, Terrell AR, Laybourn PJ, et al.: Intrinsic structural disorder of the C-terminal activation domain from the bZIP transcription factor Fos. Biochemistry. 2000; 39(10): 2708–2713. PubMed Abstract | Publisher Full Text\n\nSunde M, McGrath KC, Young L, et al.: TC-1 is a novel tumorigenic and natively disordered protein associated with thyroid cancer. Cancer Res. 2004; 64(8): 2766–2773. PubMed Abstract | Publisher Full Text\n\nZeng Y, He Y, Yang F, et al.: The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein. J Biol Chem. 2011; 286(16): 13985–13994. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGlenner GG, Wong CW: Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Commun. 1984; 122(3): 1131–1135. PubMed Abstract | Publisher Full Text\n\nMasters CL, Multhaup G, Simms G, et al.: Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels. EMBO J. 1985; 4(11): 2757–2763. PubMed Abstract | Free Full Text\n\nLee VM, Balin BJ, Otvos L Jr, et al.: A68: a major subunit of paired helical filaments and derivatized forms of normal Tau. Science. 1991; 251(4994): 675–678. PubMed Abstract | Publisher Full Text\n\nUeda K, Fukushima H, Masliah E, et al.: Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc Natl Acad Sci U S A. 1993; 90(23): 11282–11286. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWisniewski KE, Dalton AJ, McLachlan C, et al.: Alzheimer's disease in Down's syndrome: clinicopathologic studies. Neurology. 1985; 35(7): 957–961. PubMed Abstract | Publisher Full Text\n\nDev KK, Hofele K, Barbieri S, et al.: Part II: alpha-synuclein and its molecular pathophysiological role in neurodegenerative disease. Neuropharmacology. 2003; 45(1): 14–44. PubMed Abstract | Publisher Full Text\n\nPrusiner SB: Shattuck lecture--neurodegenerative diseases and prions. N Engl J Med. 2001; 344(20): 1516–1526. PubMed Abstract | Publisher Full Text\n\nZoghbi HY, Orr HT: Polyglutamine diseases: protein cleavage and aggregation. Curr Opin Neurobiol. 1999; 9(5): 566–570. PubMed Abstract | Publisher Full Text\n\nUversky VN: Amyloidogenesis of natively unfolded proteins. Curr Alzheimer Res. 2008; 5(3): 260–287. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIakoucheva LM, Brown CJ, Lawson JD, et al.: Intrinsic disorder in cell-signaling and cancer-associated proteins. J Mol Biol. 2002; 323(3): 573–584. PubMed Abstract | Publisher Full Text\n\nRadivojac P, Iakoucheva LM, Oldfield CJ, et al.: Intrinsic disorder and functional proteomics. Biophys J. 2007; 92(5): 1439–1456. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCheng Y, LeGall T, Oldfield CJ, et al.: Abundance of intrinsic disorder in protein associated with cardiovascular disease. Biochemistry. 2006; 45(35): 10448–10460. PubMed Abstract | Publisher Full Text\n\nUversky VN, Oldfield CJ, Midic U, et al.: Unfoldomics of human diseases: linking protein intrinsic disorder with diseases. BMC Genomics. 2009; 10(Suppl 1): S7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUversky VN: Intrinsic disorder in proteins associated with neurodegenerative diseases. Front Biosci (Landmark Ed). 2009; 14: 5188–5238. PubMed Abstract | Publisher Full Text\n\nMohan A, Sullivan WJ Jr, Radivojac P, et al.: Intrinsic disorder in pathogenic and non-pathogenic microbes: discovering and analyzing the unfoldomes of early-branching eukaryotes. Mol Biosyst. 2008; 4(4): 328–340. PubMed Abstract | Publisher Full Text\n\nMidic U, Oldfield CJ, Dunker AK, et al.: Protein disorder in the human diseasome: Unfoldomics of human genetic diseases. BMC Genomics. 2009; 10(Suppl 1): S12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRajagopalan K, Mooney SM, Parekh N, et al.: A majority of the cancer/testis antigens are intrinsically disordered proteins. J Cell Biochem. 2011; 112(11): 3256–3267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDunker AK, Cortese MS, Romero P, et al.: Flexible nets: The roles of intrinsic disorder in protein interaction networks. FEBS J. 2005; 272(20): 5129–5148. PubMed Abstract | Publisher Full Text\n\nOldfield CJ, Cheng Y, Cortese MS, et al.: Comparing and combining predictors of mostly disordered proteins. Biochemistry. 2005; 44(6): 1989–2000. PubMed Abstract | Publisher Full Text\n\nVucetic S, Xie H, Iakoucheva LM, et al.: Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions. J Proteome Res. 2007; 6(5): 1899–1916. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXie H, Vucetic S, Iakoucheva LM, et al.: Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J Proteome Res. 2007; 6(5): 1882–1898. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXie H, Vucetic S, Iakoucheva LM, et al.: Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins. J Proteome Res. 2007; 6(5): 1917–1932. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeng Z, Xue B, Kurgan L, et al.: Resilience of death: intrinsic disorder in proteins involved in the programmed cell death. Cell Death Differ. 2013; 20(9): 1257–1267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaczmarek A, Vandenabeele P, Krysko DV: Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity. 2013; 38(2): 209–223. PubMed Abstract | Publisher Full Text\n\nEisenberg-Lerner A, Bialik S, Simon HU, et al.: Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ. 2009; 16(7): 966–975. PubMed Abstract | Publisher Full Text\n\nHe C, Klionsky DJ: Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009; 43: 67–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu B, Cheng Y, Liu Q, et al.: Autophagic pathways as new targets for cancer drug development. Acta Pharmacol Sin. 2010; 31(9): 1154–1164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBialik S, Zalckvar E, Ber Y, et al.: Systems biology analysis of programmed cell death. Trends Biochem Sci. 2010; 35(10): 556–564. PubMed Abstract | Publisher Full Text\n\nGalluzzi L, Vanden Berghe T, Vanlangenakker N, et al.: Programmed necrosis from molecules to health and disease. Int Rev Cell Mol Biol. 2011; 289: 1–35. PubMed Abstract | Publisher Full Text\n\nOuyang L, Shi Z, Zhao S, et al.: Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 2012; 45(6): 487–498. PubMed Abstract | Publisher Full Text\n\nTan ML, Ooi JP, Ismail N, et al.: Programmed cell death pathways and current antitumor targets. Pharm Res. 2009; 26(7): 1547–1560. PubMed Abstract | Publisher Full Text\n\nXie Z, Klionsky DJ: Autophagosome formation: core machinery and adaptations. Nat Cell Biol. 2007; 9(10): 1102–1109. PubMed Abstract | Publisher Full Text\n\nHuett A, Goel G, Xavier RJ: A systems biology viewpoint on autophagy in health and disease. Curr Opin Gastroenterol. 2010; 26(4): 302–309. PubMed Abstract | Publisher Full Text\n\nChen S, Rehman SK, Zhang W, et al.: Autophagy is a therapeutic target in anticancer drug resistance. Biochim Biophys Acta. 2010; 1806(2): 220–229. PubMed Abstract | Publisher Full Text\n\nLi ZY, Yang Y, Ming M, et al.: Mitochondrial ROS generation for regulation of autophagic pathways in cancer. Biochem Biophys Res Commun. 2011; 414(1): 5–8. PubMed Abstract | Publisher Full Text\n\nLiu JJ, Lin M, Yu JY, et al.: Targeting apoptotic and autophagic pathways for cancer therapeutics. Cancer Lett. 2011; 300(2): 105–114. PubMed Abstract | Publisher Full Text\n\nHenriquez M, Armisen R, Stutzin A, et al.: Cell death by necrosis, a regulated way to go. Curr Mol Med. 2008; 8(3): 187–206. PubMed Abstract | Publisher Full Text\n\nCohen GM: Caspases: the executioners of apoptosis. Biochem J. 1997; 326(Pt 1): 1–16. PubMed Abstract | Free Full Text\n\nBrune B: Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ. 2003; 10(8): 864–869. PubMed Abstract | Publisher Full Text\n\nBrune B, Zhou J, von Knethen A: Nitric oxide, oxidative stress, and apoptosis. Kidney Int. 2003; 63(Suppl 84): S22–24. PubMed Abstract | Publisher Full Text\n\nBrune B, Schneiderhan N: Nitric oxide evoked p53-accumulation and apoptosis. Toxicol Lett. 2003; 139(2–3): 119–123. PubMed Abstract | Publisher Full Text\n\nPopov SG, Villasmil R, Bernardi J, et al.: Lethal toxin of Bacillus anthracis causes apoptosis of macrophages. Biochem Biophys Res Commun. 2002; 293(1): 349–355. PubMed Abstract | Publisher Full Text\n\nFridman JS, Lowe SW: Control of apoptosis by p53. Oncogene. 2003; 22(56): 9030–9040. PubMed Abstract | Publisher Full Text\n\nPeter ME, Krammer PH: The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ. 2003; 10(1): 26–35. PubMed Abstract | Publisher Full Text\n\nMattson MP, Chan SL: Calcium orchestrates apoptosis. Nat Cell Biol. 2003; 5(12): 1041–1043. PubMed Abstract | Publisher Full Text\n\nKroemer G, Reed JC: Mitochondrial control of cell death. Nat Med. 2000; 6(5): 513–519. PubMed Abstract | Publisher Full Text\n\nReed JC, Kroemer G: Mechanisms of mitochondrial membrane permeabilization. Cell Death Differ. 2000; 7(12): 1145. PubMed Abstract | Publisher Full Text\n\nWidlak P, Li LY, Wang X, et al.: Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I. J Biol Chem. 2001; 276(51): 48404–48409. PubMed Abstract | Publisher Full Text\n\nLi LY, Luo X, Wang X: Endonuclease G is an apoptotic DNase when released from mitochondria. Nature. 2001; 412(6842): 95–99. PubMed Abstract | Publisher Full Text\n\nParrish J, Li L, Klotz K, et al.: Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature. 2001; 412(6842): 90–94. PubMed Abstract | Publisher Full Text\n\nTsujimoto Y: Cell death regulation by the Bcl-2 protein family in the mitochondria. J Cell Physiol. 2003; 195(2): 158–167. PubMed Abstract | Publisher Full Text\n\nDegterev A, Huang Z, Boyce M, et al.: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005; 1(2): 112–119. PubMed Abstract | Publisher Full Text\n\nFestjens N, Vanden Berghe T, Vandenabeele P: Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta. 2006; 1757(9–10): 1371–1387. PubMed Abstract | Publisher Full Text\n\nBoujrad H, Gubkina O, Robert N, et al.: AIF-mediated programmed necrosis: a highly regulated way to die. Cell Cycle. 2007; 6(21): 2612–2619. PubMed Abstract\n\nEisenberg-Lerner A, Kimchi A: DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress. Cell Death Differ. 2007; 14(11): 1908–1915. PubMed Abstract | Publisher Full Text\n\nMorgan MJ, Kim YS, Liu ZG: TNFalpha and reactive oxygen species in necrotic cell death. Cell Res. 2008; 18(3): 343–349. PubMed Abstract | Publisher Full Text\n\nDiez J, Walter D, Munoz-Pinedo C, et al.: DeathBase: a database on structure, evolution and function of proteins involved in apoptosis and other forms of cell death. Cell Death Differ. 2010; 17(5): 735–736. PubMed Abstract | Publisher Full Text\n\nXue B, Dunbrack RL, Williams RW, et al.: PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim Biophys Acta. 2010; 1804(4): 996–1010. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRomero P, Obradovic Z, Li X, et al.: Sequence complexity of disordered protein. Proteins. 2001; 42(1): 38–48. PubMed Abstract | Publisher Full Text\n\nPeng K, Vucetic S, Radivojac P, et al.: Optimizing long intrinsic disorder predictors with protein evolutionary information. J Bioinform Comput Biol. 2005; 3(1): 35–60. PubMed Abstract | Publisher Full Text\n\nPeng K, Radivojac P, Vucetic S, et al.: Length-dependent prediction of protein intrinsic disorder. BMC Bioinformatics. 2006; 7: 208. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrilusky J, Felder CE, Zeev-Ben-Mordehai T, et al.: FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics. 2005; 21(16): 3435–3438. PubMed Abstract | Publisher Full Text\n\nDosztanyi Z, Csizmok V, Tompa P, et al.: IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics. 2005; 21(16): 3433–3434. PubMed Abstract | Publisher Full Text\n\nCampen A, Williams RM, Brown CJ, et al.: TOP-IDP-scale: a new amino acid scale measuring propensity for intrinsic disorder. Protein Pept Lett. 2008; 15(9): 956–963. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOldfield CJ, Cheng Y, Cortese MS, et al.: Coupled folding and binding with alpha-helix-forming molecular recognition elements. Biochemistry. 2005; 44(37): 12454–12470. PubMed Abstract | Publisher Full Text\n\nCheng Y, Oldfield CJ, Meng J, et al.: Mining alpha-helix-forming molecular recognition features with cross species sequence alignments. Biochemistry. 2007; 46(47): 13468–13477. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeszaros B, Simon I, Dosztanyi Z: Prediction of protein binding regions in disordered proteins. PLoS Comput Biol. 2009; 5(5): e1000376. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDosztanyi Z, Meszaros B, Simon I: ANCHOR: web server for predicting protein binding regions in disordered proteins. Bioinformatics. 2009; 25(20): 2745–2746. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDosztanyi Z, Csizmok V, Tompa P, et al.: The pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins. J Mol Biol. 2005; 347(4): 827–839. PubMed Abstract | Publisher Full Text\n\nPeng Z, Mizianty MJ, Xue B, et al.: More than just tails: intrinsic disorder in histone proteins. Mol Biosyst. 2012; 8(7): 1886–1901. PubMed Abstract | Publisher Full Text\n\nKirilyuk A, Shimoji M, Catania J, et al.: An intrinsically disordered region of the acetyltransferase p300 with similarity to prion-like domains plays a role in aggregation. Plos One. 2012; 7(11): e48243. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoelho Ribeiro Mde L, Espinosa J, Islam S, et al.: Malleable ribonucleoprotein machine: protein intrinsic disorder in the Saccharomyces cerevisiae spliceosome. PeerJ. 2013; 1: e2. PubMed Abstract | Publisher Full Text | Free 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): D561–568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeter ME, Krammer PH: Mechanisms of CD95(APO-1/Fas)-mediated apoptosis. Curr Opin Immunol. 1998; 10(5): 545–551. PubMed Abstract | Publisher Full Text\n\nGreen DR, Reed JC: Mitochondria and apoptosis. Science. 1998; 281(5381): 1309–1312. PubMed Abstract | Publisher Full Text\n\nGreen D, Kroemer G: The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol. 1998; 8(7): 267–271. PubMed Abstract | Publisher Full Text\n\nYin XM: Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Res. 2000; 10(3): 161–167. PubMed Abstract | Publisher Full Text\n\nMartinon F, Holler N, Richard C, et al.: Activation of a pro-apoptotic amplification loop through inhibition of NF-kappaB-dependent survival signals by caspase-mediated inactivation of RIP. FEBS Lett. 2000; 468(2–3): 134–136. PubMed Abstract | Publisher Full Text\n\nHoller N, Zaru R, Micheau O, et al.: Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000; 1(6): 489–495. PubMed Abstract | Publisher Full Text\n\nDeclercq W, Vanden Berghe T, Vandenabeele P: RIP kinases at the crossroads of cell death and survival. Cell. 2009; 138(2): 229–232. PubMed Abstract | Publisher Full Text\n\nDegterev A, Hitomi J, Germscheid M, et al.: Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008; 4(5): 313–321. PubMed Abstract | Publisher Full Text\n\nEisenberg T, Carmona-Gutierrez D, Büttner S, et al.: Necrosis in yeast. Apoptosis. 2010; 15(3): 257–268. PubMed Abstract | Publisher Full Text\n\nScaffidi P, Misteli T, Bianchi ME: Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature. 2002; 418(6894): 191–195. PubMed Abstract | Publisher Full Text\n\nZhao R, Gish K, Murphy M, et al.: Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. Genes Dev. 2000; 14(8): 981–993. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalint EE, Vousden KH: Activation and activities of the p53 tumour suppressor protein. Br J Cancer. 2001; 85(12): 1813–1823. PubMed Abstract | Publisher Full Text | Free Full Text\n\nel-Deiry WS: Regulation of p53 downstream genes. Semin Cancer Biol. 1998; 8(5): 345–357. PubMed Abstract | Publisher Full Text\n\nYu J, Zhang L, Hwang PM, et al.: Identification and classification of p53-regulated genes. Proc Natl Acad Sci U S A. 1999; 96(25): 14517–14522. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSax JK, El-Deiry WS: p53-induced gene expression analysis. Methods Mol Biol. 2003; 234: 65–71. PubMed Abstract | Publisher Full Text\n\nAnderson CW, Appella E: Signaling to the p53 tumor suppressor through pathways activated by genotoxic and nongenotoxic stress. In: Bradshaw RA Dennis EA (eds) Handbook of Cell Signaling. Academic Press, New York, 2004; pp 237–247.\n\nGottlieb TM, Leal JF, Seger R, et al.: Cross-talk between Aktp 53 and Mdm2: possible implications for the regulation of apoptosis. Oncogene. 2002; 21(8): 1299–1303. PubMed Abstract | Publisher Full Text\n\nNicholson KM, Anderson NG: The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal. 2002; 14(5): 381–395. PubMed Abstract | Publisher Full Text\n\nCollins MK, Perkins GR, Rodriguez-Tarduchy G, et al.: Growth factors as survival factors: regulation of apoptosis. Bioessays. 1994; 16(2): 133–138. PubMed Abstract | Publisher Full Text\n\nMalaney P, Pathak RR, Xue B, et al.: Intrinsic Disorder in PTEN and its Interactome Confers Structural Plasticity and Functional Versatility. Sci Rep. 2013; 3: 2035. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShieh SY, Ahn J, Tamai K, et al.: The human homologs of checkpoint kinases Chk1 and Cds1(Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 2000; 14(3): 289–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen L, Gilkes DM, Pan Y, et al.: ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage. EMBO J. 2005; 24(19): 3411–3422. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiyashita T, Krajewski S, Krajewska M, et al.: Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene. 1994; 9(6): 1799–1805. PubMed Abstract\n\nNakano K, Vousden KH: PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell. 2001; 7(3): 683–694. PubMed Abstract | Publisher Full Text\n\nOda E, Ohki R, Murasawa H, et al.: Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science. 2000; 288(5468): 1053–1058. PubMed Abstract | Publisher Full Text\n\nSax JK, Fei P, Murphy ME, et al.: BID regulation by p53 contributes to chemosensitivity. Nat Cell Biol. 2002; 4(11): 842–849. PubMed Abstract | Publisher Full Text\n\nOda K, Arakawa H, Tanaka T, et al.: p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell. 2000; 102(6): 849–862. PubMed Abstract | Publisher Full Text\n\nMeley D, Spiller DG, White MR, et al.: p53-mediated delayed NF-kappaB activity enhances etoposide-induced cell death in medulloblastoma. Cell Death Dis. 2010; 1: e41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharp AN, Heazell AE, Crocker IP, et al.: Placental apoptosis in health and disease. Am J Reprod Immunol. 2010; 64(3): 159–169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmaral JD, Castro RE, Steer CJ, et al.: p53 and the regulation of hepatocyte apoptosis: implications for disease pathogenesis. Trends Mol Med. 2009; 15(11): 531–541. PubMed Abstract | Publisher Full Text\n\nDavies L, Spiller D, White MR, et al.: PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells. Cell Death Dis. 2011; 2: e136. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUversky VN, Oldfield CJ, Dunker AK: Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. J Mol Recognit. 2005; 18(5): 343–384. PubMed Abstract | Publisher Full Text\n\nBustos DM, Iglesias AA: Intrinsic disorder is a key characteristic in partners that bind 14-3-3 proteins. Proteins. 2006; 63(1): 35–42. PubMed Abstract | Publisher Full Text\n\nRadivojac P, Vucetic S, O'Connor TR, et al.: Calmodulin signaling: analysis and prediction of a disorder-dependent molecular recognition. Proteins. 2006; 63(2): 398–410. PubMed Abstract | Publisher Full Text\n\nPatil A, Nakamura H: Disordered domains and high surface charge confer hubs with the ability to interact with multiple proteins in interaction networks. FEBS Lett. 2006; 580(8): 2041–2045. PubMed Abstract | Publisher Full Text\n\nEkman D, Light S, Bjorklund AK, et al.: What properties characterize the hub proteins of the protein-protein interaction network of Saccharomyces cerevisiae? Genome Biol. 2006; 7(6): R45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaynes C, Oldfield CJ, Ji F, et al.: Intrinsic disorder is a common feature of hub proteins from four eukaryotic interactomes. PLoS Comput Biol. 2006; 2(8): e100. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDosztanyi Z, Chen J, Dunker AK, et al.: Disorder and sequence repeats in hub proteins and their implications for network evolution. J Proteome Res. 2006; 5(11): 2985–2995. PubMed Abstract | Publisher Full Text\n\nSingh GP, Dash D: Intrinsic disorder in yeast transcriptional regulatory network. Proteins. 2007; 68(3): 602–605. PubMed Abstract | Publisher Full Text\n\nSingh GP, Ganapathi M, Dash D: Role of intrinsic disorder in transient interactions of hub proteins. Proteins. 2007; 66(4): 761–765. PubMed Abstract | Publisher Full Text\n\nBerman HM, Westbrook J, Feng Z, et al.: The Protein Data Bank. Nucleic Acids Res. 2000; 28(1): 235–242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUversky VN, Dunker AK: Understanding protein non-folding. Biochim Biophys Acta. 2010; 1804(6): 1231–1264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDunker AK, Brown CJ, Lawson JD, et al.: Intrinsic disorder and protein function. Biochemistry. 2002; 41(21): 6573–6582. PubMed Abstract\n\nDunker AK, Brown CJ, Obradovic Z: Identification and functions of usefully disordered proteins. Adv Protein Chem. 2002; 62: 25–49. PubMed Abstract\n\nTompa P: Intrinsically unstructured proteins. Trends Biochem Sci. 2002; 27(10): 527–533. PubMed Abstract | Publisher Full Text\n\nUversky VN, Oldfield CJ, Dunker AK: Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. J Mol Recognit. 2005; 18(5): 343–384. PubMed Abstract | Publisher Full Text\n\nDunker AK, Silman I, Uversky VN, et al.: Function and structure of inherently disordered proteins. Curr Opin Struct Biol. 2008; 18(6): 756–764. PubMed Abstract | Publisher Full Text\n\nDunker AK, Uversky VN: Signal transduction via unstructured protein conduits. Nat Chem Biol. 2008; 4(4): 229–230. PubMed Abstract | Publisher Full Text\n\nUversky VN: Multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes. Chem Soc Rev. 2011; 40(3): 1623–1634. PubMed Abstract | Publisher Full Text\n\nUversky VN: Disordered competitive recruiter: fast and foldable. J Mol Biol. 2012; 418(5): 267–268. PubMed Abstract | Publisher Full Text\n\nUversky VN: Intrinsic Disorder-based Protein Interactions and their Modulators. Curr Pharm Des. 2013; 19(23): 4191–4213. PubMed Abstract | Publisher Full Text\n\nDyson HJ, Wright PE: Coupling of folding and binding for unstructured proteins. Curr Opin Struct Biol. 2002; 12(1): 54–60. PubMed Abstract | Publisher Full Text\n\nDyson HJ, Wright PE: Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol. 2005; 6(3): 197–208. PubMed Abstract | Publisher Full Text\n\nWright PE, Dyson HJ: Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. J Mol Biol. 1999; 293(2): 321–331. PubMed Abstract | Publisher Full Text\n\nMohan A, Oldfield CJ, Radivojac P, et al.: Analysis of molecular recognition features (MoRFs). J Mol Biol. 2006; 362(5): 1043–1059. PubMed Abstract | Publisher Full Text\n\nVacic V, Oldfield CJ, Mohan A, et al.: Characterization of molecular recognition features, MoRFs, and their binding partners. J Proteome Res. 2007; 6(6): 2351–2366. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUversky VN: Unusual biophysics of intrinsically disordered proteins. Biochim Biophys Acta. 2013; 1834(5): 932–951. PubMed Abstract | Publisher Full Text\n\nVan Herreweghe F, Festjens N, Declercq W, et al.: Tumor necrosis factor-mediated cell death: to break or to burst, that's the question. Cell Mol Life Sci. 2010; 67(10): 1567–1579. PubMed Abstract | Publisher Full Text\n\nMeulmeester E, Jochemsen AG: p53: A guide to apoptosis. Curr Cancer Drug Targets. 2008; 8(2): 87–97. PubMed Abstract | Publisher Full Text\n\nZhivotovsky B, Kroemer G: Apoptosis and genomic instability. Nat Rev Mol Cell Biol. 2004; 5(9): 752–762. PubMed Abstract | Publisher Full Text\n\nChen H, Ma J, Li W, et al.: A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases. Mol Cell. 2007; 27(5): 717–730. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSheng Y, Saridakis V, Sarkari F, et al.: Molecular recognition of p53 and MDM2 by USP7/HAUSP. Nat Struct Mol Biol. 2006; 13(3): 285–291. PubMed Abstract | Publisher Full Text\n\nRustandi RR, Baldisseri DM, Weber DJ: Structure of the negative regulatory domain of p53 bound to S100B(betabeta). Nat Struct Biol. 2000; 7(7): 570–574. PubMed Abstract | Publisher Full Text\n\nMittl PR, Chene P, Grutter MG: Crystallization and structure solution of p53 (residues 326–356) by molecular replacement using an NMR model as template. Acta Crystallogr D Biol Crystallogr. 1998; 54(Pt 1): 86–89. PubMed Abstract | Publisher Full Text\n\nKussie PH, Gorina S, Marechal V, et al.: Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science. 1996; 274(5289): 948–953. PubMed Abstract | Publisher Full Text\n\nBochkareva E, Kaustov L, Ayed A, et al.: Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A. Proc Natl Acad Sci U S A. 2005; 102(43): 15412–15417. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeng H, Jenkins LM, Durell SR, et al.: Structural basis for p300 Taz2-p53 TAD1 binding and modulation by phosphorylation. Structure. 2009; 17(2): 202–210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu HH, Wu PY, Huang KF, et al.: Structural delineation of MDC1-FHA domain binding with CHK2-pThr68. Biochemistry. 2012; 51(2): 575–577. PubMed Abstract | Publisher Full Text\n\nCzabotar PE, Westphal D, Dewson G, et al.: Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis. Cell. 2013; 152(3): 519–531. PubMed Abstract | Publisher Full Text\n\nPeyerl FW, Dai S, Murphy GA, et al.: Elucidation of some Bax conformational changes through crystallization of an antibody-peptide complex. Cell Death Differ. 2007; 14(3): 447–452. PubMed Abstract | Publisher Full Text\n\nCzabotar PE, Lee EF, Thompson GV, et al.: Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis. J Biol Chem. 2011; 286(9): 7123–7131. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1841",
"date": "26 Sep 2013",
"name": "Martin Vabulas",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBy means of several computational tools Uversky et al. investigates the level of disorder of key members in the cellular network of apoptosis, autophagy, and necroptosis. The study represents an extension of their previous work (Peng et al. 2013) and the two are best read together. I found the study very interesting since it implicates structural disorder as a cell death network building/sustaining principle. Since individual components of the network are analyzed and the results of disorder prediction and connectivity (according to the STRING database) are presented back-to-back the study represents a handy knowledge resource and lays ground for the future experimental validation.",
"responses": [
{
"c_id": "596",
"date": "28 Oct 2013",
"name": "Vladimir Uversky",
"role": "Author Response F1000Research Advisory Board Member",
"response": "We are thankful to Dr. Vabulas for his high evaluation of our work and hope that the readers of F1000Research will find this study useful."
}
]
},
{
"id": "1839",
"date": "23 Oct 2013",
"name": "Oxana Galzitskaya",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 by Uversky et al. describes the proteins involved in programmed cell death pathways (apoptosis, autophagy, and necroptosis). Overall it is an interesting paper deserving of publication and contains very exciting illustrative material. The authors have performed a study with a particular accent on the intrinsic disordered status of these proteins. To predict the status of disordered residues, the authors used two programs: PONDR FIT and PONDR® VLXT. It should be noted that there are now programs that work with the same or better accuracy of prediction such as SPINE-D, MFDp, MD, Disopred2, IsUnstruct (version 2.02) and DISOclust (for more details see Table 5 in Lobanov, Sokolovskiy, and Galzitskaya Journal of Biomolecular Structure & Dynamics 31(10), 1034-1043, 2013). PONDR®VLXT was the least accurate at disorder prediction according to the AUC of 14 programs that were compared in Lobanov et al.",
"responses": [
{
"c_id": "595",
"date": "28 Oct 2013",
"name": "Vladimir Uversky",
"role": "Author Response F1000Research Advisory Board Member",
"response": "First of all, we would like to thank this reviewer for their positive evaluation of our work.We do agree that the number of available disorder predictors is very large and many of them have accuracies close to that of PONDR FIT. PONDR FIT is a metapredictor representing a consensus of artificial neural network prediction tools, which was developed by combining the outputs of PONDR-VLXT, PONDR-VSL2, PONDR-VL3, FoldIndex, IUPred, and TopIDP. Since PONDR FIT possesses improved prediction accuracy compared to its individual component predictors, and the overall accuracy of this tool is comparable to the accuracies of other meta-predictors, we do not think that the use of additional computational tools is needed for this study. We also know that PONDR VLXT is not the most accurate disorder predictor. However, this predictor is extremely sensitive to the local peculiarities of amino acid sequences and therefore, is very useful for finding potential disorder-based interaction sites. This point was specifically discussed in our paper dedicated to the development of the related computational tool, alpha-MoRF-PredII (Cheng Y, Oldfield CJ, Meng J, Romero P, Uversky VN, Dunker AK. Mining alpha-helix-forming molecular recognition features with cross species sequence alignments. Biochemistry. 2007; 46(47): 13468-77. PMID: 17973494)."
}
]
}
] | 1
|
https://f1000research.com/articles/2-190
|
https://f1000research.com/articles/2-186/v1
|
13 Sep 13
|
{
"type": "Short Research Article",
"title": "Pediatric chronic kidney disease rates in Southern Israel are higher than reported",
"authors": [
"Daniel Landau",
"Ruth Schreiber",
"Anya Kleinman",
"Alina Vodonos",
"Hannah Shalev",
"Ruth Schreiber",
"Anya Kleinman",
"Alina Vodonos",
"Hannah Shalev"
],
"abstract": "Background: The incidence and prevalence of pediatric chronic kidney disease (p-CKD) in developed countries has previously been estimated to be 12 and 75 cases/106 population respectively, much lower than reports in young adults (age 20-40) (40,000/106). Thus, the extent of p-CKD may be underestimated.Methods: Being the only Pediatric Nephrology center in Southern Israel, we reviewed all detected cases of p-CKD (stages 1-5) between 1994-2008. We then prospectively summarized the incidence and prevalence of CKD between 2009-2010. Results: We retrospectively identified 192 children (53.9% of Bedouin origin, 53.4% males, median diagnosis age: 1 year) with CKD. The prevalence in December 2008 was 795 cases/106 for all CKD stages and 331/106 for CKD stage >2. Calculated incidence for the study period (1994-2008) was 46/106/year. The main CKD etiologies were: hypodysplasia: 35%; obstructive uropathy: 13%; genetic renal diseases: 28% and glomerulonephritis: 15%. The proportions of children in each CKD stage were as follows: stage 1: 50%; stages 2-4: 30%; stage 5: 20%. During a subsequent two-year study period we identified 26 new CKD cases (incidence: 54 cases/106/year). Conclusions: p-CKD rates in our area are higher than reported and maybe even higher if asymptomatic populations are screened. Fifty percent of detected cases have CKD stage 1. This may contribute significantly to CKD beyond the pediatric age.",
"keywords": [
"Chronic kidney disease",
"epidemiology",
"genetic renal diseases",
"renal hypodysplasia",
"urinary bladder",
"neurogenic"
],
"content": "\n\nIn chronic kidney disease (CKD) there is permanent bilateral kidney damage, structural or functional, with or without a decrease in glomerular filtration rate. Reported childhood CKD rates are far lower than that of young adults and depend on discovered cases, possibly missing asymptomatic cases.\n\nIn our population-based study, we found pediatric CKD rates 10 times higher than previous studies, 50% of them CKD stage 1. This may explain the CKD burden beyond pediatric age.\n\n\nIntroduction\n\nChronic kidney disease (CKD) is defined as evidence for bilateral kidney damage for more than 3 months, which can be structural or functional, with or without a decrease in glomerular filtration rate1. The presence of CKD at any stage is a strong risk factor for renal function deterioration during lifetime, many times beyond the pediatric age2.\n\nThe prevalence of pediatric CKD (p-CKD; stage ≥2) in developed countries (such as in Italian3 and Spanish studies4) has been estimated to be as high as 75 cases/106 population at risk. However, until recently, such estimates were based on CKD retrospectively detected by tertiary care centers, which does not allow the determination of true incidence as patients who do not have advanced CKD (\"overt CKD\") may not be identified. In addition, these pediatric numbers are much lower than reports from American young adults (up to 40,000/106 for ages 20–40 by the National Health and Nutrition Examination Survey (NHANES studies)5. Thus, the extent of p-CKD may be underestimated.\n\nThe Negev population receives tertiary care services through a single medical institution, the Soroka University Medical Center (SUMC), which serves a population (including referrals) of 300,000 children and takes care of more than 15,000 deliveries a year. The SUMC Pediatric Nephrology service is the only caregiver for children in the area affected by kidney and urinary tract diseases. The population served by this hospital is composed of two major subpopulations, usually living in separate settlements: Jews (75%) and Arab-Bedouins (25%). The consanguinity rate among Jewish couples is nowadays low and estimated as 2.3%, including 0.8% first cousin marriages6. The Negev Bedouins' current estimated population is 200,000. The total fertility rate in the Bedouin-Muslim population in 2006 was 7.3, compared with to 3.3–4.1 in other Muslim populations in Israel and 2.62 in the Jewish population in southern Israel. In Bedouin society, cousin marriage is the preferred type of marital union and traditionally involves marriage between first-degree cousins7,8. Currently, 60% of this population is under 19-years-of-age9. More than half of the Bedouin population live in unrecognized villages and shanty towns, and suffer from a high rate of unemployment. However, all Israelis are entitled to medical care as a result of a National Health Insurance plan introduced in 1995. The reported pediatric CKD stage 5 end-stage renal disease (ESRD) incidence for 2006 in this area was 19 cases/106/year10, similar to reports from developed countries11. Previous studies from this center provide reliable population-based epidemiological data12,13. The purpose of this study was to assess the prevalence and etiology of p-CKD in Southern Israel.\n\n\nMaterial and methods\n\nThe study protocol was approved by the hospital's ethics committee (approval number: 10568). We reviewed all recorded cases of p-CKD among inhabitants of the Negev area (and not referrals from other country areas) during the January 1994-December 2008 period, based on a review of medical records of children detected by the Pediatric Nephrology service to have this condition. This was double-checked by screening the hospital's Medical Records department discharge diagnoses to look for additional children (age 0–19 years) with CKD diagnosis. For the retrospective analysis, we graded based on the worst CKD stage reached by the children. We calculated CKD prevalence and stage according to the living child’s status (CKD stage 1–5) as at Dec 31, 2008. In addition, we prospectively collected all new CKD cases diagnosed in our hospital, from January 1, 2009 until December 31, 2010. Data from medical records were entered into standardized forms, including demographics, age of onset, underlying renal disease, etc. The standardized form used is provided in the data file.\n\nEstimated glomerular filtration rate (eGFR) was calculated by the original Schwartz formula14. Hypodysplasia was defined by imaging studies as a congenital reduced kidney size and maldevelopment of the renal tissue (including renal scars), with or without associated malformations in the urinary tract (hydronephrosis, hydroureter, vesicoureteric reflux), in both urinary tract systems. Unilateral urinary tract anomalies and/or a single kidney were not included unless an additional feature of CKD (such as proteinuria or hypertension) was present. Obstructive uropathy was divided between neurogenic bladder and congenital obstruction. Genetic renal disease was diagnosed only for monogenic diseases that are known to be associated with a decreased renal function with age. Therefore, diseases such as Bartter syndrome or nephrogenic diabetes insipidus were not included. CKD stage 1 was defined as an eGFR of equal or more than 90 ml/min/1.73 m2 (a normal GFR value) but with the presence of either persistent albuminuria (as may exist for reflux or diabetic nephropathy) or bilateral structural renal anomalies (as in polycystic kidney disease, hypodysplasia, or bilateral renal scars). CKD stage 2 was defined as a eGFR between 60–90 ml/min/1.73 m2; CKD stage 3 was a eGFR between 30–60 ml/min/1.73 m2, CKD stage 4 was a eGFR between 15–30 ml/min/1.73 m2 and CKD stage 5 was defined as a GFR of < 15 ml/min/1.73 m2.\n\nA direct age adjustment method was performed to compare the prevalence and incidence of p-CKD between the Jewish and Bedouin populations using children under the age of 19 as standard population. Population statistics were obtained from The Israeli Central Bureau of Statistics national census. Statistical analysis was performed using the SPSS software, version 18.\n\n\nResults\n\nWe identified 192 living p-CKD patients at December 31, 2008 (46.1% of Jewish origin, 53.4% males) during the retrospective study period. The population at risk at that time was 241,400, as provided by the Israeli Census Bureau (http://www1.cbs.gov.il/reader/shnaton/templ_shnaton_e.html?num_tab=st02_10x&CYear=2010), providing a point prevalence of 795.4 (95% confidence interval (CI): 690.6–916.0) per million (Table 1). The age-adjusted Jewish p-CKD prevalence rate was 628.2 (95% CI: 549.2–751.9) per million, whereas the age-adjusted Bedouin p-CKD prevalence rate was 850.2 (95% CI: 764.4–1000.8) per million. Incidence for the whole study period (1989–2009) was 45.4 (95% CI: 39.7–53.0) per million. When different pediatric age groups were analyzed separately, a higher rate was found in younger children (age 0–4 yrs) of Bedouin origin than children of Jewish origin. Rates were similar between the two groups at ages 5–14 and 15–19 years (Figure 1).\n\nX axis: age groups (years). Black bars: children of Bedouin origin. White bars: children of Jewish origin. No p values for comparison are provided since the total population (and not a sample) is analyzed.\n\n* Refers to living patients aged < 19 years on December 2008. Prevalence is adjusted for age. ** Mostly autosomal dominant polycystic kidney disease (ADPKD); *** Mostly atypical hemolytic uremic syndrome and infantile nephronophtisis. No p values for comparison are provided since the total population (and not a sample) is analyzed.\n\nThe mean age of diagnosis was 4.9±6 years, but the median age of diagnosis was 1 year. The main p-CKD etiologies were: hypodysplasia: 35%; obstructive uropathy: 13%; genetic renal diseases: 28% and glomerulonephritis: 15%.\n\nThe proportions of children in each CKD stage were as follows: stage 1: 50%; stages 2–4: 30%; stage 5: 20%. One hundred thirty-one children were diagnosed with CKD stage 1, which included 44 children with bilateral renal hypodysplasia (with or without renal scars), 10 children with obstructive uropathy (five of them with congenital obstruction of both kidneys and five with neurogenic bladder and bilateral renal scars), 35 children with genetic renal diseases (14 with autosomal dominant polycystic kidney disease (ADPKD) and 10 with the autosomal recessive form (ARPKD)), and 28 children with chronic glomerulonephritis (10 with IgA nephropathy, five children with Henoch Schoenlein purpura nephropathy and residual proteinuria). Miscellaneous diagnoses14 included seven children and adolescents with diabetic nephropathy.\n\nChildren of Bedouin origin had a lower median age of diagnosis (0.3 vs 4 years in Jews, p < 0.01). Twelve children died during the retrospective study period, 11 of them of Bedouin origin, six of them reached ESRD due to two genetic renal diseases: atypical hemolytic uremic syndrome and infantile nephronophtisis. The other five children had hypodysplasia, four of them as part of multiple congenital anomalies, and only one of these 5 children reached ESRD. The one child of Jewish origin that died had Lowe syndrome and died of neurologic complications at CKD stage 2. The same rate of Jewish and Bedouin children (15%) had a renal transplant.\n\nThe prevalence of CKD at stage 2 or above was 331/106 (95% CI: 266.3–412.4), a higher number than that reported from the series in Spain and Italy 71/106 and 75/106 (Table 2). The male: female ratio and age of diagnosis were similar between the series. However, the fractions of CKD children who had a genetic renal disease or obstructive uropathy were higher in our area.\n\n* Age at registration: 6.9±5.4. ** Including congenital obstructive uropathy.\n\nDuring a subsequent 2 year period (January 2009 – December 2010), we identified 26 new children with CKD, providing a yearly incidence of 53.8 (95% CI: 31.5–92.1) new cases/106/yr (half of them at CKD stage 2 or above). The mean age of diagnosis for CKD (stage 2 or above) in the prospective cohort was 2.7±4.8 years (interquartile range: 0–6 yrs, median age: 0.5 yrs).\n\n\n\n\nDiscussion\n\nThe p-CKD prevalence reported in this study is higher than that reported for other recent series from developed countries, such as Italy and Spain (Table 2). We chose to compare our data with these two European countries because of the similarities in accessibility to medical services. A Universal Health Care system by law has been implemented in Israel since 1995 and provides comprehensive medical care to all Israelis. Infant mortality has dropped in Israel in the past decades and is now comparable to most developed countries15. In addition, access to prenatal diagnosis by different screening tests including frequent fetal ultrasound studies has become the standard of care for the great majority of the Israeli population: 90% of Israeli pregnant women have at least one fetal ultrasound performed (Drs Shochat and Romano-Zelicha, Israeli Ministry of Health survey, personal communication). This may allow the detection of many congenital anomalies of the kidneys and urinary tract as well as some of the genetic renal diseases that cause CKD (such as polycystic kidney disease). It may also be the reason for the low median age of detection of p-CKD in our cohort (1 year for the general cohort and as low as three months for the Bedouin population) (Table 1). The lower mean age of diagnosis for the Bedouin population may reflect the higher rate of genetic renal diseases in our area (28% in Southern Israel vs. 14% and 15.6% in Spain and Italy respectively)11. In addition, we found a higher rate of obstructive uropathy due to neurogenic bladder, most of these cases in the Bedouin population, similar to reports from Turkey16, again raising the possibility of the contribution of consanguinity or poverty (such as lower access to folic acid supplementation). However, a similar male predominance and high prevalence of renal hypodysplasia as the main etiology for CKD is seen in these three series.\n\nIt must be remembered that this apparent higher p-CKD rate is still an underestimation of the probable true p-CKD prevalence that may be detected if population screening studies are performed. Such studies have been performed in recent years in Australia, where persistent albuminuria or hypertension were found in 1–2% of prospectively screened school-age children17. Similar reports come from Japan, Taiwan and Korea18. Furthermore, the striking difference in CKD rates between pediatric and young adult populations needs to be addressed. The overall prevalence of CKD stages 1 to 4 in adults increased significantly in the 1999 to 2004 period compared with 1988 to 1994 (13.1 versus 10.0 percent respectively)19. Data from the NHANES 2003–2006 survey, with GFR estimated by the CKD-Epidemiology Collaboration study (CKD-EPI) equation, showed that the overall prevalence of CKD stages 1 through 5 among adults is 14.2%20. In other countries, CKD prevalence ranges between 1 to 30%21. This is not only true for the aged population, since a CKD (stage ≥2) prevalence rate as high as 4% (or 40,000/106 has been reported for the 20–40 year old population22.\n\nThere are fewer reports on the incidence of CKD among adults. In the Framingham cohort (n = 2580), after a mean follow-up of 18.5 years, 244 participants (9.4%) had developed CKD stage 3 and above23, which is a yearly incidence of about 500 new patients/million/year. If one assumes an overall CKD prevalence of 10% (or 100,000 per million) this provides a prevalence-to-incidence ratio of 200 in adults, which is expected given the chronic nature of CKD.\n\nA major cause of mortality in adult CKD is increased cardiovascular comorbidity, in addition to ESRD progression24. When compared to the pediatric population (which includes the first two decades of life vs. the subsequent average of six decades in adults), where no clinical cardiovascular morbidity has yet developed and mortality is lower than adults, one would expect a higher than reported prevalence-to-incidence ratio, which has been only around 6:1 in various previous studies. In our study, we report a ratio of around 15:1. This suggests an underdiagnosis of CKD in the pediatric population. In addition, all studies performed to-date exclude CKD stage 1, which may be incorrect given the more long-term follow-up required for pediatric patients. The potential for gradual clinical progression, even from CKD stage 1, has been demonstrated for renal hypodysplasia in Italian25 and Dutch26 surveys, and is a well recognized requirement for diabetic nephropathy, in both type I27 and, more importantly, type II diabetes mellitus28. Supporting all these observations is a recent series of publications based on a true population frequency in Turkey, which demonstrate that the prevalence of childhood CKD approaches 1%, a number in the same order of magnitude as that of the younger adult cohort in the United States29.\n\nIn summary, we report on a higher prevalence and incidence for p-CKD in Southern Israel and propose that, if proper population-based screening studies are performed, then higher p-CKD values should be found in other developed countries. This may expand the contribution of pediatric renal disease to CKD even beyond the pediatric age.",
"appendix": "Author contributions\n\n\n\nDL conceived the study design, participated in data collection and elaboration, as well as writing the final manuscript version. RS participated in data collection and reviewed the manuscript draft. AK participated in data collection and analysis. AV an epidemiologist, reviewed the data quality, helped in the validation of data and its adjustment for local population data. HS participated in data collection, conceived and reviewed the manuscript draft. All authors reviewed and agreed on the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHogg RJ, Furth S, Lemley KV, et al.: National Kidney Foundation's Kidney Disease Outcomes Quality Initiative clinical practice guidelines for chronic kidney disease in children and adolescents: evaluation, classification, and stratification. Pediatrics. 2003; 111(6 Pt 1): 1416–21. PubMed Abstract\n\nNeild GH: What do we know about chronic renal failure in young adults? I. Primary renal disease. Pediatr Nephrol. 2009; 24(10): 1913–9. PubMed Abstract | Publisher Full Text\n\nArdissino G, Daccò V, Testa S, et al.: Epidemiology of chronic renal failure in children: data from the ItalKid project. Pediatrics. 2003; 111(4 Pt 1): e382–7. PubMed Abstract | Publisher Full Text\n\nAreses Trapote R, Sanahuja Ibáñez MJ, Navarro M: Epidemiology of chronic kidney disease in Spanish pediatric population. REPIR II Project. Nefrologia. 2010; 30(5): 508–17. PubMed Abstract | Publisher Full Text\n\nCenters for Disease Control and Prevention (CDC). Prevalence of chronic kidney disease and associated risk factors--United States, 1999–2004. MMWR Morb Mortal Wkly Rep. 2007; 56(8): 161–5. PubMed Abstract | Publisher Full Text\n\nCohen T, Vardi-Saliternik R, Friedlander Y: Consanguinity, intracommunity and intercommunity marriages in a population sample of Israeli Jews. Ann Hum Biol. 2004; 31(1): 38–48. PubMed Abstract | Publisher Full Text\n\nRaz AE, Atar M, Rodnay M, et al.: Between acculturation and ambivalence: knowledge of genetics and attitudes towards genetic testing in a consanguineous bedouin community. Community Genet. 2003; 6(2): 88–95. PubMed Abstract | Publisher Full Text\n\nShoham-Vardi I, Weiner N, Weitzman D, et al.: Termination of pregnancy: attitudes and behavior of women in a traditional society. Prenat Diagn. 2004; 24(11): 869–75. PubMed Abstract | Publisher Full Text\n\nFriger M, Shoham-Vardi I, Abu-Saad K: Trends and seasonality in birth frequency: a comparison of Muslim and Jewish populations in southern Israel: daily time series analysis of 200 009 births, 1988–2005. Hum Reprod. 2009; 24(6): 1492–500. PubMed Abstract | Publisher Full Text\n\nFiner G, Shalev H, Landau D: Genetic Kidney Diseases in the Pediatric Population of Southern Israel. Pediatr Nephrol. 2006; 21(7): 910–6. PubMed Abstract | Publisher Full Text\n\nHarambat J, van Stralen KJ, Kim JJ, et al.: Epidemiology of chronic kidney disease in children. Pediatr Nephrol. 2012; 27(3): 363–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreenberg D, Givon-Lavi N, Newman N, et al.: Intussusception in children in Southern Israel: disparity between 2 populations. Pediatr Infect Dis J. 2008; 27(3): 236–40. PubMed Abstract | Publisher Full Text\n\nColes CL, Levy A, Gorodischer R, et al.: Subclinical vitamin A deficiency in Israeli-Bedouin toddlers. Eur J Clin Nutr. 2004; 58(5): 796–802. PubMed Abstract | Publisher Full Text\n\nSchwartz GJ, Gauthier B: A simple estimate of glomerular filtration rate in adolescent boys. J Pediatr. 1985; 106(3): 522–6. PubMed Abstract | Publisher Full Text\n\nAmitai Y, Haklai Z, Tarabeia J, et al.: Infant mortality in Israel during 1950–2000: rates, causes, demographic characteristics and trends. Paediatr Perinat Epidemiol. 2005; 19(2): 145–51. PubMed Abstract | Publisher Full Text\n\nBek K, Akman S, Bilge I, et al.: Chronic kidney disease in children in Turkey. Pediatr Nephrol. 2009; 24(4): 797–806. PubMed Abstract | Publisher Full Text\n\nHaysom L, Williams R, Hodson E, et al.: Risk of CKD in Australian indigenous and nonindigenous children: a population-based cohort study. Am J Kidney Dis. 2009; 53(2): 229–37. PubMed Abstract | Publisher Full Text\n\nLin CY, Sheng CC, Lin CC, et al.: Mass urinary screening and follow-up for school children in Taiwan Province. Acta Paediatr Taiwan. 2001; 42(3): 134–40. PubMed Abstract\n\nCoresh J, Selvin E, Stevens LA, et al.: Prevalence of chronic kidney disease in the United States. JAMA. 2007; 298(17): 2038–47. PubMed Abstract | Publisher Full Text\n\nLevey AS, Stevens LA, Schmid CH, et al.: CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration). A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009; 150(9): 604–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJha V, Garcia-Garcia G, Iseki K, et al.: Chronic kidney disease: global dimension and perspectives. Lancet. 2013; 382(9888): 260–72. PubMed Abstract | Publisher Full Text\n\nCoresh J, Astor BC, Greene T, et al.: Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third national health and nutrition examination survey. Am J Kidney Dis. 2003; 41(1): 1–12. PubMed Abstract | Publisher Full Text\n\nFox CS, Larson MG, Leip EP, et al.: Predictors of new-onset kidney disease in a community-based population. JAMA. 2004; 291(7): 844–50. PubMed Abstract | Publisher Full Text\n\nGo AS, Chertow GM, Fan D, et al.: Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N Engl J Med. 2004; 351(13): 1296–305. PubMed Abstract | Publisher Full Text\n\nSanna-Cherchi S, Ravani P, Corbani V, et al.: Renal outcome in patients with congenital anomalies of the kidney and urinary tract. Kidney Int. 2009; 76(5): 528–33. PubMed Abstract | Publisher Full Text\n\nWestland R, Schreuder MF, Bökenkamp A, et al.: Renal injury in children with a solitary functioning kidney--the KIMONO study. Nephrol Dial Transplant. 2011; 26(5): 1533–41. PubMed Abstract | Publisher Full Text\n\nMöllsten A, Svensson M, Waernbaum I, et al.: Cumulative risk, age at onset, and sex-specific differences for developing end-stage renal disease in young patients with type 1 diabetes: a nationwide population-based cohort study. Swedish Childhood Diabetes Study Group; Diabetes Incidence Study in Sweden; Swedish Renal Registry. Diabetes. 2010; 59(7): 1803–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPavkov ME, Bennett PH, Knowler WC, et al.: Effect of youth-onset type 2 diabetes mellitus on incidence of end-stage renal disease and mortality in young and middle-aged Pima Indians. JAMA. 2006; 296(4): 421–6. PubMed Abstract | Publisher Full Text\n\nSoylemezoglu O, Duzova A, Yalçinkaya F, et al.: Chronic renal disease in children aged 5–18 years: a population-based survey in Turkey, the CREDIT-C study. Nephrol Dial Transplant. 2012; 27(Suppl 3): iii146–51. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1819",
"date": "16 Sep 2013",
"name": "Craig Langman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhat shall we learn from this report by Landau and colleagues from southern Israel? It is clear that when one looks at the incidence of CKD and ESRD in the United States in young adults (<39 years), there has been tremendous increase of theses diseases over the past two decades. In part, this is due to paediatric CKD manifesting itself in young adulthood due to loss of nephrons from the very conditions uncovered by the Landau group. Certainly with survival of very premature infants deprived of adequate in utero nephron development, and loss of nephrons for a multitude of reasons in neonates and older children (including acute kidney injury, multiple nephrotoxic medications, and chronic diseases that lead to increased patient survival but at the expense of nephron health) we have a true epidemic. This report goes a long way to beginning to warn paediatricians world-wide that rates of paediatric CKD cannot be left to those referral centers alone, but should be sought for in general visits to the paediatricians’ office with a careful history and physical examination followed by a consideration for testing that might include a kidney ultrasound to look for hypo-dysplasia, and performance of a test for low-grade albuminuria (termed microalbuminuria) as early signs. Identification of such early disease (CKD1-2) may lead to useful strategies to preserve functional nephron mass as long as possible. The current report provides excellent evidence in this regard.",
"responses": []
},
{
"id": "1817",
"date": "03 Oct 2013",
"name": "Uri Alon",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA note about the possible reason(s) for the high incidences like consanguinity and poverty should be added to the abstract's conclusion for completion, otherwise I approve this study.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-186
|
https://f1000research.com/articles/2-185/v1
|
13 Sep 13
|
{
"type": "Research Article",
"title": "Polarized localization and borate-dependent degradation of the Arabidopsis borate transporter BOR1 in tobacco BY-2 cells",
"authors": [
"Noboru Yamauchi",
"Tadashi Gosho",
"Satoru Asatuma",
"Kiminori Toyooka",
"Toru Fujiwara",
"Ken Matsuoka",
"Noboru Yamauchi",
"Tadashi Gosho",
"Satoru Asatuma",
"Kiminori Toyooka",
"Toru Fujiwara"
],
"abstract": "In Arabidopsis the borate transporter BOR1, which is located in the plasma membrane, is degraded in the presence of excess boron by an endocytosis-mediated mechanism. A similar mechanism was suggested in rice as excess boron decreased rice borate transporter levels, although in this case whether the decrease was dependent on an increase in degradation or a decrease in protein synthesis was not elucidated. To address whether the borate-dependent degradation mechanism is conserved among plant cells, we analyzed the fate of GFP-tagged BOR1 (BOR1-GFP) in transformed tobacco BY-2 cells. Cells expressing BOR1-GFP displayed GFP fluorescence at the plasma membrane, especially at the membrane between two attached cells. The plasma membrane signal was abolished when cells were incubated in medium with a high concentration of borate (3 to 5 mM). This decrease in BOR1-GFP signal was mediated by a specific degradation of the protein after internalization by endocytosis from the plasma membrane. Pharmacological analysis indicated that the decrease in BOR1-GFP largely depends on the increase in degradation rate and that the degradation was mediated by a tyrosine-motif and the actin cytoskeleton. Tyr mutants of BOR1-GFP, which has been shown to inhibit borate-dependent degradation in Arabidopsis root cells, did not show borate-dependent endocytosis in tobacco BY-2 cells. These findings indicate that the borate-dependent degradation machinery of the borate transporter is conserved among plant species.",
"keywords": [
"Boron is one of the essential nutrients for plants",
"and boron deficiency is a major cause of reduced crop production1. Large quantities of boron are toxic to plants",
"and boron toxicity is a worldwide problem in food production2. Two different classes of borate transporters were discovered in Arabidopsis thaliana3. One of them",
"a transporter named BOR1",
"is a plasma membrane borate exporter in Arabidopsis root cells",
"and is essential for efficient xylem loading of boron4. BOR1 and its paralogs are also involved in boron toxicity tolerance in plants5",
"6."
],
"content": "Introduction\n\nBoron is one of the essential nutrients for plants, and boron deficiency is a major cause of reduced crop production1. Large quantities of boron are toxic to plants, and boron toxicity is a worldwide problem in food production2. Two different classes of borate transporters were discovered in Arabidopsis thaliana3. One of them, a transporter named BOR1, is a plasma membrane borate exporter in Arabidopsis root cells, and is essential for efficient xylem loading of boron4. BOR1 and its paralogs are also involved in boron toxicity tolerance in plants5,6.\n\nIt was reported that the level of BOR1 is tightly regulated by the concentration of borate in the growth environment7. At low concentrations of borate, BOR1 is stably localized to the proximal side of plasma membrane in root cells but is degraded upon application of high concentrations of borate7–9. This degradation occurred after endocytosis of the transporter from the plasma membrane and the endocytosed transporter was transported from early endosome to multivesicular body, ubiquitinated and finally targeted to vacuoles for degradation8–11. A similar boron-dependent decrease in borate transporter levels was also observed in rice12 although in this case the mechanism of the reduction was not elucidated. Thus it was not clarified whether the borate-induced endocytotic degradation of BOR1 that is found in Arabidopsis root cells is conserved among different plant species and different types of plant cells.\n\nThe tobacco BY-2 cell line is widely used as a model for the analysis of the cell cycle and protein trafficking in plant cells. This cell line is advantageous for conducting pharmacological studies because of the small size of its cell clumps as well as its ability to grow in liquid suspension13. To obtain an insight into the regulation of borate transporter levels and borate sensing machinery, we investigated the localization and degradation of BOR1-green fluorescent protein (GFP) fusion in tobacco BY-2 cells in the presence of high concentrations of borate and analyzed the effect of inhibitors for protein synthesis, protein degradation and intracellular trafficking on its degradation.\n\n\nResults\n\nWe expressed the Arabidopsis BOR1-GFP fusion construct7 in tobacco BY-2 cells under the control of the cauliflower mosaic virus 35S RNA promoter. Examination of transformed cells at growing phase using an epifluorescence microscope indicated that the fluorescence localized at the most peripheral part of the cells as well as intracellular dots (Figure 1A). In contrast, cells grown to the stationary-phase showed faint punctate distribution of the fluorescence in the cell (Figure 1B). To examine whether the peripherally-localized BOR1-GFP in rapidly-growing cells actually localized to the plasma membrane we stained the cells with FM4-64 for 15 min on ice and compared the pattern of FM4-64 and the GFP fluorescence using a spinning disk confocal microscope (Figure 1C, 0 min). The peripheral staining pattern of FM4-64 is almost completely identical to that of the fluorescence of GFP. When the FM4-64 incubated cells were further incubated at room temperature for 30 min, we observed internalization of the FM4-64 signal. Under this condition we did not observe the internalization of BOR1-GFP signal (Figure 1C, 30 min). These observations suggest that BOR1-GFP is localized to the plasma membrane in transformed tobacco BY-2 cells.\n\nA. Fluorescence pattern of rapidly growing tobacco cells expressing BOR1-GFP was detected using an epifluorescence microscope. Plasma membranes as well as some intracellular structures showed GFP fluorescence. B. GFP fluorescence of tobacco cells expressing BOR1-GFP at stationary phase of growth. Images were collected as in A. C. Colocalization of plasma membrane and endomembrane marker FM4-64 and BOR1-GFP. Cells expressing BOPR1-GFP were suspended in medium containing FM4-64 and incubated for 15 min on ice. Fluorescence images were collected immediately after the incubation or 30 min after incubation at room temperature using a spinning disk confocal microscope. D. Membrane-association of BOR1-GFP. Cell lysate was subjected to two rounds of centrifugation and soluble and sedimentable fractions containing membranes was obtained. T, total cell lysate. P10, pellet of 10,000 x g 10 min centrifugation. P100, pellet of 100,000 x g 60 min centrifugation. S, soluble fraction after 100,000 x g 60 min centrifugation.\n\nWe also analyzed the distribution of the fluorescence protein after fractionation of the cell lysate into membranous organelle and soluble fractions. As shown in Figure 1D, BOR1-GFP was enriched in the precipitated fractions. This confirmed that BOR1-GFP was targeted to the membranes in tobacco BY-2 cells. Taken together, we concluded that a significant portion of BOR1-GFP expressed in tobacco BY-2 cells is targeted to the plasma membrane as in the case of Arabidopsis root cells.\n\nIn order to address whether BOR1-GFP is degraded upon supply of high concentrations of borate in tobacco cells as observed in Arabidopsis, we first analyzed the effect of borate on the growth of tobacco cells that expressed the BOR1-GFP. Tobacco cells were grown for a week in medium containing varied concentrations of borate (0.1 to 20 mM) and the volume of cells was measured. No difference in the response to cell growth was observed between non-transformed and BOR1-GFP expressing cells. Medium containing up to 5 mM borate caused little defect in the growth of cells (Figure 2).\n\nSeven-day old tobacco BY-2 cells were subcultured into medium containing the indicated concentration of borate and shaken for a week. The volume of cells in the culture was measured. Gray bar; standard borate concentration medium. Error bars, SD.\n\nWe then treated the transformed cells with 5 mM borate for up to 90 min and the fluorescence of BOR1-GFP was monitored under an epifluorescence microscope (Figure 3A). We found that the BOR1-GFP signal at the peripheral part of the cells decreased significantly during incubation. The same decrease in signal was also observed in the presence of 3 mM borate. Interestingly, lower concentrations of borate (0.1 mM) did not cause a loss of the BOR1-GFP signal. This decrease is specific for BOR1-GFP as the same treatment did not decrease the fluorescence of plasma membrane intrinsic protein (PIP)-GFP, which is a plasma membrane aquaporin fused with GFP.\n\nA. Fluorescence images of transformed cells expressing BOR1-GFP in medium containing a high concentration of borate (5 mM; +Borate) and a normal concentration of borate (0.1 mM, -Borate). Images were collected with identical exposure conditions using a confocal microscope. B. Time course of the decrease in BOR1-GFP signal in the presence of borate. Upper gel; cells expressing BOPR1-GFP were incubated in medium with normal or high borate concentrations for the indicated times. Lower gel; cells expressing PIP-GFP were incubated in high borate medium for the indicated times. Fluorescence of GFP fusion proteins after separation by SDS-PAGE was detected by scanning the gel with an image analyzer. A representative gel image is shown. C. Quantification of the degradation of BOR1-GFP. Means of three independent experiments are shown. The gray line is a best-fit curve of the exponential decrease of BOR1-GFP.\n\nWe also analyzed the fate of BOR1-GFP after separation of cellular proteins by SDS/PAGE and recorded the fluorescence (Figure 3B). We found that BOR1-GFP signal was decreased in the presence of higher concentrations of borate. This decrease was specific for BOR1-GFP as this protein was not decreased without exposure to borate and the level of PIP-GFP was not changed even with high concentrations of borate. Quantification of the intensities of the BOR1-GFP band allowed us to estimate the kinetic parameters of the decline in the signal intensity. The decrease ratio fitted well into an index recurrence curve with a lag time of 11.7 min and half life of 62 min (Figure 3C), suggesting that the degradation kinetics is first order. The presence of a lag time suggested that several steps of cellular events must have taken place before degradation of the BOR1-GFP.\n\nAs described above, BOR1-GFP but not PIP-GFP was degraded upon addition of borate. In Arabidopsis roots BOR1-GFP behavior differs from some other plasma membrane proteins in both borate-dependent degradation and polarized localization in cells7,8. To address whether localization of BOR1-GFP and PIP-GFP differs in tobacco BY-2 cells, we compared the localization of these proteins in tobacco BY-2 cells at the early log-phase stage (Figure 4).\n\nA. Confocal images of PIP-GFP and BOR1-GFP at the four-cell stage. About half to two-thirds of cell clumps are shown. GFP fluourescence of PIP-FGP at the plasma membrane that faces to the medium showed much higher intensities than that of BOR1-GFP. B. Schematic representation of each cell membrane face in the cell clump. Only half of the 4-cell stage cell clump was indicated. A1, A2, E1 and E2 indicate the face of cells defined for the analysis of polarized localization. C. Comparison of the relative density of fluorescence at each face of PIP-GFP and BOR1-GFP expressing cells. Means (with SD) density relative to the E1 face (n=8) are shown.\n\nWith our culture conditions, BY-2 cells at the stationary phase are largely unicellular. Cells at this stage are approximately three-times longer than cells at mid-log phase. When such cells are transferred to fresh medium, they start to divide at the center resulting in two daughter cells which elongate slightly and further divide at the center of each daughter cell without separation. At this stage, a linearly attached clump of cells contain four cells. The clump has four cell walls with a distinct location and age; the one between the two cells that generated at the first cell division, the one between the two cells that generated at the second cell division, the one exposed to the culture medium in the center of two cells, and the one exposed to medium of the two extreme cells containing tips of the cell wall clump. We name these four faces of cells as A1, A2, E1 and E2, respectively (Figure 4B).\n\nWe measured the density of the green fluorescence of these four faces from cells expressing BOR1-GFP and PIP-GFP and the distribution of their relative density (relative to the E1 face). In both cases, the fluorescence density at E1 and E2 was almost the same but in the case of PIP-GFP, the densities at A1 and A2 were approximately 1.5- and 2-fold higher than that of E1, respectively (Figure 4C). These fluorescence intensities between cells suggest that PIP-GFP is distributed nearly evenly in the plasma membrane as the A1 and A2 faces have two sheets of plasma membrane. In contrast, the density of BOR1-GFP at both A1 and A2 faces were approximately 5-fold higher than that of E1 (Figure 4C). This observation indicated the polarized localization of BOR1-GFP and clearly showed that this fusion protein accumulates to the plasma membrane that is facing neighboring cells in growing tobacco BY-2 cells.\n\nThe observation that BOR1-GFP is degraded upon addition of borate in the medium (Figure 3) suggest two possible scenarios for the borate-dependent decrease in BOR1-GFP. The first possible case is that the decrease of BOR1-GFP in the presence of high concentrations of borate is the result of inducible degradation. Another possibility is that the rate of the degradation of BOR1-GFP is rapid in BY-2 cells even in the absence of borate but a high rate of biosynthesis of BOR1-GFP in the normal medium maintained the level of the transporter.\n\nTo address the likely scenario causing the BOR1-GFP decrease in tobacco cells under high borate conditions, we treated the transformed cells with a protein biosynthesis inhibitor cycloheximide (CHX) in either normal or high-boron medium and monitored the level of BOR1-GFP (Figure 5). In the normal borate medium, addition of CHX did not change the level of BOR1-GFP significantly over 2 hours. This observation indicated that the turnover of BOR1-GFP in normal medium is slow and most of the BOR1-GFP showing the green fluorescence is the result of the accumulation of this protein. This also indicated that the decrease in BOR1-GFP in high borate medium is a result of inducible degradation.\n\nWe observed partial inhibition of BOR1-GFP degradation in the presence of CHX (Figure 5). This observation indicated that continuous synthesis of degradation machinery is necessary for the continuation of the degradation. The partial suppression of the degradation also indicated that the machinery for the degradation is already present in cells that were cultured in the normal medium.\n\nCells were incubated with normal (0.1 mM) or high (5 mM) concentrations of borate with or without cycloheximide for 2 hours. A. Image of BOR1-GFP fluorescence after separation by SDS-PAGE. CHX: cycloheximide. B. Quantified result of A. Means of three independent triplicated experiments plus standard deviations are shown a, b and c above the error bars indicate that values in each letter showed significant difference whereas marked with the same letter showed no significant difference (p<0.01) using Tukey’s test.\n\nIt was shown previously that endocytosis is the first step of BOR1-GFP degradation in Arabidopsis7,8. To assess whether similar machinery operates in tobacco BY-2 cells in the presence of high concentrations of borate, we first analyzed the BOR1-GFP signal in cells cultured for 1 to 2 hours under high borate conditions using a confocal laser scanning microscope (CLSM). We found intracellular puncta in cells treated with high borate (Figure 6A). We then analyzed the colocalization of the BOR1-GFP signal with an early endosome that can be marked with FM4-64. Tobacco cells expressing BOR1-GFP were incubated for 2 hours in a medium containing 3 mM borate and then the cells were stained with FM4-64 on ice, washed with cold medium containing 3 mM borate, after which they were returned to room temperature to resume intracellular trafficking as described14. Five to 10 min after FM4-64 incubation, dotted structures emitting red fluorescence, which represent an early endosome distinct from the trans-Golgi network (TGN) in tobacco cells14, was observed (Figure 6B, left). Most of the early endosome signal colocalized with intracellular dots of BOR1-GFP, suggesting that BOR1-GFP passes through early endosome during degradation as in the case of Arabidopsis cells.\n\nA. Two hours after incubating the cells in medium containing 3 mM borate, cells were incubated with FM4-64 for 30 min and analyzed with a confocal fluorescent microscope. B. Many, but not all intracellular BOR1-GFP dots colocalized with puncta of FM4-64. C. BOR1-GFP did not pass through the trans-Golgi network (TGN) during degradation. BOR1-GFP was transiently expressed in cells expressing SCAMP2-mRFP, which is a marker for TGN and the secretory vesicle cluster14. Co-expressed cells were incubated in medium containing 3 mM borate for 2 hours and the fluorescence was analyzed by confocal laser scanning microscopy. No clear overlap of the intracellular puncta of SCAMP2-mRFP and BOR1-GFP was seen.\n\nIt is also known that FM4-64 is transported from the early endosome to the TGN, and thereafter targeted to the vacuolar membrane. To evaluate whether BOR1-GFP passes through the TGN, we expressed SCAMP2-mRFP14 in cells expressing BOR1-GFP. SCAMP2-mRFP localizes in the plasma membrane, the TGN and the secretory vesicle cluster, which is an exocytotic compartment derived from the TGN14. No colocalization of green and red intracellular dots was observed when cells were treated with 3 mM borate for 2 hours (Figure 6C). These findings suggested that the BOR1-GFP is internalized to endosomes and then sorted to a degradation pathway from the endocytotic route, which is directed to the TGN.\n\nIt was previously reported that the treatment of Arabidopsis roots with brefeldin A (BFA), which is an inhibitor for both endocytosis and exocytosis, prevents the borate-dependent degradation of BOR1-GFP7. To address whether this compound is also effective in tobacco BY-2 cells and to assess the mechanism of the degradation, we analyzed borate-induced degradation in the presence of various inhibitors (Figure 6). We used BFA, 2,3,5-triiodobenzoic acid (TIBA), a compound that stabilizes the actin bundle15; tyrphostin A23, which is a tyrosine kinase inhibitor, which can inhibit endocytosis by competing with the interaction of the AP-2 adaptor complex and Tyr-containing endocytosis motifs in mammalian cells and also can inhibit the endocytosis of rice SCAMP1-YFP and FM4-64 in tobacco BY-2 cells16,17; ikarugamycin, which is an inhibitor of clathrin-mediated endocytosis in tobacco18; colchicine, which is an inhibitor of microtubule depolymerization; latrunculin B, which inhibits actin polymerization; 2,3-butanedione monoxime (BDM), which is a general myosin ATPase inhibitor and inhibits the endocytosis of SCAMP2-mRFP in tobacco BY-2 cells14; MG-132, which is an inhibitor of proteasomes at low concentrations and also inhibits other proteases; and K-252a, which is a general Ser/Thr type protein kinase inhibitor.\n\nThree hours after application of inhibitor solution to the tobacco cell culture, borate was added at a final concentration of 5 mM and further incubated for 2 hours. Thereafter, the amount of BOR1-GFP was quantified (Figure 7A). We found that BFA and TIBA inhibited degradation almost completely (>80% remained) at a concentration that inhibits the endocytosis of SCAMP2-YFP14. A slightly weaker effect of tyrphostin A23 was observed at 400 μM, which is a concentration similar to that used in the works of Leborgne-Castel et al.16 and Lam et al.17. In this case, approximately 70% of BOR1-GFP remained in the cell. We also found partial inhibition of degradation by BDM at 5 mM, the concentration that inhibits the endocytosis of SCAMP2-YFP14. We also observed partial inhibition by MG-132 at 400 μM. In these two cases, approximately 40% of BOR1-GFP remained in the cell. Other inhibitors tested did not cause any detectable effect on the extent of degradation.\n\nA. Cells incubated with or without inhibitors for 30 min were further incubated in medium containing 5 mM of borate for two hours in the presence of the same inhibitors. The relative intensity of BOR1-GFP fluorescence was quantified as described in the Figure 3 legend. The final concentration of inhibitors in the culture and the number of replicates were; brefeldin A (BFA), 25 μM, n=8; 2,3,5-triiodobenzoic acid (TIBA), 50 μM, n=5; tyrphostin n=5; Tyrphostin A23, 100 μM, n=5; MG-132, 400 μM, n=5; 2,3-butanedione monoxime (BDM), 40 mM, n=3; K-252a, 4 μM, n=3; Ikarugamycin, 20 μM, n=3; Latrunculin A, 100 μM, n=3; colchicin, 200 μM, n=3. Error bars, SD. ** and * represent the significant difference relative to no inhibitor (p<0.01 and <0.05 respectively). B. Time-course analysis of the degradation of BOR1-GFP in the presence of either TIBA, tyrphostin A23 or in the absence of an inhibitor. C. Changes in the fluorescence pattern of BOR1-GFP in the absence and presence of BFA or TIBA in the presence of 5 mM borate. D. Timing of the addition of inhibitors did not affect the subsequent degradation. Cells were incubated in medium containing 5 mM of borate. At the same time (0 min) or the other indicated times, either BFA, tyrphostin A23 or TIBA was included in the medium and further incubated for up to 120 min. The intensity of the BOR1-GFP band relative to that at the time when the inhibitor was included is shown. Error bars, SD.\n\nTime-course analysis of the decrease of BOR1-GFP in the presence of TIBA and tyrphostin A23 (Figure 7B) clearly showed that most of the degradation was inhibited up to 3 hours. We next addressed whether the inhibition occurred at the point of endocytosis from the plasma membrane or at intermediate compartments. Fluorescence images were collected in transformed tobacco cells in the presence of both inhibitors and borate. We found that the images were not significantly different up to 3 hours in the presence of either BFA or TIBA (Figure 7C). These observations suggested that both BFA and TIBA inhibited the early event of endocytosis. We could not analyze fluorescence images in the presence of tyrphostin A23 because treatment of BY-2 cells with this compound increased cellular fluorescence significantly and such fluorescence was independent of the expression of BOR1-GFP.\n\nTo address whether the inhibition of endocytosis required BY-2 cells to be pretreated with BFA, tyrphostin A23 or TIBA, we investigated the timing of adding the inhibitors (Figure 7D). We compared the level of BOR1-GFP without inhibitors with BOR1-GFP levels when these inhibitors were added into the culture. We found that addition of inhibitors at the same time, in the lag time as well as during the degradation phase, prevented the decrease in BOR1-GFP almost completely. These observations indicated that these inhibitors have a direct effect against the endocytosis of BOR1-GFP and were not a side effect of these chemicals. These observations also suggested that actin dynamics, GDP-GTP exchange of Arf small GTPase, and either tyrosine phosphorylation or AP-2 adaptor interaction are necessary for the degradation of BOP1-GFP in tobacco BY-2 cells.\n\nIt was shown that both polarized localization and borate-dependent degradation are mediated by specific Tyr residues in BOR1-GFP in Arabidopsis root cells8. To address whether these Tyr residues also contribute to the localization and borate-induced degradation of BOR1-GFP in tobacco BY-2 cells, we expressed Y373A/Y398A/Y405A mutants of BOR1-GFP in BY-2 cells (Figure 8). Confocal images of the mutant BOR1-GFP displayed higher intensity of fluorescence signals between attached cells as in the case of wild-type BOR1-GFP (Figure 8A). Quantification of the intensities of different faces indicated that this protein also showed a polar localization, although the polarized nature seems somewhat weaker than the wild-type (Figure 8B). This observation indicated that the polarized localization of BOR1-GFP in tobacco cells is largely independent of the tyrosine residues in the BOR1 protein.\n\nA. Confocal images of 4-cell stage tobacco cells expressing wild-type and mutant BOR1-GFP. B. Comparison of the relative density of fluorescence at each face of the cells expressing either PIP-GFP, wild-type BOR1-GFP or mutant BOR1-GFP. Mean densities relative to the E1 face (n=8) are shown. Error bars, SD. ** represents significance at p<0.01. C. Mutant BOR1-GFP is stable in the presence of high-concentrations of borate. Epifluorescence images of cells incubated with normal (-B, 0.1 mM) or high borate (+B, 5 mM) for 2 h are shown.\n\nWhen tobacco cells expressing the mutant protein were incubated in medium containing 5 mM borate, we still observed strong fluorescence of GFP signals. We did not detect any difference in the pattern and intensity of the fluorescence signals between cells incubated in normal medium and high borate medium (Figure 8C). Under the same condition, most of the wild-type BOR1-GFP was degraded in the cell (Figure 3, Figure 8C). This observation indicated that Y373, Y398, and/or Y405 residues of BOR1-GFP are involved in borate-induced degradation in tobacco BY-2 cells as in the case in Arabidopsis root cells.\n\n\nDiscussion\n\nWe found that the Arabidopsis borate transporter BOR1-GFP fusion protein was targeted to the plasma membrane of rapidly growing tobacco BY-2 cells. The plasma membrane localization of BOR1-GFP was different from that of PIP-GFP. BOR1-GFP was more abundant in the plasma membranes that face neighboring cells (Figure 4A, B). This observation indicates that BOR1-GFP showed a polarized localization in tobacco BY-2 cells. This observation further indicates that plasma membranes of BY-2 cells facing the medium and the neighboring cells are distinct in nature. It was shown in Arabidopsis root cells that BOR1-GFP is localized in the proximal region of the plasma membrane. However, polarized localization in BY-2 cells and Arabidopsis root cells seems to be mediated by distinct mechanisms because the latter, but not the former, is sensitive to the mutation of Tyr residues in the cytoplasmic loop of BOR1 protein (Figure 8A, B)8.\n\nThe concentrations of borate that caused the degradation of BOR1-GFP in BY-2 cells was >3 mM, which is higher than that in Arabidopsis plants (100 μM)7. This difference might correlate with the difference of high-boron sensitivity of the growth between the two species. BY-2 cells can grow normally up to 5 mM borate (Figure 2D) whereas 300 μM borate caused about 50% reduction of the growth of Arabidopsis19. Thus it will be interesting to investigate in the future what attributes of plants including tobacco cells and Arabidopsis plants determine sensitivity to high borate levels.\n\nThe borate-induced degradation of BOR1-GFP was inhibited by the protein synthesis inhibitor CHX (Figure 5). We observed previously that CHX treatment did not inhibit endocytosis of SCAMP2-YFP in tobacco BY-2 cells14. Therefore, the effect of CHX on the inhibition of BOR1-GFP is not a general response in tobacco cells. The inhibition by CHX on BOR1-GFP degradation was not complete and approximately 50% of the protein was degraded under CHX treatment (Figure 5). This observation suggested that BY-2 cells used pre-existing machinery for the degradation of BOR1-GFP. However, the degradation the machinery was limited in the cell and de novo synthesis of the machinery is required for the continuation of the degradation.\n\nThe plasma membrane accumulation of BOR1-GFP was abolished when cells were incubated in medium containing high concentrations of borate. The decline in the BOR1-GFP was mediated by endocytosis and further degradation (Figure 3 and Figure 6) and was dependent on the tyrosine residues (Figure 8C) as observed in Arabidopsis root cells. These observations suggest that boron sensing mechanisms as well as the borate-dependent induction of the endocytosis of BOR1-GFP is conserved in different plant species. However, there are some differences in the endocytotic trafficking between tobacco cells and Arabidopsis root cells. The most pronounced difference is the organization of organelles in these cells. In tobacco BY-2 cells, early endosomes and the TGN are separated14 and BOR1-GFP passed through the early endosome but not the TGN during degradation (Figure 6B, C). In contrast, early endosomes cannot be distinguished from the TGN in Arabidopsis root cells9. Therefore, differentiation of secretory organelles is not completely identical in these two species. Further analysis of the degradation of BOR1-GFP in either cultured Arabidopsis cells or tobacco root cells will give a clearer picture as to whether the difference in trafficking paths originate from differences between species or the differentiation state of the cells.\n\nThe endocytosis-dependent degradation of BOR1-GFP was inhibited almost completely upon addition of the inhibitors BFA, TIBA and tyrphostin A23. BFA is known as an inhibitor of the GDP-GTP exchange factor for ADP-ribosylation factor and has many effects on different plant cells19. On tobacco BY-2 cells, we have shown that BFA inhibits both endocytosis of SCAMP2-YFP, Golgi protein localization and export from the endoplasmic reticulum14,21,22. It was also shown that BFA inhibits the endocytosis of BOR1-GFP in Arabidopsis root cells7. The fact that BFA inhibited the endocytosis of BOR1-GFP in tobacco BY-2 cells in the presence of borate (Figure 7) indicates that some machinery for the borate-dependent endocytotic degradation is conserved between Arabidopsis root cells and tobacco BY-2 cells.\n\nTIBA is known to inhibit the endocytosis of the auxin transporter and its function is to stabilize the actin cytoskeleton at 25 μM in tobacco BY-2 cells15. The fact that TIBA at a similar concentration range inhibited the degradation of BOR1-GFP almost completely (Figure 7) suggested that the actin cytoskeleton is involved in the endocytotic degradation of BOR1-GFP. The partial inhibition of degradation by BDM (Figure 7), which is an inhibitor of myosin motors7 also supports the idea that an actin-dependent mechanism is a key part of the machinery for the endocytotic degradation of BOR1-GFP.\n\nAnother inhibitor that prevented borate-dependent degradation was tyrphostin A23. This chemical is known to inhibit Tyr-signal mediated endocytosis by inhibiting the recognition of the AP-2 adaptor complex signal in mammalian cells. The requirement of Tyr residues in BOR1 for endocytosis (Figure 8C) agrees well with the action of tyrphostin A23 in mammalian cells. This compound is also known to inhibit endocytosis in several plant species16,17,23,24. It was reported recently that this compound inhibited elicitor-induced endocytosis in tobacco BY-2 cells and this event seems related to the induction of clathrin-mediated endocytosis16. However, ikarugamycin, which is known to inhibit clathrin-mediated endocytosis in both animal cells25 and tobacco protoplasts18, did not inhibit the degradation of BOR1-GFP. It was reported recently that AP-2 is involved not only in clathrin-mediated endocytosis but also in clathrin-independent endocytosis in mammalian cells26. Therefore further investigation is necessary to elucidate whether clathrin is involved in the endocytosis of BOR1-GFP.\n\nIn addition to these inhibitors, we observed that MG-132 also inhibited the degradation of BOR1-GFP. It was reported that this compound also inhibits the degradation of interleukin-2 through the inhibition of endocytosis of its receptor in mammalian cells27. As MG-132 is a potent inhibitor of proteasomes, our finding raises the possibility that a proteasome system is somehow involved in BOR1-GFP degradation. However, our data does not rule out the possibility that the target of MG-132 on BOR1-GFP degradation is different from proteasomes as we needed a very high concentration (400 μM) of this compound to prevent BOR1 degradation and MG-132 can inhibit other proteases, such as calpain in animals, at a high concentration28. Thus, future experiments will be necessary to ascertain whether proteasomes are involved in the degradation of BOR1-GFP and to show which molecule is the real target of MG-132 on BOR1-GFP degradation in tobacco cells.\n\n\nMaterials and methods\n\nPlasmids for the expression of wild-type and mutant BOR1-GFP, and SCAMP2-mRFP have been described previously7,8,14. The expression plasmid for PIP-GFP was a kind gift from Dr. M. Maeshima (Nagoya University). This plasmid contains radish plasma membrane type aquaporin PAQ129 and GFP fusion protein under an enhancer-duplicated cauliflower mosaic virus 35S promoter30.\n\nThe following chemicals were used to test the inhibition of the degradation: BFA (Wako Pure Chemicals, Osaka, Japan), CHX (Wako Pure Chemicals, Osaka, Japan), TIBA (Aldrich Chem. Co. USA), Tyrphostin A23 (Sigma-Aldrich, Inc, Missouri, USA), colchicine (Tokyo Kasei Co., Tokyo, Japan), K-252a (Sigma-Aldrich, Inc, Missouri, USA), BDM (Sigma-Aldrich, Inc., Missouri, USA), Latruncurin A (Calbiochem, San Diego, USA), ikarugamycin (BIOMOL Research Labs Inc. USA), MG-132 (Calbiochem Inc. San Diego USA), wortmannin (Calbiochem Inc. San Diego USA), 3MA (Sigma-Aldrich, Inc., Missouri, USA) and E-64 (Peptide Institute, Osaka, JAPAN).\n\nThe culture of tobacco BY-2 cells (a kind gift from Drs. K. Nakamura (Nagoya University) and T. Nagata (University of Tokyo) and the transformation of this cell line were as described30. Cells were cultured weekly and 3 or 4 day-old cultures were used unless otherwise stated. For transient transformation of cells expressing BOR1-GFP to express SCAMP2-mRFP in the same cell, Agrobacterium tumefaciens EHA10531 harboring a binary plasmid was co-cultured with BOR1-GFP expressing tobacco cells for 2 days, washed with fresh medium and further incubated in medium containing 200 mg/L cefotaxime for a day. Thereafter fluorescence of cells was visualized using CLSM.\n\nCells expressing GFP fusion proteins were lysed with equal volumes of buffer consisting of 0.4 M Sorbitol, 0.1 M KOAc, 6 mM EGTA, 4 mM EDTA, 40 mM HEPES-KOH, 2 mM DTT, 1 drop/100 ml protease inhibitor tablet (Complete EDTA-free, Roche), pH 7.3 at room temperature. Cells were lysed by sonication and the resulted cell lysate was centrifuged at 300 × g for 10 min. The resulting supernatant was used as the cell lysate. Proteins in the cell lysate was mixed with 1/5 volume of a buffer consisting 0.25 M Tris-HCl, 0.5 M dithiothreitol, 50 % glycerol, 10 % SDS, 0.025 % bromophenol blue, pH 6.8 and loaded on SDS-polyacrylamide gels without heating. Fluorescence images of the gels were recorded using Typhoon 9600 scanner (GE Healthcare, Tokyo, Japan) at the following setting; filter 520SPCy2, excitation 488 nm blue laser at PMT 650, medium sensitivity). The intensities of bands were quantified using Image Quant software (GE Healthcare).\n\nFor epifluorescence detection, cells were mounted on glass slides and observed using IX40 inverted microscope with an LCPlanFL 20X lens using NBY excitation/emission filter set and a DP-70 camera (Olympus Co. Tokyo Japan). For time-lapse image collection, cell suspension was placed into in a well of glass-bottom 96 well micro-plates (EZVIEW® Glass Bottom Culture Plates LB, Asahi Techno Glass Co. Tokyo, Japan). Cells in the plate wells were observed using an IX80 inverted fluorescence microscope equipped with DSU spinning disk confocal unit and an Uplan SApo 40×/0.90 lens using NBY or RFP excitation/emission filter sets (Olympus). Images were collected using an iXON+-DU888E-CS0-BV camera (Andor) under the control of Meta Imaging Series 7.6.3 software (Molecular Devices Inc. Sunnyvale, CA, USA).\n\nFor the colocalization analysis, cells were mounted on slides with culture medium. These cells were observed using a CLSM system (LSM510 META, Axioplan2 Imaging; Carl Zeiss) with a Plan-Apochromat lens (633 1.4 oil differential interference contrast; optical slices of 1 mm). We used a 25-mW argon laser (power, 5%) with 488-nm excitation and a 505–530-nm band-pass filter for GFP8. For FM4-64 (Molecular Probes, Eugen, USA) we used a 650-nm long-pass filter and 488-nm excitation. A 560-nm long-pass filter and 1-mW He-Ne laser at 80% power with 543-nm excitation was used for Alexa Fluor 568 and mRFP. Crosstalk was prevented using a multitrack configuration with line sequential scanning. Composite figures were prepared using Zeiss LSM Image Browser software.\n\nFor the measurement of intensity, confocal images at 256 bit tiff file was quantified using ImageJ software. If required, relative density to the E1 face was calculated in each cell clump.\n\nAn aliquot of 0.5 M boric acid in water was added to 96.5 ml of suspension culture of three-day old tobacco cells expressing BOR1-GFP in a 300 ml Erlenmeyer flask at a concentration of 3 or 5 mM (as indicated in the legends to figures) and further cultured with the same condition of regular culture. For the control, cells grown in a normal culture medium, which contains 0.1 mM borate, was used. At the indicated time, a 0.5 or 1 ml aliquot of the culture was removed from the flask and used for the analysis.\n\nFor the comparison of the two values student’ t-test was employed using Microsoft Excel software. For the comparison of difference with multi values, Tukey’s test was used.",
"appendix": "Author contributions\n\n\n\nTF and KM conceived the study and designed the experiments. NY, TG, SA, KT and KM carried out the research. TG, NY and KM prepared the first draft of the manuscript and TF contributed the preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content. NY and TG contributed equally to the work.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was partially supported by the Ministry of Education, Science, Sports and Culture, Japan for Scientific Research on Priority Areas Nos. 17078009 and 17078004 to KM and TF, respectively, and for Scientific Research (B) No. 21380208 to KM.\n\n\nAcknowledgements\n\nWe thank Drs. K. Nakamura (Nagoya University) and T. Nagata (Univ. of Tokyo) for tobacco BY-2 cell line, Dr. M. Maeshima (Nagoya University) for the expression plasmid for PIP-GFP, Dr. Junpei Takano (Hokkaido University) for discussion and Dr. Moses O. Abiodun (Kyushu University) for improving the manuscript.\n\n\nReferences\n\nMarschner H: Mineral nutrition of higher plants, 2nd Ed. Academic Press, San Diego. 1995. Reference Source\n\nNable R, Banuelos G, Paull J: Boron toxicity. Plant Soil. 1997; 193: 181–191. Reference Source\n\nTakano J, Miwa K, Fujiwara T: Boron transport mechanisms: Collaboration of channels and transporters. Trends Plant Sci. 2008; 13(8): 451–457. PubMed Abstract | Publisher Full Text\n\nTakano J, Noguchi K, Yasumori M, et al.: Arabidopsis boron transporter for xylem loading. Nature. 2002; 420(6913): 337–340. PubMed Abstract | Publisher Full Text\n\nMiwa K, Takano J, Omori H, et al.: Plants tolerant of high boron levels. Science. 2007; 318(5855): 1417. PubMed Abstract | Publisher Full Text\n\nSutton T, Baumann U, Hayes J, et al.: Boron-toxicity tolerance in barley arising from efflux transporter amplification. Science. 2007; 318(5855): 1446–1449. PubMed Abstract | Publisher Full Text\n\nTakano J, Miwa K, Yuan L, et al.: Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability. Proc Natl Acad Sci U S A. 2005; 102(34): 12276–12281. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTakano J, Tanaka M, Toyoda A, et al.: Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways. Proc Natl Acad Sci U S A. 2010; 107(11): 5220–5225. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoshinari A, Kasai K, Fujiwara T, et al.: Polar localization and endocytic degradation of a boron transporter, BOR1, is dependent on specific tyrosine residues. Plant Signal Behav. 2012; 7(1): 46–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nViotti C, Bubeck J, Stierhof YD, et al.: Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell. 2010; 22(4): 1344–1357. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKasai K, Takano J, Miwa K, et al.: High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana. J Biol Chem. 2011; 286(8): 6175–6183. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakagawa Y, Hanaoka H, Kobayashi M, et al.: Cell-type specificity of the expression of Os BOR1, a rice efflux boron transporter gene, is regulated in response to boron availability for efficient boron uptake and xylem loading. Plant Cell. 2007; 19(8): 2624–2635. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatsuoka K: Protein sorting and protein modification along the secretory pathway in BY-2 cells. In. T Nagata., S Hasezawa., D Inze., eds Tobacco BY-2 cells: Biotechnology in Agriculture and Forestry, Springer-Verlag, Heidelberg, 2004; 53. : 283–298. Publisher Full Text\n\nToyooka K, Goto Y, Asatsuma S, et al.: A mobile secretory vesicle cluster involved in mass transport from the Golgi to plant cell exterior. Plant Cell. 2009; 21(4): 1212–1229. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDhonukshe P, Grigoriev I, Fischer R, et al.: Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes. Proc Natl Acad Sci U S A. 2008; 105(11): 4489–4494. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeborgne-Castel N, Lherminier J, Der C, et al.: The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with reactive oxygen species production in Bright Yellow-2 tobacco cells. Plant Physiol. 2008; 146(3): 1255–1266. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLam SK, Cai Y, Tse YC, et al.: BFA-induced compartments from the Golgi apparatus and trans-Golgi network/early endosome are distinct in plant cells. Plant J. 2009; 60(5): 865–881. PubMed Abstract | Publisher Full Text\n\nOnelli E, Prescianotto-Baschong C, Caccianiga M, et al.: Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold. J Exp Bot. 2008; 59(11): 3051–3068. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoguchi K, Yasumori M, Imai T, et al.: bor1-1, an Arabidopsis thaliana mutant that requires a high level of boron. Plant Physiol. 1997; 115(3): 901–906. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson DG, Langhans M, Saint-Jore-Dupas C, et al.: BFA effects are tissue and not just plant specific. Trends Plant Sci. 2008; 13(8): 405–408. PubMed Abstract | Publisher Full Text\n\nMatsuoka K, Watanabe N, Nakamura K: O-glycosylation of a precursor to a sweet potato vacuolar protein, sporamin, expressed in tobacco cells. Plant J. 1995; 8(6): 877–889. PubMed Abstract | Publisher Full Text\n\nYuasa K, Toyooka K, Fukuda H, et al.: Membrane-anchored prolyl hydroxylase with an export signal from the endoplasmic reticulum. Plant J. 2005; 41(1): 81–94. PubMed Abstract | Publisher Full Text\n\nDhonukshe P, Aniento F, Hwang I, et al.: Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis. Curr Biol. 2007; 17(6): 520–527. PubMed Abstract | Publisher Full Text\n\nKonopkaa CA, Backuesb SK, Bednarek SY: Dynamics of Arabidopsis dynamin-related protein 1C and a clathrin light chain at the plasma membrane. Plant Cell. 2008; 20(5): 1363–1380. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuo T, Fredericksen B, Hasumi K, et al.: Human immunodeficiency virus type 1 Nef-induced CD4 cell surface downregulation is inhibited by ikarugamycin. J Virol. 2001; 75(5): 2488–2492. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLau AW, Chou MM: The adaptor complex AP-2 regulates post-endocytic trafficking through the non-clathrin Arf6–dependent endocytic pathway. J Cell Sci. 2008; 121(Pt 24): 4008–4017. PubMed Abstract | Publisher Full Text\n\nYu A, Malek TR: The proteasome regulates receptor-mediated endocytosis of interleukin-2. J Biol Chem. 2001; 276(1): 381–385. PubMed Abstract | Publisher Full Text\n\nMailhes JB, Hilliard C, Lowery M, et al.: MG-132, an inhibitor of proteasomes and calpains, induced inhibition of oocyte maturation and aneuploidy in mouse oocytes. Cell Chromosome. 2002; 1(1): 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuga S, Imagawa S, Maeshima M: Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs. Planta. 2001; 212(2): 294–304. PubMed Abstract | Publisher Full Text\n\nMatsuoka K, Nakamura K: Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting. Proc Natl Acad Sci U S A. 1991; 88(3): 834–838. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHood EE, Kusnadi A, Nikolov Z, et al.: Molecular farming of industrial proteins from transgenic maize. Adv Exp Med Biol. 1999; 464: 127–147. PubMed Abstract"
}
|
[
{
"id": "2106",
"date": "23 Oct 2013",
"name": "Tomomichi Fujita",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 demonstrates that in a heterologous plant system the Arabidopsis borate transporter BOR1 was polarly localized in tobacco BY-2 cells and was degraded in a borate-dependent manner as has been reported in Arabidopsis roots. Pharmacological analysis suggested both common and distinct routes for the BOR1 degradation between Arabidopsis roots and tobacco BY-2 cells. The authors also explored Tyr mutants of the BOR1-GFP in BY-2 cells and found an interesting, distinct response from Arabidopsis. Thus I think this article has enough quality of science to be approved in F1000Research. I have some comments and questions that should be considered and explained for the improvement of the article.\n\nFig.1 A: I can see some green dots from BOR1-GFP in the cytosol. According to the rest of the results (e.g. Fig. 1C, 3A (-Borate), 4A (Bor1-GFP)) the fluorescent signal in the low concentration of borate media and in the log phase of the BY-2 line is mainly detected in plasma membranes but not in the cytosol; could you explain why you think this difference occured?Fig.1 D: The authors fractionated the cell lysate to show that BOR1-GFP is targeted to the membrane fractions; it would be interesting to know whether either fraction between P10 and P100 contains more plasma membranes or more microsomes. It would also be helpful if the authors could add control experiments for each fraction using protein markers that are known to localize in plasma membranes, microsomes, or the soluble fraction.p.4 left column: The authors used PIP-GFP, and then later SCAMP2-mRFP. It would be better to describe the origins (Arabidopsis or tobacco) of these in the Materials and Methods section.Fig.2: The authors measured the cell volume of BY-2 cells. Please add the methods used and how many times the measurement was replicated (n=?).Fig. 6A: In the picture, please add the note ‘Bor1-GFP’ as indicated in Fig. 6B and C.Fig. 6C legend: Please check if the sentence ‘BOR1-GFP was transiently expressed in cells expressing SCAMP2-mRFP…’ is not instead supposed to read ‘SCAMP2-mRFP was transiently expressed in cells expressing BOR1-GFP.’Fig. 6C: The authors examined the endocytic route for BOR1 degradation. Based on the observation in the merged picture, they concluded that since no overlapping of green and red dots was observed, co-localization of SCAMP2 and BOR1 was unlikely. However, I see only a few dots in the original picture of BOR1-GFP that they presented, and I wonder if this is why in the merged picture there were few yellow signals indicating an overlap of SCAMP2 and BOR1. The authors should show an appropriate BOR1-GFP image with more dots of BOR1-GFP, and the SCAMP2 image should be merged so that the colocalization can be observed. Additionally, even in the current merged picture, I can see at least one yellow dot at the periphery of the bottom-side plasma membrane; how is this dot explained?p.6 right column, line-16: (Figure 6) should be (Figure 7).Fig. 7C: The authors examined whether inhibition occurred at the point of endocytosis from the plasma membrane or at intermediate compartments. However, it is hard to distinguish if there are any intermediate compartments or not at the presented magnification/resolution of the images. Especially in the images for the TIBA treatment, as the relatively strong fluorescent signal was probably recognized in a surrounding region of the nucleus. Please show better images and explain if this is related to the intermediate compartments or something else.p.11 left column, line 7 from below: ‘GFP8’ could be ‘GFP’?",
"responses": []
},
{
"id": "2177",
"date": "28 Oct 2013",
"name": "Liwen Jiang",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study on the degradative pathway and dynamics of BOR1-GFP in transgenic tobacco BY-2 cells. The conclusions are largely supported by the provided data. I welcome its publication as it is informative and useful for the study of endocytosis in plant cells.Some minor suggestions:Add scale bars to some of the Figures (e.g. Figures 1C, 3A, 4A, 7C, 8A).Please indicate the molecular weight (kDa) of the detected proteins in Figures 1D, 3B and 5A.",
"responses": []
},
{
"id": "2107",
"date": "06 Nov 2013",
"name": "Agustín González-Fontes",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 an extension and confirmation of findings on the degradation of the borate transporter BOR1 in Arabidopsis thaliana tobacco BY-2 cells. From elegant and conclusive techniques used in previous works by the Fujiwara and Matsuoka groups, the authors demonstrated that, under high B supply (3-5 mM), the BOR1 transporter in transformed tobacco BY-2 (BOR1-GFP) cells was also transported from the plasma membrane to the vacuole via endocytosis for degradation, this borate-dependent process being specific for BOR1-GFP. However, unlike in Arabidopsis, BOR1-GFP did not pass through the trans-Golgi network during degradation.Interestingly, the authors showed that de novo synthesis of the borate-dependent degradation machinery of the borate transporter BOR1 is required for maintaining the degradation. However, it is noteworthy that the threshold B concentration for the onset of borate-dependent degradation in tobacco BY-2 cells was 3 mM, but in Arabidopsis root cells is only 0.1 mM B. The authors should give some additional explanation for this significant discrepancy, other than that the plant species have different high-boron sensitivity.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-185
|
https://f1000research.com/articles/2-184/v1
|
13 Sep 13
|
{
"type": "Method Article",
"title": "Bayesian prediction of microbial oxygen requirement",
"authors": [
"Dan B. Jensen",
"David W. Ussery",
"David W. Ussery"
],
"abstract": "Background: Prediction of the optimal habitat conditions for a given bacterium, based on genome sequence alone would be of value for scientific as well as industrial purposes. One example of such a habitat adaptation is the requirement for oxygen. In spite of good genome data availability, there have been only a few prediction attempts of bacterial oxygen requirements, using genome sequences. Here, we describe a method for distinguishing aerobic, anaerobic and facultative anaerobic bacteria, based on genome sequence-derived input, using naive Bayesian inference. In contrast, other studies found in literature only demonstrate the ability to distinguish two classes at a time.\nResults: The results shown in the present study are as good as or better than comparable methods previously described in the scientific literature, with an arguably simpler method, when results are directly compared. This method further compares the performance of a single-step naive Bayesian prediction of the three included classifications, compared to a simple Bayesian network with two steps. A two-step network, distinguishing first respiring from non-respiring organisms, followed by the distinction of aerobe and facultative anaerobe organisms within the respiring group, is found to perform best.\nConclusions: A simple naive Bayesian network based on the presence or absence of specific protein domains within a genome is an effective and easy way to predict bacterial habitat preferences, such as oxygen requirement.",
"keywords": [
"Comparative genomics",
"oxygen requirements",
"prediction",
"Bayesian inference"
],
"content": "Background\n\nIdentification of microbial organisms with specific habitat adaptations is important for a range of purposes, such as specifying organisms as likely producers of industrially or scientifically relevant enzymes. An easy-to-make prediction of adaptations to specific habitats based on genome sequences, independent of time consuming laboratory tests, would therefore be of value to researchers for narrowing down a list of potential organisms of interest for their particular purpose. In addition, a list of genomic features that effectively predicts the environmental preference of a group of organisms would aid scientific researchers in gaining a mechanistic understanding of the requirements a given environment imposes on its microbial inhabitants.\n\nTo demonstrate a method for making such predictions, this study aims to predict bacterial oxygen requirements. This choice was made in part because prediction and description of genomic characteristics relevant for oxygen requirements are relatively absent in the literature, in spite of a many characterized genomes available. Furthermore, when prediction of oxygen requirement has been attempted in the literature, the authors generally invoke the false dichotomy of a bacterium being either an aerobe or anaerobe1,2. Similar unfairly dichotic approaches are often seen with respect to other habitat classifications, e.g. salinity and thermophilicity3–5. In contrast, this study aims to distinguish between three different classifications: aerobe, anaerobe and facultative anaerobe. These oxygen requirement classifications can be found at the NCBI list of sequenced genomes (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) and have simple and specific definitions. Obligate aerobes are organisms that require the presence of oxygen for respiration, while the presence of oxygen is detrimental to the growth of obligate anaerobes. Non-obligate, or aero tolerant, anaerobes may grow in the presence of oxygen, but are unable to use it in respiration. In this study, we did not distinguish between these two types of anaerobes. Facultative anaerobic bacteria can use oxygen for respiration, but will also grow in the absence of oxygen, although typically more slowly6.\n\nSpecific living-conditions will naturally impose selective pressures on the optimal set of protein functions, and a sensible basis for prediction would thus be the genomic make-up with respect to an organism protein domain profile. This idea has been the basis of a number relatively successful attempts at predicting different types of habitat adaptations1.\n\nFor the purpose of classification prediction, this study implements a naive Bayesian classifier. This is a relatively simple method, but it has in the past been shown to be effective prediction tool in a vast range of areas, including bacterial thermophilicity prediction7,4, genetic risk factors for disease8,9 and taxonomic classification of fungi10.\n\n\nMethods\n\nThe genomes included in this study were selected from the NCBI genome database based on the oxygen requirement classifications in the NCBI Iproks table (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). To avoid overestimation of the predictive performance, only one member of each genus was randomly selected to be included within each classification. Thus the overall dataset configuration was as show in Table 5.\n\nThe included genomes where translated to predicted proteomes using the Prodigal tool11 with default settings. The predicted proteomes were searched for the presence of the protein domain Pfam-A12. This search was performed using hmmscan3 with default settings, a tool which is part of the HMMR3 package13. The presence or absence of all Pfam-A domains found in the sum of proteomes was stored in a presence/absence matrix (Additional file 6). Based on this matrix, Pfam-A domains overrepresented in any one specific class were identified. Similarly to a previous study7, overrepresentation is here defined as the domain being present in at least 65% of the members of a given class, and that the frequency in that class is significantly (p < 0.05) different from the frequency in all other classes, given a two-tailed independent t-test. In this manner, a list of class-associated Pfam-A domains, along with their frequency of occurring in each of the three classifications, was created. This list contained the observed likelihood of a given Pfam-A domain being present, given the classification, and will be referred to as the ‘likelihood file’. The script used to construct the model can be found in Additional file 7. All scripts can additionally be found at https://github.com/danbjensen/Oxygen_requirement_prediction. The scripts are also permanently available at http://dx.doi.org/10.5281/zenodo.7099.\n\nPredictions were based on the above described likelihood file. A flat prior was used, meaning that initially the probability of an arbitrary genome being any of the three classifications was considered to be 1/NClasses ~ 0.333. If a given domain was found to be present in a given genome, the probability of that genome belonging to each of the included classifications was updated by a factor of the observed likelihood for the individual groups, p(family|class). If the family or domain was found not to be present, the probability was updated by a factor of 1-p(family|class). The posterior probability for a given genome belonging to the various classifications, C, given the observed presence or absence of a specific domain, Oi, was then calculated using Bayes rule:\n\n\n\nA pseudo-count (PC) of 0.1 was used for all likelihoods to prevent the probabilities from plummeting to zero. The three included classifications of oxygen requirements were predicted using a one-step and two-step naive Bayesian inference network, as illustrated in Figure 1.\n\na) In the one-step prediction method the genomes in the test set are assigned a posterior probability for each of the three included classifications, given their protein domain profile. The genomes are predicted to belong to the classification to which they have the highest posterior probability. b) The genomes in the test set are first assigned posterior probabilities for being able or unable to respire, based on their protein domain profile. Using a second model, those genomes found most likely to be capable of respiration are assigned a posterior probability of belonging to the classifications Aerobe or Facultative.\n\nIn the one-step approach, each genome is assigned the single classification it is considered most likely to have, based on its protein domain profile. The likelihood file is based on a training matrix containing the protein domain profiles of all genomes with their associated classification, with the exception of the genome being predicted (N-fold cross-validation).\n\nIn the two-step approach, every genome is first predicted to be able or unable to use oxygen for respiration. This is done based on a training matrix, containing every included genome, marked as either respiration-capable (aerobe and facultative anaerobe) or not respiration-capable (anaerobe). The genome being predicted is excluded from the training matrix. Any genome predicted to not be respiration-capable is considered to be a predicted anaerobe, while genomes predicted to be respiration-capable go through a second round of predictions. These predictions are based on the likelihood files derived from a matrix containing the protein domain profiles of aerobes and facultative bacteria only. Based on this, every genome predicted to be respiration-capable is predicted to be either an aerobe or facultative anaerobe. If a genome under prediction is present in the training matrix, the profile of this genome is excluded from the training. The script used to make the described predictions can be found as Additional file 8.\n\nTo evaluate predictive performance, Matthew's Correlation Coefficient (MCC)14 was used. As three categories were included in this study, the predictions for each category were evaluated individually by forcing the three classes into two; the one a given genome belongs to, and every other class. This is a common method for adapting the MCC method to prediction data with more than two possible classifications15. The script used to calculate the performances can be found in Additional file 9.\n\n\nResults and discussion\n\nTo evaluate the efficiency of class-associated Pfam-A domains, i.e. Pfam-A domains found significantly more frequently in one specific oxygen requirement class compared to any other, as an input for a naive Bayesian classification of bacterial oxygen requirements, the Matthew's Correlation Coefficient (MCC)14 was used. In the context of the MCC, a value of 1 indicates perfect correlation between predicted and actual class, a value of -1 indicates a perfect anti-correlation and a value of 0 is expected when the predictions are perfectly random. Two strategies were attempted: one where prediction of all three classifications was attempted in a single step and another where a simple Bayesian network was implemented, describing the oxygen requirement classifications as two nested dichotomies.\n\nOne-step predictions. Table 1 shows the predictive performance achieved when the three classifications are predicted in a single step, based on the relative abundance of the various Pfam-A domains in the different classes. The performance is clearly best for the prediction of aerobes and anaerobes, which perform with an MCC value well above 0, although not fully 1. The performance for predicting facultative anaerobes, although higher than 0, are not satisfyingly above what one might expect from random clustering of the data to be considered truly meaningful. The exact predictions and correct classification of the individual genomes are listed in Additional file 1.\n\nPredictions of all classes are performed better than random chance, although aerobe and anaerobe bacteria clearly show the best performance compared to facultative anaerobe bacteria.\n\nTo further examine the conditions behind the above performances, Table 2 shows the distribution of class-predictions for genomes of each of the three actual classifications. As can be seen, the vast majority of aerobe and anaerobe genomes are predicted correctly. For the facultative subsection of the dataset, however, many genomes are erroneously predicted to be aerobes. By contrast, the rate of erroneous prediction of genomes in these an aerobe or facultative anaerobe of being an anaerobe is rather low. This explains why the prediction performance of aerobe genomes appeared lower than for anaerobes; the false positive value in the MCC equation becomes larger for aerobe genomes, thus causing the overall value to drop.\n\nAerobe bacteria are correctly predicted to aerobe in 87% of the cases and are mis-predicted to be facultative anaerobes in 11% of the cases. Similarly anaerobe bacteria are correctly predicted in 88% of the cases, and are mis-prediction of anaerobes as aerobe or facultative anaerobes happen equally frequently, in 6% of the cases. Facultative anaerobes are most commonly mis-predicted to be aerobes, in 44% of the cases. The facultative anaerobes are only correctly predicted in 35% of the cases.\n\nThis finding arguably makes sense in light of the fact that facultative anaerobes possess the ability to respire using oxygen, which is a feature missing in strict anaerobic organisms. It thus makes sense to assume that a specific list of enzymatic characteristics are required or useful for the organism to perform respiration, which one might thus expect to find in aerobes as well as facultative anaerobes. The same characteristics would likely not be useful in anaerobe bacteria, which would result in an enzymatic profile of aerobe and facultative anaerobe bacteria, which would stand out as separate from anaerobes.\n\nTwo-step predictions. Inspired by the findings described above, a reasonable prediction strategy would be to first separate the respiration-capable organisms from the anaerobes, and subsequently attempt to further distinguish between the two kinds of respiring bacteria. Here, the initial prediction of distinguishing anaerobe from non-anaerobe bacteria is based on the Pfam-A presence/absence data, with all aerobe and facultative anaerobe bacteria simply considered as the same classification. The secondary prediction is based on the Pfam-A presence/absence data from known respiration-capable genomes only, disregarding the anaerobe portion of the dataset. This two-step approach yields the overall predictive performances shown in Table 3. The exact predictions and correct classification of the individual genomes can be found in Additional file 2. It should be noted that a considerable improvement is found in the performance of prediction of facultative anaerobes. These improvements can be understood by how the members of the three classes are actually predicted, as shown in Table 4. Many facultative anaerobe genomes are still erroneously predicted to be aerobes. However, the percentage of correct predictions of aerobes and anaerobes has clearly increased compared to the one-step method, indicating that the two-step network offers some advantage.\n\nThe performance for aerobe and anaerobe predictions are the same as for the one step prediction method, but the performance for prediction of facultative anaerobes have increased from 0.31 to 0.39.\n\nNotice that the frequency of correctly predicted facultative anaerobes have not increased compared with the one-step method (33% vs. 35%), but that the fraction of erroneous predictions of aerobe and anaerobe bacteria have been decreased (5% vs. 11% for aerobes, 4% vs. 6% for anaerobes). Thus the better performance of the prediction of facultative anaerobe genomes is due to an increased accuracy in predicting aerobe and anaerobe bacteria rather than an increased accuracy in predicting facultative anaerobe bacteria.\n\nAs described above, the most effective method for predicting oxygen requirement attempted in the present study was the two-step Bayesian network, where anaerobes were first distinguished from the respiring bacteria (aerobes and facultative anaerobes). This study found a total of 252 protein domains to be consistently over-represented in anaerobe genomes, compared to the respiration-capable genomes. The specific likelihoods of these domains being present given an anaerobe or aerobe/facultative genome, are listed in Additional file 3.\n\nAerobe genomes were consistently distinguished from facultative anaerobe genomes by 402 domains, while facultative genomes consistently had 122 specific domains over-represented in their genomes, compared to aerobe genomes. The specific likelihoods of these 524 domains being present given that the genome is from an aerobe or facultative anaerobe bacterium, respectively, are listed in Additional file 4.\n\nThus, by applying the information provided in Additional file 3 and Additional file 4 in a two-step Bayesian estimation, as described in the Method section, it is possible to calculate the most likely oxygen requirement class of an arbitrary bacterial genome, provided a Pfam-A profile is available for said genome.\n\nVery few studies that attempt to predict microbial oxygen requirements can be found in the literature. Two examples are the studies by Wu & Moore and Lingner et al.1,2. In both of these studies they distinguish just two classes of oxygen requirement at a time. Although Wu & Moore look at three classes (aerobe, anaerobe and facultative anaerobe), they only attempt dichotic predictions, always leaving out one class entirely. They are thus uninformative about the reliability of predictions where the genome in question can be any of the possible classifications. In contrast, the method described in the present study offer more realistic estimations of how well the prediction of any of the three included classifications will perform.\n\nIn their distinction of aerobic and anaerobic organisms, Wu & Moore report an average misclassification rate, when distinguishing between aerobe and anaerobe genomes, of 15% and 13% when basing predictions on Clusters of Orthologous Groups and KEGG Orthology groups, respectively. To allow for a direct comparison, the two-step method described in the present study shows an average misclassification rate of a slightly less than 8% (MCC = 0.84) when distinguishing between aerobe and anaerobe genomes alone (Additional file 5). This suggest that the two-step method described here, along with being more simple to perform, is actually almost twice as accurate, when directly compared to the methods presented by Wu & Moore.\n\nSimilar to the present study, Lingner et al. attempted to predict oxygen requirements based on protein domain profiles; however only the distinction between aerobe and anaerobe genomes was described. For this purpose, Lingner et al. reported a performance in the form of sensitivity multiplied by specificity, of 0.88, which is comparable to the 0.84 achieved for aerobe/anaerobe distinction when using the method described here (Additional file 5). To construct the protein domain profiles used by Lingner et al., the number of each of the Pfam-A domains present in a given genome was used. In contrast, this study looked only on the presence or absence of the various Pfam-A domains in the individual genome. The comparable performances thus indicate that the presence of specific protein domains is indicative of oxygen requirements, regardless of the copy-number of those domains. Furthermore, it should be noted that Lingner et al. performed their predictions based on genomes available from NCBI 2009. They do not specifically specify the number of genomes labeled with respect to oxygen requirement at that time, but given the continuous additions of new genome sequences, it can reasonably be assumed to be fewer than the genomes available for the present study.\n\nOne of the main basic premises of the naive Bayesian inference method is that the various inputs, on which the inference is made, are mutually independent. In this study, the various inputs were different Pfam-A domains16. Protein domains by definition consist of compact sequences that will fold, perform functions and even evolve independently of the rest of the protein in which they reside17. Based on this fact, the basic premise of independence seems reasonable, and the method should thus be applicable.\n\nFurthermore, even in situations where the premise of independence is invalid, the naive Bayesian classifier can be shown to produce excellent performance18,19. This means that even if certain protein domains might be found together regardless of classification, it would still be reasonable to expect the two-step method described here to be effectively applicable.\n\n\nConclusions\n\nThe results presented in this study show that bacterial oxygen requirements can be accurately predicted without considering protein domain copy number. Although facultative anaerobes could be predicted with a performance significantly better than random guessing, further optimization is still desired so as to make the distinction meaningful in practice. Such optimization would include additional biologically meaningful markers, e.g. the presence of specific transcription factors. However, the distinction between aerobe and anaerobe organisms, are as good, or better than what is achieved by other methods published in the scientific literature1,2.\n\nThe best performances were achieved when using a simple Bayesian network, first distinguishing respiration-capable bacteria (aerobes and facultative anaerobes) from anaerobic bacteria, and subsequently distinguishing the aerobes from the facultative anaerobes. The respiration-capable bacteria could be distinguished from anaerobic bacteria with a Matthews' Correlation Coefficient of 0.76, while pure aerobes could be distinguished from anaerobes with a Matthews' Correlation Coefficient of 0.84.\n\nGiven the success with respect to distinguishing respiring bacteria from anaerobes, a reasonable follow up would be to study the class-associated Pfam-A domains identified in this study in more detail. They offer a logical first step for supplying a mechanistic model, explaining the genetic adaptations necessary for a bacterium given certain environmental oxygen exposures. Furthermore, we plan to test this method for prediction of other types of habitats, including cases with more than two categories. Based on the findings of this study, we would recommend that the categories of such cases be divided into biologically meaningful sets of dichotic super-classes, followed by two or more rounds of predictions.",
"appendix": "Author contributions\n\n\n\nConceived the project: DWU, DBJ.\n\nPerformed data analysis: DBJ.\n\nWrote the first draft of the manuscript: DBJ.\n\nPaper feedback and analysis suggestions: DWU.\n\nBoth authors have read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe thank the Technical University of Denmark and the Danish Research Council for funding this work. All grants were assigned to David Ussery.\n\n\nAcknowledgements\n\nThe authors would like to thank Tammi Vesth for help in this project.\n\n\nReferences\n\nLingner T, Mühlhausen S, Gabaldón T, et al.: Predicting phenotypic traits of prokaryotes from protein domain frequencies. BMC bioinformatics. 2010; 11: 481. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu H, Moore E: Association analysis of the general environmental conditions and prokaryotes’ gene distributions in various functional groups. Genomics. 2010; 96(1): 27–38. PubMed Abstract | Publisher Full Text\n\nSmole Z, Nikolic N, Supek F, et al.: Proteome sequence features carry signatures of the environmental niche of prokaryotes. BMC Evol Biol. 2011; 11(1): 26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGromiha MM, Suresh MX: Discrimination of mesophilic and thermophilic proteins using machine learning algorithms. Proteins. 2008; 70(4): 1274–1279. PubMed Abstract | Publisher Full Text\n\nHurst LD, Merchant AR: High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes. Proc Biol Sci. 2001; 268(1466): 493–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMadigan MT, Martinko JM: Biology of Microorganisms 11th ed. Benjamin Cummings. 2006; 161. Reference Source\n\nJensen DB, Vesth TC, Hallin PF, et al.: Bayesian prediction of bacterial growth temperature range based on genome sequences. BMC genomics. 2012; 13(Suppl 7): S3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSebastiani P, Solovieff N, Sun JX: Naïve Bayesian Classifier and Genetic Risk Score for Genetic Risk Prediction of a Categorical Trait: Not so Different after all! Front Genet. 2012; 3: 26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOkser S, Lehtimäki T, Elo LL, et al.: Genetic variants and their interactions in the prediction of increased pre-clinical carotid atherosclerosis: the cardiovascular risk in young Finns study. PLoS Genet. 2010; 6(9): e1001146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu KL, Porras-Alfaro A, Kuske CR, et al.: Accurate, rapid taxonomic classification of fungal large-subunit rRNA genes. Appl Environ Microbiol. 2012; 78(5): 1523–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHyatt D, Chen GL, Locascio PF, et al.: Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC bioinformatics. 2010; 11: 119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSonnhammer EL, Eddy SR, Durbin R: Pfam: a comprehensive database of protein domain families based on seed alignments. Proteins. 1997; 28(3): 405–20. PubMed Abstract | Publisher Full Text\n\nEddy SR: Accelerated Profile HMM Searches. PLoS Comput Biol. 2011; 7(10): e1002195. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatthews BW: Comparison of the predicted and observed secondary structure of t4 phage lysozyme. Biochim Biophys Acta. 1975; 405(2): 442–451. PubMed Abstract | Publisher Full Text\n\nGorodkin J: Comparing two K-category assignments by a K-category correlation coefficient. Comput Biol Chem. 2004; 28(5–6): 367–74. PubMed Abstract | Publisher Full Text\n\nSonnhammer EL, Eddy SR, Durbin R: Pfam: a comprehensive database of protein domain families based on seed alignments. Proteins. 1997; 28(3): 405–20. PubMed Abstract | Publisher Full Text\n\nPonting CP, Russell RR: The natural history of protein domains. Annu Rev Biophys Biomol Struct. 2002; 31: 45–71. PubMed Abstract | Publisher Full Text\n\nPazzani PDM: Beyond independence: Conditions for the optimality of the simple Bayesian classifier. Machine Learning. 1997; 29: 103–130.\n\nZhang H: The Optimality of Naive Bayes. Proceedings of the 17th International FLAIRS conference (FLAIRS2004). 2004. Reference Source"
}
|
[
{
"id": "2198",
"date": "28 Oct 2013",
"name": "Kazuhiro Takemoto",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 propose a prediction method for bacterial oxygen requirements using Bayesian instance with protein domain profiles. Although there is no originality in the context of prediction methods, the prediction accuracy of the proposed method is higher than that of the previous study. This suggests the validity of this method. This method is expected to be useful for estimating the oxygen requirement of newly isolated bacteria. The manuscript is generally well constructed, and it is readable. However, I have the following concerns:This study is limited to bacterial species. Thus, the authors have to emphasize this limitation, and the title should be “…bacterial oxygen requirements”. The authors have to discuss the prediction of archaeal oxygen requirement. For example, can this method be applicable to archaea? The authors should provide a clear description of the relationship protein domains and oxygen requirement. What protein properties are dominant for predicting oxygen requirement? A previous study (e.g. Kim KM et al., 2012) may be helpful. I recommend that the authors mention some applications of the proposed method. The denominator in the first stand alone equation is incorrect: 0_i => O_i Are the numbers of included genomes in Table 5 correct? For example, Tables 3 and 4 include 157 aerobes, 108 anaerobes, and 98 (97 in Table 4) facultative anaerobes. This is inconsistent with Table 5.",
"responses": []
},
{
"id": "3018",
"date": "30 Jan 2014",
"name": "Anita Krisko",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Jensen and Ussery entitled 'Bayesian prediction of microbial oxygen requirement' describes a novel improved method of distinguishing aerobic, anaerobic and facultative anaerobic bacteria. While previously published methods only demonstrate the ability to distinguish two classes at a time, the method described herein first distinguishes respiring from non-respiring bacteria, followed by distinguishing aerobic and facultative anaerobic bacteria within the respiring group.There are several issues that require additional comments.Looking at Additional Table 3 and Additional Table 4, I noticed that the definition of over-representation of domains may be a problem. For example, Additional file 3, line 6: domain ADK_lid is 86% represented in respiring bacteria and 94% in anaerobes. The authors decide that this domain is over-represented in anaerobes. However, isn't it over-represented in both? The authors state at the beginning of the manuscript that the over-represented domain is defined as the domain being present in at least 65% of the members of a given group. Have the authors considered defining over-represented domains as over-represented in one group and under-represented in the other? In any case, both have to be analyzed and commented on in the manuscript. In addition to the abbreviation of each domain, it may be useful to include the function of each domain. Also, the authors need to discuss the functions of over-represented and under-represented (maybe even completely lacking) within each group of bacteria. If the authors would like to claim this method for all organisms, the analysis needs to be performed also in archaea as well as the available eukaryotes. However, if the authors decide to present only the results on bacteria, this will have to be pointed out in the text.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-184
|
https://f1000research.com/articles/2-183/v1
|
13 Sep 13
|
{
"type": "Case Report",
"title": "Mania induced by varenicline",
"authors": [
"Ping-Tsun Chang",
"Cheng-Ta Li",
"Ping-Tsun Chang"
],
"abstract": "Varenicline is sometimes used in the treatment of smoking addiction. Side effects such as nausea, vomiting, and headaches have been well documented. In addition, depressed mood and suicidal thoughts relating to varenicline usage have been previously reported. We report a case of mania in a patient with refractory depression whose first manic episode developed under the use of varenicline in combination with original antidepressant medications.",
"keywords": [
"Mr. L",
"a 55-year-old male",
"was diagnosed with major depressive disorder based on DSM-IV TR criteria in December 2010. The presenting symptoms included depressed mood",
"irritability",
"anxiety",
"somatic worries",
"difficulty falling asleep",
"ideations of death",
"anhedonia",
"and an impaired working ability for two months. Therefore",
"we prescribed standard antidepressant treatment",
"and also attended psychotherapeutic intervention sessions following his visit to our outpatient department."
],
"content": "Case report\n\nMr. L, a 55-year-old male, was diagnosed with major depressive disorder based on DSM-IV TR criteria in December 2010. The presenting symptoms included depressed mood, irritability, anxiety, somatic worries, difficulty falling asleep, ideations of death, anhedonia, and an impaired working ability for two months. Therefore, we prescribed standard antidepressant treatment, and also attended psychotherapeutic intervention sessions following his visit to our outpatient department.\n\nHe showed a poor response to both antidepressant monotherapy and combination therapy with a selective serotonin reuptake inhibitor (SSRI), serotonin/norepinephrine reuptake inhibitor (SNRI) and norepinephrine/dopamine reuptake inhibitor (NDRI) combined with lamotrigine. In detail, he received sertraline monotherapy (50 mg, 3 months), venlafaxine monotherapy (112.5 mg, 4 months), duloxetine monotherapy (30 mg, 2 months) and combination therapy of bupropion and lamotrigine (300 mg/50 mg, 6 months). Repetitive transcranial magnetic stimulation was also tried with only a partial antidepressant response. Finally, he maintained mild to moderate depression with the use of a melatonergic agent (agomelatine 25 mg) and lamotrigine (50 mg).\n\nThe patient reported being addicted to smoking for the past 40 years, 1 pack per day, with a Fagerstrom nicotine dependence score of 6. In March 2013, he was prescribed varenicline 0.5 mg/day for smoking cessation. He had been treated with agomelatine (25 mg/day) for three months prior to the initiation of varenicline treatment. After six days of varenicline treatment, there was no reported nausea or other side effects. The dosage of varenicline was subsequently titrated to 2 mg/day. During the first month of varenicline treatment, persistent elevated mood, high irritability and other symptoms of mania such as hyper-talkativity, grandiosity, decreased need for sleep were reported. In addition, increased verbal and physical aggressions toward strangers were noted for one week. Thus, varenicline-related mania was diagnosed and after discontinuation of varenicline, the manic symptoms disappeared rapidly within a few days. His mood returned back to its original status between mild to moderate depression.\n\n\nDiscussion\n\nThe occurrence of the mania in the present case seemed to be dose-dependent and the adverse effect rapidly disappeared after discontinuation of varenicline. Depressed mood and suicidal ideation have been listed in varenicline’s black box warning1. In addition, a previous case of aggressive behavior in a patient with schizophrenia induced by varenicline has been reported2. However, the mechanism by which varenicline could induce mania is not clear.\n\nA prominent reduction in the expression of mRNA for several nicotinic subunit isoforms has been reported in bipolar disorders. Nicotinic cholinergic receptors play an important role in regulating the activity of GABA inhibitory interneurons3.\n\nSelectively binding to the nicotinic acetylcholine α4β2 subunit may cause an imbalance of the inhibitory controls in the mood circuit, since the nicotinic acetylcholine receptor α7 and α4β2 subtypes have a different degree of GABAergic inhibition in target neurons4. Furthermore, an increase in nicotinic receptor α7-dependent signaling has been suggested to be critically involved in the pathophysiology of bipolar disorder from a study on post-mortem patients5. Varenicline seems to be safe in patients with bipolar disorders6, yet a few case reports have demonstrated a hypomanic or manic relapse in patients with identified bipolar disorders7–9. Varenicline is a partial agonist at the nicotinic acetylcholine α4β2 subunit and a full agonist at the α7 subunit10. Potent α7-dependent activation and weak α4β2-depedent activation caused by varenicline might lead to a central inhibitory dysfunction that might, in turn, lead to manic symptoms in patients with bipolar disorders or bipolar diathesis. Some patients with major depression may harbor bipolar disorders later, particularly if they show high resistance to antidepressant treatment11. Although central mechanisms of varenicline induced mania remain elusive, the present case report highlights the importance of monitoring for potential manic side effects with the use of varenicline in patients with major depression, especially when used in combination with antidepressants.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nConceived the case: Cheng-Ta Li. Analyzed the data: Ping-Tsun Chang and Cheng-Ta Li. Wrote the paper: Ping-Tsun Chang. Both authors critically revised the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nFood and Drug Administration. Information for Healthcare Professions: Varenicline (marketed as Chantix). 2008. Reference Source\n\nFreedman R: Exacerbation of schizophrenia by varenicline. Am J Psychiatry. 2007; 164(8): 1269. PubMed Abstract | Publisher Full Text\n\nBenes FM: Nicotinic receptors and functional regulation of GABA cell microcircuitry in bipolar disorder and schizophrenia. Handb Exp Pharmacol. 2012; (213): 401–17. PubMed Abstract | Publisher Full Text\n\nAlkondon M, Albuquerque EX: Nicotinic acetylcholine receptor alpha7 and alpha4beta2 subtypes differentially control GABAergic input to CA1 neurons in rat hippocampus. J Neurophysiol. 2001; 86(6): 3043–55. PubMed Abstract\n\nThomsen MS, Weyn A, Mikkelsen JD: Hippocampal α7 nicotinic acetylcholine receptor levels in patients with schizophrenia, bipolar disorder, or major depressive disorder. Bipolar Disord. 2011; 13(7–8): 701–7. PubMed Abstract | Publisher Full Text\n\nWu BS: Smoking and Co-occurring Disorders: Implications for Smoking Cessation Interventions for Adolescents in Residential Addiction Treatment. Journal of Dual Diagnosis. 2012; 8(2): 131–132.\n\nKohen I, Kremen N: Varenicline-induced manic episode in a patient with bipolar disorder. Am J Psychiatry. 2007; 164(8): 1269–70. PubMed Abstract | Publisher Full Text\n\nMorstad AE, Kutscher EC, Kennedy WK, et al.: Hypomania with agitation associated with varenicline use in bipolar II disorder. Ann Pharmacother. 2008; 42(2): 288–9. PubMed Abstract | Publisher Full Text\n\nAlhatem F, Black JE: Varenicline-induced mania in a bipolar patient. Clin Neuropharmacol. 2009; 32(2): 117–8. PubMed Abstract | Publisher Full Text\n\nMihalak KB, Carroll FI, Luetje CW: Varenicline is a partial agonist at alpha4beta2 and a full agonist at alpha7 neuronal nicotinic receptors. Mol Pharmacol. 2006; 70(3): 801–805. PubMed Abstract | Publisher Full Text\n\nLi CT, Bai YM, Huang YL, et al.: Association between antidepressant resistance in unipolar depression and subsequent bipolar disorder: cohort study. Br J Psychiatry. 2012; 200(1): 45–51. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "2395",
"date": "19 Nov 2013",
"name": "Eva Kralikova",
"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\nVarenicline is commonly used in the treatment of tobacco dependence. Smokers have a very important and large psychiatric comorbidity. The paper presents no evidence that the status was caused by varenicline, including missing major publications on this topic - so I would evaluate it as not acceptable.",
"responses": []
},
{
"id": "2637",
"date": "10 Dec 2013",
"name": "Jonathan Foulds",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a case report of a man who had been suffering from treatment-resistant depression who developed symptoms of mania. The main problem with this paper is that although the patient was prescribed varenicline for smoking cessation, the report contains no information on any changes in his smoking around the time he was prescribed varenicline and thereafter…none! We are left to guess about this, and it is a reasonable guess that the patient significantly reduced his smoking. In that case why are the manic symptoms being attributed to varenicline rather than to smoking cessation? Some of the symptoms described (high irritability, decreased sleep) are known symptoms of nicotine withdrawal. There is also a lack of information on other drug/substance use that may help to interpret the patient's mood changes. For example, did he consume much caffeine? Caffeine metabolism slows down significantly on smoking cessation; could that have played a role?Overall there is insufficient provision of key information to help the reader interpret this potentially interesting case report. In addition, this report fails to cite recent key studies on large samples that are relevant to the question of whether varenicline may cause neuropsychiatric side effects (Gibbons & Mann 2013; Foulds et al, 2013; Thomas et al, 2013).The paper needs to be revised to include a detailed description of the timing of symptoms relative to cigarette consumption and to address any other potential causes of mania.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-183
|
https://f1000research.com/articles/2-165/v1
|
31 Jul 13
|
{
"type": "Commentary",
"title": "The gastric H, K-ATPase system also functions as the Na, K-ATPase and Ca-ATPase in altered states",
"authors": [
"Tushar Ray"
],
"abstract": "This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump) seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump) and/or Ca-ATPase (Ca-pump) depending on cellular needs. This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM) fraction exhibits a (Ca or Mg)-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF), the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg)-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM) shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM) and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.",
"keywords": [
"At the peak of acid secretion gastric juice has a pH close to 0.1 compared to blood (pH",
"7.4). Based on this the parietal cells transport protons against a concentration gradient of over a million fold mediated by the gastric H",
"K-ATPase system. This member of the P-2 ATPase family has been the most extensively studied along with the Na",
"K-ATPase and Ca-ATPase families due to their prominent roles in health and disease. Major developments in the field occurred following the single topology scheme for the Na",
"K-ATPase reaction proposed by Post and Albers in the early 1960s",
"which was subsequently extended to the H",
"K-ATPase system. The Post-Albers (PA) scheme visualizes Na-dependent phosphorylation of the 100 k Da α-subunit by ATP (a kinase step) and a sequential K-dependent dephosphorylation (a phosphatase step) during each reaction cycle. The activity of K-dependent p-nitrophenyl phosphatase (K-pNPPase)",
"which is always co-purified with the Na",
"K-ATPase system was assumed to represent the phosphatase step of the total ATPase reaction. However this assumption was subsequently proven to be erroneous since K-pNPPase activity reflects the ion channel activity across the membrane rather than being a partial reaction of the ATPase1. Based on the orientation of the ATP hydrolytic sites and the associated pNPPase sites together with their corresponding K regulatory sites (of high and low affinity respectively) across the membrane1",
"2 a mirror image orientation of the two α-subunits (within the functional H",
"K-ATPase complex) was proposed. The dual topology model fits well with numerous reports in the literature3."
],
"content": "Introduction\n\nAt the peak of acid secretion gastric juice has a pH close to 0.1 compared to blood (pH, 7.4). Based on this the parietal cells transport protons against a concentration gradient of over a million fold mediated by the gastric H, K-ATPase system. This member of the P-2 ATPase family has been the most extensively studied along with the Na, K-ATPase and Ca-ATPase families due to their prominent roles in health and disease. Major developments in the field occurred following the single topology scheme for the Na, K-ATPase reaction proposed by Post and Albers in the early 1960s, which was subsequently extended to the H, K-ATPase system. The Post-Albers (PA) scheme visualizes Na-dependent phosphorylation of the 100 k Da α-subunit by ATP (a kinase step) and a sequential K-dependent dephosphorylation (a phosphatase step) during each reaction cycle. The activity of K-dependent p-nitrophenyl phosphatase (K-pNPPase), which is always co-purified with the Na, K-ATPase system was assumed to represent the phosphatase step of the total ATPase reaction. However this assumption was subsequently proven to be erroneous since K-pNPPase activity reflects the ion channel activity across the membrane rather than being a partial reaction of the ATPase1. Based on the orientation of the ATP hydrolytic sites and the associated pNPPase sites together with their corresponding K regulatory sites (of high and low affinity respectively) across the membrane1,2 a mirror image orientation of the two α-subunits (within the functional H, K-ATPase complex) was proposed. The dual topology model fits well with numerous reports in the literature3.\n\nThis unified dual topology model helped to clarify the demonstrated lack of Na, K-ATPase activity in parietal cell fractions so far using the conventional assay procedure even though ouabain (a Na-pump inhibitor) has long been known to eliminate gastric acid secretion4. However, gastric H, K-ATPase activity shows stimulation5 by sodium manifesting a Na, K-ATPase activity when assayed at pH 8.5. In addition, the purified cytosolic activator protein (of 80 k Da mass) for the H, K-ATPase system stimulates the gastric H, K-ATPase and the renal Na, K-ATPase to the same degree6. These data revealed that the catalytic α-subunit (which faces cytosol) of the functional dual topology H, K-ATPase can also bind Na at an alkaline pH thus acting like a Na, K-ATPase. Such encouraging high pH neighboring the H, K-ATPase catalytic site would be attained during peak gastric acid secretion. This paper reviews the evidence pointing to the conclusion that the cytosolic regulation of the active transport of H/K, Na/K and Ca/H occurs in a tissue-specific manner where an individual P-2 ATPase subspecies is capable of transporting any of the other cations depending on the local ionic milleu in order to maintain optimal housekeeping conditions.\n\nHAF is an 80 k Da (a dimer of two identical 40 k Da subunits) cytosolic protein that occurs universally7–13 in the parietal cells. Activation of the gastric H, K-ATPase by the HAF is rather complex, resulting in a substantial increase in the affinity of the enzyme to K. Interaction of the HAF results in both up- and down-regulation of the gastric H, K-ATPase system depending on its concentration, showing a strong positive cooperativity (Hill coefficient = 4.5) followed by rapid decline9,13. The anti-HAF antibody completely blocks acid secretion in histamine-stimulated rabbit gastric glands demonstrating the essentiality of the HAF in gastric secretory process6. Studies with phospholipase14 and mild ethanol treatment15,16 revealed that the HAF dimer is rather loosely associated with the membrane-bound H, K-ATPase system, and the phospholipid is in some way involved in this process.\n\nSuch a loose association of the HAF with the secretory membrane of the parietal cell became clear when we studied the effects of HAF on the isolated apical (APM) and tubulovesicular (TV) membranes from rabbit gastric glands and observed characteristic differential effects17. The APM showed very high basal (Ca or Mg-ATPase) activity with a negligible K-stimulated component (H, K-ATPase activity). When assayed with Mg, K and HAF, the K-stimulated component was greatly stimulated (over 100-fold) by the HAF17. In contrast, the pure TV membranes exhibited a very low or negligible basal activity, but the very high K-stimulated ATPase activity only required a small amount of stimulation (only about 60%) by the HAF17. These studies revealed that the HAF is not only loosely bound to the APM but also plays an essential role in gastric acid secretion thus supporting our earlier conclusion.\n\nSuch differential association between HAF and the APM and TV membranes was also reflected in their lipid profiles which were qualitatively similar but quantitatively very different. Thus, the phosphatidyl choline content of APM and TV was 67 and 33 µ moles/mg protein respectively with corresponding phosphatidyl choline/phosphatidyl ethanolamine ratios being 1.38 and 0.87 for APM and TV. Also, the phosphatidyl inositol and phosphatidyl serine content of APM were 24 and 8 µ moles/mg protein, respectively, about twice as much as that of TV17.\n\nDuring the activation of the H, K-ATPase system, the activator molecules demonstrate strong positive cooperativity (Hill coefficient = 4.5) followed by a rapid decline to zero suggesting the binding of the HAF with the H, K-ATPase oligomer occurs over a small activator concentration range9. In other words, the bound HAF interacts in some way with the empty sites on the cytosolic domain of the H, K-ATPase to increase their affinity for the activator molecules. Similar to the sigmoidal activation and dramatic inhibition of the H, K-ATPase with increasing HAF, varying calcium concentrations (µM) also showed dual effects on the HAF-stimulated component of the H, K-ATPase. Low concentrations of calcium showed a small but consistent stimulation (about 20%) with a range of 0–1 µM followed by a dramatic inhibition abolishing the HAF-stimulated activity at 4 µM Ca17,18. Such positive cooperativity and down regulation with varying HAF and µM Ca concentrations are the marks of a delicate control mechanism inherent in the living system. The dramatic Ca-inhibition suggests a sequestration of Ca within the catalytic (cytosolic) domain of the gastric ATPase system. Such sequestration would depend primarily on the surface charge of the complex formed between the HAF and the H, K-ATPase catalytic domain and to a lesser extent on the nature of the neighboring phospholipid microdomain. This information suggests a combined role of calcium and cytosolic HAF in the intracellular regulation of gastric H transport.\n\nIt is obvious that an appropriate level of Ca (below 1 µM) facilitates a direct contact of the HAF with the catalytic surface of the enzyme while a higher concentration of Ca interferes presumably by building a critical barrier on the enzyme/HAF interface, thus preventing a direct interaction with the HAF. Under this condition, the apical membrane-located H, K-ATPase system would be acting as a provisional device for pumping out calcium prior to the onset of acid secretion17,18.\n\nThe suggested role of the APM-embedded H, K-ATPase as a provisional Ca-pump prior to acid secretion is fully consistent with recent reports from two different laboratories19–21. Using fluorescent-tagged antibodies against the plasma membrane Ca-ATPase (PMCA) and the gastric H, K-ATPase Caroppo et al.19 demonstrated that not only do both ATPases have a closely similar and asymmetric distribution on the APM (of oxyntic cells of bullfrog gastric mucosa) but also were found to be co-localized by superimposition. At the same time, systematic studies with rabbit gastric glands by Michelangeli and coworkers20,21 revealed a consistent but transient peak of Ca transport into the secretary lumen prior to the onset of H-secretion. Such an oscillation between the two modalities of gastric H, K-ATPase system is depicted in Figure 1.\n\nIn a similar fashion, the H, K-ATPase will also act as a Na-pump (not shown in the diagram) at the basolateral membrane depending on the local Na-concentration and pH. Following our current evidence, the critical interplay among the HAF, H, K-ATPase and Ca in parietal cells is depicted in this diagram. While the pump molecules integral to the tubulovesicle (TV) are stimulated appreciably by the HAF, those associated with the apical plasma membrane (APM) are absolutely dependent on the HAF for their function, revealing the essential nature of the HAF in gastric proton-pump function17.\n\nWhile the HAF is characteristically present in the parietal cells of the fundic mucosa, the NaAF was initially demonstrated in the cytosolic fractions of the brain and kidney from rabbits and also in pigs13 and subsequently purified22. A near homogeneous preparation of the NaAF, which has a mass of 170 k Da, was obtained by a modification of the procedure used for HAF purification22. Unlike the 80 k Da HAF dimer, the NaAF is monomeric and has a 170 k Da mass in its active state. Also, the NaAF stimulates only the Na, K-ATPase without stimulating the H, K-ATPase, while the HAF is equally effective at stimulating both6, suggesting that they share some domain(s) critical for the activation process.\n\nIn spite of such differences, some fundamental similarity was observed in the way HAF and NaAF work. Similar to the HAF-stimulated H, K-ATPase, the NaAF-stimulated Na, K-ATPase activity10,22 showed an abrupt inhibition within a relatively narrow range of Ca concentration. However, the HAF-stimulated H, K-ATPase activity was much more sensitive to Ca inhibition than the NaAF-stimulated Na, K-ATPase activity. The concentration of Ca needed for complete inhibition of the NaAF stimulation was 25–50 µM compared to the 3–4 µM for the HAF-stimulated H, K-ATPase10.\n\nA model for the cytosolic regulation of the P-2 ATPase system in parietal cells by the HAF and µM Ca is depicted in Figure 1.\n\nThe pivotal roles of Ca in HAF regulation of the pump strongly imply that Ca acts as a physiological feedback control switch in gastric H transport10,17. The Figure also shows that in the presence of around 4 µM Ca, the Ca-inhibited H-pump spontaneously changes itself into a unique Ca-pumping mechanism for promptly reversing the Ca-induced inhibition, thus bringing the local calcium concentration back down to 1 µM at which point the HAF activation of the H-pump resumes. Such intimate interplay between Ca and HAF would also help the parietal cells to conserve energy by preventing the needless accumulation of H inside the cytosolic TV prior to their destined inclusion into the APM by subsequent fusion. This unique ability of >1 µM Ca concentrations to switch the inhibited gastric H-pump spontaneously to the Ca-pumping mode is also likely to be operative in other H-pumping epithelia such as the distal colon and kidney tubules23,24.\n\n\nConclusion\n\nThe present paper proposes that the gastric H, K-ATPase, in addition to its well known role as a proton pump, may also act as a provisional Na-pump and a Ca-pump in the parietal cells where the HAF plays a critical role. Such altered modes demand immediate attention for a fresher look at the NaAF-regulated Na, K-ATPase system in various tissues. This is particularly critical for the central nervous system. The human brain, which weights only three pounds (about 2% of total body weight), consumes almost 25% of the total energy (ATP); thus it would be expected that the NaAF would play a major role in brain metabolism and function.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported in part by a grant from NIH, AM 19531. The author was a recipient of the NIH Research Career Development Award (AM00623) during the course of this investigation at SUNY-Upstate Medical Center, Syracuse, NY, USA.\n\n\nReferences\n\nRay TK, Nandi J: K+-stimulated p-nitrophenyl phosphatase is not a partial reaction of the gastric (H++ K+)-transporting ATPase. Evidence supporting a new model for the univalent-cation-transporting ATPase systems. Biochem J. 1986; 233(1): 231–238. PubMed Abstract | Free Full Text\n\nNandi J, Das PK, Levine RA, et al.: Half of the (Na++ K+)-transporting-ATPase associated K+-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior. Biochem J. 1988; 252(1): 29–34. PubMed Abstract | Free Full Text\n\nRay TK, Das PK, Zinkievich M, et al.: Dual topology of the gastric proton pump: A bidirectional molecular motor for the simultaneous uphill transport of H and K. in Gastrointestinal Disorders: Symptoms, Treatment and Prevention, Nova Science Publications, N. Y. (Editors, M. Battik and N. Grimaldi). 2012; pp. 175–200. Reference Source\n\nDavenport HW: Effect of ouabain on acid secretion and electrolyte content of frog gastric mucosa. Proc Soc Exp Biol Med. 1962; 110(3): 613–615. PubMed Abstract | Publisher Full Text\n\nRay TK, Nandi J: Modulation of gastric H+, K+-transporting ATPase function by Sodium. FEBS Lett. 1985; 185(1): 24–28. PubMed Abstract | Publisher Full Text\n\nRay TK, Nandi J: Calcium Signalling is pivotal to P2-ATPase function --- Submitted to Biochemistry. 2013.\n\nRay TK: Gastric K+-Stimulated Adenosine Triphosphatase: Demonstration of an Endogenous Activator. FEBS Lett. 1978; 92(1): 49–52. PubMed Abstract | Publisher Full Text\n\nBandopadhyay S, Ray TK: Purification and characterization of a cytosolic activator protein for the gastric H+, K+-ATPase system from dog fundic mucosa. Prep Biochem. 1986; 16(1): 21–32. PubMed Abstract | Publisher Full Text\n\nBandopadhyay S, Das PK, Wright MV, et al.: Characteristics of a pure endogenous activator of the gastric H+, K+-ATPase system: Evaluation of its role as a possible intracellular regulator. J Biol Chem. 1987; 262(12): 5664–5670. PubMed Abstract\n\nRay TK, Bandyopadhyay S, Das PK: Regulation of the gastric H+, K+-transporting ATPase by a cytosolic 80 kDa activator protein and calcium: Ca2+ as a possible physiological switch. Indian J Biochem Biophys. 1988; 25(1–2): 43–51. PubMed Abstract\n\nRay TK, Bandopadhyay S, Das PK: Endogenous activator regulation of the gastric H+, K+-ATPase: Studies with pure preparations. Annals NY Acad of Sci. 1988; 529: 88–91. Publisher Full Text\n\nBandopadhyay S, Ray TK: Purification and characterization of a cytosolic activator protein for the gastric H+, K+-ATPase system from dog fundic mucosa. Prep Biochem. 1986; 16(1): 21–32. PubMed Abstract | Publisher Full Text\n\nRay TK, Chakrabarti R: Regulation of active transport of monovalent cation across the animal cell plasma membranes by cytosolic regulatory proteins. Indian J Biochem Biophys. 1988; 25(3): 219–225. PubMed Abstract\n\nSen PC, Ray TK: Control of the potassium ion-stimulated adenosine triphosphatase of pig gastric microsomes: Effects of lipid environment and the endogenous activator. Arch Biochem Biophys. 1980; 202(1): 8–17. PubMed Abstract | Publisher Full Text\n\nNandi J, Wright MV, Ray TK: Effects of phospholipase A2 on gastric microsomal H+, K+-ATPase system: Role of “boundary lipids” and the endogenous activator protein. Biochemistry. 1983; 22(25): 5814–5821. PubMed Abstract | Publisher Full Text\n\nRay TK, Nandi J: Regulation of the gastric (H++, K+)-transporting ATPase system by the endogenous activator. Effects of phospholipase A2 treatment. Biochem J. 1983; 212(3): 887–890. PubMed Abstract | Free Full Text\n\nRay TK, Das PK, Nandi J, et al.: Characteristics of the isolated apical plasmalemma and intracellular tubulovesicles of the gastric acid secreting cells: Demonstration of secretagogue-induced membrane mobilization. Biochemistry. 1988; 27(25): 8958–8968. PubMed Abstract | Publisher Full Text\n\nRay TK, Bandopadhyay S, Das PK: Regulation of the gastric H+, K+-transporting ATPase by a cytosolic 80 kDa activator protein and calcium: Ca2+ as a possible physiological switch. Indian J Biochem Biophys. 1988; 25(1–2): 43–51. PubMed Abstract\n\nCaroppo R, Gerbino A, Debellis L, et al.: Asymmetrical, agonist-induced fluctuations in local extracellular [Ca(2+)] in intact polarized epithelia. EMBO J. 2001; 20(22): 6316–6326. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerez-Zoghbi JF, Mayora A, Ruiz MC, et al.: Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i. Am J Physiol Gastrointest Liver Physiol. 2008; 295(4): G671–G681. PubMed Abstract | Publisher Full Text\n\nPerez JF, Ruiz MC, Michelangeli F: Simultaneous measurement and imaging of intracellular Ca(2+) and H(+) transport in isolated rabbit gastric glands. J Physiol. 2001; 537(Pt 3): 735–745. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDas PK, Chakrabarti R, Bandopadhyay S, et al.: Demonstration of an endogenous activator for the Na+, K(+)-ATPase system. Mol Cell Biochem. 1989; 91(1–2): 123–129. PubMed Abstract | Publisher Full Text\n\nRajendran VM, Sangan P, Geibel J, et al.: Ouabain sensitive H,K-ATPase functions as Na,K-ATPase in apical membrane of rat distal colon. J Biol Chem. 2000; 275(17): 13035–13040. PubMed Abstract | Publisher Full Text\n\nCougnon M, Bouyer P, Planelles G, et al.: Does the colonic H,K-ATPase also act as an Na,K-ATPase? Proc Natl Acad Sci U S A. 1998; 95(11): 6516–6520. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1281",
"date": "05 Aug 2013",
"name": "Silvana Curci",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTushar Ray here provides a review of his work on the possible multiple roles of gastric H/K ATPase. Ray discusses the potentially interesting role of an endogenous activator of the H/K-ATPase, identified in the last few years by his research group. Unfortunately I did not have the chance to read the recent study (submitted) where it was found that an “anti-activator antibody blocks acid secretion in histamine-stimulated glands”. Thus I am unable to really comment on this relevant point.I was a bit confused by the title, expecting to read about “altered states” in which the H/K ATPase would assume multiple roles. Also it would be nice if the author could comment on the possibility that the function and activity of H/K ATPase and of the Na/K ATPase would vary depending on the specific location of the parietal cells in the gland (i.e. more luminal or more basal), see Fujii et al. (2008) (JBC 2008. 283, 6869-6877).Minor comments: a couple of typos at page 3: 2nd paragraph “of the functional dual topology H,K ATPase”; 4th paragraph “The APM showed very high basal( Ca2+….)”.",
"responses": [
{
"c_id": "556",
"date": "13 Sep 2013",
"name": "Tushar Ray",
"role": "Author Response",
"response": "We studied the role of the HAF in proton production using a monospecific anti-HAF antibody raised in female rabbits against a pure preparation of the HAF. The histamine-stimulated acid secretion (as measured by 14C-aminopyrene uptake) in isolated rabbit gastric glands was eliminated by the anti-HAF-antibody proving the essential role of the HAF in the production of protons at the catalytic domain inside parietal cells (DOI: 10.5281/zenodo.7093). Following the excellent suggestion of Dr. Silvana Curci, I have changed the title which now reads as. “The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states”The issue of variable gastric acid secretion depending on location of the parietal cells in a gland is an important one, and I thank the reviewer for raising this issue. Since the parietal cells at the base of the gland secrete very little acid and are known to remain practically unaffected by gastric ulcers, one would expect to see a marked reduction both in the total number of H-pumps as well as in the turnover of the existing pumps. Also, one would expect to see a proportionate reduction in the altered function of the H, K-ATPase as provisional Na-pumps in these base glandular cells. Since the activity and function of both H- and Na-pumps rely on the cytosolic HAF, a noticeable change is expected to occur at the level of HAF in these cells. Thus a comparison in the activity and turnover of the HAF in parietal cells nearest to the secretary canneliculi of gastric glands compared to those farthest at the glandular base needs to be investigated.Pondering over the turnover of HAF in parietal cells I recalled some novelty of the HAF which now appears relevant. The HAF was previously demonstrated to possess high (NH2OH-insensitive) protein-kinase activity by its ability to phosphorylate histone, but at the same time HAF is not auto-phosphorylated, and cannot be phosphorylated by heart protein kinase from Sigma (reference 13, DOI: 10.5281/zenodo.7095 ). This rare ability to phosphorylate histone suggests that the HAF is capable of regulating its own intracellular level by directing gene expression, thus raising the possibility of a similar auto-regulation by the NaAF in other cell types. This aspect has now been discussed in the revised version."
}
]
},
{
"id": "1466",
"date": "21 Aug 2013",
"name": "Gabrielle Planelles",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reflects the opinion that the gastric H,K-ATPase may transport other cations than potassium and protons, i.e., may transport sodium or calcium ions. According to the author, these different transport modes mostly depend on the local cytosolic composition (Na+, pH, and Ca2+), and on a cytosolic factor that acts in a dual manner on the pump activity. This interesting view of the mechanisms of gastric secretion is based on previous results that have been obtained by T. Ray's group. However, I would appreciate that the author further discuss the specificity of the reported assays, the purity of the preparations (apical and tubulovesicular membranes), and the possibility that other, physiologically quiescent, P-ATPases or channels may obscure the interpretation. Also, I feel that the title sounds different than the ms content, and that a change has to be considered.",
"responses": [
{
"c_id": "555",
"date": "13 Sep 2013",
"name": "Tushar Ray",
"role": "Author Response",
"response": "Following the suggestions of Dr. Gabrielle Planelle the conditions used for monitoring the effects of HAF and µM Ca on gastric H, K-ATPase activity associated with the APM and TV have now been specified. The purity and characteristic features of the isolated APM and TV membranes used in the study have also been provided. However, the possibility of interference under the conditions of our assay seems very unlikely. Also, following the excellent suggestion of the reviewer I have changed the title of the paper which now reads as, “The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states”"
}
]
},
{
"id": "1468",
"date": "21 Aug 2013",
"name": "John Geibel",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary presents an interesting yet highly controversial view of acid secretion. The literature cited is very limited and is predominantly from the author’s previous work. My second point is that there are factual errors. There is no evidence for Calcium being pumped into the lumen of the gastric gland. The paper the author cites is measuring intracellular Ca not efflux of Ca into the lumen. In that study there are changes in intracellular Ca associated with carbachol that are typical for Ca activated H,K stimulated acid secretion. The critical studies have not been preformed that are necessary to prove the theory; namely block the H,K with omeprazole, stimulate the cells with increased intracellular Calcium (thapsigargin, carbachol, etc) and show measurements of luminal Ca concentration changes. As it stands now there are small pieces of unconnected data that do not give a convincing argument for this paper.",
"responses": [
{
"c_id": "554",
"date": "13 Sep 2013",
"name": "Tushar Ray",
"role": "Author Response",
"response": "The reason for such (seemingly odd) citations as pointed out by the reviewer is to clarify to the reader various important facets of the HAF in P-2 ATPase regulation within the parietal cells. To my knowledge no other laboratory came up with any publication either supporting or refuting our work so far which I could refer to. However, I will be most happy to be corrected by the reviewer if I am wrong.Contrary to Dr. Giebel’s second statement on luminal Ca-transient, Caroppo et al (EMBO J. 20, 6316-6326, 2001) directly measured the Ca-concentration using Ca-selective microelectrodes and reported the carbachol-induced Ca-transients in the gastric lumen."
}
]
}
] | 1
|
https://f1000research.com/articles/2-165
|
https://f1000research.com/articles/2-21/v1
|
23 Jan 13
|
{
"type": "Research Article",
"title": "Correlation between miRNA-targeting-specific promotermethylation and miRNA regulation of target genes",
"authors": [
"Y-h Taguchi"
],
"abstract": "Background miRNA regulation of target genes and promoter methylation were known to be the primary mechanisms underlying the epigenetic regulation of gene expression. However, how these two processes cooperatively regulate gene expression has not been extensively studied. Methods Gene expression and promoter methylation profiles of 271 distinct human cell lines were obtained from gene expression omnibus. P-values that describe both miRNA-targeting-specific promoter methylation and miRNA regulation of target genes were computed with the MiRaGE method proposed recently by the author. Results We found that promoter methylation was miRNA-targeting-specific. In other words, changes in promoter methylation were associated with miRNA binding at target genes. It was also found that miRNA-targeting-specific promoter hypomethylation was related to miRNA regulation; the genes with miRNA-targeting-specific promoter hypomethylation were downregulated during cell senescence and upregulated during cellulardierentiation. Promoter hypomethylation was especially enhanced for genes targeted by miR-548 miRNAs, which are non-conserved, and primate-specific miRNAs that are typically expressed at lower levels than the frequently investigated conserved miRNAs. Conclusions It was found that promoter methylation was affected by miRNA targeting. Furthermore, miRNA-targeting-specific promoter hypomethylation was suggested to facilitate gene regulation by miRNAs that are not strongly expressed (e.g., miR-548 miRNAs).",
"keywords": [
"cell lines",
"methylation"
],
"content": "Introduction\n\nThe epigenetic regulation of gene expression1 has recently attracted the interests of many researchers. The mechanism is known to regulate gene expression without modifying DNA sequences. Some examples of epigenetic regulation include promoter methylation2, histone modification3, the binding of transcription factors to gene promoter regions4, and miRNA regulation of target genes5.\n\nAmong them, the promoter methylation and the miRNA regulation of target genes are particularly important in the epigenetic regulation of gene expression. Promoter methylation is relatively stable, long term, and in some cases, heritable. It is generally believed that genes with hypermethylated promoters are repressed. In addition, there is mounting evidence that DNA methylation is involved in the development and progression of certain disease states. For example, aberrant methylation in cancer is frequently observed6 and the distinct patterns of promoter methylation between monozygotic (MZ) twin pairs have also been found to result in different health conditions7. In contrast to DNA methylation, miRNA regulation of target genes is more flexible and rapidly changing. miRNA expression can change even during cellular differentiation8. miRNA expression is often tissue-specific, and similar to DNA methylation, miRNA expression has been linked to human disease9. Thus, although miRNA-directed gene regulation is thought to result in subtle changes, it is generally believed that miRNAs are involved in many important biological processes ranging from cell division to aging.\n\nAlthough DNA methylation of miRNA promoters has been studied extensively (e.g., in accordance with tumor formation10), the relationship between promoter methylation and miRNA regulation of target genes has not been thoroughly investigated. One likely reason for this is that the regulation of gene expression by promoter methylation is a form of pre-transcriptional control, whereas miRNA regulation of target genes is a form of post-transcriptional control, with the former taking place inside the nucleus and the latter outside the nucleus (cytoplasm). Thus, these two mechanisms are separated by both time and space, and as a result, to date, there have not been plausible biological reasons to suspect that promoter methylation and miRNA-mediated gene regulation operate in concert.\n\nHowever, Su et al.11 recently found that miRNAs have a tendency to target genes with hypomethylated promoters. To my knowledge, their study was the first report suggesting cooperative regulation of gene expression by promoter methylation and miRNAs. In addition, Sinha et al. reported that genes promoters with high CpG content were more often targeted by miRNAs12. Saito and Sætrom also dicussed the relationship between miRNA-mediated gene regulation and various features of target genes, but they did not consider the methylation status of target genes promoters13. Although the study of Su et al represents the first evidence of a direct link between promoter methylation and miRNA regulation, the biological significance of their findings is not clear. In this study, I report that promoter methylation is miRNA-targeting-specific; that is, the amount of methylation observed at a given gene promoter is dependent on whether that gene is also a target of miRNA regulation. Furthermore, the miRNA-targeting-specific promoter methylation is also related to how miRNAs regulate target gene expression. Especially, I find that miR-548 miRNAs target genes with highly hypomethylated promoters.\n\n\nMethods\n\nIn this study, I used publically available promoter methylation profiles from various resources, obtained from GEO ID: GSE3065314. This included 283 human promoter methylation profiles for distinct cell lines, ranging from hESC to various somatic samples, measured by HumanMethylation27 BeadChip (Illumina), which provides an efficient approach for surveying genome-wide DNA methylation profiles. The HumanMethylation27 panel targets CpG sites located within the proximal promoter regions of transcription start sites (TSS). Thus, it was suitable for our purpose. Promoter methylation profiles (GEO ID: GSE30653) also included data from both IMR90 and MRC5 cell lines, which were used to investigate relationships between promoter methylation and previously reported miRNA regulation and miRNA expression profile data15,16. Promoter methylation profiles in both BG02 and BG03 were also included in this study, and were compared to miRNA regulation and miRNA expression profile data (see below).\n\nAdditional promoter methylation profiles in IMR90 cell lines were obtained from GEO ID: GSM86800814, GEO ID: GSM73994017, and GEO ID: GSM37544218. They were compared to IMR90 promoter methylation profiles, GEO ID: GSM760387 within GEO ID: GSE30653. I also used promoter methylation profiles for IMR90 from GEO ID: GSE31848, which includes GSM868008; these data were generated using the Illumina HumanMethylation450 BeadChip. This BeadChip allowed us to interrogate > 485000 methylation sites per sample at single-nucleotide resolution. In addition, because this array also includes CpG sites outside of promoter regions, I restricted probes to a subset labeled as either TSS200 or TSS1500. Data from GSM739940 includes IMR90 promoter methylation profiles measured by the Illumina HumanMethylation27 BeadChip. However, because these data were generated by a different research group than that of GSE30653, I tested profiles from this dataset to confirm that obtained results were not research group dependent. Finally, I also used methylation profile data from GSM375442, which was generated using next generation sequencing (NGS). CpG methylation profiles from promoter regions were extracted using Bismark Software (Ver. 0.7.4)19 (see below); promoter regions were defined as nucleotide positions between -200 and +1200 basepairs (bp) from the transcription start site (TSS).\n\nIn order to compare miRNA-targeting-specific promoter methylation with target gene miRNA regulation and miRNA expression profile data from BG02 and BG03 cell lines, both miRNA and mRNA profiles were obtained from GEO ID: GSE1447320. Gene (mRNA) expression profiles of undifferentiated and differentiated BG02 and BG03 cell lines were obtained from the GEO IDs GSM551204 and GSM551206, and GSM551216 and GSM551218, respectively. Corresponding miRNA expression profiles of these two cell lines were obtained from the GEO IDs GSM361147 and GSM361271 (BG02) and GSM361288 and GSM361289 (BG03). Raw data files were downloaded from the site for further analysis and were normalized so as to have a mean of 0 and a variance of 1.\n\nIn order to infer miRNA-targeting-specific promoter methylation, I employed the MiRaGE method21 (see below). The MiRaGE method, which was implemented on a public domain MiRaGE server and Bioconductor MiRaGE package, was first used to infer miRNA regulation of target genes. This software was designed to infer the differential expression of miRNA target genes between two experimental conditions based on the expression profiles of the target genes in question; however, in this study, I used this method to infer miRNA-targeting-specific promoter methylation by substituting gene expression profiles with the promoter methylation profiles of each gene.\n\nI first prepared a control dataset (pseudo) in which the expression level of all genes assigned a value of 1. Then, the amount of methylation at each gene was used as values of treatment data set. Although, usually, the ratio of number of methylated sites to the total number of methylated and non-methylated sites is used to describe promoter methylation levels, I employed a method in which total methylation values were used. I did this because I found that P-values computed when using methylation data were more strongly correlated to the P-values calculated from target gene miRNA regulation data (see below), which is likely due to the fact that the frequency of CpGs is also related to miRNA targeting12; i.e., genes with promoters that contain more CpGs were more often targeted by miRNAs as mentioned above. Using this procedure, I attributed two P-values to each miRNA, one expressing the degree of promoter hypermethylation, and the other expressing the degree of promoter hypomethylation. For this purpose, depending on the methodology and/or type of deposited data set, I computed P-values that describe promoter methylation as follows:\n\nIn this case, the promoter methylation profiles used to replace \"gene expression\" values within the MiRaGE method were\n\n\n\n\n\nwhere M0i represents the scaled values of signal_B (intensity estimated of methylated DNA), which are expressed as the amount of promoter methylation of ith gene,\n\n\n\n\n\n\n\nwhere N is the total number of genes considered and Mi is the raw value of signal_B. This signifies that the amount of promoter methylation is scaled so as to have a mean 〈M0i〉 of zero and standard deviation σM0i of 1. exp is applied in this instance because I want to consider the amount of methylation rather than the ratio of the amount of promoter methylation. Because P-values are computed after the pair of input values are transformed to a logarithmic ratio, by substituting 1 in the control dataset and an exponential value in the treatment dataset based on raw values results in the usage of raw values as differential expression/promoter methylation (see below).\n\nIn this case, the promoter methylation profiles used to replace \"gene expression\" values within the MiRaGE method were\n\n\n\n\n\nwhere Ci takes 1 only when Mi = 0; otherwise, it takes on 0, so as to avoid infinite values after transformation to the logarithmic ratio.\n\nIn this case, the promoter methylation profiles used to replace \"gene expression\" values within the MiRaGE method were\n\n\n\n\n\nwhere βi is the ratio of methylated sites to unmethylated sites,\n\n\n\nwhere Ui is the signal from unmethylated sites (signal_A) and C is the regulation constant, which typically takes on the value of 100. Since only β values were deposited in the public datasets used, we could not avoid using them; however, the correlation with target gene miRNA regulation was substantially decreased. An explanation for this is noted above.\n\nIn this case, the promoter methylation profiles used to replace \"gene expression\" values within the MiRaGE method were\n\n\n\n\n\nwhere max(Mi) is the maximum value of Mi and Mi is computed in this case as follows:\n\n\n\nwhere yj, 0 ≤ yj ≤ 100 is the percentage of methylation at site j, which was computed by the Bismark Software19 (see below). The summation was taken over the length of the promoter region as defined above (i.e., between -200 bp and +1200 bp from the TSS).\n\nHere are the command line inputs used for generating the methylation values of CpG sites within the Bismark Software package19.\n\n% bismark_genome_preparation \\\n\n–path_to_bowtie bowtie_dir \\\n\n–verbose ./hg19/ &\n\n% R\n\n>x <- scan(\"GSM375442_CpgMIP-IMR90.seq.txt\",\n\nsep=\"\\n\",what=character(0))\n\n>write.table(file=\"sequence.fa\",\n\npaste(paste(\">p\",1:length(x),sep=\"\"),\n\nx,sep=\"\\n\"),sep=\"\\n\",row.names=F,\n\nquote=F,col.names=F)\n\n>q()\n\n% bismark ,/hg19/ \\\n\n–path_to_bowtie bowtie_dir \\\n\n–bowtie2 -f sequence.fa\n\n% methylation_extractor -s –comprehensive \\\n\nsequence.fa_bt2_bismark.sam\n\n% genome_methylation_bismark2bedGraph_v3.pl \\\n\nCpG_content_sequence.fa_bt2_bismark.sam.txt \\\n\n> sequence.fa_bt2_bismark.sam.bed\n\nwhere bowtie_dir is the directory where bowtie2 is installed. We also assumed that R22 was installed but the part executed by R can be perfomed by any other alternative script languages. GSM375442_CpgMIP-IMR90.seq.txt is a file downloaded from GEO and sequence.fa_bt2_bismark.sam.bed includes methylation percentages of each CpG sites, yj, which was explained above.\n\nThe inference of miRNA-targeting-specific promoter methylation/miRNA regulation of target genes was carried out using the MiRaGE method, which has been described previously21.\n\nAlthough the MiRaGE method is typically used for datasets with two experimental conditions, each of which contains more than one replicate, I used this method for instances in which each condition consisted of only a single replicate. In each case, I based our analysis on the premise that for a given gene i, there were a pair of gene expression datasets or promoter methylation profile datasets, which were measured under a control condition xcontrol,i and a treatment condition xtreat,i. From this, I computed the logarithmic ratio\n\n\n\nIn such an example, when a difference between raw values is favorable, exponential values exp(xi) can be used instead of xi, in which case I get\n\n\n\nWhen computing P-values that reject the null hypothesis by using the alternative hypothesis that Δxis of the target genes of the miRNA m are less (greater) than those that are off-target but form a target of any other miRNAs, I compute\n\n\n\nwhere P[A < (>)B] is P-values computed by statistical tests when two set A and B are compared. The tests implemented within the MiRaGE Server/package are the t-test, Wilcoxon rank sum test, and Kolmogorov-Smironov test.\n\nThus, judging A < (>)B depends upon the selected statistical test used. Gm represents the set of genes targeted by miRNA m and G’m is the intersection of the set of off-target genes of miRNA m and the set of target genes of all other miRNAs. It should be noted that genes that were not the targets of any miRNAs were totally excluded from the analysis; however, all miRNAs were considered and no exclusions based on conservation were applied. When inferring promoter methylation, xcontrol,i = 1 and xtreat,i were used to represent the amount of promoter methylation. When inferring miRNA regulation of target genes during cell senescence in IMR90 and MRC5 cell lines, xcontrol,i was used to represent gene expression of young cell line and xtreat,i was used to represent gene expression in senescent cell lines. When inferring regulation of target genes during differentiation in BG02 and BG03 cell lines, xcontrol,i was used to represent gene expression in undifferentiated cell lines and xtreat,i for gene expression in differentiated cell lines.\n\nI have used two types of P-values, P<(>)m:methy, which corresponded to the miRNA-targeting-specific promoter methylation, and P<(>)m:regul which corresponded to the miRNA regulation of target genes of miRNA m. When P<(>)m:methy[regul] is small enough, the target genes of miRNA m are significantly hypermethylated or hypomethylated and thus downregulated or upregulated. In order to see if these two types of P-values were correlated, I computed various correlation coefficients:\n\n\n\n\n\n\n\nand accompanied P-values to reject null hypothesis that ρ = 0 by using the alternative hypothesis that ρ ≠ 0. ρ[a, b] is the Pearson’s correlation coefficients between a and b and rank(xm) is the rank order of xm among {xm}. Pm:z where z ∈ {methyl, regul} is either P<m, P>m, 1 – P<m or 1 – P>m. Thus, there are 4 × 4 = 16 possible combinations of Pm:methy and Pm:regul. ρs of Kolmogorov-Smirnov test and ρPearsonlogs for all tests can change when P<(>) is replaced with 1 – P>(<) because P<(>) ≠ 1 – P>(<) for Kolmogorov-Smirnov test and log(P<(>)) = log(1 – P>(<)) for all tests. Thus, optimal combinations with the maximum absolute correlation coefficients were employed.\n\nIn contrast to the cell senescence study15 in which miRNA expression was investigated by NGS, only microarray measurements were available for differentiation in BG02 and BG03 cell lines. Due to issues related to the accuracy and quality of microarray data and the relatively small amount of miRNA expression, very few miRNAs were found to be differentially expressed between undifferentiated and differentiated cell lines. Thus, miRNAs differentially expressed between undifferentiated and differentiated cell lines were selected based on two criteria before being subjected to further analyses;\n\nAbsolute differential expression |xdiff,i – xundiff,i|> Δxc, where xdiff,i and xundiff,i are the normalized expression of gene i of differentiated and undifferentiated cell lines, respectively. Δxc is the threshold value that can be used to select genes associated with significant differential expression during differentiation.\n\nFor this method, it was required that adjusted P-values based on the BH criterion23 were less than 0.05 for significant upregulation or downregulation. Here, P-values were computed using a t test between two sets of probe values attributed to gene i.\n\nAfter Δxc was suitably selected, the correlation coefficient between differential expression xdiff,i – xundiff,i and log (P<m:regul) were computed. Positive values were taken to indicate a reciprocal relationship between miRNA expression and miRNA regulation of target genes; smaller P<m:regul were assumed to signify that target genes were upregulated (and vice versa); thus, a reciprocal relationship required that miRNA should be downregulated, i.e., xdiff,i – xundiff,i < (>)0, which should result in a positive correlation between xdiff,i – xundiff,i and log (P<m:regul). Since miRNA names in GSE14473 were old (miRBase, release 9.1), miRNA names were converted to miRNA names used in the present version of the MiRaGE software package (miRBase release 18) and by the miRConverter implemented in miRSystem24.\n\nmiRNAs were ranked based on P>m in each of 283 samples in GSE30653, after excluding 12 control samples. Each miRNA was ranked based within each sample, and then, the cumulative rank was determined for 271 samples.\n\nmiRNAs were ranked based on P-values for either downregulation during cell senescence (IMR90 and MRC5) or upregulation during differentiation (BG02 and BG03) for each statistical test. Then, each miRNA was ranked based on order summed up over three statistical tests.\n\n\nResults and discussion\n\nBased on the inference from the data produced by the MiRaGE method21 (see Methods), the promoters of genes that are targets of 70–90% of human miRNAs were significantly hypomethylated, dependent on the statistical tests used, and the definition of promoter methylation levels: the β-value or the amount of methylation (see Table 1 and Additional files). This finding was consistent with conclusions made by Su et al.11, who stated that miRNAs had a tendency to target genes with hypomethylated promoters. Given that promoter methylation patterns do not change drastically between certain cell types, the amount of miRNA-targeting-specific promoter methylation is also highly cell-type-independent. Mean correlation coefficients of P>m range between 0.8 and 0.9, again depending on the statistical test used and the definition of promoter methylation levels (Table 2).\n\nThe summation of the number of miRNAs that target genes with significantly hypomethylated promoters in each cell line. The total numbers of miRNAs and cell lines were 1921 and 271, respectively (the number of cell lines was out of 283 in GSE30653, excluding 12 control samples). Thus, the total number of combinations of an miRNA and a cell line is 1921 × 271 = 520591. Promoter methylation levels were defined as either the amount of methylation or the β-values. Observed P-values based on the BH criterion23, P>m of < 0.05 were used to signify the promoters of the genes targeted by miRNAs that significantly hypomethylated. P-values were adjusted using the p.adjust function in R22. A complete list of raw (not adjusted) P-values is available in additional file 1. Cell line names can be also identified as column names of the additional file 1.\n\nMean Pearson’s correlation coefficients of P>m between pairs of cell lines.\n\nIn order to see if miRNA-targeting-specific promoter methylation was genuinely related to miRNA regulation of target genes, I compared P-values of miRNA-targeting-specific promoter methylation to P-values of miRNA regulation of target genes during the senescence of IMR90 and MRC5 cell lines15,16 and during the differentiation of BG02 and BG03 cell lines20. It was clear that promoter methylation and miRNA regulation of target genes were significantly correlated during both cell senescence and differentiation (Table 3), despite the fact that correlation coefficients exhibited opposite directionalities in cell senescence and differentiation. This means that genes with miRNA-targeting-specific promoter hypomethylation are downregulated during cell senescence, but upregulated during differentiation.\n\nVarious correlation coefficients between P-values of promoter methylation and regulation of target genes in IMR90 and MRC5 during cell senescence and in BG02 and BG03 during differentiation; P-values associated with the correlation coefficients are indicated. P>ms were employed for cell senescence while P>ms of promoter methylation and 1 – P>m of regulation of target genes were employed for differentiation as shown, as these combinations exhibited the most significant correlations. Genes with miRNA-targeting-specific promoter hypomethylation were recognized to be downregulated during cell senescence based on positive correlations and upregulated during differentiation based on negative correlations (for details, see Methods).\n\n* Indicates < 2.2 × 10-16.\n\nTo confirm that the significant correlation noted above between miRNA-targeting-specific promoter methylation and miRNA regulation of target genes was not a false-positive finding, I also tested for this association in IMR90 cell lines by using a different microarray and, NGS approach, as described by another group of researchers18 (Table 4). Again, I observed a significant correlation between promoter methylation status and miRNA regulation, suggesting that the relationship found between these two mechanisms was independent of experimental conditions.\n\nCorrelation coefficients between miRNA-targeting-specific promoter methylation and miRNA regulation of target genes during cell senescence shown in Table 3 were evaluated by a comparison with the results produced by different array design (GSM868008), distinct research group, alternative measures of promoter methylation (GSM739940), and NGS (GSM375442). Independent of these factors, miRNA-targeting-specific promoter methylation was found to be always positively correlated to miRNA regulation of target genes (i.e., genes with miRNA-targeting-specific promoter hypomethylation were downregulated during cell senescence).\n\nIn addition to these analyses, I also confirmed a significant reciprocal relationship between miRNA expression and target gene expression. Because it is usually believed that miRNAs downregulate target gene expression, downregulated genes should be targeted by upregulated miRNAs and vice versa. In fact, the reciprocal relationship during the senescence process of IMR90 cells has been reported15,16. In this study, I observed a similar reciprocal relationship during the differentiation of BG02 and BG03 cells20 (Table 5).\n\nThe reciprocal relationship between target gene regulation by miRNA and miRNA expression is evaluated in BG02 and BG03 cell lines during differentiation. Given that only a limited number of miRNAs are differentially expressed during differentiation, miRNAs were screened as described in the Methods section before computing correlation. Then the correlation coefficients between differential expression of miRNA and logarithmic value of P-values of miRNA regulation of target genes were computed A positive correlation implied the reciprocal relationship, that is, if the target genes are expressive, then, the miRNA itself should be suppressive. (for details, see Methods).\n\nThus, I conclude that miRNA-targeting-specific promoter methylation is not an artifact, but a genuine biological process. One may doubt this conclusion given the finding that miRNA-targeting-specific promoter methylation is cell-line-independent and miRNAs are believed to regulate target gene expression in a cell-line-dependent manner. Although this implies an apparent discrepancy, the correlation coefficients obtained, which were at most 0.7 to 0.8, indicate that miRNA-targeting-specific promoter methylation governs at most 50% to 60% of miRNA regulation of target genes. Thus, cell line specific miRNA regulation of target genes can act within the remaining 50% to 40%. In actuality, as can be seen in Table 3, the correlations between miRNA-targeting-specific promoter methylation and miRNA regulation of target genes have opposite signs dependent on the biological processes being considered. Therefore, the fact that cell-line-independent and miRNA-targeting-specific promoter methylation partially governs cell line dependent miRNA regulation of target genes is not a discrepancy.\n\nIn order to better understand the biological significance of our findings regarding miRNA-targeting-specific promoter methylation, I sought to identify the specific miRNAs that target genes with hypomethylated promoters. I found that most gene promoters targeted by miR-548 miRNAs were highly hypomethylated (Table 6; here, I define miRNAs called \"miR-548\" as miR-548 miRNAs). At present, little is known about miR-548 miRNAs, which likely results from the fact that miR-548 miRNAs are primate-specific miRNAs (ps-miRNAs). Because of this, ps-miRNAs were not expected to play critical roles in fundamental biological processes like cell differentiation and development, and it has so far been widely assumed that such basic biological processes are conserved among mammals. This assumption has resulted in the exclusion of ps-miRNAs from consideration as candidates for basic and important biological processes. Thus, it is thought that the roles of these ps-miRNAs may be limited to primate-specific biological processes. Another reason that miR-548 miRNAs have not been extensively investigated is that they are generally expressed at low levels; miRNAs that play critical roles are thought to be highly expressed. For example, although miRBase stores the amount of short reads detected by next generation sequencing (NGS) for various miRNAs, miR-548 miRNAs have at most 100 short reads; this is in comparison to more abundant miRNAs like those from the let-7 family, which are typically represented by several millions reads. This makes it difficult to detect miR-548 miRNAs using microarray technology or sequencing. Actually, although there have been a few reports noting that miR-548 family members, including miR-548 miRNAs, play critical roles, these data come exclusively from quantitative PCR (qPCR) experiments25,26.\n\n68 miR-548 miRNAs were ranked as described in the Methods (promoter methylation was defined as the amount of methylation). Independent of the statistical methods used for computation of P>m, 43 out of 68 miR-548 miRNAs were ranked within the top-ranked 100 miRNAs. The mean rank of all miR-548 miRNAs is ~200 independent of the statistical method used. Given that the total number of miRNAs considered was 1921, these values are highly significant, P = 2 × 10-35.\n\nIn this study, here I found that promoters of genes targeted by miR-548 miRNAs were hypomethylated. Because it is generally assumed that genes with hypomethylated promoters are expressed, the fact that promoters of target genes of miR-548 miRNAs are hypomethylated may mean that, compared to genes that are targets of other miRNAs, target genes of miR-548 miRNAs are more sensitive to changes in miRNA expression. This could explain why promoters of target genes of non-expressed miR-548 mIRNAs are specifically hypomethylated.\n\nmiR-548 miRNAs represent a large set of miRNAs, with 68 family members; thus, although these miRNAs exhibit low levels of expression, the fact that there are many family members may imply that they have important biological roles. Indeed, it has been reported that potential target genes of miR-548 miRNAs play critical roles in various biological processes27,28. Since it is also suggested that they originate from transposable elements (TEs), miR-548 miRNAs exhibit high sequence similarities while maintaining significant sequence diversity27.\n\nDuring senescence processes of IMR90 and MRC5 cell lines, the target genes of miR-548 miRNAs were significantly downregulated (Table 7), whereas during differentiation of the BG02 cell line, the target genes of miR-548 miRNAs were significantly upregulated (Table 7). The fact that I observed significant changes in expression of target genes without significant changes in expression of miRNAs that target these genes implicates a role miRNA-targeting-specific promoter hypomethylation.\n\nmiRNAs were ranked as described in the Methods. Target genes of miR-548 miRNAs were significantly downregulated during cell senescence (IMR90 and MRC5 cell lines) and upregulrated during differentiation (BG02). Insignificant upregulation of target genes of miR-548 miRNAs in the BG03 cell line was weakly correlated to miRNA expression and miRNA regulation of target genes (Table 5). Given that the total number of miRNAs considered was 1921, the values for IMR90, MRC5, and BG02 cell lines are highly significant.\n\n\nConclusions\n\nFigure 1 schematically summarises the principal findings of this study. To the best of my knowledge, this is the first time that miRNA-targeting-specific promoter methylation in many types of cell lines have been observed (Table 1 and Table 2). miRNA-targeting-specific hypomethylation was correlated (Table 3 and Table 4) with regulation of miRNA target genes, which had a reciprocal relationship with miRNA expression (Table 5); genes with miRNA-targeting-specific hypomethylated promoters were downregulated during cell senescence and upregulated during differentiation. I also found that genes with miRNA-targeting-specific hypomethylated promoters were specific targets of miR-548 miRNAs (Table 6 and Table 7). Regulation of target genes by miR-548 miRNAs, which were typically expressed at low levels are likely the result of miRNA-targeting-specific promoter hypomethylation.\n\nPromoters are methylated in a miRNA-targeting-specific manner (Table 1 and Table 2). Genes with miRNA-targeting-specific hypomethylated promoters are downregulated during cell senescence and upregulated during cellular differentiation (Table 3 and Table 4), whereas miRNAs that target genes with hypomethylated promoters were upregulated during cell senescence17,18 and downregulated during differentiation (Table 5). miR-548 miRNAs, which are expressed at low levels, were suggested to regulate target genes with the assistance of miRNA-targeting-specific promoter hypomethylation (Table 6 and Table 7).\n\n\nList of abbreviations\n\nTSS: transcription start sites;\n\nGEO: gene expression omnibus;\n\nbp: basepair;\n\nmiRNA: microRNA;\n\nps-miRNA: primate-specific miRNAs;\n\nTE: transposable elements;\n\nNGS: next generation sequencing.",
"appendix": "Competing interests\n\nNo relevant competing interests disclosed.\n\n\nAuthor’s contributions\n\nConceived and designed the experiments: YHT. Analyzed the data: YHT. Wrote the paper: YHT.\n\n\nAcknowledgements\n\nThe author thanks the anonymous reviewer16 who suggested the investigation of the promoter methylation of miRNA target genes. This study was supported by KAKENHI (23300357).\n\n\nAdditional files\n\nAdditional file 1 – Full list of P-values from the analysis of miRNA-targeting-specific promoter hypomethylation: t test\n\nt test.xlsx: List of P-values from analysis of promoter hypomethylation of miRNA target genes calculated using the t test based on the amount of methylation.\n\nAdditional file 2 – Full list of P-values from the analysis of miRNA-targeting-specific promoter hypomethylation: Wilcoxon rank sum test\n\nWilcoxon rank sum test.xlsx: List of P-values from the analysis of promoter hypomethylation of miRNA target genes calculated using the Wilcoxon rank sum test based on the amount of methylation.\n\nAdditional file 3 – Full list of P-values from the analysis of miRNA-targeting-specific promoter hypomethylation: Kolmogorov-Smirnov test\n\nKolmogorov Smirnov test.xlsx: List of P-values from the analysis of promoter hypomethylation of miRNA target genes calculated using the Kolmogorov-Smirnov test based on the amount of methylation.\n\nAdditional file 4 – Full list of P-values from the analysis of miRNA-targeting-specific promoter hypomethylation: t test\n\nbeta t test.xlsx: List of P-values from the analysis of promoter hypomethylation of miRNA target genes calculated using the t test based on β-values.\n\nAdditional file 5 – Full list of P-values from the analysis of miRNA-targeting-specific promoter hypomethylation: Wilcoxon rank sum test\n\nbeta Wilcoxon rank sum test.xlsx: List of P-values from the analysis of promoter hypomethylation of miRNA target genes calculated using the Wilcoxon rank sum test based on β-values.\n\nAdditional file 6 – Full list of P-values from the analysis of miRNA-targeting-specific promoter hypomethylation: Kolmogorov-Smirnov test\n\nbeta Kolmgorov Smirnov test.xlsx: List of P-values from the analysis of promoter hypomethylation of miRNA target genes calculated using the Kolmogorov-Smirnov test based on β-values.\n\n\nReferences\n\nGolbabapour S, Abdulla MA, Hajrezaei M, et al:A concise review on epigenetic regulation: insight into molecular mechanisms. Int J Mol Sci 2011, 12(12): 8661–8694.\n\nSuzuki MM, Bird A: DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet. 2008, 9(6): 465–476.\n\nBartova E, Krejci J, Harnicarova A, et al:Histone modifications and nuclear architecture: a review. J Histochem Cytochem. 2008, 56(8): 711–721.\n\nPabo CO, Sauer RT: Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992, 61: 1053–1095.\n\nHe L, Hannon GJ: MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004, 5(7): 522–531.\n\nDas PM, Singal R: DNA methylation and cancer. J Clin Oncol. 2004, 22(22): 4632–4642.\n\nBoks MP, Derks EM, Weisenberger DJ, et al:The relationship of DNA methylation with age, gender and genotype in twins and healthy controls. PLoS One 2009, 4(8): e6767.\n\nBerardi E, Pues M, Thorrez L, et al:microRNAs in ES Cell Differentiation. Am J Physiol Heart Circ Physiol. 2012303(8): H931–9 in press.\n\nEtheridge A, Lee I, Hood L, et al:Extracellular microRNA: A new source of biomarkers. Mutat Res. 2011717(1-2): 85–90.\n\nLopez-Serra P, Esteller M: DNA methylation-associated silencing of tumor-suppressor microRNAs in cancer. Oncogene 2012, 31(13): 1609–1622.\n\nSu Z, Xia J, Zhao Z, et al:Functional complementation between transcriptional methylation regulation and post-transcriptional microRNA regulation in the human genome. BMC Genomics 2011, 12Suppl 5: S15.\n\nSinha S, Adler AS, Field Y, et al:Systematic functional characterization of cis-regulatory motifs in human core promoters. Genome Res. 2008, 18(3): 477–488.\n\nSaito T, Sffitrom P: Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments. Silence 2012, 3: 3.\n\nNazor KL, Altun G, Lynch C, et al:Recurrent variations in DNA methylation in human pluripotent stem cells and their differentiated derivatives. Cell Stem Cell 2012, 10(5): 620–634.\n\nTaguchi YH: Inference of Target Gene Regulation via miRNAs during Cell Senescence by Using the MiRaGE Server. Aging Dis, 2012; 3(4): 301–6 in press.\n\nTaguchi YH: Inference of target gene regulation via miRNAs during cell cenescence by using the MiRaGE Server. In Emerging Intelligent Computing Technology and Applications. Edited by Huang DS, Gupta P, Zhang X, Premaratne P, Springer: Heidelberg 2012: 441–446.\n\nOhm JE, Mali P, Van Neste L, et al:Cancer-related epigenome changes associated with reprogramming to induced pluripotent stem cells. Cancer Res. 2010, 70(19): 7662–7673.\n\nDeng J, Shoemaker R, Xie B, et al:Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol. 2009, 27(4): 353–360.\n\nKrueger F, Andrews SR: Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 2011, 27(11): 1571–1572.\n\nStadler B, Ivanovska I, Mehta K, et al:Characterization of microRNAs involved in embryonic stem cell states. Stem Cells Dev. 2010, 19(7): 935–950.\n\nYoshizawa M, Taguchi YH, Yasuda J, et al:Inference of Gene Regulation via miRNAs During ES Cell Differentiation Using MiRaGE Method. Int J Mol Sci. 2011, 12: 9265–9276.\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria 2012.\n\nBenjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 1995, 57: 289–300.\n\nLu TP, Lee CY, Tsai MH, et al:miRSystem: An Integrated System for Characterizing Enriched Functions and Pathways of MicroRNA Targets. PLoS One 2012, 7(8): e42390.\n\nMarasa BS, Srikantan S, Martindale JL, et al:MicroRNA profiling in human diploid fibroblasts uncovers miR-519 role in replicative senescence. Aging (Albany NY) 2010, 2(6): 333–343.\n\nYang C, Wang C, Chen X, et al:Identification of seven serum microRNAs from a genome-wide serum microRNA expression profile as potential noninvasive biomarkers for malignant astrocytomas. Int J Cancer 2012132(1): 116–27.\n\nLin S, Cheung WK, Chen S, et al:Computational identification and characterization of primate-specific microRNAs in human genome. Comput Biol Chem 2010, 34(4): 232–241.\n\nLiang T, Guo L, Liu C, et al:Genome-wide Analysis of mir-548 Gene Family Reveals Evolutionary and Functional Implication. J Biomed Biotechnol. 20122012: 679563 in press."
}
|
[
{
"id": "748",
"date": "04 Feb 2013",
"name": "Wenqian Hu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reports interesting correlations between the regulation of genes by miRNAs and the methylation status of the miRNA target genes. Although the two processes occur in two different compartments of the cell, these correlations suggest that there may be some potential connections in these two regulatory events of gene expression.",
"responses": []
},
{
"id": "751",
"date": "06 Feb 2013",
"name": "Denis Dupuy",
"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 that the often-used term of ‘miRNA-targeting-specific promoter methylation’ (sic) should be replaced by a less confusing expression, e.g. ‘miRNA-targeted-gene promoter methylation’, as ‘targeting’ and ‘specific’ are redundant and confuse the point. Specifically, the grammatical construction would seem to indicate that there are hypomethylated promoter specific targeting miRNAs (title of a subsection) which doesn't seem to be what the author means. Otherwise, the term ‘specific’ could also lead to believe that there is a dedicated mechanism for demethylation of these subset of (miRNA targeted) promoters that would be distinct from other methylation pathways. As there is no way to know if it is the case or whether there is a unique activity biased toward genes submitted to post transcriptional regulation.Moreover, the use of both: ‘miRNA-targeting-specific promoter hypomethylation’ and ‘miRNA-targeting-specific promoter methylation’ adds to the confusion as these should indicate opposite trends or mechanisms.The author states that; ‘[MiRaGE] was designed to infer the differential expression of miRNA target genes between two experimental conditions based on the expression profiles of the target genes in question’, this is not exactly right: as stated on the MiRaGE page, ‘MiRaGE method is the method used to infer gene expression regulation via miRNA based upon target gene expression.’ Thus, it doesn't infer the expression of target genes! It infers the contribution of miRNA to the measured expression levels. So by replacing the expression levels by the methylation status of the promoter the author is trying to uncover a link between miRNA expression and the methylation of their target genes.I feel that a much needed control would be required to see how the correlation between methylation status and miRNA target gene expression differs from the correlation between non-targeted genes with similar expression levels.Another potentially damning issue is raised in Table 3: ‘It was clear that promoter methylation and miRNA regulation of target genes were significantly correlated during both cell senescence and differentiation’. Another way to interpret this would be that the expression level of those genes is correlated with the methylation level of their promoter (which is well reported in the literature), this could be considered an alternative explanation to the modification of expression levels observed in those genes (i.e. not through miRNA regulation but through chromatin remodeling). This should warrant the use of a control set of non-miRNA targeted genes to check if the promoter methylation is truly ‘specific’ of miRNA targeting. More troubling is this statement: ‘correlation coefficients exhibited opposite directionalities in cell senescence and differentiation. This means that genes with miRNA-targeting-specific promoter hypomethylation are downregulated during cell senescence, but upregulated during differentiation’. It seems that there should be a more in depth discrimination between genes that are up- or downregulated in different conditions. Is the point of this paper to indicate that miRNA downregulation is associated with hypomethylation of the promoter? If so, how do you interpret an inverted correlation in different conditions in this framework? It would seem that the expression of the ‘miRNA target genes’ is more correlated to the status of their promoter than to the presence of the miRNA.The author states that this inverse correlation ‘is not a discrepancy’ but it is actually highlighting the discrepancy that has to be explained rather than brushed off.Similarly the following statement by the author doesn't seem to be so obvious to me: ‘0.7 to 0.8, indicate that miRNA-targeting-specific promoter methylation governs at most 50% to 60% of miRNA regulation of target genes. Thus, cell line specific miRNA regulation of target genes can act within the remaining 50% to 40%.’ An alternate explanation could be that the methylation doesn't govern the expression status of the genes but reflects it partially (i.e. a degraded messenger could trigger an unknown pathway that would lead the modification of the promoter methylation and stabilize the downregulation at the genomic level.A similar logical leap is made further down in the text: ‘The fact that I observed significant changes in expression of target genes without significant changes in expression of miRNAs that target these genes implicates a role miRNA-targeting-specific promoter hypomethylation.’ It could also mean that these genes are regulated transcriptionally without the intervention of miR-548 miRNAs, there are probably other gene groups that are differently expressed in these cell lines.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-21
|
https://f1000research.com/articles/2-181/v1
|
10 Sep 13
|
{
"type": "Observation Article",
"title": "A salp bloom (Tunicata, Thaliacea) along the Apulian coast and in the Otranto Channel between March-May 2013",
"authors": [
"Ferdinando Boero",
"Genuario Belmonte",
"Roberta Bracale",
"Simonetta Fraschetti",
"Stefano Piraino",
"Serena Zampardi",
"Genuario Belmonte",
"Roberta Bracale",
"Simonetta Fraschetti",
"Stefano Piraino",
"Serena Zampardi"
],
"abstract": "Between March-May 2013 a massive Salpa maxima bloom was recorded by a citizen science study along the Ionian and Adriatic coast of the Salento peninsula (Italy). Citizen records were substantiated with field inspections along the coast and during an oceanographic campaign in the Otranto Channel.Salps clogged nets, impairing fishing activities along the coast. Swimmers were scared by the gelatinous appearance of the salps, and thought they were jellyfish. At the end of the bloom the dead bodies of the colonies, that were up to 6-7 m long, were accumulated along the coast and stirred by the waves, forming foams along dozens of kilometers of coast. The bloom also occurred at the Tremiti Islands, north of the Gargano Peninsula. The possible impacts of such events on the functioning of pelagic systems are discussed.",
"keywords": [
"The phylum Chordata is divided into three subphyla",
"the invertebrate Tunicata and Cephalochordata",
"and the vertebrate Vertebrata. The tunicates comprise the popular class Ascidiacea",
"the sea squirts",
"the inconspicuous Appendicularia and Sorberacea",
"and the conspicuous Thaliacea. Thaliaceans are mostly planktonic and are part of gelatinous macrozooplankton (Bone",
"1998)1. They are usually open water animals and are rarely seen from the shore. They are filter feeders and their colonies can become rather big",
"reaching several metres in length. Salps are colonial thaliacea thriving in all oceans",
"their chains of zooids can be very conspicuous."
],
"content": "Observation\n\nThe phylum Chordata is divided into three subphyla, the invertebrate Tunicata and Cephalochordata, and the vertebrate Vertebrata. The tunicates comprise the popular class Ascidiacea, the sea squirts, the inconspicuous Appendicularia and Sorberacea, and the conspicuous Thaliacea. Thaliaceans are mostly planktonic and are part of gelatinous macrozooplankton (Bone, 1998)1. They are usually open water animals and are rarely seen from the shore. They are filter feeders and their colonies can become rather big, reaching several metres in length. Salps are colonial thaliacea thriving in all oceans, their chains of zooids can be very conspicuous.\n\nDuring the spring of 2013, a citizen science study (http://meteomeduse.focus.it/), aimed at documenting the presence of gelatinous plankton in Italian waters (Boero 2013a)2, recorded about sixty sightings of Salpa maxima Forskål, 1775. The first, scattered records of salps arrived from the Apulian coasts in March 2013. Records increased in April and reached a peak in May. In April-May 2013, some of the authors had several chances to document the bloom, from both the coast (FB, RB, SF, SP), and from an oceanographic vessel (GB) during a cruise for the project CoCoNet (http://www.coconet-fp7.eu/), between 8–21 May 2013. In mid-May 2013 the colonies experienced massive degeneration and, in the following days, became stirred and stranded along parts of the Apulian coast where they formed foam belts, each several kilometers long.\n\nFigure 1 shows the distribution of the highest number of recorded salp chains of the citizen science study and of original observations by the authors in the first half of May, 2013. Figure 1 also shows the records of salp chains at stations of the CoCoNet cruise in the Otranto Channel. Dozens of colonies were visible from a single point of observation and, while moving, the same pattern persisted in a rather homogeneous fashion, leading to the perception of a single, large bloom, probably involving several million individuals. The bloom was more apparent from the coast, since the colonies were concentrated by the wind and currents, whereas in the open sea they were more scattered.\n\nCoastal presences from both citizen science records and authors’ own observations were aggregated with the software Fishery Analyst, offshore records derive from onship observations during the CoCoNet Cruise. Yellow lines: trajectories of the main currents.\n\nThe colonies were impressively long (Figure 2) reaching up to 6–7 m in length and were seen by hundreds of people, including fishermen, who alerted the local authorities. At the end of the bloom, in mid-May 2013, the dissolving colonies were seen along the whole coast of the Salento pensinsula (the southernmost part of Apulia) (Figure 3).\n\nPicture by R. Bracale.\n\nPicture by S. Fraschetti.\n\nMerging the citizen science records with our direct observations from the coast and onboard the CoCoNet cruise, it is clear that the bloom was not a localized event, and that it characterized the plankton of a vast body of water for a prolonged period of time. During the same period of the reported bloom, citizens delivered just one salp record from the northern Adriatic and four from the Ligurian Sea, even though citizens sent many records of other gelatinous plankters, mostly jellyfish. The small number of salp records, and the massive presence of other gelatinous plankters along the whole coast of Italy (more than 5000 records from March to August 2013), are rather strong indications that salps were rare elsewhere beyond the Apulian coast. Since salp colonies ranged between 2 and 6–7 m in length, it is rather unlikely that organisms of that size might have passed unnoticed while much less conspicuous gelatinous plankters had been recorded.\n\n\nThe public reaction\n\nMany people thought that salps were jellyfish. Ocean literacy is rather limited, and very few people know what a salp is. People took pictures of them and reports started to appear in the local media, from local televisions to local newspapers and web sites. Eventually, the news reached the national press as well, with mentions in magazines such as Internazionale and Venerdì di Repubblica. A picture of a large colony, taken during a field inspection, became rather popular and was published several times (Figure 2). Fishermen started to complain, because their nets were clogged and they could not operate. They documented the events with pictures, to support their requests of compensation.\n\nThe foam formed at the end of the bloom was at first attributed to a massive malfunction of sewage treatment plants, inexplicably occurring simultaneously in several plants that are independent of each other. The local media covered this event with some alarm. F. Boero made several interventions on local TVs and newspapers, explaining the reason of the appearance of the foam in such a wide area.\n\n\nWhy a salp bloom can be important\n\nSalps are gelatinous filter feeders; they are usually rather rare in Apulian waters, especially in coastal waters, and decades can pass without any salp observations. Records of salps in the scientific literature are rather scant, suggesting a similar pattern of occurrence also elsewhere. On June 5th 1998, F. Boero saw a bloom similar to the present one while crossing the Adriatic sea from Dubrovnick to Bari. During the six-hour journey, salps were constantly present in high densities, forming chains whose estimated length was 5–6 m. No report of that event was ever published. On that occasion, however, the colonies did not reach the coast, and remained off shore, with only relatively low quantities being washed ashore (the majority of them had ended their life cycle while sinking to the bottom).\n\nThe attention the 2013 salp bloom received in the media suggests that it is not likely that events of this magnitude occurred often in the past, since they would have been covered by the media as well. The surprise expressed by lay people and fishermen, indicates that, even if people are used to jellyfish, they are not used to salps, and the reason is simple: salps are rarely encountered in Italian waters. Such conspicuous animals do not pass unnoticed, and if they are around they are seen and recorded.\n\nSalps have extremely high clearing rates and can feed on all sizes of phytoplankton, from viruses to protists, competing with crustacean filter feeders (Bone, 1998)1. The presence of a massive bloom of salps likely depletes phytoplankton production, impairing the phytoplankton-crustacea-fish larvae and juveniles pathway (see Boero et al. 2008)3. The match of fish recruitment with a salp proliferation, could lead to a decrease in fish reproductive success due to food depletion. If this phenomemon was not reported, it could result in fishery scientists who, after a few months, might record anomalies in the age classes of some fish species while being unable to link them to any particular cause.\n\nSince salps tend to disgregate and precipitate as marine snow, they tend to fuel the detritus food chain on the sea bottom. Salp blooms, thus, might redirect the functioning of marine ecosystems in a sudden and dramatic way, leaving little evidence of the cause of changes that, indeed, might become apparent long after the end of the bloom.\n\nAs remarked by Boero (2013 b)4, the availability of records of unusual events such as this salp proliferation, might help us to understand anomalies in the population structure of some species or even anomalies in the functioning of whole ecosystems.\n\n\nPredictability of salp blooms\n\nDeibel and Paffenhöfer (2009)5, whilst analysing a long time series of plankton records, identified a correlation between specific oceanographic conditions and the occurrence of thaliacean blooms, especially in correspondence with eddies, since these currents presumably concentrate salps. The area where the presently recorded salp bloom occurred is, indeed, characterized by a large gyre (Figure 1). The current that flows along the Italian coast, and outflows from the Adriatic into the Ionian Sea is compensated by an inflow current that brings Ionian waters into the Adriatic sea on the East coast of the Adriatic (i.e. Greece, Albania, Montenegro, Bosnia and Croatia). These two opposite currents often generate a gyre in the Southern Adriatic, connecting the Eastern Adriatic coast to the Western one, immediately below the Gargano Peninsula, whereas, in correspondence of the Otranto Channel, a current connects the Italian Adriatic coast to the Albanian coast. The salp bloom, however, was also recorded at the Tremiti Islands, north of the Gargano Peninsula, and along the Ionian coast of the Salento Peninsula (e.g. Porto Cesareo) so outside the southern Adriatic gyre. Unfortunately, this area of the Adriatic is not systematically monitored for gelatinous plankton blooms, so the regularities identified by Deibel and Paffenhöfer (2009)5, which occured along the Atlantic coast of the USA, cannot be searched for in Adriatic historical records. Licandro et al. (2006)6, however, identified some correlations between hydrographic conditions and thaliacean presence along the Mediterranean coast of France, thus showing that the presence of thaliacean blooms is far from rare, under a given set of conditions, in spite of the scantiness of records. Both Licandro et al. (2006)6 and Deibel and Paffenhöfer (2009)5 ascribed the occurrence of thaliacean blooms to the occurrence of a given set of abiotic conditions, disregarding biotic interactions.\n\n\nGelatinous plankton, ecosystem functioning and natural history\n\nThaliacean specialists, as Bone (1998)1 summarized, concur that the ecological role of pelagic tunicates is much underestimated in plankton studies and that their impact on ecosystem functioning is much larger than commonly perceived.\n\nThe functioning of planktonic ecosystems is one of the most important ecological processes on the planet both in terms of oxygen production and as a carbon sink. The whole functioning of what is commonly called “nekton” (e.g. marine vertebrates and large squids), furthermore, depends on phytoplankton primary production and on the efficiency of energy transfer by grazers such as crustaceans or, as in this case, thaliaceans.\n\nThe presences of gelatinous plankton species, either carnivorous (jellyfish and ctenophores) or herbivorous (thaliaceans), commonly occurs in pulses that are almost invariably perceived as episodic and irregular events (Boero et al. 2008)3 and so considered of negligible importance. This perception is simply wrong and, as Ricklefs (2012)7 convincingly showed, the understanding of the functioning of ecosystems would gain much from the practicing of natural history. Regularities, such as the massive presence of crustacean plankton after phytoplankton blooms, do greatly attract ecologists, whereas irregularities such as gelatinous plankton blooms that, usually, are of short duration and might even be overlooked by standard monitorings, tend to be disregarded. If only regularities were responsible of the functioning of ecological systems, however, there would be no change. Irregularities are the motor of change and cannot be considered as negligible freaks, they are, instead, the key for the understanding of change, i.e. of the history of natural systems.",
"appendix": "Author contributions\n\n\n\nF. Boero coordinated the citizen science study and wrote the article\n\nG. Belmonte made the onboard observations during the CoCoNet cruise\n\nR. Bracale made observations along the Adriatic coast and the picture in Figure 2\n\nS. Fraschetti made observations along the Ionian coast and the picture in Figure 3\n\nS. Piraino made observations along the Adriatic and Ionian coast\n\nS. Zampardi collected the citizen science observations, treated the data with the software fishery analyst and produced the map in Figure 1\n\nAll authors critically revised the manuscript and approved it for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThese observations were made while working at a series of projects financed by the European Union and the Italian Ministry of Research (RITMARE, COCONET, VECTORS OF CHANGE, MED-JELLYRISK, PERSEUS, PRIN).\n\n\nAcknowledgements\n\nFabio Tresca produced Figure 1; Paolo D’Ambrosio and Sergio Fai helped in treating citizen science data with Fishery Analyst (http://www.mappamondogis.it/fisheryanalystonline.htm).\n\n\nReferences\n\nBone Q: The biology of pelagic tunicates. Oxford: Oxford University Press, 1998. Reference Source\n\nBoero F: Review of jellyfish blooms in the Mediterranean and Black Sea. GFCM Studies and Reviews. 2013a; 92: 53pp. Reference Source\n\nBoero F, Bouillon J, Gravili C, et al.: Gelatinous plankton: irregularities rule the world (sometimes). Mar Ecol Progr Ser. 2008; 356: 299–310. Publisher Full Text\n\nBoero F: Observational articles: a tool to reconstruct ecological history based on chronicling unusual events [v1; ref status: indexed, http://f1000r.es/1kg] F1000Research. 2013b; 2. : 168. Publisher Full Text\n\nDeibel D, Paffenhöfer GA: Predictability of patches of neritic salps and doliolids (Tunicata, Thaliacea). Journ Plankton Res. 2009; 31: 1571–1579. Publisher Full Text\n\nLicandro P, Ibanez F, Etienne M: Long-term fluctuations (1974–1999) of the salps Thalia democratica and Salpa fusiformis in the Northwestern Mediterranean Sea: relationships with hydroclimatic variability. Limnol Oceanogr. 2006; 51: 1832–1848. Publisher Full Text\n\nRicklefs RE: Naturalists, natural history, and the nature of biological diversity. Am Nat. 2012; 179(4): 423–35. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1771",
"date": "26 Sep 2013",
"name": "Claudia Mills",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSalp blooms are medium-scale oceanographic events that happen normally throughout the world oceans. We can only appreciate the regularity, extent, and significance of these events if there is a record of their occurrence. This paper localizes an extensive salp bloom in time and space along the Apulian coast, and also mentions a single anecdotal observation of an unrecorded bloom in the same area in 1998, giving us perhaps an idea of the rarity of such an event.",
"responses": []
},
{
"id": "1768",
"date": "27 Sep 2013",
"name": "Priscilla Licandro",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and well written article, which reports a large bloom of gelatinous plankton (i.e. salps) observed in Spring 2013 along the Apulian coast (southern Adriatic). Such an exceptional event, which occurred for more than 2 months over a very large region, likely had a significant impact on the functioning of the marine ecosystem of the southern Adriatic, in particular on the proportion of sinking carbon as opposed to the carbon transferred across the pelagic food web. Records of salp blooms are quite scant in the literature. For various reasons gelatinous zooplankton have been often overlooked so that, as rightly mentioned by the authors, blooms such as this have likely occurred but not been reported in the past.\nCitizen science records, used in this study, have the advantage of providing real time information over a large spatial scale, a scale which is not covered by any monitoring program ongoing around the southern Adriatic or in other regions of the Mediterranean. Even though those records are not always rigorous measurements, and should be considered with care, I recommend that the authors provide more information on the range of Salpa maxima abundance observed in Spring 2013. Ranges of abundance (i.e. <10, 10-100, 100-500, 500-1000, >1000) available from the online portal http://meteomeduse.focus.it/ and similar - if not more precise information from the CoCoNet cruise - should be indicated, as they might enable researchers to assess whether S. maxima swarms were evenly distributed inshore and offshore, or whether they were instead concentrated in a particular area. I suggest some amendments in the text (see below for details), to clarify that salps are commonly found in Italian waters (see for instance the time series off Chiavari (Ligurian Sea) in Licandro & Ibanez, 2000 and off Naples (Tyrrhenian Sea) in Mazzocchi et al., 2011), although salp blooms are not regular events. In particular Salpa maxima, as well as other species of salps, had been previously found in the southern Adriatic (see ‘Checklist of the Flora and Fauna in Italian Seas-II’, 2010, Biologia Marina Mediterranea, Vol 17 (suppl.1)). The authors might want to mention that records of salps available in other regions of the southern Adriatic (e.g. Lokrum, the monitoring station off Dubrovnik, where gelatinous plankton has been regularly sampled every month since the mid-1990s) could possibly provide background information, to clarify the recurrence of salp blooms in the southern Adriatic and the environmental conditions associated with those events. Detailed comments:Fig. 1 - I suggest indicating the average range of Salpa maxima abundance using, if necessary, different colours for different classes of abundance.First paragraph, page 4 - I suggest the following modification: \"The small number of salp records, and the massive presence of other gelatinous plankters along the whole coast of Italy (...) suggest that salps were rare elsewhere beyond the Apulian coast...\" Fourth paragraph, page 4 - I suggest the following modification: “Salps are gelatinous filter-feeders; they tend to be less abundant in Apulian waters..”Fifth paragraph, page 4 - I suggest the following modification: “ ..blooms of salps are not regularly encountered in Italian waters..”",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-181
|
https://f1000research.com/articles/2-85/v1
|
12 Mar 13
|
{
"type": "Case Report",
"title": "Tectal etiology for irrepressible saccades: a case study in a Rhesus monkey",
"authors": [
"James W Gnadt",
"Christopher T Noto",
"Jagmeet S Kanwal",
"James W Gnadt"
],
"abstract": "Brain circuits controlling eye movements are widely distributed and complex. The etiology of irrepressible square wave saccades is not fully understood and is likely different for different neuropathologies. In a previous study, spontaneously occurring irrepressible saccades were noted after a cerebrovascular accident that damaged the rostral superior colliculus (SC) and its commissure in a Rhesus monkey. Here, we tracked and quantified the development of similar symptoms in a Rhesus monkey caused by a lesion in the rostromedial SC and its commissure. We documented the changes in these saccadic intrusions while the monkey attempted fixation of a target on three consecutive days post-onset. On the first day, eye jerk amplitude was ~10 degrees and the direction was ~30 degrees above the left horizontal meridian. On the second day, the amplitude decreased to 6.5 degrees and the direction shifted towards vertical, ~20 degrees to the left of the vertical meridian. Size, but not direction, of the eye jerks continued to decrease until intrusions dissipated within one month. Histological examination after ~6 months from the first appearance of the intrusions revealed a lesion in the commissure of the SC. Results from this and the previous study confirm the involvement of the commissure of the SC as the common target for triggering this neuropathy. Our data suggest that commissural fibers play an important role in maintaining normal visual stability. Interrupting the commissure between the two superior colliculi causes saccadic intrusions in the form of irrepressible jerking of the eyes, probably by disrupting inhibitory signals transmitted through the commissure. Furthermore, disappearance of the symptoms suggests that inhibitory fields within the SC are plastic and can expand, possibly via inputs from inter-collicular and nigrotectal pathways.",
"keywords": [
"Tectal",
"saccades",
"Rhesus monkey",
"vision",
"eye movements"
],
"content": "Introduction\n\nWe observe the world by making successive fixations disrupted by periods of eye movements. During the periods of fixation, we gather and integrate relevant visual information about our environment and decide where to look next. However, even during these periods of fixation, our eyes are not completely still. Involuntary microsaccades at regular intervals, 1–2 movements per second, move the eyes by ~0.08–0.5 degrees to, refresh the retinal image and thus preserve normal vision1. Also, macrosaccadic activity of the eye (e.g. square wave jerks) has been observed across a broad age spectrum of normal human subjects2–8, and possibly plays a role in preventing adaptation of visual neurons during continuous exposure to the same image. Square wave eye jerks observed in the majority of subjects showed small (0.5–3 degrees) saccades, which disrupt fixation and are followed by a second saccade, about 200–500 ms later that return eye gaze to the target. These eye jerks (normal instability) can occur at low rates (far fewer than 12 per minute), in any direction, but are usually present in the horizontal direction. These movements happen infrequently and there remain questions regarding their role and necessity during fixation.\n\nPathologies of the central nervous system (CNS) can result in abnormal instability of the eye. Eye jerks and other irrepressible breaks in fixation caused by saccadic intrusions, saccade oscillations, and saccade dysmetria have been noted as indicators of CNS disease. For example, the well-known basal ganglia disorder, Parkinson’s disease, presents with many types of saccadic intrusions when a patient attempts to fixate9–11, task-dependent reaction time impairments10,12,13, and saccade dysmetria10,14–17. Deep brain stimulation in the subthalmic nucleus (STN) diminishes the frequency of saccadic intrusions11 and improves both reaction time12 and the accuracy of memory-guided saccades18. This is important because we know that the basal ganglia project bilaterally to the superior colliculus (SC) via crossed and uncrossed projections of the substantia nigra pars reticulata (SNr) (STN-SNr-SC pathway)19,20. Via this pathway, the influence of the SNr has been postulated to act as an inhibitory regulator of saccade onset, suggesting that appropriate inhibition of SC neurons by the SNr is a major contributing factor to the stability of the eye during fixation11,20,21. Indeed, Carasig22 demonstrated that a lesion made through the tectum and commissure of a monkey’s superior colliculus (SC), presumably disrupting SNr signaling, led to a case of irrepressible saccadic intrusions and square-wave eye jerks of approximately 20 degrees. In particular, they described intrusive eye jerk movements that gradually diminished over the course of about 3 weeks.\n\nIn addition to Parkinson’s disease and tectal lesions, unilateral lesions of the globus pallidus in Parkinsonian patients22 and a unilateral lesion of the raphe interpositus nucleus (RIP)23 have been shown to cause saccadic intrusions. Pallidotomy caused square wave jerks that varied in direction, though in one case the preponderance of eye jerks were directed contraversive to the lesion22. The lesion of the RIP, which contains saccade-related omnipause neurons (OPNs), caused saccadic oscillation of the eyes with ipsiversive-directed primary saccades, though these oscillations may have been caused by mechanisms secondary to interrupting the pathway to the RIP from the contralateral SC23. Here we describe intrusions of the type noted above that were created by a serendipitous lesion. We also provide histological evidence for localizing the lesion primarily to the commissure of the SC in the Rhesus monkey. The data reported here confirm a previous observation of the same etiology24, strengthening the significance of our finding and its interpretation.\n\n\nMaterials and methods\n\nThe study presented here describes data collected from one, seven-year old, female Rhesus monkey (Macacca mulatta) obtained from Stony Brook University (Stony Brook, New York). The animal was instrumented with a scleral search coil and recording chambers directed towards the superior colliculus (SC) for obtaining neurophysiological recording. During a regular recording session using a micro-wire, the animal developed saccadic intrusions. As soon as the monkey began exhibiting eye jerks, we recorded horizontal and vertical eye position under normal room lighting and in the dark while the monkey attempted to fixate on a small LED target located straight ahead. At the end of the recording session, we performed a standard neurological examination of the monkey24 to test for cranial nerve and long-tract motor symptoms. Aside from the oculomotor difficulties, there were no neurological impairments of the limbs, posture, mobility or cranial nerve functions. Further neural recording experiments were suspended and for three days post-discovery, we continued to record eye movements while the monkey was fixating a target in ambient light and in the dark.\n\nAll surgical and experimental protocols were approved by the Georgetown University Animal Care and Use Committee (protocol #09-025). The animal was handled in a manner which ameliorated distress or suffering, as suggested in the Weatherall report25, and cared for in accordance with institutional guidelines as put forth by AAALAC and United States federal law.\n\nMonkeys are housed individually in grooming contact cages or in groups in customized primate cages of 1.6 or 2.5 m3 with view of colony mates in a large open room. Cages are continuously equipped with swings, mirrors, foraging devices and/or small toys. Animals are provided with daily care and medical maintenance, including a balanced diet of dry food formula, vegetables and fruit. Environmental enrichment for the monkeys included playing of natural sounds, radio or TV and daily handling, mock grooming and socialization by laboratory personnel.\n\nEach monkey is first acclimated to handling and interaction with the human investigators over a period of weeks to months and taught to transfer from the home cage to a portable, enclosed chair, which is wheeled into the laboratory for experiments. Once the monkey becomes acclimated to handling, it undergoes aseptic surgical procedures under general anesthesia monitored by veterinary support staff to prepare them for behavioral training of eye movements during visual psychophysics tasks, and neurophysiological recording.\n\nAnimals are prepared for participation in experiments by performing two surgeries. For the first surgery, we implant a head restraining device and one scleral eye coil. With the head secured in a stereotaxic device, a 5 cm midline incision is made in the scalp. Periosteum and muscle is retracted using blunt techniques and the calvarium is freed of soft tissue. A 3 cm stainless steel bar, which fits a head restraining apparatus of the primate chair, is attached vertically to the calvarium using surgical stainless steel screws and a mound of acrylic bone cement (e.g. PALACOS, Heraeus Medical)24,26. The screws are mounted into small burr holes in the bone and buried in the bone acrylic along with the head post and electrical connectors. A scleral eye coil is implanted on one eye. Briefly, the conjunctiva is cut near the limbus and reflected to expose the sclera. A coil made of three turns of Teflon-insulated wire is sutured to the sclera using 6-0 Vicryl (e.g. polyglactin 910, Ethicon Inc.), and the conjunctiva is sutured back over the coil. The ends of the coil wire are led out of the orbit subdermally to the acrylic cap where they are attached to a small electrical connector. One week post-surgery, we begin a daily task specific training regimen. Once training has proceeded to an acceptable level, generally within a few months, another aseptic surgery is performed to implant an eye coil on the second eye and one or two stainless steel recording cylinder(s) are mounted into the head cap under stereotaxic guidance. The acrylic overlaying the appropriate portion of the skull is removed using dental burrs in a hand-held, dental drill and we make a 15 mm craniotomy. Stainless steel recording cylinders are placed over the craniotomy and cemented into place with acrylic bone cement. The sterile interior of each cylinder is secured with a threaded Teflon cap having a pressure-release vent.\n\nUsing standard behavioral shaping procedures, the animals are trained to fixate and to follow small visual stimuli by rewarding them with a drop of fruit juice from a gravity-fed “straw” for successfully completing each series of eye movements defined by the presentation of the stimuli. Training and experimental procedures generally are performed for 1–3 hours.\n\nPrior to the incident we describe and following full recovery, painless extracellular neuronal recordings in the SC were made using standard electrophysiological methods while our subject tracked a target presented on a video monitor located 24” in front of them using fine wire tungsten microelectrodes (0.5 to 1.2 MOhm, 31 gage, Microprobe, Inc., USA) mounted in a guide tube of stainless steel hypodermic tubing several times a week. Trans-dural penetrations are made by a hydraulic microdrive (FHC, Inc., USA) through the bore of a 21 gauge hypodermic needle mounted in a micropositioner that attaches to the outside of the chronic recording cylinders on the subject's head. Neuronal activity is recorded by a high impedance amplifier system (AMC Systems, Inc., USA) and a laboratory computer that also controls and monitors the behavioral tasks.\n\nDuring post-incident monitoring, we recorded the eye position by means of current induced on the scleral search coil27 that was amplified and offset by a phase detector (Riverbend Instruments, Inc., USA) before being passed to a dedicated PC for data sampling at 1 kHz. To document the eye jerks, we collected approximately 100 seconds of data in both the light and dark on four consecutive days while the monkey fixated a central green LED target (0.14 degrees). A post-hoc script read the binary file and differentiated eye position into velocity, allowing us to easily identify the beginning and end of saccades using a 30 degrees/second threshold. The script wrote a text file containing specific information about each movement (e.g. saccade size and direction, peak eye velocity, movement duration, and other measures), which was later used to quantify the metrics of the observed eye jerks. Eye jerks collected in the light and dark were statistically indistinguishable for each day so we pooled these data.\n\nSix months after the monkey first started exhibiting saccadic intrusions, we humanely euthanized it with pentobarbital anesthesia and perfused it through the left cardiac ventricle with saline, sodium nitrate, and heparin solution followed by 4% paraformaldehyde. After the perfusion, the brain was removed from the skull and placed in a buffered 20% glycerin bath for 10 days, then sliced into 40 µm sections using a freezing microtome and mounted onto slides. We mounted every 6th section from the level of the anterior commissure to the dorsal column nuclei of the medulla, stained the mounted sections by cresyl violet using standard histological methods (e.g. Nissl staining) to reveal cell bodies, and using a light microscope with an attached high-resolution digital camera created photomicrographs of the tissue to illustrate the extent of the lesion.\n\n\nResults\n\nFigure 1 illustrates examples of the four fixation deficits observed in this monkey: 1) square wave movements away from the target and back (i.e., square wave jerks), 2) doublets of consecutive eye movements away from the target, 3) multiple, rapid, saccadic “staircase” movements, and 4) saccade oscillations consisting of overshooting saccades across the target. The preponderance of the saccadic intrusions across all days were the square wave eye jerks. The ratio of this type of eye jerk to all other types of saccadic intrusions was nearly 9:1 on the day of discovery and 4:1, 6:1, and 4:1 on successive days. At the first observation of fixation deficits, no doublet eye jerks were made, but we observed a number of staircase saccades. Over the course of the following three days, the number of doublet eye jerks increased until day 3, when we found nearly equal numbers of staircase saccades and doublet eye jerks. Saccades made before the appearance of the syndrome fit well with established main sequence criteria for saccadic quick phases, and so did the fixation-breaking movements of all types of saccadic intrusions. These observations provide convincing evidence for the presence of irrepressible eye movements or saccades that are generated by the brainstem saccade circuit.\n\nGrey traces show the horizontal component (Left) and black traces the vertical component (Up) of representative irrepressible saccades collected on the third post-discovery day.\n\nThe frequency of square wave jerk eye movements made by the monkey changed each day. The frequency of movements ranged from 7.8 to 42 per minute with average intersaccadic intervals between 75 ± 11 ms and 143 ± 81 ms. On the first day of the occurrence of intrusions, saccade size averaged 10.1 ± 2.8 degrees and the average direction was 27 ± 15 degrees above the left horizontal meridian. Peak velocities averaged 534 ± 114 degrees per second. On the next day, we observed a significant change. Average saccade size decreased to 7.1 ± 1.2 degrees and the direction moved closer to vertical, 17 ± 8 degrees to the left of the vertical meridian. Velocities decreased to 326 ± 60 degrees per second. On the two succeeding days, saccade amplitude continued to decrease until, on the last day tested, it averaged 4.7 ± 1.8 degrees. Saccade direction did not change further and on the last day was 28 ± 17 degrees to the left of the vertical meridian. Peak velocities averaged 257 ± 83 degrees per second. The decrease in saccade peak velocity was secondary to the decrease in mean saccade size.\n\nThe square wave eye jerks discussed above consisted of a single saccade away from the fixation point followed by a second saccade returning the eye to the target. The saccadic intrusions we describe here consisted of two (doublets) or multiple (staircases) saccades away from the fixation point followed by a saccade that returned the eye to the target many hundreds of milliseconds later. We considered the doublet and staircase saccades as a single population during the analysis of our data because they seemed to be variations of the same phenomenon that are at different points on a continuum. The general trend across all days was for the monkey to make a large initial saccade with a large vertical component followed by smaller saccades with prominent horizontal components. During their initial occurrence, all saccadic intrusions were directed leftward, and their size averaged <2.5 degrees. They were made in approximately the same direction (~36 degrees above the horizontal meridian). In the days following the lesion, a much larger initial leftward-directed saccade with a large vertical component (4.5–6.5 degrees at 23 degrees to the left of the vertical meridian) was noted. This was followed by smaller, more oblique or horizontally directed saccades. On the first day of its occurrence, staircases of saccades occurred with a one-time maximum of ten consecutive saccades away from the fixation point. Commonly, 3–6 movements occurred during each staircase pattern. On the first day of occurrence of abnormal jerk eye movements, the interval between successive movements averaged 61.5 ± 24.6 ms, an interval about 1/4 that of the normal intrasaccadic latency. On the three following days, we observed a daily increase in the intrasaccadic interval toward normal: first 114 ± 41, then 142 ± 26, and finally 161 ± 80 ms (within the lower range of normal saccade latency, 150–350 ms).\n\nSaccade oscillations occurred infrequently, but were more frequent in the presence of ambient lighting. Most oscillations occurred as two or three consecutive saccade pairs, within a 400–600 ms period, but in severe instances the oscillation could last for up to 1 second or more, presumably severely disrupting visual perception. The saccades within a given oscillation varied in amplitudes of <10 degrees, tended to be slightly smaller later in the series, and repeatedly overshot to one side of the fixation point and then the other. The initial movement of the oscillations occurred in the same direction as the previously described saccadic intrusions, leftward and upward.\n\nExamination of Nissl-stained brain sections under a bright-field microscope revealed a dorsoventral lesion through the rostral commissure of the SC and rostromedial tectum. Figure 2A–C illustrate the location and nature of this lesion. Figure 2A shows three consecutive rostral-to-caudal tracings to illustrate the lesion observed at the medial edge of the right SC. This panel reveals essentially a parasagittal “knife cut” lesion at the level of the pineal gland. The lesion ran for approximately 720 µm (3 × 6 × 40 µm sections; one series sampled out of 6) through rostromedial SC and the collicular commissure on the right. Figure 2B and C show photomicrographs of the top two images shown in Figure 2A & C shows the lesion as it extends caudally bisecting the commissure and ~85% of the dorsal-to-ventral extent of the rostral-medial SC. Less extensive gliosis from two other smaller electrode tracts can be also seen in this panel; a lateral one in the right SC, and another that is central and deep within the left SC. These electrode tracts represent penetrations presumably made with either stimulation or recording electrodes at a later date. Figure 2D and E show that two important inhibitory nuclei of the saccadic system, the raphe interpositis pontis (RIP) and the substantia nigra pars reticulata (SNr) remained intact.\n\nPanel A shows tracings of the superior colliculus (SC) and other structures in the brainstem relative to the lesion we found bisecting the commissure and the right rostral medial SC. Panels B & C show photomicrographs taken of the lesion as indicated by arrows. Two other electrode tracts are indicated by asterisks. Panels D & E show that the RIP and the SNr, respectively, of this monkey remained intact and undisturbed. Abbrevations: SC) superior colliculus, IC) inferior colliculus, PAG) periaqueductal gray, III) oculomotor nerve, IV) trochlear nerve, bc) brachium conjunctivum, RIP) raphe interpositus, SNr) substantia nigra reticulata.\n\nThe lesion reported here is very similar in size and extent to the one described by Carasig et al.24 that caused similar saccadic intrusions. A video of the Carasig subject is available, with permission, as supplemental data. The lesion in the prior study was examined histologically soon (6 weeks) after it occurred, and encompassed roughly 1 mm3 of tissue. In the present case study, the histological assessment of the lesion occurred after a much longer time (6 months), and the long recovery time undoubtedly included the effects of healing that occurred before euthanasia.\n\n\nDiscussion\n\nThis paper reports the tectal etiology for irrepressible saccades observed after a tungsten wire made a lesion during a routine recording procedure. This wire interrupted the commissure and damaged the rostral-medial part of a Rhesus monkey’s SC. Post lesion, the monkey exhibited four types of saccadic intrusions while trying to fixate on a green LED presented before the animal. The amplitude and direction of these intrusions matched well with what could be expected based on the location of the lesion within the SC motor map. Histological examination of the tectum confirmed that the damaged tissue was located exclusively in the rostromedial part of the SC, which contains neural circuitry for contraversive, upward saccades. No other regions were damaged. Square wave saccadic jerks of the eye were the most frequently displayed abnormal behavior, though we routinely observed doublets of these saccadic movements and even staircases of saccades. Less frequently, the instability in fixation became so great that saccades oscillated about the fixation point for >400 ms. Observations across consecutive days showed a rapid rotation in direction of about 50 degrees from predominately horizontal to predominately vertical, between onset and one day after onset of saccadic intrusions. These intrusions were accompanied by a gradual decrease in eye jerk saccade size.\n\nThe data presented in this case study showed generally the same saccadic intrusions in terms of the direction of initial saccade of the eye jerk, from up-right to up-left within 24 hours of onset as reported previously24. Also, the amplitude of the eye jerks decreased over time until about three weeks later when abnormal saccadic intrusions were no longer easily detected.\n\nOur monkey’s saccadic intrusions were also similar to clinical observations of square wave saccadic eye jerks and saccade oscillations in humans. Both human Parkinson’s disease patients11 and our monkeys made eye jerks much greater in size and frequency than considered as normal by many researchers3–8.\n\nEye jerks and saccade oscillations are a primary indicator for a wide array of neurodegenerative diseases in addition to Parkinson’s disease (e.g. progressive supranuclear palsy9, multiple sclerosis28, and cerebellar degeneration29). However, two conspicuous differences exist between saccadic intrusions induced by lesions of the SC and those caused by CNS disease. First, the square wave jerks and oscillations made following the SC lesion were oriented obliquely, which is uncommon in CNS degeneration where eye jerks occurred only along one axis, usually horizontal5,29,30. Note however that vertical saccadic intrusions have been observed in clinical settings30. A second difference is that the size and frequency of saccadic intrusions made following the SC lesion gradually diminished over about a one-month period. After that time, we observed normal fixation. We speculate that this occurs due to plastic rebalancing of excitatory and inhibitory influences within the tectal, inter-collicular and nigrotectal pathways following this small and discrete neural insult, which like the case of Carasig et al.24 involved less than a cubic millimeter of tissue. Saccadic intrusions caused by CNS degeneration never diminish or go away without surgical or drug therapies. For example, deep brain stimulation reduced the frequency of eye jerks caused by Parkinson’s disease11 while administration of diazepam, thiamylal, phenobarbital, and clonazepam were shown to nearly stop saccade oscillations in a patient with cerebellar and brainstem atrophy31. The eye jerks and oscillations, however, return without ongoing intervention. This suggests that the plastic-adaptive mechanisms of the saccade system are also degraded during these types of CNS degeneration, or that the adaptability of the system has been taxed maximally before appearance of symptoms. Therefore, further compensation of the instability of the eye during fixation is no longer possible.\n\nLesions of the globus pallidus22 or RIP23 also cause square wave jerks and saccadic oscillations, respectively. However, inspection of histology from our subject showed that these structures were intact. The square-wave jerks most likely were caused by unbalanced excitation and inhibition at a focused site in the SC, as illustrated by the preferentially-directed, amplitude-specific saccades generated by our subject following the lesion. This is in contrast to the varied direction of square wave jerks observed by general disinhibition of the SC following unilateral pallidotomy. Doublets and staircases were most likely caused by an unbalanced and prolonged disinhibition of a specific site within the SC motor map. Experimental studies to remove19 or augment20 the modulating inhibitory influence of the nigrotectal system reveals a likely mechanism for the etiology of this site-specific, pathologic disruption in ocular muscle tone. In particular, Liu and Basso20 reveal a crossed nigrotectal inhibition that could be disrupted by transection of fibers transversing the commissure of the SC. In addition, disruption of descending inhibition from the lesioned area of the SC to RIP could also contribute to the observed etiology32.\n\n\nConclusion\n\nWe have shown that a lesion through the SC commissure and rostral SC was coincident with the onset of an eye jerk syndrome in monkeys. Current literature describing the role of the commissure and rostral SC, in both monkey and human, states their importance in the inhibition of saccade onset11,12,20,21. In other words, both areas are involved in the permissive side of saccade generation. Presumably by disrupting the balance of excitation and inhibition within a specific region of the rostral-medial SC, motor maps become biased and cause saccades matching the vector space corresponding to the location of the lesion.",
"appendix": "Author contributions\n\n\n\nJWG conceived the experimental design, assisted with recordings and wrote the first draft of the manuscript. CTN participated in recordings, data acquisition and writing the manuscript. JSK assisted with facilitating the experiments and with writing and communicating the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWork supported in part by NIH grants EY015870 to JWG and JSK from the National Eye Institute (NEI), and grants EY08217, and NS039407 to JWG. Additional support was provided by the Biomedical Graduate Research Organization (BGRO) of Georgetown University and by Howard University. The content is sole responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\n\n\nAcknowledgements\n\nWe thank the staff at Georgetown University’s Division of Comparative Medicine. We especially thank Dolphus Truss for his dedication to the care and well being of our animals and his exceptional work in support of our research efforts, and Dr. Farrel Robinson for his editorial assistance and suggestions while preparing this manuscript.\n\n\nReferences\n\nMartinez-Conde S, Macknik SL, Hubel DH: The role of fixational eye movements in visual perception. Nat Rev Neurosci. 2004; 5(3): 229–240. PubMed Abstract | Publisher Full Text\n\nSchweigart G, Hoffmann KP: Pretectal jerk neuron activity during saccadic eye movements and visual stimulations in the cat. Exp Brain Res. 1992; 91(2): 273–83. PubMed Abstract | Publisher Full Text\n\nHerishanu YO, Sharpe JA: Normal square wave jerks. Invest Opthalmol Vis Sci. 1981; 20(2): 268–272. PubMed Abstract\n\nShallo-Hoffmann J, Petersen J, Muhlendyck H: How normal are \"normal\" square wave jerks? Invest Ophalmol Vis Sci. 1989; 30(5): 1009–1011. PubMed Abstract\n\nShallo-Hoffmann J, Sendler B, Muhlendyck H: Normal square wave jerks in differing age groups. Invest Ophalmol Vis Sci. 1990; 31(8): 1649–1652. PubMed Abstract\n\nShaffer DM, Krisky CM, Sweeney JA: Frequency and metrics of square-wave jerks: influences of task-demand characteristics. Invest Opthalmol Vis Sci. 2003; 44(3): 1082–1087. PubMed Abstract | Publisher Full Text\n\nAbadi RV, Gowen E: Characteristics of saccadic intrusions. Vision Res. 2004; 44(23): 2675–2690. PubMed Abstract | Publisher Full Text\n\nSalman MS, Sharpe JA, Lillakas L, et al.: Square wave jerks in children and adolescents. Pediatr Neurol. 2008; 38(1): 16–19. PubMed Abstract | Publisher Full Text\n\nRascol O, Sabatini U, Simonetta-Moreau M, et al.: Square wave jerks in Parkinsonian syndromes. J Neurol Neurosurg Psychiatry. 1991; 54(7): 599–602. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoti P, Kulashekhar S, Behari M, et al.: Impaired inhibitory oculomotor control in patients with Parkinson’s disease. Exp Brain Res. 2007; 177(4): 447–457. PubMed Abstract | Publisher Full Text\n\nWark HA, Garell PC, Walker AL, et al.: A Case report on fixation instability in Parkinson’s disease with bilateral deep brain stimulation implants. J Neurol Neurosurg Psychiatry. 2008; 79(4): 443–447. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSauleau P, Pollak P, Krack P, et al.: Subthalamic stimulation improves orienting gaze movements in Parkinson’s disease. Clin Neurophysiol. 2008; 119(8): 1857–1863. PubMed Abstract | Publisher Full Text\n\nVan Stockum S, MacAskill M, Anderson T, et al.: Don’t look now or look away: two sources of saccadic disinhibition in Parkinson’s disease? Neuropsychologia. 2008; 46(13): 3108–3115. PubMed Abstract | Publisher Full Text\n\nHodgson TL, Dittrich WH, Henderson L, et al.: Eye movements and spatial working memory in Parkinson’s disease. Neuropsychologia. 1999; 37(8): 927–938. PubMed Abstract | Publisher Full Text\n\nLe Heron CJ, MacAskill MR, Anderson TJ: Memory-guided saccades in Parkinson’s disease: long delays can improve preformance. Exp Brain Res. 2005; 161(3): 293–298. PubMed Abstract | Publisher Full Text\n\nChan F, Armstrong IT, Pari G, et al.: Deficits in saccadic eye-movement contol in Parkinson’s disease. Neuropsychologia. 2005; 43(5): 784–796. PubMed Abstract | Publisher Full Text\n\nGurvich C, Georgiou-Karistianis N, Fitzgerald PB, et al.: Inhibitory control and spatial working memory in Parkinson's disease. Mov Disord. 2007; 22(10): 1444–1450. PubMed Abstract | Publisher Full Text\n\nRivaud-Pechoux S, Vermersch AI, Gaymard B, et al.: Improvement of memory guided saccades in parkinsonian patients by high frequency subthalamic nucleus stimulation. J Neurol Neurosurg Psychiatry. 2000; 68(3): 381–384. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHikosaka O, Wurtz RH: Visual and oculomotor functions of monkey substantia nigra pars reticulata. IV. Relation of substantia nigra to superior colliculus. J Neurophysiol. 1983; 49(5): 1285–1301. PubMed Abstract\n\nLiu P, Basso MA: Substantia Nigra stimulation influences monkey superior colliculus neuronal activity bilaterally. J Neurophysiol. 2008; 100(2): 1098–112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMunoz DP, Wurtz RH: Fixation cells in monkey superior colliculus. II. Reversible activation and deactivation. J Neurophysiol. 1993; 70(2): 576–589. PubMed Abstract\n\nAverbuch-Heller L, Stahl JS, Hlavin ML, et al.: Square-wave jerks induced by pallidotomy in parkinsonian patients. Neurology. 1999; 52(1): 185–8. PubMed Abstract | Publisher Full Text\n\nAverbuch-Heller L, Kori AA, Rottach KG, et al.: Dysfunction of pontine omnipause neurons causes inpaired fixation: macrosaccadic oscillations with a unilateral pontine lesion. Neuroophthalmology. 1996; 16(2): 99–106. PubMed Abstract | Publisher Full Text\n\nCarasig D, Paul K, Fucito M, et al.: Irrepressible saccades from a tectal lesion in a Rhesus monkey. Vision Res. 2006; 46(8–9): 1161–1169. PubMed Abstract | Publisher Full Text\n\nWeatherall DJ: The Use of Non-Human Primates in Research: A Working Group Report. London: Wellcome Trust, 2006. Reference Source\n\nRamcharan EJ, Gnadt JW, Sherman SM: Single-unit recording in the lateral geniculate nucleus of the awake behaving monkey. Methods. 2003; 30(2): 142–151. PubMed Abstract | Publisher Full Text\n\nRobinson DA: A method of measuring eye movement using a scleral search coil in a magnetic field. IEEE Trans Biomed Eng. 1963; 10: 137–145. PubMed Abstract | Publisher Full Text\n\nDell’osso LF, Troost BT, Daroff RB: Macro square wave jerks. Neurology. 1975; 25(10): 975–979. PubMed Abstract | Publisher Full Text\n\nHotson JR: Cerebellar control of fixation eye movements. Neurology. 1982; 32(1): 31–36. PubMed Abstract | Publisher Full Text\n\nFahey MC, Cremer PD, Aw ST, et al.: Vestibular, saccadic and fixation abnormalities in genetically confirmed Friedreich ataxia. Brain. 2008; 131(Pt 4): 1035–1045. PubMed Abstract | Publisher Full Text\n\nFukazawa T, Tashiro K, Hamada T, et al.: Multisystem degeneration: drugs and square wave jerks. Neurology. 1986; 36(9): 1230–1233. PubMed Abstract | Publisher Full Text\n\nKim JS, Choi KD, Oh SY, et al.: Double saccadic pulses and macrosaccadic oscillations from a focal brainstem lesion. J Neurol Sci. 2007; 263(1–2): 118–123. PubMed Abstract | Publisher Full Text"
}
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[
{
"id": "850",
"date": "18 Mar 2013",
"name": "Michele A Basso",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 straightforward case observation with a clear result and sound interpretation.I would prefer the authors to use ‘superior colliculus’ instead of ‘tectal’, since the optic tectum generally refers to the structure in non-mammalian species.Could the oscillations be due to encroachment of the lesion on the pre-tectum? (I know, it probably should be called pre-colliculus). Also, some comparison with the oscillations observed after muscimol injections in SC might be useful.",
"responses": []
},
{
"id": "962",
"date": "23 May 2013",
"name": "Farrel Robinson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate and the methods and analysis are appropriate to this article’s conclusions. The conclusions are mostly fine. My one comment is that the authors cite reference 23 at least twice to support the statement that lesions of the RIP cause saccadic intrusions. Ref 23 is a report of one clinical case whose brain damage is almost certainly not restricted to RIP and not even very well defined. In contrast, a careful study which specifically damaged the RIP in 3 monkeys reports a result consistent across all 3 animals and very different from that in ref 23 (Kaneko, 1996, J Neurophysiol. 75:2229-2242.)The authors clearly present enough data here to support their conclusions.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-85
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https://f1000research.com/articles/2-128/v1
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20 May 13
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{
"type": "Commentary",
"title": "Discovery of continental-scale travelling waves and lagged synchrony in geometrid moth outbreaks prompt a re-evaluation of mountain birch/geometrid studies",
"authors": [
"Olle Tenow"
],
"abstract": "The spatio-temporal dynamics of populations of two 9-10 year cyclic-outbreaking geometrids, Operophtera brumata and Epirrita autumnata in mountain birch forests in northern Fennoscandia, have been studied since the 1970´s by a Swedish-Norwegian research team and, during the last decade, by Norwegian and Finnish research teams. Some of the early results have been challenged by the Norwegian team. To examine the base for disagreements, five of the papers published by the Norwegian team (2004-2011) are reviewed. It is found that conclusions in these papers are questionable or data could not be interpreted fully because two decisive traits in the spatio-temporal behaviour of outbreaks of the two species were not considered.",
"keywords": [
"Operophtera brumata",
"Epirrita autumnata",
"defoliation",
"Scandinavia",
"population dynamics"
],
"content": "Introduction\n\nTwo recent observations of the spatio-temporal outbreak pattern of some extensively studied geometrid moth species necessitate a re-evaluation of previously published findings. Firstly, it has been demonstrated that defoliating outbreaks on deciduous trees of the winter moth (Operophtera brumata L.) and associated geometrids every 9–10 years travel as a wave across Europe from east to west with the front of the wave stretching from high to low latitudes1. Secondly, long-term studies on the temporal behaviour of Epirrita autumnata, an associated geometrid, and O. brumata in the mountain chain of Scandinavia (the Scandes) and northern Finland show that outbreaks of O. brumata have been synchronized with those of E. autumnata with a time lag of about 1–2 years2–4.\n\nTemporal dynamics of populations of cyclic geometrids have been studied in some parts of central Europe (e.g. Raimondo et al. 20045; Glavendekić 20026; Kula 20087). However, research on spatio-temporal behaviour seems to have been undertaken only in Fennoscandia (Norway, Sweden and Finland), i.e., on O. brumata, E. autumnata and Agriopis aurantiaria on birch (Betula pubescens and B. p. czerepanovii=mountain birch). These studies started with a historical survey of outbreaks on birch of E. autumnata and O. brumata that covered the years 1862–1968 in the geographical area of Fennoscandia8. It revealed that 12 outbreak periods had occurred during the surveyed period, with an average interval of 9–10 years. The survey was followed-up for the years 1969–20019, adding three more outbreak periods, this time with quantitative data (including for A. aurantiaria) for the period 1990–20013. In these studies, it was found that outbreaks sometimes occurred contemporaneously along the Scandes and sometimes moved as a wave from north to south along the Scandes or from south to north. Although true within the scope of these studies, it has now been demonstrated that the patterns described are illusions caused by continental-scale outbreak waves passing the Scandes either in parallel or obliquely from north or south1. Another study, also restricted to Fennoscandia, showed that waves of E. autumnata outbreaks during the four outbreak periods from 1970–2004 have, on average, travelled across Fennoscandia from about NE/E to SW/W1. This aligns with the results in Klemola et al. (2006)10.\n\nIn Tenow et al. (2013)1, it is argued that local and even regional population dynamics of a species cannot be properly understood if large-scale waves in cyclic populations occur without them being recognized. The prime examples of such misinterpretations on a regional scale are the descriptions of outbreak waves in the above-mentioned papers8,9. The purpose of this paper is to expose further examples of similar shortcomings.\n\nIn 1999, a Norwegian project was launched to cover one cycle of the population ecology of O. brumata and E. autumnata in northern Fennoscandia. The project has hitherto (2013) produced 22 papers (http://www.birchmoth.com/) of which nine focus on local and regional spatio-temporal population behaviour of mainly O. brumata. The results and conclusions in five of them are relevant in the present context. These papers are Ims et al. (2004)11, Hagen et al. (2008)12, Jepsen et al. (2009a)13, Jepsen et al. (2009b)14 and Jepsen et al. (2011)15. The papers are interconnected, and to a large extent written by the same authors, and are therefore considered together. They are commented on in order of publication year.\n\nFor information on the investigation region, area and sites, see maps, Figure 1, modified from Tenow et al. (2007, Figure 3)3 and Jepsen et al. (2011, Figure 3)15.\n\na) Map of northern Fennoscandia where defoliations of birch forests during outbreaks of E. autumnata and O. brumata in 2000–2008 were mapped from satellite imagery (Jepsen et al. 2009a13; 2009b14). Framed area marks the area for intensive studies of O. brumata occurrence in 1999–2009 (Ims et al. 200411; Hagen et al. 200812; Jepsen et al. 201115). b) Map of intensive study area with study sites and indicated movement directions of the wave of the 1990s O. brumata occurrence (Ims et al. 200411; Hagen et al. 200812; Jepsen et al. 201115). Pink = the wave has passed the sites and populations are in the low phase; green = crash phase; blue = peak and crash phases. Island sites: 12 (Årøy), 10 (Karlsøy), 8 (Dåvøy), 6 (Vengsøy), 4 (Rekvik), 2 (Tussøy). Mainland sites: 11 (Lyngen), 9 (Reinøy), 7 (Dåfjord), 5 (Skulsfjord), 3 (Tromvik), 1 (Sandvik). Numbers of sites are from Jepsen et al. (2011, Figure 3)15; corresponding names from Ims et al. (2004, Figure 1)11.\n\nIms et al. (2004) Do sub-Arctic winter moth populations in coastal birch forest exhibit spatially synchronous dynamics? Journal of Animal Ecology 73: 1129–113611.\n\nThis paper deals with the spatio-temporal behaviour of O. brumata populations in north-western-most Norway (Troms County; Figure 1a) in 1999–2002 (Figure 1 in the paper; cf. Jepsen et al. 2011 below15). E. autumnata was omitted from the study because of insufficient numbers in this coastal area. In an early study8 based on historical documentation, it was suggested that climatic forcing (the Moran effect) may have synchronized the 9–10-year cyclic outbreaks of O. brumata (and E. autumnata) occurring in the Scandes and northern Finland. The actual study raised the question as to whether this suggestion withstands closer scrutiny in terms of more detailed quantitative data. Commenting on the short duration of the study, it is said\n\n“…although studies of dynamics of cyclic populations for many purposes need to span a longer time period than the time period of a cycle (i.e., 10 years for the focal moth species) the degree of synchronous dynamics can be evaluated from more short termed data (.....)\", and, \"for cyclic populations, identifying and comparing the phase among sites at any given time would be sufficient to verify the prevalence of synchrony. Here we use the latter approach on our 4-year data set…”.\n\nThe female O. brumata moth has stunted wings and cannot fly. Physical barriers, such as sea water between mainland and islands at the coast, may therefore prevent the spread of females and isolate island populations. On the understanding that the climate is identical, differing population dynamics on the mainland and nearby islands should imply the disruption of the Moran effect. Six pairs of sites were selected from east to west in the north-western coastal part of Troms, each with one site on the mainland (or a large island) and one on a medium-sized island, separated by >1.5 km of open sea (Figure 1b). At each site, the number of caterpillars per sampled twig at a number of stations up-slope was recorded over four years (1999–2002).\n\nIt was found that population densities were low on all islands, except for one (site 2, Tussøy), and that populations there were maximally out of phase with peaking populations on the mainland, or populations that had recently peaked, except for one (site 1, Sandvik). The authors concluded that\n\n“…the distinct asynchrony between adjacent sites (<10 km apart) clearly belonging to the same climatic domain rules out the possibility that climate or, for that matter any other supposedly large-scale phenomenon (.....) could be responsible for the population phase differences”.\n\nThus, the notion of a large-scale spatial synchrony due to a Moran effect was rejected, at least for coastal birch forest.\n\nLater, it was shown3 that a large-scale E. autumnata/O. brumata outbreak wave arrived at northern Fennoscandia from the east in 1991–1992. The wave travelled westward across the region and reached the eastern part of Troms in 1994–1995, and finally the west coast of Troms in about 2000 where the mainland/island study was performed. It was stated in Tenow et al. (2007)3 that this wave must be considered when the results of that study are to be interpreted.\n\nSince then, Jepsen et al. (2011)15 have added seven more years to the data set, which now comprises the years 1999–2009. This offers an opportunity to re-evaluate the 2004 results. The low density island sites, seen in Figure 1 in the paper, may be subdivided into three groups from east to west within the investigation area (cf. enlargement of population curves for 1999–2002 in Jepsen et al. 2011, Figure 3, and comments to that paper below15): one group consisting of the easternmost site and the two northernmost sites that experienced an evident low during the study time (1999–2002) (Figure 1b: sites 8, 10, 12) and one group of the next two island sites to the south-west that underwent the greatest crash phase during the same period (sites 4, 6). Finally, the south-western-most island site (2) exhibited a full peak and a crash. The mainland sites may also be divided into three groups; the easternmost site with an evident low during the study (11), the two northernmost sites with a crash (7, 9; although site 9 shows a deviating population curve), and the three south-western-most sites with a full peak and a crash (1, 3, 5). This demonstrates that the wave of the 1990s, which arrived to eastern Troms in 1994–1995 and then continued westward, had already passed across the northern part of the investigation area before the study, i.e., about the middle of the 1990s. Then, in 1999–2002, the wave was recorded at the south-western island sites in its crash phase and finally, in 2000–2004, it was caught up lingering at the south-western-most mainland sites in its peak and crash phases. Hence, by first reaching the island sites in the north in the direction E-W, then the island sites along the west coast in the direction N-S, and finally the mainland sites inside the islands, it behaved like a wave \"breaking\" successively over the mainland part of the investigation area (Figure 1b). Of the six pairs of sites, only one (9, 10) indicates populations that may have been maximally out of phase-one branch of the wave passed south of the investigation area already in 1994–19953.\n\nIn conclusion, there were no distinct, maximally out of phase of population dynamics between sites on mainland and nearby islands. Hence, the Moran effect cannot be discarded. Instead, there was a continuous progression over the investigation area of the large-scale wave of the 1990s, first via the island sites then to the mainland sites. Therefore, at any given time, populations separated in space in the direction of the wave had been in continuous change in larval density. Furthermore, the continuation of the study (cf. Jepsen et al. 201115) clearly demonstrates that a four-year study in this case was not sufficient to allow a correct interpretation.\n\nHagen et al. (2008) Anisotropic patterned population synchrony in climatic gradients indicates nonlinear climatic forcing. Proceedings of the Royal Society B 275: 1509–151512.\n\nThe paper gives detailed data on spatio-temporal changes in larval abundance of O. brumata and E. autumnata in vertical and horizontal directions on a coastal mountain slope (Figure 1b: site 9, Reinøya) in 2001–2007. It is assumed that the air temperature gradients decrease linearly with increasing altitude whereas the temperature is approximately the same in a horizontal direction at each altitude. It is predicted that the varying sensitivity of different insect life stages to climate can cause a non-linear forcing on population synchrony along altitudinal air temperature gradients on slopes. If there is a nonlinear forcing in this case, a synchronisation caused by a Moran effect could be rejected. It is stated that the `global spline correlogram´, applied for each species, was based on all population time series (i.e., 2001–2007). In the beginning of the results section, the authors stated:\n\n“The 7-year time series of both species exhibit sufficient temporal variation to provide an useful basis for investigating patterns of population synchrony (….). That is, all populations had crashed at the end of the study, enabling us to highlight synchrony of the crash phase of the population cycle”.\n\nIt is shown that E. autumnata went through all stages of a regular cycle (increase, peak, and crash during 5 years, followed by a low) during the 7 years. The changes of the O. brumata population showed more variation but the overall trend was less steep and often lagged behind E. autumnata. There is no mention of cycle phases other than the crash phase (O. brumata), nor mention of successive population cycles or of outbreak waves (cf. Tenow 19728; Tenow et al. 20073) only of “more complex spatial patterns”. Directional (anisotropic) correlograms for O. brumata showed that the synchrony declined steeply in altitudinal direction but not in the horizontal direction of the slope. E. autumnata did not exhibit any anisotropy in either direction.\n\nThese results can be compared with Schott et al. (2010)16, a study based on the same data. That study clearly points out a crash phase for O. brumata, for the cycle of the 1990s, in addition to that of the first decade of the twenty-first century. The crash of the 1990s occurred in 2001 or 2000–2001, followed by a population low in 2002 (Schott et al. 2010, Figure 316, most distinct at higher altitudes). The same low is visible, although faint, for the same site in Jepsen et al. (2011; Figure 315). For E. autumnata, the crash phase of the cycle of the 1990s had occurred before the start of the project. The low occurred in 2001 or 2000–2001, i.e., 1–2 years ahead of O. brumata. Evidently, the low for each species separated the cycle/wave of the 1990s (cf. Ims et al. 2004 above11) from the following one in the 2000s (cf. Jepsen et al. 2011 below15). For O. brumata, the peak of the latter cycle occurred in 2004–2005 at the two highest altitudes, followed by a crash at all altitudes in 2006–2007. For E. autumnata, the peak of the same cycle occurred contemporaneously at all altitudes in 2002–2003, two years ahead of O. brumata at the two highest altitudes (Schott et al. 2010, Figure 316). The crash followed in 2005–2006, also at all altitudes.\n\nThe O. brumata low in 2002, shown in Schott et al. (2010, Figure 316), means that 2002 was a year of transition between the O. brumata cycle of 1990s and that of the 2000s. In the analysis of the spatially directed (anisotropic) synchrony patterns, this low is not acknowledged, although visible with an expected time lag after E. autumnata in Figure 4 of the paper (cf. Schott et al. 2010, Figure 316), and the 7-year time series for O. brumata seems to have been treated as one single long crash phase. It is important here to point out that no specific cycle (increase, peak and crash) lasts 7 years, not to mention the crash phase, which used to be the most rapid of the three phases. As a consequence, two successive O. brumata cycles became involved in the analysis, the crash in 2001 of the cycle of the 1990s, the low in 2002 and the full cycle of the 2000s. According to Figure 3 in Schott et al. (2010)16, populations at the same altitudes (240, 170 and 100 m above sea level, respectively) were much less synchronized during the crash of the cycle of the 1990s than during phases of the cycle of the 2000s, including the crash. Despite the fact that these fluctuations constitute a minor part in the analysis, they may have created undue variation. In addition, to be comparable, the analysis for the two geometrids should probably have covered the same phases of the outbreak cycle (increase, peak, crash), or the years 2002–2004 (2005) for E. autumnata and, with the time lag, the years 2003–2005 (2006) for O. brumata. It is obvious from Figure 4 in the paper that the spatio-temporal population behaviour during the same phases was similar for the two species. These circumstances cast doubt on the claim that there was an “anisotropic patterned population synchrony in climatic gradients” for O. brumata.\n\nThe “target” concept is mentioned in the paper; implying that the target, the Moran effect, will be aimed at with precision and accuracy. However, the precision of advanced statistical methods is in vain when accuracy is poor, as can be suspected in this case. The study did not account for the cycles in two successive outbreak waves that passed the site. This makes it open to questions. Does this omission imply that the analysis of the O. brumata data became biased and that the result therefore is unreliable?\n\nJepsen et al. (2009a) Monitoring the spatio-temporal dynamics of geometrid moth outbreaks in birch forest using MODIS-NDVI data. Remote Sensing of Environment 113: 1939–194713.\n\nThis study monitored the defoliations of mountain birch forests across northern Fennoscandia (Figure 1a) in 2000–2008. This was achieved by satellite imagery and the different contributions from the two geometrids E. autumnata and O. brumata were not specified. One result identified was that the defoliation had spread north-eastward along the mountain chain. Thereby, the conclusion in Tenow et al. (2007)3 is challenged namely that outbreaks, like that during the preceding period 1990–1999, obey a consistent pattern by moving in a wave-like fashion broadly from east to west. Instead, it is stated that\n\n“Even without a formal analysis of the spatial dynamics of defoliation, our data indicate that this is not so. Future analysis of the spatio-temporal dynamics of defoliation patterns will substantiate this”.\n\nIn any case, here the existence of unidirectional outbreak movements in northern Fennoscandia seems to be accepted (cf. below).\n\nThe surveyed region stretches from Varangerfjord in north-eastern-most Norway to the outer coast of Troms County in north-western Norway, a distance of about 400 km (Figure 1a). Visible defoliations appeared first at the inner part of Varangerfjord in 2002 where they peaked in 2003. In 2004, widespread defoliations could be seen in the middle and the south-western-most parts of the surveyed region (south-west Troms and adjoining parts of Sweden and Finland, Figure 1a). In 2005, they faded away farthest to the southwest while expanding in the middle part. In 2005, defoliations were again visible at Varangerfjord. They peaked in 2006–2007 and ceased in 2008 (Figure 7 in the paper).\n\nIt is known that the defoliations at Varangerfjord in 2002–2003 were caused by E. autumnata (Klemola, T. et al. 2008, Figure 14). It is also known that the 2004–2005 defoliations in the middle, inner parts, e.g. Kilpisjärvi-Enontekiö (Forststyrelsen, Finland; Finlands Natur 4/5/2005) and Karasjok-Kautokeino areas in Norway and Finland (Skogskader på Internet 22/6/2005) and in the inner, south-western-most part, e.g. in Dividalen in Norway (Skogskader på Internet 6/7/2004) and the Lake Torneträsk-Kiruna area in Sweden (Entomologiska Sällskapet i Lund 24/9/2004; Dagens Nyheter 26/7/2004) were caused by E. autumnata. Finally, there were again defoliations at Varangerfjord in 2005–2007, however, this time by O. brumata4. It is stated that\n\n\" Where the two species occur sympatrically within their outbreak range, there is a partial interspecific synchrony in the timing of the outbreaks, but with winter moth often lagging 1–2 years behind autumnal moth…\".\n\nIf this well-known 1–2 year lag (e.g. Hagen 200717; Tenow et al. 20073; Klemola et al. 20084) is applied to the satellite mapping (in addition to the acceptance of waves), it is evident that there was also a lagged defoliation pattern in this case, i.e., defoliations by E. autumnata in 2002–2003 at Varangerfjord and then by O. brumata in 2005–2007 in the same area. In the meantime, the E. autumnata defoliations had spread westward to the middle and south-western parts in 2004. In 2005, the E. autumnata defoliations faded away in the south-western-most part while expanding in the middle part of the region. Therefore, when the species and time lag were not recognized, the pattern seen by satellite in 2004–2007 was erroneously interpreted as outbreaks moving from the south-western part in 2004 via the middle part in 2005 to Varangerfjord in 2005–2007, that is from SW to NE. As has been shown, the consistent pattern of outbreak movements is the reverse; that outbreak waves travel broadly from east to west in northern Fennoscandia (1, 3, 10). In accordance with a broadly E-W wave, O. brumata peaked at outbreak levels at inner coast sites of Troms in 200617 and at low densities further west at outer coast sites in 2006–2008 (Jepsen et al. 2011, Figure 3, site 1, 3, 5; cf. Figure 1b15), with a subsequent outbreak here in 2007–2008 (Skogfjord, Ringvassøya; cf. also Jepsen et al. 2009b below14), i.e., with a delay of about 1 and 2 years, respectively, compared with Varangerfjord.\n\nIt can be concluded that the outbreak wave of the 2000s travelled broadly E-W over northern Fennoscandia as waves have done as far back in time as can be surveyed and not occasionally in the opposite direction as stated in the paper.\n\nJepsen et al. (2009b) Phase-dependent outbreak dynamics of geometrid moth linked to host plant phenology. Proceedings of the Royal Society B 276: 4119–412814.\n\nThis study is based on the same 2000–2008 satellite imagery mapping of defoliations across northern Fennoscandia (Figure 1a) as used by Jepsen et al. (2009a; above)13. The satellite mapping did not discriminate between E. autumnata and O. brumata defoliations because, again, their different contributions could not be separated. The authors applied two analytical approaches to the spatio-temporal defoliation data. The first approach was focused on population synchrony, namely to assess how synchrony depends on distance. Synchrony can take the form of phase-dependent synchrony, meaning that the degree of synchrony may differ between the increase, peak and crash phases (here evidently implying that phases for the two species coincided). The second approach focused on the rate of spread of defoliation. It was found that defoliation could be divided in three phases. Thus, the analyses comprised estimation of the area of defoliated forest during each of the incipient (2000–2002), epidemic (2003–2005) and crash (2006–2008) phases (Figure 2 in the paper). The analyses revealed that the incipient phase was characterized by high regional synchrony and long defoliation spread distances per year, whereas the epidemic and crash phases were characterized by much lower regional synchrony and a much shorter spread of defoliation. This suggests two independent spread-steps, one long-range process and one very short-range diffusion-like process. As for defoliation, the incipient phase of the outbreak showed a higher regional synchrony in the onset of plant growth (budburst) than the later phases of the outbreak.\n\nThere is no discussion about potential outbreak waves crossing the region (cf. Klemola. 200610; Tenow et al. 20073). It is assumed (in the Introduction) that the spatio-temporal synchrony of large-scale outbreaks may be explained by a putative regionalized Moran effect, e.g. a match/mismatch between moth and host phonologies\n\n“Such a phenological mismatch-driven Moran effect could also be responsible for the more complex dynamics recently reported for birch forest moths (Klemola et al. 2006; Tenow et al. 2007)”.\n\nWe now know that geometrid outbreak wave travelled from south-eastern Europe to the Atlantic coast in the 2000s and in the 1990s (as well as in earlier decades1). It is evident from comments to papers above that both waves crossed the region. During the investigation period (2000–2008), the wave of the 1990s still lingered during 2000–2004 in the coastal area farthest to the west (Figure 1b: sites 1–3, 5; Jepsen et al. 2011, Figure 315; cf. below). It also embraced the low between the two waves, which should have been in 2000–2002, as a mean for this particular region (Figure 2 a in Tenow et al. 20131), that is, during the incipient phase. At the end of a low, local populations become released from delayed density-dependent constraints. In older literature, this phase is named the latent phase, with populations still low but prone to increase, for the geometrids according to their 9–10-year cyclicity. The average travel speed of the wave during the 2000s can be approximated to about 300 km/year (Figure 2 a in Tenow et al. 20131). When the low between the two waves travelled westward at about 300 km year by year, the populations passed by the low should have been in the latent/increase phase at that point. This should apply for most of the populations within the approximately 400 km wide region mapped during the 2000–2002 low (cf. Jepsen et al. 2009a above13).\n\nThe average speed of about 300 km/year implies that the wave should have travelled the distance from Varangerfjord to the coast of Troms in about 2 years, allowing for delay in some coastal areas (Figure 3 in Tenow et al. 20073; Figure 4 in Tenow et al. 20131). This is in accordance with the time lapse of about 2 years between E. autumnata outbreaks at Varangerfjord in 2002–2003 and in the south-western most part of the region in 2004–2005 (Jepsen et al. 2009a above13), a distance of about 400 km, and of about 2 years between O. brumata outbreaks at Varangerfjord in 2005–2007 and at Skogfjord at the coast in 2007–2008 (Jepsen et al. 2009a above13).\n\nA time lag between the defoliations is not discussed despite being acknowledged in previous papers12,17. Instead, it is stated that E. autumnata and O. brumata\n\n“…exhibit largely synchronous dynamics, with winter moth dominating at termination of the outbreaks…”.\n\nThe lag between species was distinct for the Varangerfjord area to the east (high populations4). Also, in the western part (Figure 1b), a time lag occurred between the two species (low populations) at site 11 (Lyngenfjord)18 and was repeated one year later at site 9 (Reinøya) to the northwest16.\n\nBecause of the wave and the lag, the phase-dependent outbreak dynamics depicted was in reality two separate dynamics, one for each outbreak species. The incipient phase (2000–2002) that occurred between the two waves should mainly have consisted of latent and slowly increasing populations of E. autumnata over part of the region first, followed later by O. brumata. Released in 2000–2001, the E. autumnata populations at Varangerfjord peaked in 2002–2003, partly within the incipient phase, with increasing areas being defoliated without any significant participation of O. brumata4. They then crashed in 2004–2005 during the epidemic phase. The O. brumata defoliations did not begin to increase in the Varangerfjord area until 2003–2004 where they peaked in 2005–20064 at the transition between the epidemic and crash phases, thus dominating in defoliated areas during the first year of the crash phase (cf. Figure 7 in Jepsen et al. 2009a13). Finally, O. brumata crashed in 2007–20084 (Figure 7 in Jepsen et al. 2009a13) at the end of the crash phase. As the wave of outbreaks proceeded westward, E. autumnata peaked over most of the region in 2004–2005 and thereby completely dominated in defoliated areas during the epidemic phase. On the other hand, O. brumata came to peak at the west coast of Troms in 200617 and 2007–2008 (Jepsen et al. 2011, Figure 315) during the crash phase. In this area, the satellite did not record any extensive defoliation despite severe outbreaks. The topography is steep and the forest is fragmented. Because of this, the area of O. brumata defoliation is most likely to have been underestimated in the western coastal region compared with the area in the Varanger region with its smoother topography and more continuous forests13. This is supported by reports on large O. brumata outbreaks across northern Norway in 200617, i.e., in areas that were closer to the coast than the E. autumnata outbreaks (cf. Jepsen et al. 200819). When summarising the time scales, it is obvious that E. autumnata dominated defoliation that moved progressively from east to west during the incipient and epidemic phases (2002–2005) and O. brumata, with some overlap, in a similar way during the crash phase (2005–2008), all according to the time lag between the species. Thus, neither dynamic fitted the applied presumed periods of the phase-dependent synchrony.\n\nAccording to the definition of a wave, defoliations that were widely separated in space can hardly have been contemporaneous during the incipient, epidemic and crash phases. In addition, these phases are split in one and the same area due to the time lag between species. Therefore, it cannot be claimed that the spread of defoliation was determined by a stationary, step-wise process, one incipient long-range step and one later short-range.\n\nThe onset of plant growth (budburst) had a phase-dependent pattern of spatial synchrony during the incipient phase that was similar to that in the occurrence of defoliation (above; although with a much higher level of spatial synchrony for the former). This suggested to the authors that spring phenology plays a decisive role in the synchrony of moth outbreaks, i.e., an indirect Moran effect. However, contrary to defoliation, spring and budburst do not arrive to the region as a wave moving broadly from east to west. In line with a much higher level of spatial synchrony for budburst than for defoliation (above), budburst may occur over large areas at the same time and, probably, does not have much to do with the degree of defoliation (cf. Bylund 199920; Nilssen et al. 20079). Defoliation events in northern Fennoscandia (as well as in Fennoscandia as a whole8), are cyclic and tightly connected to the outbreak waves that pass broadly from east to west approximately every 9–10 years1,3,10. Why these waves travel as they do and if/how they force cyclicity upon local populations is not known. What can be said is that there is no known direct or indirect effect of climate forcing on any match/mismatch of plant/insect synchrony that, firstly, can cause a 9–10-year cyclicity locally and, secondly, travels across Europe from east to west every 9–10 years1. In addition, in northern Fennoscandia such an effect should act twice, first on E. autumnata and then on O. brumata, with a lag of 1–2 years. Should the match/mismatch apply only to E. autumnata or to both species?\n\nIn conclusion, the division of outbreak dynamics in phases with different synchrony and spread speed must be questioned. Instead, it is the wave and the time lag that are important. Furthermore, contrary to what is suggested, there can be no climate-mediated Moran effect that drives the 9–10 year passages of continental travelling waves over the region and/or drives the local 9–10-year population cycles.\n\nJepsen et al. (2011) Rapid northwards expansion of forest insect pest attributed to spring phenology matching with sub-Arctic birch. Global Change Biology 17: 2071–208315.\n\nThis paper surveys the time period 1999–2009 and offers interesting quantitative data on the occurrence of O. brumata at the same 6 pairs of sites in the western, coastal part of northern Fennoscandia as mentioned above (Figure 1b; Ims et al. 2004 above11). The main purpose of the study was to describe and explain a recent invasion of A. aurantiaria in the area; outbreak waves were not considered.\n\nTaken together, comments on the papers above reveal that waves of O. brumata population peaks have travelled broadly from east to west in northern Fennoscandia. This is further substantiated by the population curves depicted in Figure 3 in the paper. There is only one comment on the representation of O. brumata in these curves\n\n“Curiously, O. brumata displayed a second, much smaller, peak in abundance during the years and sites where A. aurantiaria was most abundant (2005–2009)”.\n\nIn fact, Figure 3 in the paper catches two successive travelling waves, the waves of the 1990s and the 2000s, which together shed light on the “curious” peak. The O. brumata population curves for the eastern-most and western-most sites reveal this most clearly. At the eastern-most inland pair of sites (11, 12) and the north-eastern-most island sites (8, 10), the wave of the 1990s had already passed at the time of the study (cf. Ims et al. 2004 above11). Instead, the wave of the 2000s had arrived and culminated in 2004–2006 after a distinct low in 2000–2002. Farthest to the west, at sites 1–3 and 5, both waves are represented, i.e., the wave of the 1990s culminating in 2000 and then crashing to a low (cf. Ims et al. 2004 above11; see also Tenow et al. 20073), followed by a weaker peak (= the “curious” peak) that must be identified as the wave of the 2000s. It culminated in 2006–2008, with a delay of 1–2 years since it stood at the eastern sites 11 and 12. Where A. aurantiaria occurred (as an associated geometrid), it culminated in about the same years as O. brumata at most sites. At the remaining, intermediary sites (4, 6, 7, 9), the crash of the wave of the 1990s remains visible plus the wave of the 2000s. The two waves are distinctly separated by a population low around (2002) 2004–2005 for sites 1–3, 5 (Figure 3 in the paper). Hence, during the study period, the two waves crossed the area in continuous movements from east to west, first the wave of the 1990s, then that of the 2000s. The interval between the peaks was short, about 7–9 years compared with the overall average of 9–10-years; however, short intervals have not been uncommon in northern Fennoscandia8,9.\n\nIn conclusion, as demonstrated with the “curious” O. brumata peak as an example, it is not possible to interpret long-term data fully if the continental-scale outbreak waves of O. brumata (and associated geometrids) that pass northern Fennoscandia are neglected or not known (however, see Hagen et al. 201018, referring to Tenow et al. 20073).",
"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 am grateful to Finn Williams for correcting the English in this article.\n\n\nReferences\n\nTenow O, Nilssen AC, Bylund H, et al.: Geometrid outbreak waves travel across Europe. J Anim Ecol. 2013; 82(1): 84–95. PubMed Abstract | Publisher Full Text\n\nHogstad O: Numerical and functional responses of breeding passerine species to mass occurrence of geometrid caterpillars in a subalpine birch forest: a 30-year study. Ibis. 2005; 147(1): 77–91. Publisher Full Text\n\nTenow O, Nilssen AC, Bylund H, et al.: Waves and synchrony in Epirrita autumnata/Operophtera brumata outbreaks. I. Lagged synchrony: regionally, locally and among species. J Anim Ecol. 2007; 76(2): 258–268. PubMed Abstract | Publisher Full Text\n\nKlemola T, Andersson T, Ruohomäki K: Fecundity of the autumnal moth depends on pooled geometrid abundance without a time lag: implications for cyclic population dynamics. J Anim Ecol. 2008; 77(3): 597–604. PubMed Abstract | Publisher Full Text\n\nRaimondo S, Turcáni M, Patoéka J, et al.: Interspecific synchrony among foliage-feeding forest Lepidoptera species and the potential role of generalist predators as synchronizing agents. Oikos. 2004; 107(3): 462–470. Publisher Full Text\n\nGlavendekic MM: (The wintermoths (Lepidoptera Geometridae) in oak forests in Serbia). Andrejevic ´Endowment, Dissertation. 2002; 144: 130. Reference Source\n\nKula E: The seasonal population dynamics of moth larvae in birch stands of the Krusne´ Hory Mountains (the Czech Republic) from 1986–2004. Ekolo´logia (Bratislava). 2008; 27(1): 119–129. Reference Source\n\nTenow O: The outbreaks of Oporinia autumnata Bkh. and Operophthera spp. (Lep., Geometridae) in the Scandinavian mountain chain and northern Finland 1862–1968. Zoologiska Bidrag från Uppsala. 1972; Supplement 2: 107. Reference Source\n\nNilssen AC, Tenow O, Bylund H: Waves and synchrony in Epirrita autumnata/Operophtera brumata outbreaks. II. Sunspot activity cannot explain cyclic outbreaks. J Anim Ecol. 2007; 76(2): 269–275. PubMed Abstract | Publisher Full Text\n\nKlemola T, Huitu O, Ruohomäki K: Geographically partitioned spatial synchrony among cyclic moth populations. Oikos. 2006; 114(2): 349–359. Publisher Full Text\n\nIms RA, Yoccoz NG, Hagen SB: Do sub-Arctic winter moth populations in coastal birch forest exhibit spatially synchronous dynamics? J Anim Ecol. 2004; 73(6): 1129–1136. Publisher Full Text\n\nHagen SB, Jepsen JU, Yoccoz NG, et al.: Anisotropic patterned population synchrony in climatic gradients indicates nonlinear climatic forcing. Proc Biol Sci. 2008; 275(1642): 1509–1515. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJepsen JU, Hagen SB, Högda KA, et al.: Monitoring the spatio-temporal dynamics of geometrid moth outbreaks in birch forest using MODIS-NDVI data. Remote Sens Environ. 2009; 113(9): 1939–1947. Publisher Full Text\n\nJepsen JU, Hagen SB, Karlsen SR, et al.: Phase-dependent outbreak dynamics of geometrid moth linked to host plant phenology. Proc Biol Sci. 2009; 276(1676), 4119–4128. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJepsen JU, Kapari L, Hagen SB, et al.: Rapid northwards expansion of a forest insect pest attributed to spring phenology matching with sub-Arctic birch. Glob Chang Biol. 2011; 17(6): 2071–2083. Publisher Full Text\n\nSchott T, Hagen SB, Ims RA, et al.: Are population outbreaks in sub-arctic geometrids terminated by larval parasitoids? J Anim Ecol. 2010; 79(3): 701–708. PubMed Abstract | Publisher Full Text\n\nHagen SB, Jepsen JU, Ims RA, et al.: Shifting altitudinal distribution of outbreak zones of winter moth Operophtera brumata in sub-arctic birch forest: a response to recent climate warming? Ecography. 2007; 30(2): 299–307. Publisher Full Text\n\nHagen SB, Jepsen JU, Schott T, et al.: Spatially mismatched trophic dynamics: cyclically outbreaking geometrids and their larval parasitoids. Biol Lett. 2010; 6(4): 566–569. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJepsen JU, Hagen SB, Ims RA, et al.: Climate change and outbreaks of the geometrids Operophtera brumata and Epirrita autumnata in subarctic birch forest: evidence of a recent outbreak range expansion. J Anim Ecol. 2008; 77(2): 257–264. PubMed Abstract | Publisher Full Text\n\nBylund H: Climate and the population dynamics of two insect outbreak species in the north. Ecological Bulletins. 1999; 47: 54–62 Copenhagen 1999. Reference Source"
}
|
[
{
"id": "965",
"date": "24 May 2013",
"name": "Judith H Myers",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPopulation cycles of two species of forest caterpillars, winter moth Operophtera brumata and the autumnal moth Epirrita autumnata have been studied extensively over both spatial and temporal scales in Norway, Sweden and Finland by a number of workers. Two major findings are that the outbreaks of winter moth tend to lag those of autumnal moth by one to two years, and waves of population outbreaks of both species occur from east to west across Fennoscandia with a ten-year periodicity (Tenow 2013). Over the years there has been much interest in the synchronous population dynamics of cyclic species and whether this is associated with some environmental cue, wide ranging dispersal, or the movement of natural enemies among populations. The lag in outbreaks of winter moth and autumnal moth and the waves of outbreaks suggest that a single environmental cue is unlikely to explain the outbreak dynamics.Here Tenow reinterprets the results of 5 publications of population outbreaks of winter moth and autumnal moth in Norway to show how interpretations could be different if the lag between the two species and the waves of outbreaks were considered. The descriptions are complex and the paper would be clearer if the figures from the original papers were included so that it was evident what the population trends were that were being discussed. Given the extensive amount of work on this system it is useful to reevaluate earlier studies in light of more recent findings as is done here. Remaining interesting questions are 1. What factors determine the geographical waves of outbreaks? And 2. Why does this apparently consistent lag occur in outbreaks of two species feeding on the same host trees?",
"responses": [
{
"c_id": "546",
"date": "06 Sep 2013",
"name": "Olle Tenow",
"role": "Author Response",
"response": "Professor Meyers, many thanks for your referee report of 24 May! In it you asked for figures from the papers I treated to be inserted in my article to make it easier for the reader to follow the text. I can only say that I asked the copyright-holders for permission to use some specified figures but they, sorry to say, did not allow me to do that."
}
]
},
{
"id": "967",
"date": "28 May 2013",
"name": "Patrick Tobin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study of forest insect population dynamics has a long and storied history, and many forest insect species have served as model systems for studying conceptual processes of population biology and ecology. This is particularly the case for two geometrid species undergoing cyclical populations in Fennoscandia. A recent paper by Tenow et al. (2013)1, recommended in F1000Prime2) revealed the presence of regular travelling waves for the winter moth (Operophtera brumata) across the European continent, providing evidence of the importance of continental processes in understanding regional and local dynamics of cyclical populations.In this commentary paper, Tenow contends that due to the presence of these continental-scale travelling waves, other studies within this system that do not account for these patterns could be in fact missing an important driver of population dynamics. Tenow critiques five papers to challenge their respective conclusions. This commentary should stimulate a thoughtful dialogue by all involved in these studies, which undoubtedly would a benefit to others involved in the study of insect spatial and temporal dynamics.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-128
|
https://f1000research.com/articles/2-155/v1
|
11 Jul 13
|
{
"type": "Research Article",
"title": "Prevalence of antibiotic-resistant E. coli in retail chicken: comparing conventional, organic, kosher, and raised without antibiotics",
"authors": [
"Jack M Millman",
"Kara Waits",
"Heidi Grande",
"Ann R Marks",
"Jane C Marks",
"Lance B Price",
"Bruce A Hungate",
"Jack M Millman",
"Kara Waits",
"Heidi Grande",
"Ann R Marks",
"Jane C Marks"
],
"abstract": "Retail poultry products are known sources of antibiotic-resistant Escherichia coli, a major human health concern. Consumers have a range of choices for poultry, including conventional, organic, kosher, and raised without antibiotics (RWA)-designations that are perceived to indicate differences in quality and safety. However, whether these categories vary in the frequency of contamination with antibiotic-resistant E. coli is unknown. We examined the occurrence of antibiotic-resistant E. coli on raw chicken marketed as conventional, organic, kosher and RWA. From April – June 2012, we purchased 213 samples of raw chicken from 15 locations in the New York City metropolitan area. We screened E. coli isolates from each sample for resistance to 12 common antibiotics. Although the organic and RWA labels restrict the use of antibiotics, the frequency of antibiotic-resistant E. coli tended to be only slightly lower for RWA, and organic chicken was statistically indistinguishable from conventional products that have no restrictions. Kosher chicken had the highest frequency of antibiotic-resistant E. coli, nearly twice that of conventional products, a result that belies the historical roots of kosher as a means to ensure food safety. These results indicate that production methods influence the frequency of antibiotic-resistant E. coli on poultry products available to consumers. Future research to identify the specific practices that cause the high frequency of antibiotic-resistant E. coli in kosher chicken could promote efforts to reduce consumer exposure to this potential pathogen.",
"keywords": [
"The use of antibiotics in livestock production may pose health risks to humans",
"as such usage has been correlated with the occurrence of antibiotic-resistant bacteria isolated from human infections1",
"2. Methods of livestock production differ in antibiotic use",
"and this can influence the frequency of antibiotic-resistant bacteria on retail meats. For example",
"antibiotic-resistant Escherichia coli has been shown to be less common on poultry raised without antibiotics (RWA) as compared to poultry raised conventionally3. Likewise",
"organic poultry can have lower frequencies of antibiotic-resistant bacteria than poultry raised conventionally4–10",
"although this is not always the case11–13. Organic",
"RWA",
"and kosher food products supply a growing market niche14. Consumers perceive that they offer health benefits14–21 and are willing to pay a premium for them22–24. The actual health benefits of organic food are not always clear25",
"and the health benefits of kosher foods are largely anecdotal. Little is known about the frequency of antibiotic-resistant microorganisms on kosher products."
],
"content": "Introduction\n\nThe use of antibiotics in livestock production may pose health risks to humans, as such usage has been correlated with the occurrence of antibiotic-resistant bacteria isolated from human infections1,2. Methods of livestock production differ in antibiotic use, and this can influence the frequency of antibiotic-resistant bacteria on retail meats. For example, antibiotic-resistant Escherichia coli has been shown to be less common on poultry raised without antibiotics (RWA) as compared to poultry raised conventionally3. Likewise, organic poultry can have lower frequencies of antibiotic-resistant bacteria than poultry raised conventionally4–10, although this is not always the case11–13. Organic, RWA, and kosher food products supply a growing market niche14. Consumers perceive that they offer health benefits14–21 and are willing to pay a premium for them22–24. The actual health benefits of organic food are not always clear25, and the health benefits of kosher foods are largely anecdotal. Little is known about the frequency of antibiotic-resistant microorganisms on kosher products.\n\nThe organic and RWA labels require specific production methods as stipulated in US federal regulations, whereas the kosher label adheres to religious requirements that are regulated privately. The RWA label requires that “livestock have never received antibiotics from birth to harvest”26. The United States Department of Agriculture (USDA) organic standard is only slightly less strict, stipulating that “The producer of an organic livestock operation must not sell, label, or represent as organic any animal or edible product derived from any animal treated with antibiotics”, but also that “Poultry or edible poultry products must be from poultry that has been under continuous organic management beginning no later than the second day of life”26,27. Therefore, injecting antibiotics into eggs or administering them during the first 24 hours of the chick’s life will not violate the letter of the USDA organic standard28,29. Kosher production differs from organic and RWA in that it is inherently predicated on religious requirements. For kosher meat, the major requirements are that it must be from animals that have split hooves and chew their cud, it must not be mixed with dairy products, and all equipment used must be used exclusively for kosher food19. Animals must be slaughtered “humanely”, and meat is typically salted to remove blood rapidly, a practice that has been shown to reduce the microbial load30. Unlike for organic and RWA, kosher poultry is not regulated by Federal laws but rather by private certification organizations, and thus the specific practices vary19.\n\nHere, we compared four major types of poultry-conventional, kosher, organic, and RWA-in order to assess the frequency of contamination with antibiotic-resistant E. coli. We focused on poultry products from a major metropolitan center (the greater New York City area) and products available to typical consumers by studying multiple brands of chicken from multiple stores. Our goal was to compare the frequency of antibiotic-resistant E. coli. in these four categories of chicken.\n\n\nMethods\n\nDuring April–June 2012, raw chicken was purchased from supermarkets, butcher shops, specialty stores, and food distributors in the greater New York City area. A variety of widely available brands were procured in four categories: conventional, kosher, organic and RWA. Some samples included more than one category (e.g., kosher and organic). Five collections occurred resulting in 213 total samples. Samples were drumsticks or samples from which drumsticks were removed for analysis (all with skin). After purchase, each chicken sample was placed in a labeled, ziplock bag, and placed in a cooler with ice packs. Three coolers with ice packs were shipped overnight to T-Gen North within two days of collection.\n\nChicken samples arrived at the laboratory in their original packaging and were refrigerated at 4°C until processed. One putative E. coli strain was isolated and screened from each sample using standard methods for assaying for antimicrobial resistance described by the Clinical and Laboratory Standards Institute (CLSI)31. The use of one strain per sample enabled efficient testing among a population of chicken samples for differences in the frequency of antibiotic resistance.\n\nOne whole drumstick was selected from each package or removed from each whole chicken sample using a sterilized knife. Each sample was transferred aseptically to a Stomacher Bag (VWR, Radon, PA, USA, catalog number 11216–902) containing 250 ml MacConkey broth (Alpha Biosciences, Baltimore, MD) and agitated at speed 7 for 3 min on a rocking platform shaker (VWR, Radon, PA, USA, model no. 40000–302) and incubated overnight at 44°C. A 10 μl loop was used to inoculate a VRBA+MUG (Teknova, Hollister, CA) plate with the enriched broth. The plate was incubated at 37°C for 2 h and then at 44°C for 22 h, along with QA/QC strains ATCC E. coli 35218, Klebsiella pneumoniae, Hafnia alvei, Citrobacter freundii, and Serratia plymuthica. QA/QC strains not listed as ATCC were isolated and identified using the BD Phoenix at Flagstaff Medical Center. From each VRBA+MUG plate, four putative E. coli colonies were streaked to CHROMagar (Hardy Diagnostics, Santa Maria, CA) and incubated 20 to 24 h at 37°C. One putative E. coli colony, appearing pink to rose, was streaked to a second CHROMagar plate and incubated 20 to 24 h at 37°C. For each sample, a putative E. coli isolate was inoculated into an assigned well of a 96-well plate containing 75 µl of Tris EDTA (TE) buffer. DNA was released from cell suspension with a thermal cycler (Bio-Rad, Hercules, CA) using the following parameters: heated lid, 95°C; block temperature, 90°C for 15 min. To confirm the identity of putative E. coli isolates, a uidA qPCR assay and a universal bacterial qPCR (BactQuant32) were used. For each reaction, 2 μl of DNA was added into 8 μl of master mix, with the final reaction containing 1.8 μM of each forward and reverse uidA primer, 0.25 μM uidA-VIC probe, 0.90 μM of each forward and reverse Pan16S primer, 0.25 μM Pan16S-FAM probe, 1X QuantaPerfeCTa® Multiplex qPCR SuperMix w⁄ROX (Quanta Biosciences, Gaithersburg, MD) and molecular-grade water. All samples were run in triplicate and each experiment included a standard curve and no-template controls. The 7900HT Real-Time PCR System (Applied Biosystems, Carlsbad, CA) was used to run the reactions with following conditions: 3 min at 50°C for UNG treatment, 10 min at 95°C for Taq activation, 15 s at 95°C for denaturation and 1 min at 60°C for annealing and extension × 40 cycles. Six isolates were excluded from further analysis because they were not confirmed as E. coli using the qPCR assay.\n\nGuidelines from the Clinical and Laboratory Standards Institute (CLSI) for disk diffusion methods31 were used to test each strain for resistance to antibiotics. Some strains did not grow under assay conditions (n=23) and were excluded from further analysis. Twelve antibiotics were tested, representing seven classes of drugs: tetracycline (class, tetracyclines); ampicillin and ampicillin sulbactam (class, penicillins); cefazolin, cefoxitin, and ceftriaxone (class, cephalosporins); gentamicin and amikacin (class, aminoglycosides); nalidixic acid and ciprofloxacin (class, quinolones); trimethoprim sulfamethoxazole (class, folate pathway inhibitors); and imipenem (class, carbapenems) (VWR, Radon, PA). Breakpoint guidelines from the CLSI M100 Tables 2A through 2J for E. coli31 were used to classify strains into “resistant”, “intermediate” or “susceptible”; designations of “intermediate” were lumped with “resistant” for purposes of statistics and inference, a conservative approach with respect to consumer safety.\n\nAnalysis of variance (ANOVA) was used to test whether antibiotic resistance varied among the brands of chicken sampled, using SYSTAT 13.1. Effects of brand within each category were tested (i.e., using all the data within conventional, organic, kosher, RWA). For each drug, Microsoft Excel for Mac Version 14.1.0 was used to conduct chi-square tests to determine whether the frequency of resistance varied among categories of chicken: conventional, organic, kosher and RWA.\n\nThe total number of drugs and drug classes to which each strain was resistant were enumerated. One-way ANOVA was used to compare the average number of drugs to which strains were resistant among categories, using samples with only one category designation (n=120). This test captures the effect of a consumer’s choice whether to purchase chicken in one category over another on the likelihood of exposure to antibiotic-resistant E. coli.\n\nMulti-factor ANOVA was used to test whether trends held across the broader dataset (n=184), including samples with multiple category designations. The collection of samples included adequate replication (>14) for every possible two-way combination of labels (organic & kosher, RWA & organic, and RWA & kosher). Replication for the three-way combination (organic, kosher & RWA) was low (n=5), and all samples were from one brand. To avoid bias, these samples were excluded from the ANOVA. Each of the three labeling categories was included as a factor in three-way ANOVAs (organic, RWA, and kosher, each with two levels), with the number of drugs and drug classes exhibiting resistance as response variables. This tests for the effect of each category and for interactive effects of combining categories.\n\n\nResults\n\nAcross the entire dataset, resistance to cefazolin was most common (41.3%), followed by ampicillin (31.5%), tetracycline (30.4%), and ampicillin sulbactam (19.6%). Some resistance was detected for cefoxitin, (12.5%) and gentamicin (10.9% of strains), but no strain was resistant to amikacin, the other aminoglycoside tested. For the quinolones, some (3.3%) of strains were resistant to nalidixic acid, but none was resistant to ciprofloxacin. Resistance was low (3.3%) for trimethoprim sulfamethoxazole, the one folate pathway inhibitor tested, and was absent for imipenem, the one carbapenem tested.\n\nWithin categories of chicken purchased, brands did not vary in the extent of antibiotic resistance (Table 1). By contrast, categories of chicken differed in the number of drugs to which strains of E. coli were resistant (Figure 1). Strains of E. coli isolated from kosher chicken were resistant to more drugs than were strains from the other categories (Tukey’s HSD comparisons: kosher vs. conventional, P=0.023; kosher vs. organic, P=0.041; kosher vs. RWA, P=0.002).\n\nThe response variable was the number of drugs to which strains of E. coli exhibited resistance. N indicates numbers of brands within each category. The P-values are for the effect of brand, tested for each category. N indicates numbers of brands within each category. The P-value is for the effect of brand in the ANOVA.\n\nA. The percentage of resistant strains of E. coli as a function of the number of drugs tested for each of the four categories of chicken sampled. Values shown on the x-axis are cumulative. For example, the percentage of strains resistant to five or more drugs includes strains resistant to five to seven drugs. B. The average number of drugs to which strains of E. coli exhibited resistance in each of the four categories of chicken sampled. Values shown are means ± standard errors of the mean. Category was a significant factor in a one-way ANOVA (P=0.003). Bars with different letters are significantly different at P<0.05 (Tukey’s HSD). RWA-raised without antibiotics.\n\nThese patterns held when analyzing the broader dataset, including the samples with multiple designations. Strains of E. coli isolated from kosher chicken samples were resistant to more drugs compared to the other categories (Figure 2). Strains of E. coli isolated from samples in the RWA category tended to be resistant to fewer drugs but the difference was not significant versus conventional and organic which did not differ from each other.\n\nValues shown are means ± standard errors of the mean. Kosher was a significant factor in the analysis of variance (P=0.00374), whereas ‘raised without antibiotics’ (RWA) (P=0.122), organic (P=0.874), and all interactions (P<0.050) were not significant.\n\n\n\n\nDiscussion\n\nAntibiotic use is widespread in the production of chicken both for therapeutic and non-therapeutic purposes (e.g., growth promotion). The use of antibiotics in poultry production selects for antibiotic-resistant microorganisms including Salmonella, Campylobacter, Enterococcus, and extra-intestinal pathogenic E. coli33. Studies of E. coli from bloodstream infections in Europe suggest that poultry are an important source of antibiotic-resistant infections34. Use of antibiotics is restricted in production of chicken carrying the USDA organic and USDA RWA labels. Like conventional chicken, chicken with a certified kosher label does not indicate any special restrictions in the use of antibiotics.\n\nOur finding that the frequency of antibiotic resistant strains of E. coli on organic poultry did not differ significantly from conventional (Figure 1 and Figure 2) reflects some past studies in this area that have found no difference in antibiotic resistance between organic and conventional practices11–13. Others found that pathogens on organic or RWA poultry products had lower resistance to antibiotics compared to conventional products4,10,35–38, which was the trend we observed for RWA. The distinction between USDA organic from USDA RWA may be important, given that organic chicks can receive antibiotics via in ovo injections and during the first day of life. Previous studies have provided unequivocal evidence that even in ovo injection of antibiotics can affect the susceptibility of the bacteria that contaminate poultry products2. With a larger sample, the tendency for E. coli isolated from RWA samples to have lower frequency of antibiotic resistance than other categories (P=0.122; Figure 1 and Figure 2) may emerge as significant.\n\nCross-contamination is another possible source of antibiotic resistance39. Shared facilities for product and slaughter could promote cross-contamination and antibiotic strains could be spread among organism and environments40,41. Poultry could then be inadvertently exposed to antibiotic-resistant E. coli. For example, companies with both conventional and organic products may slaughter in the same facilities, promoting cross-contamination. Production facilities that convert from one practice to another could also experience residual contamination, though there is evidence that converting from conventional to organic can reduce frequency of resistance8. The identification of possible cross-contamination is outside the scope of this study, but these possibilities would need to be considered when investigating the sources of antibiotic resistance.\n\nThe increased resistance of E. coli in kosher chicken compared to conventional was surprising, because, while kosher does not stipulate anything about antibiotic use, kosher is perceived as clean and safe to consume19. The higher resistance found in isolates from kosher chicken (Figure 1 and Figure 2), and the distinct antibiotic-resistance profile (Table 2) suggests that use of antibiotics in the kosher production chain is common and that it may be more intensive than use of antibiotics among conventional, organic, or RWA practices. It is not immediately obvious where in the kosher chicken production process antibiotic use might be more prevalent, or where exposure to antibiotic-resistant organisms is more likely. Consumers perceive organic, kosher and RWA products to be healthier14–21, though the real health benefits from organic products are unclear10, and, to our knowledge, the actual health benefits of kosher have not been assessed. Our findings are consistent with the suggestion that some ‘niche market’ products, while perceived to be safer, may have higher incidence of foodborne pathogens compared to conventional products42.\n\nBold text denotes significant differences among categories according to one-way ANOVA.\n\nOur study was limited in geographic and temporal scale, as we focused on the New York metropolitan area over a three-month time period. Yet, the region is large and populous, we focused on the most widely available brands in all categories, and this area particularly offered multiple kosher brands. Our sample size was limited (n=184) but not atypical for the field43–46. Finally, we only assayed for generic E. coli and did not assess virulence or virulence group assignments for each sample. However, E. coli is a useful focal organism because it is widespread and an important potential pathogen.\n\nMore studies are needed to test whether antibiotic resistance among kosher products is consistently higher than conventional and other categories. Nevertheless, our study offers insight into another area of the food production system increasing the exposure of people to microorganisms that are resistant to antibiotics. In addition to regulation, more consistent surveillance or auditing would add an additional layer of consumer protection, enabling improved purchase decisions based on price and health benefits guided by meaningful labels.",
"appendix": "Author contributions\n\n\n\nJMM, ARM, JCM, LBP, and BAH designed the study. JMM and ARM collected the samples. KW and HG carried out the laboratory analyses. JMM and BAH analyzed the data. All authors contributed to writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the Merriam-Powell Center for Environmental Research and the Ecosystem Science & Society Center at Northern Arizona University.\n\n\nReferences\n\nSilbergeld EK, Graham J, Price LB: Industrial food animal production, antimicrobial resistance, and human health. Annu Rev Public Health. 2008; 29: 151–169. PubMed Abstract | Publisher Full Text\n\nDutil L, Irwin R, Finley R, et al.: Ceftiofur resistance in Salmonella enterica serovar Heidelberg from chicken meat and humans, Canada. Emerg Infect Dis. 2010; 16(1): 48–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang J, Massow A, Stanley M, et al.: Contamination rates and antimicrobial resistance in Enterococcus spp., Escherichia coli, and Salmonella isolated from “no antibiotics added”-labeled chicken products. Foodborne Pathog Dis. 2011; 8(11): 1147–1152. PubMed Abstract | Publisher Full Text\n\nMiranda JM, Guarddon M, Mondragon A, et al.: Antimicrobial resistance in Enterococcus spp. strains isolated from organic chicken, conventional chicken, and turkey meat: a comparative survey. J Food Prot. 2007; 70(4): 1021–1024. PubMed Abstract\n\nMiranda JM, Mondragon A, Vazquez BI, et al.: Microbiological quality and antimicrobial resistance of Escherichia coli and Staphylococcus aureus isolated from conventional and organic “Arzua-Ulloa” cheese. Cyta-J Food. 2009; 7(2): 103–110. Publisher Full Text\n\nMiranda JM, Vazquez BI, Fente CA, et al.: Antimicrobial resistance in Escherichia coli strains isolated from organic and conventional pork meat: A comparative survey. Eur Food Res Technol. 2008; 226(3): 371–375. Publisher Full Text\n\nYoung I, Rajic A, Wilhelm BJ, et al.: Comparison of the prevalence of bacterial enteropathogens, potentially zoonotic bacteria and bacterial resistance to antimicrobials in organic and conventional poultry, swine and beef production: a systematic review and meta-analysis. Epidemiol Infect. 2009; 137(9): 1217–1232. PubMed Abstract | Publisher Full Text\n\nSapkota AR, Hulet RM, Zhang G, et al.: Lower prevalence of antibiotic-resistant Enterococci on U.S. conventional poultry farms that transitioned to organic practices. Environ Health Perspect. 2011; 119(11): 1622–1628. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlvarez-Fernandez E, Cancelo A, Diaz-Vega C, et al.: Antimicrobial resistance in E. coli isolates from conventionally and organically reared poultry: A comparison of agar disc diffusion and Sensi Test Gram-negative methods. Food Control. 2013; 30(1): 227–234. Publisher Full Text\n\nSmith-Spangler C, Brandeau ML, Hunter GE, et al.: Are organic foods safer or healthier than conventional alternatives?: a systematic review. Ann Intern Med. 2012; 157(5): 348–66. PubMed Abstract | Publisher Full Text\n\nLeJeune JT, Christie NP: Microbiological quality of ground beef from conventionally-reared cattle and “raised without antibiotics” label claims. J Food Prot. 2004; 67(7): 1433–1437. PubMed Abstract\n\nRoesch M, Perreten V, Doherr MG, et al.: Comparison of antibiotic resistance of udder pathogens in dairy cows kept on organic and on conventional farms. J Dairy Sci. 2006; 89(3): 989–997. PubMed Abstract | Publisher Full Text\n\nLuangtongkum T, Morishita TY, Ison AJ, et al.: Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry. Appl Environ Microbiol. 2006; 72(5): 3600–3607. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCohen E, Schwartz Z, Antonovski R, et al.: Consumer perceptions of kosher products. J Foodserv Bus Res. 2002; 5(3): 69–88. Publisher Full Text\n\nJolly DA, Schutz HG, Diazknauf KV, et al.: Organic foods - consumer attitudes and use. Food Technol. 1989; 43: 60. Reference Source\n\nJolly DA: Differences between buyers and nonbuyers of organic produce and willingness to pay organic price premiums. J Agribusiness. 1991; 9: 97–111. Reference Source\n\nWilliams PR, Hammitt JK: A comparison of organic and conventional fresh produce buyers in the Boston area. Risk Anal. 2000; 20(5): 735–746. PubMed Abstract | Publisher Full Text\n\nWilliams PR, Hammitt JK: Perceived risks of conventional and organic produce: Pesticides, pathogens, and natural toxins. Risk Anal. 2001; 21(2): 319–330. PubMed Abstract\n\nCampbell H, Murcott A, MacKenzie A: Kosher in New York City, halal in Aquitaine: challenging the relationship between neoliberalism and food auditing. Agric Human Values. 2011; 28(1): 67–79. Publisher Full Text\n\nPino G, Peluso AM, Guido G: Determinants of Regular and Occasional Consumers' Intentions to Buy Organic Food. J Consum Aff. 2012; 46(1): 157–169. Publisher Full Text\n\nShafie FA, Rennie D: Consumer Perceptions towards Organic Food. In: Abbass MY, Bajunid AFI, editors. Proceedings of the 1st National Conference on Environment-Behaviour Studies. Amsterdam: Elsevier Science Bv. 2012; 49. : 360–367. Publisher Full Text\n\nBatte MT, Hooker NH, Haab TC, et al.: Putting their money where their mouths are: Consumer willingness to pay for multi-ingredient, processed organic food products. Food Policy. 2007; 32(2): 145–159. Publisher Full Text\n\nKrystallis A, Chryssohoidis G: Consumers' willingness to pay for organic food: Factors that affect it and variation per organic product type. Br Food J. 2005; 107(5): 320–343. Publisher Full Text\n\nYiridoe EK, Bonti-Ankomah S, Martin RC: Comparison of consumer perceptions and preference toward organic versus conventionally produced foods: A review and update of the literature. Renew Agric Food Syst. 2005; 20(4): 193–205. Publisher Full Text\n\nBrandt K, Leifert C, Sanderson R, et al.: Agroecosystem Management and Nutritional Quality of Plant Foods: The Case of Organic Fruits and Vegetables. CRC Crit Rev Plant Sci. 2011; 30(1–2): 177–197. Publisher Full Text\n\nUSDA United States Standards for Livestock and Meat Marketing Claims. In: Agriculture USDo, editor. FR 79552: Federal Register. 2002. Reference Source\n\nCode of Federal Regulations t, part 205, subpart C. National Organic Program, Organic Production and Handling Requirements. In: Regulations CoF, editor. 7. Code of Federal Regulations. 2012. Reference Source\n\nCrandall PG, Seideman S, Ricke SC, et al.: Organic poultry: Consumer perceptions, opportunities, and regulatory issues. J Appl Poult Res. 2009; 18(4): 795–802.Publisher Full Text\n\nFanatico AC, Owens CM, Emmert JL: Organic poultry production in the United States: Broilers. J Appl Poult Res. 2009; 18(2): 355–366. Publisher Full Text\n\nShin D, Kakani G, Molina VA, et al.: Effect of kosher salt application on microbial profiles of poultry carcasses. Poult Sci. 2012; 91(12): 3247–3252. PubMed Abstract | Publisher Full Text\n\nClinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing M100–S21. 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087–1898, USA: Clinical and Laboratory. 2011; Standards Institute. pp. 178. Accessed 21 June 2013 from. Reference Source\n\nLiu CM, Aziz M, Kachur S, et al.: BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay. BMC Microbiol. 2012; 12: 56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavis MF, Price LB, Liu CM, et al.: An ecological perspective on U.S. industrial poultry production: the role of anthropogenic ecosystems on the emergence of drug-resistant bacteria from agricultural environments. Curr Opin Microbiol. 2011; 14(3): 244–250. PubMed Abstract | Publisher Full Text\n\nVieira AR, Collignon P, Aarestrup FM, et al.: Association between antimicrobial resistance in Escherichia coli isolates from food animals and blood stream isolates from humans in Europe: an ecological study. Foodborne Pathog Dis. 2011; 8(12): 1295–1301. PubMed Abstract | Publisher Full Text\n\nCui S, Ge B, Zheng J, et al.: Prevalence and antimicrobial resistance of Campylobacter spp. and Salmonella serovars in organic chickens from Maryland retail stores. Appl Environ Microbiol. 2005; 71(7): 4108–4111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiranda JM, Guarddon M, Vazquez BI, et al.: Antimicrobial resistance in Enterobacteriaceae strains isolated from organic chicken, conventional chicken and conventional turkey meat: A comparative survey. Food Control. 2008; 19(4): 412–416. Publisher Full Text\n\nHan F, Lestari SI, Pu S, et al.: Prevalence and antimicrobial resistance among Campylobacter spp. in Louisiana retail chickens after the enrofloxacin ban. Foodborne Pathog Dis. 2009; 6(2): 163–171. PubMed Abstract | Publisher Full Text\n\nLestari SI, Han F, Wang F, et al.: Prevalence and antimicrobial resistance of Salmonella serovars in conventional and organic chickens from Louisiana retail stores. J Food Prot. 2009; 72(6): 1165–1172. PubMed Abstract\n\nSmith JL, Drum DJ, Dai Y, et al.: Impact of antimicrobial usage on antimicrobial resistance in commensal Escherichia coli strains colonizing broiler chickens. Appl Environ Microbiol. 2007; 73(5): 1404–1414. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoodley A, Guardabassi L: Clonal spread of methicillin-resistant coagulase-negative staphylococci among horses, personnel and environmental sites at equine facilities. Vet Microbiol. 2009; 137(3–4): 397–401. PubMed Abstract | Publisher Full Text\n\nGraham JP, Leibler JH, Price LB, et al.: The animal-human interface and infectious disease in industrial food animal production: Rethinking biosecurity and biocontainment. Public Health Rep. 2008; 123(3): 282–299. PubMed Abstract | Free Full Text\n\nFox JT, Reinstein S, Jacob ME, et al.: Niche marketing production practices for beef cattle in the United States and prevalence of foodborne pathogens. Foodborne Pathog Dis. 2008; 5(5): 559–569. PubMed Abstract | Publisher Full Text\n\nCui S, Ge B, Zheng J, et al.: Prevalence and antimicrobial resistance of Campylobacter spp. and Salmonella serovars in organic chickens from Maryland retail stores. Appl Environ Microbiol. 2005; 71(7): 4108–4111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLestari SI, Han F, Wang F, et al.: Prevalence and antimicrobial resistance of Salmonella serovars in conventional and organic chickens from Louisiana retail stores. J Food Prot. 2009; 72(6): 1165–1172. PubMed Abstract\n\nMiranda JM, Guarddon M, Mondragon A, et al.: Antimicrobial resistance in Enterococcus spp. strains isolated from organic chicken, conventional chicken, and turkey meat: a comparative survey. J Food Prot. 2007; 70(4): 1021–1024. PubMed Abstract\n\nMiranda JM, Guarddon M, Vázquez BI, et al.: Antimicrobial resistance in Enterobacteriaceae strains isolated from organic chicken, conventional chicken and conventional turkey meat: a comparative survey. Food Control. 2008; 19(4): 412–416. Publisher Full Text"
}
|
[
{
"id": "1186",
"date": "23 Jul 2013",
"name": "Marilyn Roberts",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of interest because it compares chickens raised by conventional, organic, raised without antibiotics, and kosher chickens for the prevalence of antibiotic resistant E. coli. The finding that organic and conventionally raised chickens were statically indistinguishable while chickens raised without antibiotics tended to be slightly lower once again raises the question of the tangible and potential health advantages to the consumer of eating organically raised/raised without antibiotic vs. conventionally raised poultry. Why kosher products had higher prevalence of antibiotic resistant E. coli and E. coli which were multi-resistant is a new finding and certainly needs further study. Whether statistical differences in the raised without antibiotic animals would have been found if larger numbers were tested is not clear. However this study continues to fuel the debate on whether the use of antibiotics as growth promoters in poultry production leads to selection of antibiotic resistant and multi-resistant bacteria; which ultimately may have consequences for treatment of diseases in both man and animals. This issue has been settled in the EU which has banned the practice, but is of major discussion currently in the US Congress where “The Strategies to Address Antimicrobial Resistance (STAAR)” (which would take important steps to strengthen the US federal response to the public health crisis of antimicrobial resistance) is currently being considered.",
"responses": [
{
"c_id": "538",
"date": "02 Sep 2013",
"name": "Bruce Hungate",
"role": "Author Response",
"response": "We very much appreciate Dr. Roberts' comments on our study."
}
]
},
{
"id": "1506",
"date": "16 Aug 2013",
"name": "Irene Hanning",
"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 study is of interest as this type of data is necessary to fully understand the use of antibiotics in animal production. The manuscript is well written and easy to understand. There are a few points that the authors need to address:Methods:Please list the number of brands per category that were sampled as in Table 1. Also, please list the number of samples collected per brand. This information would be helpful and may have some impact on the data. Results:The total number of E. coli isolates collected per type or brand is not stated. It would be helpful to know what percentage of each were positive.Discussion:The first sentence of the discussion section is very inflammatory to the industry and is not absolutely true. I would suggest refining this greatly or deleting it. Similarly, the second sentence is quite definitive and implies that antibiotic usage always creates antibiotic resistance which may not be true. I suggest modifying this sentence with a qualifying word such as “may select for” or “can select for”.One variable the authors did not address is the fact that chickens produced by the same brand most likely came from different farms. Because there seems to be a large number of brands sampled, this further adds to the total number of farms that were likely to be sampled. The farm environment does have some impact on the quality of the food. Further, birds from multiple farms may be processed within the same processing plant and this too can impact the microbiological quality of the carcass due to cross-contamination. These are confounding variables that may have impacted the author’s data.",
"responses": [
{
"c_id": "537",
"date": "02 Sep 2013",
"name": "Bruce Hungate",
"role": "Author Response",
"response": "We appreciate Dr. Hanning’s comments and suggestions and have revised the manuscript to address each point raised. The number of samples collected per brand is now indicated in the text. The number of brands per category is listed as 'N' in Table 1. We now state, by type of chicken collected, the percentages of isolates positive for some degree of antibiotic resistance in the fifth sentence of the Results section. We have revised a few sentences in the Discussion for clarity and to remove potentially inflammatory or unsupported claims. We have added several citations that provide support for our statements, and at the same time, we removed potentially inflammatory adjectives. We also add a statement about one specific estimate of the extent of antibiotic usage for growth promotion. We realize such estimates are controversial, but we feel that these provide important and useful context for readers in the field. For the second sentence in the original, we have followed Dr. Hanning’s suggestion to modify the claim by changing 'select' to 'can select'. We appreciate the point that farm-to-farm variability could play a role in our results, and have added a sentence acknowledging this explicitly. Also, the third paragraph of the Discussion discusses the potential for cross-contamination within shared production facilities to influence our results. We note that our design was developed to test for significant effects of type of chicken from the perspective of the consumer making decisions about which chicken to purchase. Thus, while farm-to-farm variability and cross-contamination are important potential sources of variation in antibiotic resistance, incorporating this variance is an important part of our design. Future, more exhaustive surveys could attempt to partition the influence of these factors, but doing so was beyond the scope of the current study."
}
]
}
] | 1
|
https://f1000research.com/articles/2-155
|
https://f1000research.com/articles/2-129/v1
|
21 May 13
|
{
"type": "Research Article",
"title": "Co-implanting orthotopic tissue creates stroma microenvironment enhancing growth and angiogenesis of multiple tumors",
"authors": [
"Per Borgstrom",
"Phil Oh",
"Malgorzata Czarny",
"Brian Racine",
"Jan E Schnitzer",
"Per Borgstrom",
"Phil Oh",
"Malgorzata Czarny",
"Brian Racine"
],
"abstract": "Tumor models are needed to study cancer. Noninvasive imaging of tumors under native conditions in vivo is critical but challenging. Intravital microscopy (IVM) of subcutaneous tumors provides dynamic, continuous, long-term imaging at high resolution. Although popular, subcutaneous tumor models are often criticized for being ectopic and lacking orthotopic tissue microenvironments critical for proper development. Similar IVM of orthotopic and especially spontaneous tumors is seldom possible. Here, we generate and characterize tumor models in mice for breast, lung, prostate and ovarian cancer by co-engrafting tumor spheroids with orthotopic tissue in dorsal skin window chambers for IVM. We use tumor cells and tissue, both genetically engineered to express distinct fluorescent proteins, in order to distinguish neoplastic cells from engrafted tissue. IVM of this new, two-colored model reveals classic tumor morphology with red tumor cell nests surrounded by green stromal elements. The co-implanted tissue forms the supportive stroma and vasculature of these tumors. Tumor growth and angiogenesis are more robust when tumor cells are co-implanted with orthotopic tissue versus other tissues, or in the skin alone. The orthotopic tissue promotes tumor cell mitosis over apoptosis. With time, tumor cells can adapt to new environments and ultimately even grow better in the non-orthotopic tissue over the original orthotopic tissue. These models offer a significant advance by recreating an orthotopic microenvironment in an ectopic location that is still easy to image by IVM. These “ectopic-orthotopic” models provide an exceptional way to study tumor and stroma cells in cancer, and directly show the critical importance of microenvironment in the development of multiple tumors.",
"keywords": [
"Relevant animal models are vital to understand the processes involved in tumor progression and to develop new therapies1–4. Solid tumors can be followed in their native tissue when tumor cells are administered properly into their orthotopic tissue",
"or when tumors are chemically induced or arise spontaneously",
"for instance in genetically engineered mouse models. Though in vivo imaging systems are advancing rapidly",
"imaging orthotopic tumors within the animal",
"especially at the cellular level for extended periods of time dynamically and continuously",
"remains a significant challenge. Subcutaneous models that implant rodent or human tumor cells directly into the skin of animals have quickly become the most commonly used tumor models5–7",
"in part because these tumors are convenient",
"easy to implant",
"and are readily imaged outside the body4",
"8."
],
"content": "Introduction\n\nRelevant animal models are vital to understand the processes involved in tumor progression and to develop new therapies1–4. Solid tumors can be followed in their native tissue when tumor cells are administered properly into their orthotopic tissue, or when tumors are chemically induced or arise spontaneously, for instance in genetically engineered mouse models. Though in vivo imaging systems are advancing rapidly, imaging orthotopic tumors within the animal, especially at the cellular level for extended periods of time dynamically and continuously, remains a significant challenge. Subcutaneous models that implant rodent or human tumor cells directly into the skin of animals have quickly become the most commonly used tumor models5–7, in part because these tumors are convenient, easy to implant, and are readily imaged outside the body4,8.\n\nRecent advances in intravital microscopy (IVM) have made subcutaneous tumors even easier to image dynamically at higher resolutions in live rodents through dorsal skinfold window chambers7,9,10. Standard light and fluorescence microscopy in this system can distinguish individual cells in the tumor so that many cellular events, such as cell migration, mitosis, pyknosis, apoptosis, and the growth of blood vessels, can be readily quantified. Intravital microscopy can also be particularly powerful for evaluating tumor imaging probes and therapeutic agents by visualizing at high resolution and quantifying tumor targeting, delivery, processing and efficacy in vivo, dynamically and continuously.\n\nTumor cell interactions with the surrounding tissue stromal environment, including extracellular matrix, local enzymes and proteases, vasculature, inflammatory cells, growth factors and hormones, can significantly affect tumor development11–13 and are, to a large extent, extensively altered or even missing when tumors are grown in ectopic environments such as skin14–18. Most orthotopic tumor models and especially spontaneous tumors are not readily amenable to IVM except possibly acutely for very short periods after surgical exposure, which frequently can be quite invasive. Moreover, injecting tumor cells properly to maintain an orthotopic tissue microenvironment can be quite difficult, in part because the orthotopic organ to be injected can be so very tiny in the mouse. Making sure that all of the injected cells enter and stay inside tiny organs can be quite challenging. Microsurgical techniques with stereomicroscopic imaging can help but greatly increase the labor per mouse.\n\nRecently, we have successfully engrafted donor tissue from healthy rat organs and mouse prostate tissue with hormonally sensitive prostate tumor cells into the dorsal skinfold of mice carrying a window chamber for dynamic and continuous IVM imaging in vivo19,20. The implanted tissue maintained both tissue and species-specificity, even expressing key organ-specific biomarkers19. Here, we expand this tissue transplantation and revascularization model to multiple cancers by engrafting different donor tissues with various tumor spheroids to create novel ectopic-orthotopic (EO) tumor models that permit dynamic imaging by IVM while attempting to provide and maintain an orthotopic stroma microenvironment for the tumor cells. Comparative IVM analysis of these tumors directly shows the critical incorporation of the co-engrafted tissue into the stroma of the growing tumor and ultimately the pronounced importance of this stroma and unique microenvironment for tumor growth and angiogenesis.\n\n\nMethods\n\nAll materials were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.\n\nAll animal experiments were performed in accordance with Institutional Animal Care and Use Commitee guidelines at Sydney Kimmel Cancer Center and Proteogenomics Research Institute for Systems Medicine. Athymic Nude-Foxn1nu, Balb/c, C57BL/6J and FVB mice from either Charles River Laboratories (Wilmington, MA) or Jackson Laboratories (Bar Harbor, ME) GFP C57BL/6J mice were a kind gift of Dr Christa Mueller-Seiburg (Burnham Institute). Tg(TIE2GFP)287Sato/J mice were purchased from Jackson Laboratories. Female (80) and male (20) Athymic Nude-Foxn1nu, Balb/c (5 female), C57BL/6J (10 male), GFP C57BL/6J (5 female), Tg(TIE2GFP)287Sato/J (5 female) and FVB mice (20 female) were used for the dorsal skinfold implantations and donor tissues. Once the mice were ~25g (10–14 weeks old), the chambers were placed on the dorsal skinfold and the mice were segregated into separate cages and monitored daily.\n\nAll cell lines were grown at 37°C in 5% CO2 in air. N202 (gift from Joseph Lustgarten, Mayo Clinic, Scottsdale, AZ), MOVCAR-16 (gift from Denise Connolly, Fox Chase Cancer Center, Philadelphia, PA), TrampC2 (ATCC, Manassas, VA) and Lewis Lung Carcinoma (LLC-ATCC, Manassas, VA) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) high glucose supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 U/ml), sodium pyruvate (1 mM) (Invitrogen, Carlsbad, CA) and 10% heat-inactivated Fetal Bovine Serum (Omega Scientific, Tarzana, CA). BT474 cells (ATCC, Manassas, VA) were maintained in Hybridoma-SFM supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 U/ml), sodium pyruvate (1 mM) (Invitrogen, Carlsbad, CA) and 10% heat-inactivated FBS (Omega Scientific, Tarzana, CA). The histone H2B-GFP was subcloned into the SalI/HpaI sites in the LXRN vector (Clontech, Palo Alto, CA) using SalI and blunted NotI sites from the BOSH2BGFPN1 vector21. The mCherry vector was created from the H2B-GFP vector by cloning the mCherry gene (Dr Roger Tsien, UCSD) to replace the green fluorescent protein (GFP) gene. GP2-293 cells were infected with vesicular stomatitis virus (VSV) and the H2B-GFP or H2B-mCherry-containing virus to produce viable virus. N202, BT474, TrampC2, MOVCAR-16 and LLC cells were transduced with the viable virus to stably incorporate the H2B-GFP or H2B-mCherry gene. The transduced cells were sorted twice using fluorescence-activated cell sorting (FACs) to ensure 100% of the cells stably expressed the H2B-GFP or H2B-mCherry protein.\n\nWe used the classic IVM tumor model20 with modifications. The mice, usually Athymic Nude-Foxn1nu mice (25–30g body weight), were anesthetized (7.3 mg ketamine hydrochloride and 2.3 mg xylazine per 100g body weight, intraperitoneal injection) and placed on a heating pad. As per the standard IVM tumor model20,22, a titanium frame was placed onto the dorsal skinfold of the mice to sandwich the extended double layer of skin. A 15 mm diameter full-thickness circular layer of skin was then excised. The superficial fascia on top of the remaining skin was carefully removed to expose the underlying muscle and subcutaneous tissue which was then covered with another titanium frame with a glass coverslip to form the window chamber. After a recovery period of 1–2 days, tumor spheroids were implanted.\n\nTumor spheroids were formed by plating 50,000 cells (N202, LLC, TrampC2 and MOVCAR-16) onto 1% agar-coated 96-well non-tissue culture treated flat bottom dishes (Becton Dickinson, Franklin Lakes, NJ) (20 μl cells in 100 μl medium) and centrifuging 4 times at 1200g for 15 min, rotating the dish after every centrifugation. The cells were incubated an additional 3–7 days (depending on cell type) at 37°C in 5% CO2 in air to form tight 3-dimensional spheroids. BT474 cells required 500,000 cells in the presence of Matrigel (BD Bioscience, San Diego) (2:1 cell volume dilution-cells to matrigel) to form spheroids in culture.\n\nThe tumor spheroids were implanted in the window chamber directly onto the exposed dorsal skin either alone to created standard, classic, subcutaneous model or with lung, liver, mammary (lactating female mammary fat pad) or prostate tissue which was excised from a donor mouse and minced into small pieces in penicillin (10,000 U/ml) – streptomycin (10,000 μg/ml) solution. One animal was usually enough to supply donor tissues for an experimental set of 15 animals except for the EO model in the case of prostate tissue when 3 animals were needed. Typically, the tumor spheroid was placed in the center of a bed of 1–2 mm of flattened minced tissue onto the subcutaneous tissue of each mouse. Tumors were allowed to re-vascularize over 7–14 days depending on model. For the BT474 cells, in some cases 10 μl of 5 mg/ml human 17β-estradiol (University of California, San Diego pharmacy) was injected subcutaneously twice weekly.\n\nFor adaptation to a new microenvironment, the tumors were allowed to re-vascularize as above. The tumor was removed and the fluorescent tumor cells were separated from non-tumor cells. New tumor spheroids were formed and re-implanted with donor mouse tissue as above. This was repeated two more times to reprogram the tumor to its new microenvironment.\n\nAfter implantation, tumor spheroids were allowed to revascularize (12–14 days) and tumors were imaged with intravital fluorescence video microscopy, as described20. The tumors were imaged with a FITC or Texas Red filter using an integrated frame grabber. Confocal microscopy was used to acquire dual fluorescence images via a Nikon E2000 microscope (20× and 60× objective lens) equipped with a Perkin Elmer UltraView 5ERS confocal system with an Hamamatsu Orca ER camera (Hamamatsu Corporation, Bridgewater, NJ). To construct movies, dual color images were taken every second; exposures for a single fluorophore were kept under 400 msec. Z-stacks were acquired every 0.5 µm and then resolved for 3D construction with Volocity LE v.3 software (Perkin Elmer).\n\nTumors were imaged using IVM, as described above. Tumor growth was analyzed off-line from the recorded, digital, grayscale 0–to–256 images using Image-Pro Plus (Media Cybernetics, Bethesda, MD). Tumor growth was determined in two ways: by measuring the area with fluorescence signal from the GFP- or CFP-expressing tumor cells or by quantifying the cumulative fluorescence signal for the tumor over time. Tumor area was measured by counting the number of pixels with a grayscale intensity above 75, thereby making it easier to reliably follow irregularly shaped tumors. The cumulative tumor fluorescence signal was measured by signal summation of all pixels over 75. All growth curves are normalized to the tumor on day 0. In all cases, growth measured by area or aggregate fluorescence signal was found to be very similar so only one of the results is usually shown.\n\nTo determine mitotic and apoptotic indexes, two peripheral and two central fields (using 20x objective) from three different animals for six random fields from the growing tumor in the dorsal skinfold chamber for each tumor/tissue combination was used. Only mitotic figures in metaphase-telophase are included in the mitotic index (MI) to exclude potential artifact of nuclear membrane distortion. Apoptotic/pyknotic nuclei are defined as H2B-GFP labeled nuclei with a cross sectional area < 30 μm2. Nuclear karyorrhexis, easily distinguishable by the vesicular nuclear condensation and brightness of H2B-GFP, is included within this apoptotic index (AI).\n\nThe vascular density of tumors was measured from digital images (obtained using 10× objective) that were “flattened” to reduce the intensity variations in the background pixels and cropped to eliminate distorted areas. The thresholding feature was used to segment the picture into objects and background. The picture was also skeletonized to measure vascular length. Vascular density was calculated as vascular length per tumor area.\n\nSigmaStat (Systat Software, San Jose, CA) was used to determine statistical significance. Ranked ANOVAs with the Tukey post hoc test were used and a statistically significant difference delineated if p<0.05.\n\n\nResults and discussion\n\nTo develop new breast and lung tumor models that are amenable to continuous long-term, dynamic monitoring by IVM, yet maintain an “orthotopic” tumor microenvironment, we engrafted orthotopic tissue into the ectopic subcutaneous location, the dorsal skin with a window chamber already surgically attached, and then implanted tumor spheroids onto this donor tissue (see Methods). The tumor spheroids used in these EO tumors were formed from murine mammary adenocarcinoma (N202) and Lewis Lung Carcinoma (LLC) cells (see Methods). To follow tumor cell growth and chromosome dynamics independent from changes in the tumor stroma and surrounding host tissue, these tumor cell lines were transduced to stably express histone H2B linked to green (GFP) or monovalent cherry (CFP) fluorescent proteins. We observed very similar growth for the parental and stably transfected fluorescent tumor cells and when tumor spheroids were implanted simultaneously with engrafted tissue or onto engrafted tissue that had already revascularized days earlier (Supplementary Figure 1a). We also assessed if the tumors implanted in syngeneic mice had a growth advantage over tumors implanted in nude mice. When we implanted syngeneic tumor cell spheroids in non-immunocompromised versus nude mice, we did not observe any noticeable differences in EO tumor growth (Supplementary Figure 1b). Both the N202 and LLC cells grew very similarly in nude mice as in FVB and C57BL/6J mice, respectively (Supplementary Figure 1b and data not shown). Consistent with this result, classic subcutaneous IVM tumor models routinely use nude mice in part because of several key advantages: i) enables implantation of a wide variety of tumor spheroids and tissues of different strains and species and ii) their abundant and hairless skin makes it easier to implant the titanium window chambers and observe the progressing tumors. Therefore, for the remaining experiments, we used fluorescent tumor spheroids implanted concurrently with other tissues in nude mice.\n\nIn the last decade or so, it has become clear that the stroma and tissue microenvironment can affect tumor development. Our ability to co-implant other tissues from normal organs with different tumor cell types in the dorsal skin window chamber provides a unique way to study the direct effects of different tissue stromas on tumor development. Moreover, this system facilitates direct imaging of the tumors over many days using IVM. To assess the effects of different tissues on the tumor growth in vivo, tumor spheroids were implanted directly onto the dorsal skin alone in the window chamber (as per the classic IVM subcutaneous tumor model22) or with different donor tissue. IVM enabled detailed visualization and quantification of tumor cell fluorescence signal as well as tumor area to assess tumor growth (see Methods). The N202 spheroids grew well on skin alone, better with each co-implanted donor tissue and most robustly with the orthotopic mammary fat pad tissue (Figure 1a and b). After 15 days, tumors grown in mammary tissue were >3 times the size of tumors grown subcutaneously. Thus, the orthotopic tissue provided the heartiest environment for tumor growth.\n\nHaving shown previously that prostate tumor cells grow better when implanted with prostate tissue that express key hormones required for tumor cell growth20, we were concerned that robust growth could similarly emanate from hormones expressed in the mammary fat pad tissue. To avoid typical hormonal effects and to show that the enhanced tumor growth with co-engrafted orthotopic tissue was not restricted to one cell type, we created a new lung tumor model by implanting LLC tumor spheroids onto skin alone or co-engrafted with lung, liver, and mammary tissue (Figure 1c). Fluorescent IVM again showed the tumors growing sooner and more rapidly with orthotopic tissue. At 14 days after implantation, tumors with orthotopic tissue were again at least three times larger than subcutaneous tumors (Figure 1d). However, it should be noted for both LLC and N202 cells that after about a 10-day lag period, the growth rate of the subcutaneous tumors increased dramatically to become more similar to that of the EO tumors.\n\n(a–d) N202 (a and b) and LLC (c and d) tumor spheroids expressing H2B-GFP were implanted directly onto the dorsal skin or with mammary, liver or lung tissue as indicated and monitored through the dorsal skin window chamber by IVM at the indicated times. (b and d) Relative tumor fluorescence signal was measured by total GFP intensity relative to day 1. Scale bar = 500 μm Mean +/- SD are shown. * = p<0.05. n=3–4 mice for all experiments.\n\nThe EO tumor model described here uses three sources of tissue, the engrafted orthotopic tissue from the donor mice, the tumor spheroids implanted onto the engrafted tissue, and the entire living host mouse. To visualize the implanted tissue cells distinctly from the neoplastic tumor cells and to determine which tissue (engrafted tissue or host tissue) gives rise to the stroma and vasculature inside the EO tumors, donor mammary tissue from GFP transgenic mice23 was implanted simultaneously with N202 tumor spheroids expressing H2B-mCherry. Confocal fluorescence microscopy showed very typical solid tumor architecture with well-separated islands of “red” tumor cells surrounded by “green” stromal cells. When the images were projected in 3D, green vasculature with other cells derived from the orthotopic stroma could also be seen weaving amid the red tumor cells (Figure 2a–d; Supplementary Movies 1 and 2). Vascular tubes with blood flow were also readily apparent in phase images as dark vessels against the lighter stroma (Figure 2e). Under fluorescent microscopy, the blood vessels within tumors were uniformly lined with cells expressing GFP (Figure 2a, b, d and f) and were clearly distinct from tumor cells expressing mCherry (Figure 2a, b, c and g). Thus, the engrafted tissue persists to become the supportive stroma for the tumor cells in this EO model. Furthermore, these images show not only that a thriving tumor has been created with very typical, quite classic morphology but that two key components of the tumor can be marked a priori to be visualized distinctly in a long-term, dynamic, continuous imaging system.\n\nN202 H2B-mCherry mammary tumor spheroids (red) were implanted with mammary tissue excised from a lactating GFP mouse (green) or from a mouse expressing GFP under the EC-specific TEK promoter (TEK-GFP). 20 days post-implantation, 3D confocal fluorescent microscopic images were constructed (a–d: GFP, h and i: TEK-GFP) as well as direct IVM fluorescent microscopic images (e-g: GFP) of the tumor and tissue stroma. Blood vessels are indicated by arrowheads. Scale bars = 10 μm (a–d), 20 μm (e–g), 5 μm (h and i).\n\nTo examine the vascular endothelium more specifically, we also implanted donor tissue excised from mice expressing GFP under the endothelial cell-specific promoter TEK24. Here again, the tumor vasculature was clearly lined with GFP-expressing endothelial cells (Figure 2h) that were clearly distinct from tumor cells (Figure 2i). The green vessels attached to host vessels lacking GFP and blood cells circulated seamlessly between the contiguous vessels. The tumor stroma and neovasculature, therefore, arose from the engrafted donor tissue and successfully revascularized by attaching to the unlabeled vessels present in the host animal.\n\nTumor growth ultimately requires vascular development to fulfill the metabolic demands of the cancer cells25. To compare the rates of revascularization, tumors growing in different tissue microenvironments were transilluminated so that dark blood vessels were readily visible against the bright tumor background (Figure 3a). The vascular development of N202 tumors grown either subcutaneously or on implanted lung tissue lagged for days behind the N202 tumors grown on orthotopic mammary tissue. Eventually, the vascular density became nearly equivalent by about 2 weeks in both models (Figure 3a and b). Blood vessels developed similarly for the LLC tumors (Figure 3c). Vascularization occurred sooner and initially was more rapid and extensive in EO tumors, likely supporting more rapid tumor growth.\n\n(a) Phase images of N202 tumor spheroids expressing H2B-GFP that were implanted directly onto the dorsal skin (sc) or with donor mammary tissue (EO) as indicated and monitored by IVM at the indicate times after implantation. N202 (b) and LLC (c) tumor vascular densities were measured at the indicated times. Scale bar = 10 μm Mean +/- SD are shown. * = p<0.05. n=3–4 mice for all experiments.\n\nWe also tested other tumor cell lines in this system to create prostate and ovarian EO tumor models and they showed quite extreme behavior with an extraordinary dependence on co-implantation of the correct orthotopic tissue. Both Tramp-C2 prostate tumor cells (Supplementary Figure 2a) and MOVCAR-16 ovarian tumor cells (Supplementary Figure 2b) did not grow at all when implanted alone subcutaneously in the dorsal skin window chambers. They actually disappeared over a 10-day period. However, when co-implanted with their proper orthotopic tissue in the EO model, they both grew very well, with vascular development clearly evident by 6 days after implantation. Co-implantation with other ectopic tissues did not prevent tumor disappearance (data not shown). Thus, it is not just the presence of any co-implanted stroma tissue to envelop the tumor spheroid that is necessary for growth. These tumor cell lines appear actually to require the co-engraftment specifically of the orthotopic tissue to grow and to develop new blood vessels in the tumor. The orthotopic tissue co-implantation can essentially rescue in vivo growth and enable tumor cell lines to create a more robust and potentially useful tumor model in vivo.\n\nWe also observed that human tumor cells can also exhibit a strong preference for orthotopic tissue co-implantation. We implanted the well-known human breast cancer cell line BT474 as tumor spheroids in the dorsal skin window chamber with and without mouse mammary tissue. First, we did so without supplementing the mice with human estrogen, which is customary for these tumor cells. Figure 4a and b show that the tumors did not grow well and substantially regressed from the original tumor spheroid, especially in the subcutaneous-only implants. However, with mammary tissue, the tumor regression was reversed after 2 weeks with modest growth thereafter. When we performed the implantations this time with estrogen supplementation, tumor growth was much more robust. Figure 4c and d show that, again, the fluorescent tumors grew more quickly in the EO model than subcutaneous model. The tumor spheroids decreased in size initially in the subcutaneous model for about 1 week and then grew modestly thereafter. The EO tumors did not regress and required about 5–7 days to begin robust growth. Angiogenesis was readily evident by 8–10 days after implantation. Vascular development lagged along with little tumor growth in the subcutaneous tumors alone until after 2 weeks. Thus, it appears that human tumor cells can also benefit from orthotopic mouse tissue implantation quite similarly to the mouse tumor cell lines. Even without human estrogen supplementation, these tumors cells did better in the orthotopic stroma milieu (Figure 4a and b). Then, also with human estrogen, the tumors grew much better when exposed to an orthotopic tissue environment. Our new IVM study of multiple tumor types subjected to tissue co-implantation clearly shows that the tissue stroma can have a very significant and even dramatic effect on tumor growth and vascular development. Ultimately, every tumor type tested grew best when co-implanted with respective orthotopic tissue.\n\nBT474 tumor spheroids expressing H2B-GFP were implanted directly onto the dorsal skin (sc) (a and b) or with mouse mammary fat pad donor tissue (EO) (c and d) and monitored by IVM. (a and c) Fluorescence IVM images on the indicated days after implantation. (b and d) Tumor growth measured by fluorescence signal over time. Mice received human estrogen supplement in c and d but not a and b. Scale bar = 500 μm Mean +/- SD are shown. * = p<0.05. n=3–4 mice for all experiments.\n\nUsing IVM with the H2BGFP-labeled tumor cells allowed us to visualize directly the growing tumor cells and their fluorescent nuclei in real time. To begin to examine the cellular mechanisms mediating growth differences in the distinct tissue microenvironments, chromatin dynamics were imaged to quantify both mitotic and apoptotic cells in the LLC spheroids implanted with lung tissue, ectopically with other tissues, or subcutaneously, directly on skin (Figure 5a). The ratio of mitotic to apoptotic tumor cells in each tumor revealed that the LLC tumors growing on orthotopic tissue had a strong bias towards mitosis (Figure 5b). LLC tumors growing in mammary tissue, liver or skin had a more balanced ratio of mitosis to apoptosis. The N202 tumors showed very similar results whereas the TRAMP-C2 and MOVCAR-16 tumors also exhibited ample mitosis in the EO model, but no mitosis and ample apoptosis and cell death, as they disappeared when implanted alone subcutaneously (Supplementary Figure 2). Thus, the orthotopic tissue could create for multiple tumor cell lines a local tissue microenvironment that favored tumor growth by promoting tumor-cell mitosis over apoptosis.\n\n(a) Higher magnification fluorescence micrographs showing LLC tumor cells growing in indicated tissues (9 days post-implantation) to assess their effect on relative tumor cell mitosis and apoptosis. Mitotic (arrows) and apoptotic (arrowheads) cells were counted to determine the ratio of mitotic cells to apoptotic cells (b). Scale bar = 100 μm Mean +/- SD are shown. * = p<05. n=3–4 mice for all experiments.\n\nIn humans, tumors are not restricted to one organ, but instead eventually reprogram to alter their phenotype often in order to metastasize to other organs. This well-known characteristic of cancer suggests tumor cells have the inherent ability to genetically adapt and maybe even to grow optimally in other non-orthotopic tissue environments. To determine the ability of tumor spheroids to adapt to different tissue microenvironments, we passaged N202 mammary tumor spheroids on donor lung tissue in the dorsal window chamber model (as described in the methods). Initially, mammary tumors grew poorly (Figure 6a and b) and revascularized more slowly (Figure 6c) when grown on lung tissue than orthotopic mammary tissue. However, after three passages of growing in lung tissue implanted in the IVM chamber followed by isolating and re-culturing the tumor cells for spheroid formation and then re-implantation, mammary tumor cells eventually grew much more robustly and revascularized faster (Figure 6a–c) on donor lung tissue. Remarkably, when lung-adapted mammary tumor cells were implanted onto mammary tissue, the tumors grew quite poorly and revascularized rather slowly (Figure 6a–c). In fact, their growth and revascularization was similar to the growth on the lung tissue prior to being trained via lung tissue passaging. Thus, interactions between tumor cells and stroma become evident, including tumor cells adapting to a new tissue microenvironment and eventually reaching a new phenotype optimized for the new stroma, but no longer flourishing in the original orthotopic tissue.\n\n(a) N202 tumor spheroids were passaged 0 or 3 times on lung tissue, as described in methods, and implanted onto engrafted lung or fat pad tissue and monitored by IVM at the indicated times. (b) Tumor size was measured by total tumor area relative to day 1. (c) Tumor vascular density of the indicated passaged N202 tumor spheroid was also determined as described in the methods. Scale bar = 500 μm Mean +/- SD are shown. * = p<0.05. n=3–4 mice for all experiments.\n\nIVM offers an unparalleled view into tumor development, allowing dynamic, high resolution, in vivo imaging of molecular and cellular events. Here, we greatly expand the relevancy of the classic IVM tumor model by introducing orthotopic tissue into the dorsal skinfold chambers, thereby creating EO tumor models allowing easy and direct manipulation of the tissue microenvironment that can now be viewed with a long-term, dynamic, continuous imaging system. We show that tumors in an orthotopic tissue microenvironment grow more robustly and develop vasculature more rapidly than subcutaneous and other ectopic tissue models. The orthotopic environment facilitates tumor cell mitosis over apoptosis. As new blood vessels are needed to support tumor growth, the faster growing blood supply observed in the EO models likely supports the greater rates of mitosis in the tumor cells growing with orthotopic tissue versus just subcutaneously. Recreating the orthotopic tumor microenvironment in the dorsal skinfold window chamber is a significant advancement that maintains the power of the IVM imaging system. This approach incorporates the more relevant orthotopic tissue microenvironment, while still being amenable to dynamic imaging by IVM. The IVM experiments show improvements over subcutaneous tumor models and provide key direct evidence that the tumor stroma and microenvironment can dramatically influence growth and angiogenesis.\n\nThough tumor models abound, one of the many strengths of this novel EO model is its ease of use. True orthotopic tumor models, in which tumors are implanted onto orthotopic tissue, can be technically difficult to create. For example, it is quite challenging to inject mammary tumor cells into the very tiny mammary tissue of the mouse, especially when the cell number or injection volume is similar to that of mouse tissue. Genetic tumor models are complicated and costly to create and are specialized for a very specific set of genetic defects. Dynamic in vivo imaging, especially at the cellular level, is also very limited in many of these models. The EO model overcomes each of these difficulties. Engrafting tissue in the dorsal skinfold chamber is fairly straightforward. Numerous types of tumors and donor orthotopic tissue can be readily implanted. This model is widely applicable to many tumor types and is amenable to dynamic imaging by IVM, which offers an unparalleled view into tumor development, allowing dynamic, high resolution, in vivo imaging of molecular and cellular events.\n\nImportantly, recreating the orthotopic tumor microenvironment in the dorsal skinfold window chamber allows researchers to focus on tumor-stroma interactions in a more controlled environment. Growing tumor spheroids in different microenvironments revealed that tumors in an orthotopic tissue microenvironment grow more robustly than subcutaneous and other ectopic tissue models. The orthotopic environment facilitates tumor cell mitosis over apoptosis. As new blood vessels are needed to support tumor growth, this faster growing blood supply likely supports greater rates of tumor-cell mitosis in tumor cells growing orthotopically versus subcutaneously. However, growth in a single microenvironment is not hardwired into the tumor cell. Tumor cells clearly have the ability to adapt to new microenvironments. Thus, these new tumor models may allow the ongoing interaction between tumor and stroma to be examined in greater detail and with more precise control than previously possible. Further experimentation comparing EO versus subcutaneous tumors may be warranted. Such studies may find additional functional and molecular distinctions that not only uncover stroma effects, but also provide contrasts to actual tumors in humans.",
"appendix": "Author contributions\n\n\n\nAll authors contributed to the planning and execution of experiments. PO, PB and BR performed the IVM experiments and MC performed the confocal experiments.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by grant funds (to J.E.S.) from the Komen Foundation, the state of California (TRDRP grant 18XT0196 and 20XT0161, CBCRP grant 16IB-0104, DOD CDMRP W81XWH-11-1-0693 and NIH (R01CA115215, P01CA104898, R01CA119378, and R01CA83989).\n\n\nAcknowledgments\n\nWe thank Jamie Huberman and Dale Winger for technical assistance and Kerri Massey for help with manuscript preparation. We would like to thank Dr Roger Tsien for the gift of the mCherry construct, Dr Denise Connolly for the gift of the MOVCAR-16 cells and Dr Joseph Lustgarden for the gift of the N202 cells.\n\n\nSupplementary materials\n\n(a) Comparison of simultaneous and sequential implantation. N202 tumor spheroids expressing H2B-GFP were implanted either simultaneously with the donor tissue or after the donor tissue was implanted and allowed to revascularize. The days represent the time the tumor spheroids were allowed to grow after they were implanted. As observed, the growth and vascularity of the tumors are very similar between simultaneous and sequential implantation. (b) Comparison of the relative tumor size of ectopic/orthotopic implantation of tumor spheroids in FVB versus nude mice. N202 tumor spheroids expressing H2B-GFP were implanted in FVB or nude mice with the donor mammary fat pad tissue, allowed to revascularize, and imaged by IVM to measure tumor size by total fluorescence signal on the indicated day post tumor implantation. Again, the growth of the tumors are observed to be very similar.\n\nFluorescence images (a and c) and growth curves (b and d) of TRAMPC2 (a and b) and MOVCAR-16 (c and d) tumor spheroids expressing H2B-GFP that were implanted directly onto the dorsal skin (sc) or with respective orthotopic donor tissue (EO) (prostate and ovary respectively) and monitored through the dorsal skin window chamber by IVM at the indicated times. Scale bar = 500 μm Mean +/- SD are shown. * = p<0.05. n=3–4 mice for all experiments.\n\n\nReferences\n\nDeVita VT Jr, Chu E: A history of cancer chemotherapy. Cancer Res. 2008; 68(21): 8643–53. PubMed Abstract | Publisher Full Text\n\nSuggitt M, Bibby MC: 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clin Cancer Res. 2005; 11(3): 971–81. PubMed Abstract\n\nEllis LM, Hicklin DJ: VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer. 2008; 8(8): 579–91. PubMed Abstract | Publisher Full Text\n\nKerbel RS: Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved. Cancer Biol Ther. 2003; 2(4 Suppl 1): S134–9. PubMed Abstract\n\nJain RK, Munn LL, Fukumura D: Dissecting tumour pathophysiology using intravital microscopy. Nat Rev Cancer. 2002; 2(4): 266–76. PubMed Abstract | Publisher Full Text\n\nTozer GM, Ameer-Beg SM, Baker J, et al.: Intravital imaging of tumour vascular networks using multi-photon fluorescence microscopy. Adv Drug Deliv Rev. 2005; 57(1): 135–52. PubMed Abstract | Publisher Full Text\n\nVajkoczy P, Ullrich A, Menger MD: Intravital fluorescence videomicroscopy to study tumor angiogenesis and microcirculation. Neoplasia (New York, N.Y.). 2000; 2(1–2): 53–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStaquet MJ, Byar DP, Green SB, et al.: Clinical predictivity of transplantable tumor systems in the selection of new drugs for solid tumors: rationale for a three-stage strategy. Cancer Treat Rep. 1983; 67(9): 753–65. PubMed Abstract\n\nEndrich B, Asaishi K, Gotz A, et al.: Technical report--a new chamber technique for microvascular studies in unanesthetized hamsters. Res Exp Med (Berl). 1980; 177(2): 125–34. PubMed Abstract | Publisher Full Text\n\nAsaishi K, Endrich B, Gotz A, et al.: Quantitative analysis of microvascular structure and function in the amelanotic melanoma A-Mel-3. Cancer Res. 1981; 41(5): 1898–904. PubMed Abstract\n\nCano P, Godoy A, Escamilla R, et al.: Stromal-epithelial cell interactions and androgen receptor-coregulator recruitment is altered in the tissue microenvironment of prostate cancer. Cancer Res. 2007; 67(2): 511–9. PubMed Abstract | Publisher Full Text\n\nChrenek MA, Wong P, Weaver VM: Tumour-stromal interactions. Integrins and cell adhesions as modulators of mammary cell survival and transformation. Breast Cancer Res. 2001; 3(4): 224–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmeichel KL, Weaver VM, Bissell MJ: Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype. J Mammary Gland Biol Neoplasia. 1998; 3(2): 201–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTalmadge JE, Singh RK, Fidler IJ, et al.: Murine models to evaluate novel and conventional therapeutic strategies for cancer. Am J Pathol. 2007; 170(3): 793–804. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKillion JJ, Radinsky R, Fidler IJ: Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev. 1998-1999; 17(3): 279–84. PubMed Abstract | Publisher Full Text\n\nMyers JN, Holsinger FC, Jasser SA, et al.: An orthotopic nude mouse model of oral tongue squamous cell carcinoma. Clin Cancer Res. 2002; 8(1): 293–8. PubMed Abstract\n\nNakamura T, Fidler IJ, Coombes KR: Gene expression profile of metastatic human pancreatic cancer cells depends on the organ microenvironment. Cancer Res. 2007; 67(1): 139–48. PubMed Abstract | Publisher Full Text\n\nOnn A, Isobe T, Itasaka S, et al.: Development of an orthotopic model to study the biology and therapy of primary human lung cancer in nude mice. Clin Cancer Res. 2003; 9(15): 5532–9. PubMed Abstract\n\nOh P, Borgstrom P, Witkiewicz H, et al.: Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat Biotechnol. 2007; 25(3): 327–337. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrost GI, Lustgarten J, Dudouet B, et al.: Novel syngeneic pseudo-orthotopic prostate cancer model: vascular, mitotic and apoptotic responses to castration. Microvasc Res. 2005; 69(1–2): 1–9. PubMed Abstract | Publisher Full Text\n\nKanda T, Sullivan KF, Wahl GM: Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol. 1998; 8(7): 377–85. PubMed Abstract | Publisher Full Text\n\nLehr HA, Leunig M, Menger MD, et al.: Dorsal skinfold chamber technique for intravital microscopy in nude mice. Am J Pathol. 1993; 143(4): 1055–62. PubMed Abstract | Free Full Text\n\nOkabe M, Ikawa M, Kominami K, et al.: 'Green mice' as a source of ubiquitous green cells. FEBS lett. 1997; 407(3): 313–9. PubMed Abstract | Publisher Full Text\n\nMotoike T, Loughna S, Perens E, et al.: Universal GFP reporter for the study of vascular development. Genesis. 2000; 28(2): 75–81. PubMed Abstract | Publisher Full Text\n\nFolkman J: Proceedings: Tumor angiogenesis factor. Cancer Res. 1974; 34(8): 2109–13. PubMed Abstract"
}
|
[
{
"id": "1001",
"date": "14 Jun 2013",
"name": "Hong Zhao",
"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 provides a novel way to image and investigate the tumor in the original similar microenvironment. That makes it much easier to investigate the tumor characteristics related to the microenvironments. However, there are some questions need to be clarified in the paper:1. After the implantation of the orthotopic tissue to the dorsal skin chamber, where is the blood supply of the tissue from? Will the angiogenesis happen in the tissue like a tumor? Usually, it’s a challenge to make the engrafted normal tissue to get good blood supply after the implantation. If the angiogenesis or vasculogenesis happened in the tissue, where did the vessels arise from? Were they from the host animal or just attached to the blood vessel of the host animal?2. For the vascular parameters, I didn’t notice any vascular mark methods, were they only identified by the dark space between the fluorescent tumor background? If so, will the stroma tissue in the tumor which has no fluorescent effect the counting of the blood vessels?3. There are no units of the tumor size in the graphs of figure 1, 4, 6 and the supplement figures.",
"responses": [
{
"c_id": "517",
"date": "08 Aug 2013",
"name": "Phil Oh",
"role": "Author Response",
"response": "We appreciate these comments and have revised the text description to be clearer. We have also added a paragraph to the discussion that describes possible process differences between EO versus subcutaneous models.We explicitly use donor tissue from GFP-mice so that we can definitively detect via fluorescence imaging, which parts of the tumor stroma its cells and vessels are from, the implanted tissue versus the host mouse. Moreover we also utilize tumor cells that express mCherry (red signal) for further distinction. It is clear from the figure 2 that there is ample donor tissue represented in the stroma of these tumors. Then when we look specifically at blood vessels which are unambiguously identified by their distinct structure and the flow of blood cells through them, we readily observed that the endothelial cells lining these blood vessels fluorescence “green” from their GFP expression. To increase certainty that the cells of the vascular wall in the tumor were indeed from the implanted donor orthotopic tissue, we performed additional experiments where the tissue was donated once again from transgenic mice expressing GFP but this time the expression was more selective to cell type because it is controlled by an endothelial cell promoter. Again the vessel wall cells expressed the GFP in the tumors. Please also see manuscript text for fig. 2 which describes this directly. For example we state: “To examine the vascular endothelium more specifically, we also implanted donor tissue excised from mice expressing GFP under the endothelial cell-specific promoter TEK24. Here again, the tumor vasculature was clearly lined with GFP-expressing endothelial cells (Figure 2h) that were clearly distinct from tumor cells (Figure 2i). The green vessels attached to host vessels lacking GFP and blood cells circulated seamlessly between the contiguous vessels. The tumor stroma and neovasculature, therefore, arose from the engrafted donor tissue and successfully revascularized by attaching to the unlabeled vessels present in the host animal.” One way to think of this process is that in classic subcutaneous tumors the implanted tumor spheroid is communicating with the surrounding tissue to induce accommodations that are required for tumor growth and that include angiogenesis. It is the only supplicant for proper adaptations and maybe lacks all the means possible in a normal tissue wound repairing process. With co-implantation, the donor tissue not only communicates its similar needs but also inherently has important elements, including key cells and blood vessels, to provide in a primed state to form the surviving tissue. As a minced, wounded tissue in need of repair, it appears to be able to revascularize in part through anastomosis of its vessels with underlying blood vessels of the skin. This donor tissue can do so on its own as shown in our 2007 Nature Biotechnology article in which we state that the donor implanted tissue’s blood vessels “maintained both tissue- and species-specificity, even expressing key organ-specific biomarkers”. This donor tissue can also do so with the tumor spheroid where the two appear to work quite well together to create a functioning robust neoplastic tissue. When comparing tumors with and without the donor orthotopic tissue, it appears clear that the co-implanted stroma helps the tumor take root more quickly with faster development of functioning blood vessels leading to a significant growth advantage at least initially. It will be interesting to see how similar or not the EO and subcutaneous tumors are over time; once the subcutaneous tumors have overcome their longer lag period and achieve similar vascular densities and growth, does the incorporated orthotopic stroma contribute to sustained, long term meaningful differences between the two models? We can readily differentiate the blood vessels from the non-fluorescent stroma because the blood vessels not only encompass morphologically distinct and obvious dark channels between the fluorescent tumor cells but also were identified by presence of blood cells and even actual circulating blood flow, which could easily be visualized in the movies from which the static images were made. For our measurement, we picked darkness thresholds that emphasized vessels with clear blood flow. As expected, when we have used various fluorescent tracers they provided a signal that readily coincided with the blood flow seen through the blood vessels. We appreciate these comments and have attempted to be clearer by adding more details on this to the methods section. The tumor size is based on the size of the tumor on Day 1 after implantation which is normalized for each tumor to be 1. Therefore, the relative tumor growth graphs do not have any units in the Y-axis. You can see from the pictures provided that the tumors were similar but not identical in size (in part because the tumor spheroids,at the time of implantation, cannot be matched perfectly in size). Because we ultimately were interested in relative tumor growth over time between the different groups and experiments, we choose to simplify the growth curves akin to many other past published studies by this standard normalization. How we measure tumor size and growth including this normalization is described in the methods section with a brief sentence in some of the figure legends. Please note that we measured size in two ways and both gave very similar results. We appreciate these comments and have adjusted the legends and methods to be clearer."
}
]
},
{
"id": "1031",
"date": "27 Jun 2013",
"name": "Erik Sahai",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 expands on long-standing observations that tumor xenografts behave differently in different anatomical locations. The hypothesis is that there are some paracrine interactions that favour tumorigenecity that can only occur with cells or matrix from the appropriate tissue location. Many studies have suggested this over the past twenty five years, for example Nakamura et al. (2007), Killion et al. (1999), Fidler et al. (1990). This study revisits the theory in a methodical manner. Tumor cell lines from different anatomical locations are grafted with or without normal tissue from the same organ. Lung, ovarian and breast cancer cells are found to grow quicker when co-implanted with normal tissue from the lung and breast, respectively. In general, the work is thorough and convincing, although there is some scope for improvement and clarification.Technical comments1. Borgstrom et al., use area of fluorescence signal and/or cumulative fluorescence signal as a measure of tumour size and growth (Figures 1a-d, 4a-d, and 6 a-b and Supplementary 1 a-b and 2a-d). Fluorescent signal will be attenuated at increasing depths, particularly when using low wavelength/low penetrance fluorophores such as GFP. This method is then inappropriate for irregularly shaped tumours that grow into the body of the mouse rather than just expanding along the plane of the window. In such instances tumour size will be underestimated. Ex-vivo analysis of tumour size/weight may have been more accurate.2. Borgstrom et al., define apoptotic cells by the size of the nucleus (upper cut off of <30µm2). Is the same threshold used for all cells lines? If so, is this appropriate? When measuring karyorrhexis is each nuclear fragment counted as one apoptotic cell? As tumour cells lack a cell membrane marker, it is impossible to know whether several nuclear fragments arose from a single apoptotic cell, or several. This would have been better supported using a fluorescent probe for phosphatidyl serine, activated caspase or loss of plasma membrane integrity. 3. Borgstrom et al., indicate that ‘green vessels (from donor orthotopic tissue) attached to host vessels lacking GFP and blood cells circulated seamless between the contiguous vessels’- 2i: this claim is not well supported by figure. The use of shadows as a measure of vascular density is also not ideal, it would have been better demonstrated by the injection of fluorescent vascular tracers in addition also ex vivo staining of thick sections with endothelial cell markers. For comparison, another study has recently reported imaging of endothelial cells in syngeneic tumor grafts: http://www.landesbioscience.com/journals/intravital/article/24790/?show_full_text=true .",
"responses": [
{
"c_id": "518",
"date": "08 Aug 2013",
"name": "Phil Oh",
"role": "Author Response",
"response": "We would like to thank the reviewers for their time and comments. We agree that tissue attenuation of fluorescence signal is likely and could contribute to an underestimation of tumor growth. But that is why we performed two different measurements, including one based on tumor area which is defined by the pixels reaching a minimum threshold fluorescence signal from the glowing tumor cells and thus will be much less sensitive to such attenuation. The results were very similar with both measurements. Consistent with this, these tumors tend to grow more in 2-dimensions and be a bit flattened and not so spherical in shape, in part because of the glass coverslip. Not sure that this issue is all that critical to our findings and conclusion; for the purposes of comparisons made in the paper, we used the same methods which quantified growth differences that were not particularly subtle but rather quite obvious from the captured images. Performing ex-vivo measurement of tumor size and weight somewhat defeats the overall purpose behind using IVM and this model system. We wish to get dynamic and continuous intravital data on the tumors at multiple scales and ultimately avoid using huge numbers of animals to get data that may only be a bit more accurate but at such a considerable cost in many different ways. There are some advantages here: because we are using tumor cells that provide the fluorescent signal specifically, we can be sure that our measurement reflects actual tumor cells and not other events that can appear to contribute to tumor size and apparent growth such as dead cells, infiltration of other cells, hemorrhage, edema, etc. Here we wish to examine effects of stroma/tissue implantation on the tumor cells themselves in-vivo and their proliferation. So our approach is more direct and maybe even better in many respects than simple tumor excision and weighing. We have compared our definition of apoptotic cells with tunnel assays and they were very similar in their assessment of apoptosis. This was reported in reference 20. We have added a sentence in this regard in the methods citing this paper. We are able to observe the blood including cells circulating through unmistakable blood vessels. The static images shown were taken from our movies. When we set up these measurements, we picked darkness thresholds that highlighted unambiguous vessels with clear blood flow. We have frequently used various fluorescent tracers which as expected provide a signal that coincides with the blood flow seen through the vessels. But this extra procedure on the mice ultimately was unwarranted for this singular purpose because it did not really augment or refine our measurement of vascularity. Again ex vivo evaluations seem contrary to noninvasive, dynamic, continuous, in vivo imaging attained here and would likely add more effort and animals but little beyond the results and conclusions provided more efficiently through IVM. We appreciate these comments and have added further description of the vascularity measurement in the methods to provide more clarity to the reader."
}
]
}
] | 1
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https://f1000research.com/articles/2-129
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https://f1000research.com/articles/2-154/v1
|
10 Jul 13
|
{
"type": "Research Article",
"title": "The first structure in a family of peptidase inhibitors reveals an unusual Ig-like fold",
"authors": [
"Daniel J Rigden",
"Qingping Xu",
"Yuanyuan Chang",
"Ruth Y Eberhardt",
"Robert D Finn",
"Neil D Rawlings",
"Daniel J Rigden",
"Qingping Xu",
"Yuanyuan Chang",
"Ruth Y Eberhardt",
"Robert D Finn"
],
"abstract": "We report the crystal structure solution of the Intracellular Protease Inhibitor (IPI) protein from Bacillus subtilis, which has been reported to be an inhibitor of the intracellular subtilisin Isp1 from the same organism. The structure of IPI is a variant of the all-beta, immunoglobulin (Ig) fold. It is possible that IPI is important for protein-protein interactions, of which inhibition of Isp1 is one. The intracellular nature of ISP is questioned, because an alternative ATG codon in the ipi gene would produce a protein with an N-terminal extension containing a signal peptide. It is possible that alternative initiation exists, producing either an intracellular inhibitor or a secreted form that may be associated with the cell surface. Homologues of the IPI protein from other species are multi-domain proteins, containing signal peptides and domains also associated with the bacterial cell-surface. The cysteine peptidase inhibitors chagasin and amoebiasin also have Ig-like folds, but their topology differs significantly from that of IPI, and they share no recent common ancestor. A model of IPI docked to Isp1 shows similarities to other subtilisin:inhibitor complexes, particularly where the inhibitor interacts with the peptidase active site.",
"keywords": [
"X-ray crystallography",
"protein structure",
"PDB",
"peptidase",
"inhibitor",
"Bacillus subtilis",
"Ig fold"
],
"content": "Introduction\n\nThe Isp1 peptidase (also known as IspA) is the major intracellular peptidase in Bacillus subtilis, accounting for more than 80% of the intracellular azocasein- or azocollagen-degrading activity1. It is a subtilisin homologue and a member of peptidase family S8. It is unclear what the physiological role of Isp1 is; it has been shown not to be important during sporulation where the majority of intracellular proteolysis occurs, even though intracellular proteolysis was correspondingly low when a defective mutant of Isp1 was created2. Potential physiological substrates that have been identified include elongation factor Tu (cleavage within the GTP binding domain, PF00009) and ClpC, a negative regulator of genetic competence (with one cleavage occurring in a disordered region and another in a coiled-coil region)3. The peptidase is synthesized as a precursor with an N-terminal, 18-residue propeptide that blocks the active site. Release of the propeptide is probably by Isp1 itself, inter-molecularly4. The structures of both the precursor and the mature peptidase have been solved and show that a proline residue (Pro8) introduces a kink in the backbone that shifts the scissile bond in the propeptide away from the catalytic serine. Once the propeptide is released, the peptidase active site forms correctly4.\n\nThe activity of a powerful endopeptidase within the cell must be controlled in order to prevent unwanted proteolysis of cellular proteins, and in 1986 Nishino et al.5 described an inhibitor of Isp1 from Bacillus subtilis known as Intracellular Protease Inhibitor (IPI). This inhibitor was effective against a number of subtilisin homologues, using casein as a substrate following pre-incubation of peptidase and inhibitor, but was ineffective against cysteine, aspartic and metallopeptidases. A Ki for inhibition of the Isp1 peptidase was estimated to be in the order of 10-9 M6. The gene for the peptidase has been cloned7, and the deduced protein sequence showed, at the time, no similarity to any other protein. There are now 149 homologues in the Pfam family PF12690 (BsuPI). The family was included in the MEROPS database (as I228), but subsequently removed, because it was not clear whether the proteins in the family were inhibitors or competing substrates. In particular, the inhibition of intracellular peptidases Isp2 and Isp3 was \"repressed\" in the presence of the substrate casein6, which suggests the activity of competing substrates.\n\nStructural similarity to other known peptidase inhibitors might argue for reinstatement of the family in the MEROPS database. However, most peptidase inhibitors are secreted and intracellular peptidase inhibitors are rare. The only examples are cystatins A and B from family I25, which inhibit cysteine peptidases9; calpastatin, which inhibits the intracellular peptidase calpain10; chagasin from the zooflagellate Leishmania (family I42), which also inhibits cysteine peptidases11; three intracellular coagulation inhibitors from the horseshoe crab Tachypleus that are serpins from family I412; and pinA from family I24, which is an inhibitor of the ATP-dependent serine endopeptidase Lon, but is of unknown structure13. Despite the inhibitors being intracellular, the known target peptidases are all extracellular, with the exceptions of calpain and endopeptidase Lon. Given the paucity of known intracellular peptidase inhibitors, it would not be a surprise if the fold of IPI were different from any known inhibitor structures, especially the secreted inhibitors which are stabilized by disulfide bridges, because intracellular proteins lack disulfides.\n\nMost serine peptidase inhibitors act as if they were super-substrates, binding so tightly to the active site that they are either not cleaved, or if cleavage occurs then the fragments are not released from the peptidase. There is a bond, known as the reactive bond, which occupies the peptidase active site with residues either side occupying the S1 and S1′ binding pockets (in the nomenclature of Schechter & Berger, 196814). This inhibitory mechanism is known as the standard or the Laskowski mechanism15. The residues that form the reactive bond will vary from inhibitor to inhibitor, according to the specificity of the peptidase that is inhibited. The chymotrypsin-like specificity of the Isp1 peptidase implies that the P1 residue in the reactive bond of the intracellular inhibitor should be a hydrophobic residue.\n\nAn NMR structure study of the intracellular peptidase inhibitor has been published16, which identified beta strands. We report the complete tertiary structure of the intracellular peptidase inhibitor from Bacillus subtilis.\n\n\nMethods\n\nThe American Type Culture Collection (ATCC) provided the genomic DNA used to clone ipi (ATCC Number: 23857D-5). Protein production and crystallization of IPI was carried out by standard JCSG protocols17. The crystal was obtained using the vapor diffusion method in a sitting drop format where sitting drops composed of 100 nl protein solution mixed with 100 nl crystallization solution were equilibrated against a 50 μl reservoir at 293 K. The crystallization reagent consisted of 48.5% polyethylene glycol 600, 0.1M CHES pH 9.7. Data were collected at wavelengths corresponding to the inflection and high energy remote of a selenium MAD (multi-wavelength anomalous dispersion) experiment at 100 K using a MARCCD 325 detector (Rayonix) at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 9_2. Data processing were carried out using XDS18 and the statistics are presented in Table S1. The structure was determined by the MAD method using programs SHELX19 and autoSHARP20, and refinement was carried out using REFMAC521. The structure was validated using the JCSG Quality Control server (http://smb.slac.stanford.edu/jcsg/QC). Atomic coordinates and experimental structure factors to 2.6 Å resolution (PDB code: 3ISY) have been deposited in the Protein Data Bank (www.pdb.org,22).\n\nSequence conservation among homologues of IPI was mapped onto the crystal structure using ConSurf (http://consurf.tau.ac.il/;23). The results were visualised with PyMOL (http://www.pymol.org/), which was also used for structural figures.\n\nA homology model of B. subtilis intracellular proteinase, IspA, was created at the Swiss-model server24 using the structure of processed, active intracellular protease from Bacillus clausii (PDB code 2XRM), around 50% identical to IspA, as the sole template. The high degree of sequence identity with the target and the small number of insertions and deletions between target and template (one six residue deletion and a single insertion of one residue, both readily accommodated) assured a high quality model. Additionally, the insertions and deletions lie distant from the catalytic site which was the main region of interest.\n\nThe new structure of IPI was docked to the model of IspA using three different webservers, GRAMM-X (http://vakser.bioinformatics.ku.edu/resources/gramm/grammx/;25), ZDOCK (http://zdock.umassmed.edu/;26) and ClusPro 2.0 (http://cluspro.bu.edu/;27).\n\n\nResults and discussion\n\nThe crystal structure of an intracellular proteinase inhibitor (IPI, gene locus BSU11130) from Bacillus subtilis was determined to 2.6 Å resolution by the MAD method. Data collection, model and refinement statistics are summarized in Table S1. The final model includes one molecule (residues 3–119), one tetraethylene glycol and 21 water molecules in the asymmetric unit. The structure is mainly composed of nine beta strands. Gly0 (which remained at the N-terminus after cleavage of the expression/purification tag), Met1 and Glu2 were disordered and not modeled. All the side chains except for Glu17, Lys60 and Lys97 were fully modeled. The Matthews coefficient (VM28) is 3.2 Å3 Da-1 and the estimated solvent content is 60.9%. The Ramachandran plot produced by MolProbity29 shows that 98.0% of the residues are in favoured regions, with no outliers.\n\n\n\nThe structure of the IPI protein is similar to the immunoglobulin (Ig) fold. This common fold consists of a beta-sandwich formed of seven strands in two sheets with a Greek-key topology and proteins with this fold are involved in a variety of functions, including cell-cell recognition, cell-surface receptors, muscle structure and the immune system. The structure of IPI shows an unusually extended loop on one edge of the Ig fold, seen on the right hand side in Figure 1A, resulting in a wedge-like or prismatic overall shape. As Figure 1B shows, this unusual feature is conserved at the sequence level among members of the I22 family, suggesting that it may harbour a functional site.\n\nA) Stereo cartoon representation of IPI coloured from blue to red, N- to C-terminus. Protruding loops on the right hand side form a wedge shape. B) Sequence conservation amongst known homologues mapped to the protein surface with red indicating high conservation and blue low. The left panel shows IPI in the same orientation as in A), the other two show the results of successive 90 degree rotations about a vertical axis so that the wedge is seen end-on in the centre panel and the right panel shows the opposite face to the left panel.\n\nFrom a comparison using the Dali website (http://ekhidna.biocenter.helsinki.fi/dali_server/30) the protein with the closest structural similarity is the RbmA protein from Vibrio cholerae (Z-score 10.0, Root-Mean-Square Deviation (RMSD) 2.9 Å, residues 38–152), which is one of the three major protein components of the biofilm matrix important for cell-to-cell contacts31. The A chain from coagulation factor XIII32 is also structurally similar, with z-scores in the range of 9.4–9.9 depending on the source species. The structural similarity with IPI is over residues 518–629 for human factor XIII, which is a domain from the transglutaminase, C-terminal Ig-like domain family (PF00927). Factor XIII is a transglutaminase important for stabilizing fibrin clots by cross-linking chains with isopeptide bonds32. These similarities probably reflect only the all-beta nature of the structures rather than any common physiological functions.\n\nThe folds of chagasin and amoebiasin (both members of inhibitor family I42) have also been described as Ig-like33,34. From a Dali pairwise comparison, the structures of IPI and chagasin (PDB code 2NNR) are similar but distant (Z-score 2.2, RMSD 4.2 Å; structural alignment over 110 residues). The structures of IPI and amoebasin (PDB: 3M86) are also similar (Z-score 3.0, RMSD 4.5 Å, over 111 residues). Figure 2 shows a secondary structure comparison of IPI, chagasin and amoebiasin. Chagasin and amoebiasin have similar topologies, but the order of the beta strands differs significantly between these two I42 family members and IPI. This implies that IPI is not closely related to the I42 family, and that the similar folds may have been acquired independently by convergent evolution.\n\nThe labels indicate the PDB accession number and the protein name.\n\nIn order to explore the hypothesis that IPI is an inhibitor of the intracellular protease IspA, structure docking was carried out. As an experimental structure for B. subtilis was not available, a homology model was created. The ~50% sequence identity shared with available templates assures a high quality model: at this level of similarity an RMSD error of around 1 Å is expected35. Since it is often difficult to pick out the true solution from the multitude of poses typically resulting from a docking experiment, we applied three cross-checks to improve confidence in our results. First, we required that the interface of the inhibitor with protease involve the conspicuously conserved wedge structure (Figure 1B), which is indicated as the functional site of the inhibitor structure. We chose not to incorporate this information in the docking runs, although several servers allow this, in order to reserve it as an independent check on the results. Secondly, we required that a solution sterically block the catalytic site cleft since this, rather than allosteric mechanisms, is the predominant mode of protease inhibition. Thirdly, we sought docking poses that were independently proposed by different algorithms; although simple structural compatibility is a principal criterion for all docking servers, they differ in their scoring functions so that poses jointly flagged by different methods may be considered more reliable.\n\nComparison of the top 10 results from each of the docking servers used highlighted a family of poses satisfying our criteria. This contained solutions ranked 1, 4 and 5 from ZDOCK, the second solution from GRAMM-X and the third from ClusPro (Supplementary figure 1).\n\nAs shown in Supplementary figure 1, these poses occlude the catalytic site cleft of the enzyme by inserting the conserved IPI wedge. They vary slightly in their rotation about an axis running along the cleft, and to a minor extent in their translation along it, but can be considered as a cluster of solutions.\n\nIt is well known that inhibitors typically inhibit cognate enzymes through interaction of extended regions36 and, furthermore, that different families of inhibitors acting on the same class of enzyme can exhibit highly similar, but convergently evolved interaction features36–38. Remarkably, we find that the region at the edge of the IPI wedge structure could interact with subtilisin in a manner strongly reminiscent of inhibition modes of other subtilisin family inhibitors. As Figure 3A shows, in a representative of the cluster of docking solutions, solution 5 from ZDOCK, residues 62–68 of IPI superimpose well on strongly similar inhibitory stretches from five distinct inhibitor classes, especially considering the potential for small conformational changes on binding and the fact that a model was used for the docking. Incidentally, the experimentally observed propeptide4, while lying across the catalytic site cleft in the same direction, is different to both IPI and the other four inhibitors in Figure 4.\n\nA) Inhibitors are drawn as ribbon and the B. subtilis subtilisin model as cartoon, with catalytic triad shown as ball-and-stick. The IPI complex is shown in mauve, others, after superposition on the enzymes structure, as green (2SIC; Streptomyces Subtilisin inhibitor), yellow (3QTL; Kazal inhibitor), black (1OYV; plant inhibitor class) or orange (1SBN; eglin inhibitor class). The inhibitory region lies immediately above the catalytic site in this view. B) Comparison of inhibitory region sequences from IPI and four distinct inhibitor classes. The sequences are derived from the structures shown in A) and coloured in Jalview (http://www.jalview.org/43) using the ClustalX scheme44.\n\nKey to domains: BsuPI, Bacillus subtilis protein inhibitor-like; S, signal peptide; Gmad2, immunoglobulin-like domain of bacterial spore germination; SLH, S-layer homology domain; TM, transmembrane helix; PG binding, peptidoglycan-binding domain; Cu amine oxidN1, copper amine oxidase N-terminal domain; LysM, lysin motif domain.\n\nThe comparison between the matching inhibitory regions also revealed suggestive sequence similarities with IPI. As shown in Figure 3B, amino acids in IPI abide by trends evident in the other inhibitors. Most notably three of four previous inhibitors have a central Thr residue, also present in IPI. The following position is the P1 residue. Ordinarily, substrate specificity would dictate conservation here among inhibitors but, as Figure 3B shows, the sequence varies surprisingly at this position, including a Glu in 3QTL, similar to the Gln borne at this position by IPI. The previous two positions and the last are conserved hydrophobic in all sequences; again IPI conforms to the consensus.\n\nIPI homologues are found in both bacteria and archaea, but no homologues have been detected in eukaryotes. All bacterial homologues are from Gram-positive bacteria (Firmicutes), with the exception of homologues from the Gemmatimonadetes bacterium Gemmatimonas aurantiaca, and the Gammaproteobacteria Shewanella oneidensis and Kangiella koreensis. All three of these organisms also possess subtilisin homologues. A subtilisin homologue from Gemmatimonas aurantiaca (UniProtKB C1AD56) is predicted to be intracellular.\n\nAll of the 30 archaean homologues are from species of the family Halobacteriaceae, which includes high salt tolerant species. The archaeans with completely sequenced genomes that possess an IPI homologue also possess at least one subtilisin homologue, which in halophilic archaeon DL31 and Haloterrigena turkmenica include proteins predicted to be intracellular. In none of the species with completely sequenced genomes is the gene for the IPI homologue in close proximity to a gene for a subtilisin homologue, and so they are not part of the same operon or regulon. In total, there are 33 species of bacteria and archaea that possess both an IPI homologue and a subtilisin homologue that is predicted to be intracellular (from absence of a signal peptide). There are 43 species from which an IPI homologue is known, but an intracellular subtilisin homologue is unknown, but of these 43 species only four have had their genomes completely sequenced (Bacillus selenitireducens, Bacillus macauensis, Clostridium sp. BNL1100 and Selenomonas sp. oral taxon 137).\n\nIt is relatively common for inhibitors to occur as multiple domains within the same protein. Figure 4 shows examples of domain architectures seen in proteins bearing an IPI-like domain. Most of the 109 proteins with IPI-like domains share the simple domain architecture of the Bacillus subtilis protein. The presence of a signal peptide shows that many members of the family are secreted rather than intracellular proteins. Even among the proteins that contain only the IPI-like domain, a large number also possess signal peptides, including homologues from Bacillus species. One homologue from a Geobacillus species is predicted to be a type II membrane protein with an N-terminal transmembrane domain. The longest protein to include an IPI-like domain is the 585-residue Chte0880 protein from Clostridium thermocellum, which has three repeats each composed of an S-layer homology (SLH) domain followed by an IPI-like domain. The presence of SLH domains indicates that the protein binds to the proteoglycan of the cell wall.\n\nIPI itself was initially characterized as intracellular. It was purified by lysing cells with lysozyme and its N-terminal sequence determined by Edman degradation5. Later, when the gene was sequenced7, no signal peptide was detected in the coding sequence. Now that the full genome for Bacillus subtilis strain 168 has been sequenced39, it is clear that a possible alternative initiating methionine exists for the ipi gene and the coding sequence could be extended at the N-terminus, adding an extra in-frame 33 residues (MKRLLVMLLPVLLLIGCGKDEQTEPDKEVSGG). The predicted protein sequences of IPI from B. subtilis strains BSn540 and QB92841 include this N-terminal extension. When the extended sequence was submitted to the SignalP (http://www.cbs.dtu.dk/services/SignalP/) a signal peptide was predicted with cleavage at the Thr23-Glu bond. The N-terminal sequence determined by Nishino et al. (1986)5 was Glu34, with the assumption that Met33 was the initiating methionine. Signal peptidase 1 is unlikely to cleave the Met33-Glu bond, because of the hydrophilic nature of the region Glu24-Gly32. This may mean that alternative initiation exists for this protein; a secreted form starting at Met1 and an intracellular form starting at Met33. It is worth adding that even if a majority of IPI is in fact secreted, a protease inhibitor function is still physiologically plausible since most subtilisins are extracellular.\n\nThe nearest structural neighbours of IPI, including the RbmA protein and coagulation factor XIII, are involved in protein-protein interactions. This may indicate that rather than functioning as an inhibitor, IPI is binding the peptidase in some non-specific way. Such an interaction might explain the peculiar results obtained with the substrate casein, which apparently \"repressed\" inhibition of the peptidase6. Binding to an intracellular subtilisin may therefore not represent the primary physiological function of IPI. However, there are similarities between the IPI protein and chagasin, and not only in terms of structure. Chagasin inhibition of cysteine peptidases is tight but reversible33. If IPI inhibition of subtilisins were also reversible, then the repression seen with casein would be explained. IPI inhibition would not be via the classical Laskowski mechanism, in which a reactive site bond permanently occupies the peptidase active site, but access to the active site would instead be reversibly physically blocked.\n\nWithout further kinetics studies it is not possible to state categorically that IPI definitely is a peptidase inhibitor. However, the tertiary structure and docking predictions illustrate how it could feasibly inhibit its cognate enzyme. That hypothetical mode bears strong similarities to those seen for other well-characterised, unrelated serine peptidase inhibitor families such as the Kazal and Kunitz groups in agreement with a substantial literature on convergent evolution in peptidase inhibition36,38. It is intriguing that IPI may exemplify a second intracellular peptidase inhibitor family, after the chagasin and amoebiasin group, with an Ig-type fold. However, structural similarity between chagasin and IPI is weak and the two groups clearly share no recent common ancestor. We hope the resolution of the structure encourages others to further characterize IPI and proteins bearing IPI-like domains to further probe their functions.",
"appendix": "Author contributions\n\n\n\nDJ Rigden, ND Rawlings and Y Chang wrote the text. DJ Rigden performed the molecular modelling. Q Xu refined the crystal structure. DJ Rigden, RY. Eberhardt and RD Finn prepared figures. All authors revised 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\nThis work was supported by the Wellcome Trust (grant number WT077044/Z/05/Z: RYE and NDR); Howard Hughes Medical Institute (RDF); National Institutes of Health (NIH) Protein Structure Initiative (U54 GM094586-03: QX and R01GM101457: YC); National Science Foundation (IIS-0646708 and IIS-1153617: NDR, RYE, QX,YC). Portions of this research were conducted under the auspices of the Joint Center for Structural Genomics (JCSG: NIH U54 GM09458) and structural determination was carried out SSRL Molecular Biology Program at the Stanford Synchrotron Radiation Lightsource (SSRL) supported by the Department of Energy and NIH (P41 GM103393: QX and YC). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of DOE, NSF, NIGMS, NCRR or NIH.\n\n\nAcknowledgements\n\nWe are grateful to the Sanford Burnham Medical Research Institute for hosting the DUF annotation jamboree in June 2013 which allowed the authors to collaborate on this work. We would like to thank all the participants of this workshop for their intellectual contributions to this work: L. Aravind, Herbert L. Axelrod, Alex Bateman, Penny Coggill, Debanu Das, Adam Godzik, William Hwang, Lukasz Jaroszewski, Alexey Murzin, Padmaja Natarajan, Marco Punta, Mayya Sedova, Anna Sheydina, John Wooley. We thank the members of the JCSG high-throughput structural biology pipeline for their contribution to this work.\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nBurnett TJ, Shankweiler GW, Hageman JH: Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation. J Bacteriol. 1986; 165(1): 139–45. PubMed Abstract | Free Full Text\n\nBand L, Henner DJ, Ruppen M: Construction and properties of an intracellular serine protease mutant of Bacillus subtilis. J Bacteriol. 1987; 169(1): 444–6. PubMed Abstract | Free Full Text\n\nLee AY, Goo Park S, Kho CW, et al.: Identification of the degradome of Isp-1, a major intracellular serine protease of Bacillus subtilis, by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics. 2004; 4(11): 3437–45. PubMed Abstract | Publisher Full Text\n\nGamble M, Künze G, Dodson EJ, et al.: Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism. Proc Natl Acad Sci U S A. 2011; 108(9): 3536–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNishino T, Shimizu Y, Fukuhara K, et al.: Isolation and characterization of a proteinaceous protease inhibitor from Bacillus subtilis. Agric Biol Chem. 1986; 50(12): 3059–3064. Publisher Full Text\n\nNishino T, Murao S: Interaction of proteinaceous protease inhibitor of Bacillus subtilis with intracellular proteases from the same strain. Agric Biol Chem. 1986; 50(12): 3065–3070. Publisher Full Text\n\nShiga Y, Yamagata H, Udaka S: Characterization of the gene encoding an intracellular proteinase inhibitor of Bacillus subtilis and its role in regulation of the major intracellular proteinase. J Bacteriol. 1993; 175(22): 7130–7. PubMed Abstract | Free Full Text\n\nRawlings ND, Tolle DP, Barrett AJ: Evolutionary families of peptidase inhibitors. Biochem J. 2004; 378(Pt 3): 705–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLenarcic B, Krizaj I, Zunec P, et al.: Differences in specificity for the interactions of stefins A, B and D with cysteine proteinases. FEBS Lett. 1996; 395(2–3): 113–8. PubMed Abstract | Publisher Full Text\n\nTodd B, Moore D, Deivanayagam CC, et al.: A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor. J Mol Biol. 2003; 328(1): 131–46. PubMed Abstract | Publisher Full Text\n\nWang SX, Pandey KC, Scharfstein J, et al.: The structure of chagasin in complex with a cysteine protease clarifies the binding mode and evolution of an inhibitor family. Structure. 2007; 15(5): 535–43. PubMed Abstract | Publisher Full Text\n\nMiura Y, Kawabata S, Iwanaga S: A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. J Biol Chem. 1994; 269(1): 542–7. PubMed Abstract\n\nSkorupski K, Tomaschewski J, Rüger W, et al.: A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease. J Bacteriol. 1988; 170(7): 3016–24. PubMed Abstract | Free Full Text\n\nSchechter I, Berger A: On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain. Biochem Biophys Res Commun. 1968; 32(5): 898–902. PubMed Abstract | Publisher Full Text\n\nLaskowski M Jr: Protein inhibitors of serine proteinases--mechanism and classification. Adv Exp Med Biol. 1986; 199: 1–17. PubMed Abstract\n\nLi Q, Chen AS, Gayen S, et al.: 1H, 13C and 15N chemical shift assignments for an intracellular proteinase inhibitor of Bacillus subtilis. Biomol NMR Assign. 2013; 7(2): 129–32. PubMed Abstract | Publisher Full Text\n\nElsliger MA, Deacon AM, Godzik A, et al.: The JCSG high-throughput structural biology pipeline. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010; 66(Pt 10): 1137–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKabsch W: XDS. Acta Crystallogr Sect D Biol Crystallogr. 2010; 66(Pt 2): 125–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSheldrick GM: A short history of SHELX. Acta Crystallogr A. 2008; 64(Pt 1): 112–122. PubMed Abstract | Publisher Full Text\n\nSmart OS, Womack TO, Flensburg C: Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr D Biol Crystallogr. 2012; 68(Pt 4): 368–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinn MD, Isupov MN, Murshudov GN: Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr D Biol Crystallogr. 2001; 57(Pt 1): 122–33. PubMed Abstract | Publisher Full Text\n\nRose PW, Bi C, Bluhm WF, et al.: The RCSB Protein Data Bank: new resources for research and education. Nucleic Acids Res. 2013; 41(Database issue): D475–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshkenazy H, Erez E, Martz E, et al.: ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 2010; 38(Web Server issue): W529–W533. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBordoli L, Schwede T: Automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal. Methods Mol Biol. 2012; 857: 107–36. PubMed Abstract | Publisher Full Text\n\nTovchigrechko A, Vakser IA: GRAMM-X public web server for protein-protein docking. Nucleic Acids Res. 2006; 34(Web Server issue): W310–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPierce BG, Hourai Y, Weng Z: Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One. 2011; 6(9): e24657. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKozakov D, Hall DR, Beglov D, et al.: Achieving reliability and high accuracy in automated protein docking: Cluspro, PIPER, SDU, and stability analysis in CAPRI rounds 13–19. Proteins. Structure, Function, and Bioinformatics 2010; 78(15): 3124–3130. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatthews BW: Solvent content of protein crystals. J Mol Biol. 1968; 33(2): 491–7. PubMed Abstract | Publisher Full Text\n\nChen VB, Arendall WB 3rd, Headd JJ, et al.: MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 1): 12–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolm L, Sander C: Mapping the protein universe. Science. 1996; 273(5275): 595–603. PubMed Abstract | Publisher Full Text\n\nGiglio KM, Fong JC, Yildiz FH, et al.: Structural basis for biofilm formation via the Vibrio cholerae matrix protein RbmA. J Bacteriol. 2013; 195(14): 3277–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYee VC, Pedersen LC, Le Trong I, et al.: Three-dimensional structure of a transglutaminase: human blood coagulation factor XIII. Proc Natl Acad Sci U S A. 1994; 91(15): 7296–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith BO, Picken NC, Westrop GD, et al.: The structure of Leishmania mexicana ICP provides evidence for convergent evolution of cysteine peptidase inhibitors. J Biol Chem. 2006; 281(9): 5821–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCasados-Vázquez LE, Lara-González S, Brieba LG: Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: functional convergence of a common protein fold. Gene. 2011; 471(1–2): 45–52. PubMed Abstract | Publisher Full Text\n\nChothia C, Lesk AM: The relation between the divergence of sequence and structure in proteins. EMBO J. 1986; 5(4): 823–6. PubMed Abstract | Free Full Text\n\nTyndall JD, Fairlie DP: Conformational homogeneity in molecular recognition by proteolytic enzymes. J Mol Recognit. 1999; 12(6): 363–70. PubMed Abstract | Publisher Full Text\n\nBode W, Huber R: Structural basis of the endoproteinase-protein inhibitor interaction. Biochim Biophys Acta. 2000; 1477(1–2): 241–52. PubMed Abstract | Publisher Full Text\n\nOtlewski J, Jelen F, Zakrzewska M, et al.: The many faces of protease-protein inhibitor interaction. EMBO J. 2005; 24(7): 1303–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKunst F, Ogasawara N, Moszer I, et al.: The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997; 390(6657): 249–56. PubMed Abstract | Publisher Full Text\n\nDeng Y, Zhu Y, Wang P, et al.: Complete genome sequence of Bacillus subtilis BSn5, an endophytic bacterium of Amorphophallus konjac with antimicrobial activity for the plant pathogen Erwinia carotovora subsp. carotovora. J Bacteriol. 2011; 193(8): 2070–2071. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu CS, Yim KY, Tsui SK, et al.: Complete genome sequence of Bacillus subtilis strain QB928, a strain widely used in B. subtilis genetic studies. J Bacteriol. 2012; 194(22): 6308–6309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMonteiro AC, Abrahamson M, Lima AP, et al.: Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi. J Cell Sci. 2001; 114(Pt 21): 3933–42. PubMed Abstract\n\nWaterhouse AM, Procter JB, Martin DM, et al.: Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009; 25(9): 1189–1191. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarkin MA, Blackshields G, Brown NP, et al.: Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23(21): 2947–2948. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1144",
"date": "29 Jul 2013",
"name": "Patricia C Weber",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThorough discussion of the inhibition mechanism and the protein's possible biological role(s) make this manuscript of interest to a wide audience. Minor changes to improve clarity include delaying (or removing) discussions of ISP in the Abstract because ISP is not the central subject of the manuscript, expanding comments on the NMR structure and possibly citing similarities beyond the common identification of beta strands, and fixing Figure 1A where greater separation between the stereo images is needed.",
"responses": []
},
{
"id": "1142",
"date": "29 Jul 2013",
"name": "Xiayang Qiu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 first crystal structure of the IPI protein from B. subtilis, a family of protease inhibitor proteins with homologues across many different species. Although being assigned to the general IG-like fold, the structure shows a distinct topology, hence could also be regarded as a novel fold and a significant contribution to the protease inhibitor field. Additional studies are needed to understand the functional aspects of this new protein and structural scaffold.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-154
|
https://f1000research.com/articles/2-153/v1
|
10 Jul 13
|
{
"type": "Commentary",
"title": "Reporting research antibody use: how to increase experimental reproducibility",
"authors": [
"Matthew A Helsby",
"Joe R Fenn",
"Andrew D Chalmers",
"Matthew A Helsby",
"Joe R Fenn"
],
"abstract": "Research antibodies are used in a wide range of bioscience disciplines, yet it is common to hear dissatisfaction amongst researchers with respect to their quality. Although blame is often attributed to the manufacturers, scientists are not doing all they can to help themselves. One example of this is in the reporting of research antibody use. Publications routinely lack key details, including the host species, code number and even the company who supplied the antibody. Authors also fail to demonstrate that validation of the antibodies has taken place. These omissions make it harder for reviewers to establish the likely reliability of the results and for researchers to reproduce the experiments. The scale of this problem, combined with high profile concerns about experimental reproducibility, has caused the Nature Publishing Group to include a section on antibody information in their recent Reporting Checklist for Life Science Articles. In this commentary we consider the issue of reporting research antibody use and ask what details authors should be including in their publications to improve experimental reproducibility.",
"keywords": [
"Research antibodies",
"experimental reproducibility",
"monoclonal antibody",
"polyclonal antibody",
"application",
"species reactivity"
],
"content": "Antibody information is routinely omitted from publications\n\nNeuroscience, cancer research, regenerative medicine, infection and immunity, cell biology and cardiovascular research are just some of the fields in which research antibodies are commonly used. The sheer scale of their use is illustrated by huge sales, estimated to be worth in excess of $1.6 billion annually1. Despite, or perhaps because of, this widespread use, it is common to hear dissatisfaction among research scientists about the quality of these antibodies. The finger of blame is often pointed at the manufacturers2, yet it is questionable whether scientists themselves are doing everything they can to help the situation; surely not all problems can be placed at the door of the antibody manufacturer. One example of scientists not helping themselves is in their reporting of antibody use. There are many cases of good practice and detailed reporting, but all too frequently authors omit key details. These include the host species and code numbers, but even the source of the antibody may be left out. This makes it harder for reviewers to establish how well characterised the antibodies are and thus how reliable the data presented are likely to be. It also makes it more difficult for other researchers to accurately reproduce experiments.\n\nFailure to report key information is not a new problem2,3, but recent developments have increased efforts to find a solution. In particular, experimental reproducibility has been thrust into the limelight by high profile cases. For example, a study of \"landmark\" cancer research papers found that scientific findings from only 11% of them could be repeated4. Taken at face value this is a shocking statistic and, in an attempt to try to improve experimental reproducibility, the Nature Publishing Group have recently introduced a reporting checklist for life science articles5. This checklist highlights research antibodies as a reagent type for which reporting could be improved. A key question is: what information to provide? In this commentary we consider what information authors should be including in their publications to help improve experimental reproducibility.\n\n\nKey details for reporting antibody experiments\n\nPublications need to report core information regarding the antibodies that were used. This should include the name of the antibody, the company/academic who produced the antibody, the host species in which the antibody was raised and whether the antibody is monoclonal or polyclonal. In addition, the catalogue or clone number needs to be mentioned. This information is commonly omitted from current publications, but is important as large antibody companies will often have multiple antibodies to the same target; a unique identifier is therefore essential to allow unambiguous identification of the antibody concerned. For this reason the first step in improving reporting should be to make it mandatory for authors to include core antibody information, including a code or clone number for the antibodies they use.\n\nA second type of information that should be reported relates to experimental details. The application the antibody was used for is of central importance. This information is normally present, but it can be hard to extract if the antibody information is listed in a ‘Materials’ section and separated from descriptions of the techniques. Having the antibody data and application data closely linked would avoid potential confusion. Furthermore, if a study uses samples from more than one species then it is also important to clearly link which antibodies were used in which species.\n\nThere are other features that could also be reported which may be particularly relevant to certain studies. For example, the antibody batch number is rarely reported, but there is evidence of variability between different antibody batches6,7. This type of variability is likely to be a particular issue with polyclonals2, but may affect monoclonals8. Reporting the final antibody concentration or dilution is another piece of information which can help other researchers, especially if optimisation was required during the study. Finally, it has been proposed that scientists should know the antigen which was used to raise the antibody3. This information may be commercially sensitive, but at least the location of the antigen within the protein should be known, as it will have implications for interpreting the results of certain studies. In these cases authors should be encouraged to report antigen location.\n\n\nAntibody validation\n\nThe Nature Publishing Group checklist requires authors to demonstrate that every antibody used in their study has been validated for use in each of the species and specific experiments used. Validating an antibody is a complex process worthy of its own review9 and reporting it can be achieved in a number of ways. Supplementary information could be included to demonstrate validation by the author or a citation could be given to highlight a previous study in which the antibody was validated. Reference to the antibody validation profile from publically available databases such as 1degreebio, Antibodypedia, CiteAb or pAbmAbs could also be used. Including this information would help reviewers and other researchers accurately assess the results.\n\n\nA simple format for reporting antibody information\n\nBased on the points discussed above we would suggest researchers use the following format for reporting antibody information:\n\n\"The following antibodies were used, Mouse anti-protein A monoclonal antibody (company E, catalogue number #1000) was used for Western blotting with human cells, as validated in (figure X or reference Y or validation profile Z) and Western blotting in mouse tissue as validated in (figure X or reference Y or validation profile Z). Goat anti-protein B polyclonal antibody (company F, catalogue number #1001) was used for ELISA in human tissue as validated in (figure X or reference Y or validation profile Z) and flow cytometry in human tissue as validated in (figure X or reference Y or validation profile Z)\".\n\nThis format is meant as a guide and could be adapted as required; for example, details of batch number, dilution or epitope could be added where particularly important. This information could also be usefully presented in a table if allowed by the journal. Adoption of these reporting guidelines will not eliminate researchers’ frustrations with antibodies, but should help improve experimental reproducibility and scientists’ productivity, something we all seek. An additional benefit for authors who include this information is that well annotated publications are easier for antibody companies and antibody search engines like CiteAb to highlight in their databases. This inclusion is likely to increase the number of researchers who access their work and so potentially the impact of the study.\n\nA final thought is that journals have a big role to play in promoting good practice by including guidelines on reporting antibody details in their instructions to authors and encouraging reviewers to consider this aspect of publications when they carry out their review.",
"appendix": "Author contributions\n\n\n\nADC conceived the idea behind the commentary and produced a draft 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\nADC is a shareholder in CiteAb Ltd, which runs CiteAb the antibody search engine.\n\n\nGrant information\n\n\n\n\nReferences\n\nBird C: Antibody User Survey. The Scientist, 2012. Reference Source\n\nCouchman JR: Commercial antibodies: the good, bad, and really ugly. J Histochem Cytochem. 2009; 57(1): 7–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaper CB: An open letter to our readers on the use of antibodies. J Comp Neurol. 2005; 493(4): 477–8. PubMed Abstract | Publisher Full Text\n\nBegley CG, Ellis LM: Drug development: Raise standards for preclinical cancer research. Nature. 2012; 483(7391): 531–3. PubMed Abstract | Publisher Full Text\n\nReducing our irreproducibility. Nature. 2013; 496(7446): 398. Publisher Full Text\n\nChalmers AD, Pambos M, Mason J, et al.: aPKC, Crumbs3 and Lgl2 control apicobasal polarity in early vertebrate development. Development. 2005; 132(5): 977–86. PubMed Abstract | Publisher Full Text\n\nPozner-Moulis S, Cregger M, Camp RL, et al.: Antibody validation by quantitative analysis of protein expression using expression of Met in breast cancer as a model. Lab Invest. 2007; 87(3): 251–60. PubMed Abstract | Publisher Full Text\n\nVoskuil J: The troubles with commercial research antibodies dissected. Everest Biotech Blog. 2013. Reference Source\n\nBordeaux J, Welsh A, Agarwal S, et al.: Antibody validation. Biotechniques. 2010; 48(3): 197–209. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1064",
"date": "16 Jul 2013",
"name": "David Soll",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary is timely and well written, but it could be shortened or tightened up a bit for the purpose of conciseness. It also should include a few points noted in this review. The major point is the problem that lack of information in publications involving research antibodies affects assessment and future use. The discussion could be more efficient in stating that if methods were reported in a previous referenced article, then referencing that article in a new publication is sufficient, unless there are nuances (i.e., new uses of the antibody). It should also be made clear that such information be mandatory when an antibody is used in a particular way for the first time. There are also a few things the author may want to include: Many antibodies work on a particular protein in a particular cell type without knowledge of the protein domain(s) found. In spite of that they may be of value, so you don’t have to identify the sequence molecule. Some antibodies identify native conformation and therefore are not on a peptide sequence per se. Such antibodies are not unusually performed on denatured proteins in western blots, but may work in nature gels. Some antibodies have not been fully characterized beyond reference to the data sheet provided by the company or source if necessary.\n\nIf the authors of a paper refer to the company, and catalog name of the antibody, prior characterization can access. Antibody validation should go in the supplementary data to a paper. Do not cite CiteAb in your paper - it sounds like an ad. But all in all, this is a reasonable commentary. It reinforces what many already are advocating. The title and abstract were fine.",
"responses": [
{
"c_id": "539",
"date": "03 Sep 2013",
"name": "Andrew Chalmers",
"role": "Author Response",
"response": "We thank Professor Soll for his positive review and helpful comments. We have now addressed them and explain our response to each one in turn below: ‘The discussion could be more efficient in stating that if methods were reported in a previous referenced article....’ The fact that previous validation can be cited has now been spelled out more clearly (Antibody Validation section). ‘...information be mandatory when an antibody is used in a particular way for the first time’The fact that if no previous validation has occurred then validation should be carried out and reported and/or submitted to a public database has been made clearer (Antibody Validation section). These are two key points and we appreciate the fact you raised them.Things we have now included to respond to the numbered points raised. More discussion of the importance of knowing the antigen for an antibody has been added, in particular raising the point that for some antibodies the antigen is not known, for example when they are raised to a complex cell or tissue lysate (key details for reporting antibody experiments section). This comment is relevant to the experimental validation of antibodies, we have increased the amount we cover on this topic but not attempted a full review as we feel such a complex topic is beyond the scope of this comment article. We have added some addition citations for readers who require more information (Antibody Validation section). We have made it clearer when validation should be carried out and how it should be reported if no previous validation has taken place (Antibody Validation section).We have now repeated the importance of including catalogue numbers in the antibody validation section. This is now made clear (Antibody Validation section). We think giving examples of available antibody databases will be useful to readers and were careful to mention more than one database, we have now added a link to a more extensive list. We have also removed the second reference to CiteAb which was in the final section."
}
]
},
{
"id": "1193",
"date": "24 Jul 2013",
"name": "John Colyer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title and abstract are clear and appropriate. The article is timely and written clearly and accessibly. It could be improved further by providing references for papers that are examples “of good practice and detailed reporting”, which might serve as a template for others. The process of antibody validation is worthy of more extensive discussion, as the research community needs to develop a clear understanding of the most appropriate tests to be performed in each experimental system, and standards which should be attained for acceptance of the status of “validated”. This data should be provided in supplementary data, or by reference to previous supplementary data if the same reagents are used in a new study. The importance of batch number is made, but could be emphasized more. Finally, the critical role of peer-reviewers in evaluating and enforcing these standards is key. Some discussion of this would enhance the manuscript.",
"responses": [
{
"c_id": "540",
"date": "03 Sep 2013",
"name": "Andrew Chalmers",
"role": "Author Response",
"response": "We thank Professor Colyer for his positive and helpful comments and explain our response to each one in turn below.A good idea, we have now added an example reference that illustrates good reporting practice (Antibody information is routinely omitted from publications’ section). Journals which already encourage good practice have also been highlighted (‘Change will require help from journals and reviewers’). We completely agree and have increased the amount we cover on this topic, but not attempted a full review as we feel such a complex topic is beyond the scope of this comment article. We have added some additional citations for readers who require more information (Antibody Validation section).The fact that if no previous validation has occurred then it should be carried out and reported and/or submitted to a public database has been made clearer. The fact that previous validation can be cited has also been spelled out (Antibody Validation section).Additional emphasis has been added regarding the problem of batch to batch variability (‘Key details for reporting antibody experiments’ section).This has been added to the ‘Change will require help from journals and reviewers’ section."
}
]
},
{
"id": "1223",
"date": "01 Aug 2013",
"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\nThis commentary is much needed in the field of life science. It is well written and concise. Andrew Chalmer’s group has contributed significantly to the use of research antibodies by creating CiteAb. When operating the CiteAb search engine, I imagine that they constantly run into problems with publications with poorly described use of research antibodies.I have two minor suggestions: In the Antibody Validation paragraph, a statement could be included in the methods section of a paper regarding if, where and under what name antibody validation information or reviews has been posted in publically available databases. This would increase the value and transparency of these databases. Unlike the previous reviewer, I think it is fine to mention CiteAb in the paper. After all, even Nature Publishing Group is a highly commercial enterprise. However, I suggest that a table could be included listing the relevant databases including CiteAb, pAbmAbs, Biobrea, Antibodypedia, 1degreebio, Antibody-Advizer etc. In this regard, I regret that the Checklist from Nature Publishing Group only refers to sites in which they have a commercial interest (1degreebio and Antibodypedia). I hope that other publishing groups are not tempted to do the same.",
"responses": [
{
"c_id": "541",
"date": "03 Sep 2013",
"name": "Andrew Chalmers",
"role": "Author Response",
"response": "We thank Professor Glerup for his helpful comments and share his concern about the Nature Publishing Group guidelines. We explain our response to each one in turn below. The fact that if no previous validation has occurred then it should be carried out and reported and/or submitted to a public database has been made clearer. This is a good point and we agree it is important to give an overview of available databases to allow readers to choose the most appropriate. For this reason we were careful to mention a range in our first version. However, we feel it would not be appropriate for us to compile a table given our clear affiliation to one database, instead we provide a link to the most complete list of databases we are aware of."
}
]
}
] | 1
|
https://f1000research.com/articles/2-153
|
https://f1000research.com/articles/2-147/v1
|
04 Jul 13
|
{
"type": "Research Article",
"title": "Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage",
"authors": [
"Abigail L Clutterbuck",
"David Allaway",
"Pat Harris",
"Ali Mobasheri",
"Abigail L Clutterbuck",
"David Allaway",
"Pat Harris"
],
"abstract": "Objective: Curcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation.Methods: Articular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG) release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB) assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days.Results: Curcumin induced chondrocyte death in primary cultures (50μM p<0.001 and 100μM p<0.001) after 24 hours. After 48 hours and five days, curcumin (≥25μM) significantly increased cell death (p<0.001 both time points). In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM) significantly reduced IL-1β-stimulated PG (p<0.05) and PGE2 release (p<0.001) from explants, whilst curcumin (≥12μM) significantly reduced MMP-3 release (p<0.01).Conclusion: Non-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.",
"keywords": [
"Osteoarthritis (OA) involves destruction of articular cartilage by a combination of mechanical injury",
"inflammatory mediators and proteolytic enzyme activity1. The high cost and potential negative side effects of conventional pharmacotherapy",
"i.e. non-steroidal anti-inflammatory drugs (NSAIDs)",
"has stimulated interest in natural plant products with anti-inflammatory properties",
"as an alternative or adjunct to conventional therapy2. These products are being investigated for potential efficacy in a wide range of disorders with an inflammatory component",
"including arthritis and cancer3",
"4."
],
"content": "Introduction\n\nOsteoarthritis (OA) involves destruction of articular cartilage by a combination of mechanical injury, inflammatory mediators and proteolytic enzyme activity1. The high cost and potential negative side effects of conventional pharmacotherapy, i.e. non-steroidal anti-inflammatory drugs (NSAIDs), has stimulated interest in natural plant products with anti-inflammatory properties, as an alternative or adjunct to conventional therapy2. These products are being investigated for potential efficacy in a wide range of disorders with an inflammatory component, including arthritis and cancer3,4.\n\nCurcumin (diferuloylmethane) is a polyphenol found in turmeric derived from the rhizomes of Curcuma longa. Curcumin is traditionally known for its powerful anti-inflammatory and anti-oxidant properties. At concentrations between 50 and 100μM it has been shown to have anti-inflammatory properties via its suppressive effects on I kappa B kinase (IKK) activity and consequently the nuclear factor-kappa B (NF-κB) signaling pathway in various cell types, including chondrocytes5,6. However, published work suggests that curcumin is cytotoxic to both primary chondrocytes7 and transformed chondrocyte cell lines8 at 50μM and above. Primary cells in their initial passages can be more phenotypically and genotypically relevant than transformed cells and are more applicable to the clinical setting9. Therefore, the cytotoxicity observed in chondrocyte cell lines may be a consequence of the transformation induced by the SV-40 virus. The hypothesis to be tested in this study was that curcumin exerts anti-inflammatory and anti-catabolic effects on interleukin-1beta (IL-1β)-stimulated cartilage explants at non-cytotoxic concentrations. Accordingly, we evaluated the concentrations at which a commercially available curcumin formulation (sourced from Sigma-Aldrich) was cytotoxic to primary equine chondrocytes in both monolayer and explant cultures and determined whether non-toxic concentrations could reduce proteoglycan (PG) loss and inflammatory mediator production in an in vitro model of early OA.\n\n\nMaterials and methods\n\nMacroscopically normal articular cartilage samples were obtained from weight-bearing regions of the metacarpophalangeal joints of eleven horses of mixed breed, age and sex. The joint tissues were sourced from two UK-based abattoirs. Animals were euthanized for non-research purposes having been stunned before slaughter for meat in accordance with Welfare of Animals (Slaughter or Killing) Regulations 1995. Full depth cartilage from six animals was taken for explant culture and thin cartilage shavings were used for chondrocyte isolation from the remaining five animals. Cartilage samples from each animal were kept separate throughout. Cartilage shavings were aseptically harvested into low glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Thermo Fisher Scientific, Loughborough, UK) containing 4% penicillin/streptomycin (Sigma-Aldrich, Gillingham, UK) before washing in phosphate-buffered saline (PBS) containing 10% penicillin/streptomycin (Sigma-Aldrich, Gillingham, UK) for 20 minutes.\n\nThin cartilage slices were digested overnight in 0.1% collagenase type I (Sigma-Aldrich, Gillingham, UK) at 37°C and 5% CO2. The resulting cell suspension was filtered and washed before undergoing first expansion in low glucose DMEM with 2% penicillin/streptomycin and 10% fetal bovine serum (FBS). Once confluency was reached, cells were passaged into 12-well plates. Only first and second passage confluent cells were used in this study.\n\nCulture media was removed and replaced with treatment media (1ml/well). Control wells contained media alone (low glucose DMEM with 2% penicillin/streptomycin and 10% FBS), which formed the base for the other treatments. The NSAID carprofen (100μg/ml; Rimadyl®, Pfizer, Sandwich, UK) was included as a positive control, due to its anti-inflammatory effects on chondrocytes10. The nitric oxide donor, sodium nitroprusside (SNP; Sigma-Aldrich, Gillingham, UK) dissolved in DMEM (50mM) was used as a positive control for inducing cell death11. Stock solutions of curcumin (100mM; C1386, Sigma-Aldrich, Gillingham, UK) were prepared in cell culture grade dimethyl sulfoxide (DMSO; Sigma-Aldrich, Gillingham, UK) and diluted in DMEM to 1mM. From this 1mM stock, experimental concentrations of curcumin (3μM, 6μM, 12μM, 25μM, 50μM and 100μM) were prepared in DMEM and added to the appropriate wells. A DMSO control containing a volume equivalent to that found in the highest curcumin concentration was included on each plate to ensure that any observed effects were not due to the carrier solvent. The plates were incubated at 37°C and 5% CO2. Cytotoxicity was assessed after 24 hours, 48 hours and five days.\n\nChondrocyte viability was assessed using a commercially available live/dead assay (Invitrogen, Paisley, Scotland, UK) that utilizes calcein AM and ethidium homodimer-1 to identify live and dead cells, respectively. Media was removed and centrifuged (Eppendorf, Eppendorf rotor) at 10,000 rpm for 25 seconds at room temperature. The resulting pellet of detached cells (if any) was washed and resuspended in 20μl PBS. Adherent cells in the wells were washed in PBS before adding 20μl of the detached cell suspension to the appropriate wells and incubating in calcein AM (2μM) and ethidium homodimer-1 (4μM) in PBS (Sigma-Aldrich, Gillingham, UK) for 30 minutes at room temperature. Fluorescence was detected and captured using an inverted contrasting microscope (Leica DM IL, Leica Microsystems Ltd, Wetzlar, Germany) with Leica Application Suite imaging software (Version 2.4.0 R1, Leica Microsystems Ltd). Six random fields of view of live and dead cells were taken per well (magnification×100). Live and dead cells were counted with Image J Software (National Institutes of Health, Bethesda, MD) and the percentage of dead cells (expressed as a percentage of the total number of cells present) was calculated at 24 hours, 48 hours and five days for each treatment.\n\nFull depth cartilage shavings were cut into 3mm discs. Three discs per well from the same animal were placed in 24-well plates containing 1ml of culture medium (serum-free low glucose DMEM supplemented with 2% penicillin/streptomycin) and allowed to equilibrate overnight at 37°C under 5% CO2. The following day, culture media was replaced with fresh media before the experiment began.\n\nCartilage from three animals was used for curcumin viability studies, and cartilage from three different animals was used for the remaining studies. All plates contained 1ml culture media, which formed the base for other treatments and acted as a control for each plate. Explant viability studies were performed by adding curcumin (12μM, 25μM and 100μM) prepared in DMEM as described above to the appropriate wells (one well per animal per treatment). SNP (50mM) was used as a positive control for cell death. The remaining 3 wells per animal per treatment were used to determine PG release from unstimulated explants in response to curcumin.\n\nExperiments aimed at assessing the anti-catabolic and anti-inflammatory effects of curcumin were conducted by incubating explants in culture media containing recombinant equine IL-1β (R&D Systems, Abingdon, UK) (10ng/ml) and various concentrations of curcumin (3μM, 6μM, 12μM, 25μM and 50μM) prepared in IL-1β-treated media. Carprofen (100μg/ml) was prepared in IL-1β-treated media and included as a positive control. DMSO controls were performed previously and found to have no effect on PG release from both IL-1β-stimulated and unstimulated cartilage explants at volumes equivalent to that found in the highest curcumin concentration (data not shown). Plates were incubated at 37°C and 5% CO2 for five days. After five days, explants were immediately assayed for cytotoxicity or frozen at -20°C with their corresponding supernatants for subsequent secretome assays.\n\nAfter five days, chondrocyte viability was assessed using the live/dead assay. Explants were washed in PBS then incubated with calcein AM (2μM) and ethidium homodimer-1 (8μM) in PBS for 30 minutes at room temperature. A confocal microscope (Leica TC SP2) was used to detect and measure fluorescence in 15μm z-sections though each explant (magnification×10).\n\nFor evaluation of matrix PG release we used the metachromatic dye 1,9 dimethylmethylene blue (DMMB) to quantify the amount of sulfated glycosaminoglycans (GAGs) into the medium. Cartilage discs were digested in papain(Sigma-Aldrich, Gillingham, UK) for 16 hours. Papain-digested cartilage and their corresponding supernatants were assayed in 96-well plates using the DMMB (Sigma-Aldrich, Gillingham, UK) method as previously described12. Shark chondroitin sulfate (Sigma-Aldrich, Gillingham, UK) was used as a standard (0–70μg). DMMB solution (200µl) was added to samples and standards (40μl). The plate was read (Multiskan Ascent, Thermo Labsystems, Basingstoke, UK) using Ascent Software (version 2.6, Thermo Labsystems, Basingstoke, UK). Total PG release was obtained from a spectrophotometric reading of the digested cartilage and its corresponding supernatant at 540nm. Percentage of PG release from the total PG content of the explants was calculated by dividing the supernatant value from the total PG release for each well.\n\nA competitive immunoassay kit (R&D Systems, Abingdon, UK) was used to measure PGE2 release according to the manufacturer’s instructions. Standards (19.6–1250pg/ml), supernatant samples and reagents were added to a 96-well plate coated in goat anti-mouse polyclonal antibody and incubated for 19 hours at 6°C. The plate was washed and developed with 200μl substrate solution per well in the dark at room temperature for 20 minutes. A stop solution was added and the plate was read immediately (Multiskan Ascent, Thermo Labsystems, Basingstoke, UK) at 450nm with wavelength correction set at 540nm using Ascent Software (version 2.6, Thermo Labsystems, Basingstoke, UK).\n\nThe protein content of explant supernatants from the PG and PGE2 assays was quantified and aliquots containing 50µg protein were freeze-dried overnight. The resultant pellets were resuspended in 37µl sample buffer (NuPAGE Lithium dodecyl sulfate sample buffer (4×) and electrophoresed on precast 4–12% Bis-Tris 10-well gels (Invitrogen, Paisley, Scotland, UK) under denaturing and reducing conditions. Proteins were transferred to 0.45µm polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Little Chalfont, UK) and blocked with 5% (w/v) non-fat milk with Tris-Buffered saline (TBS) containing 0.1% (v/v) Tween20 for 1 hour. Membranes were incubated with a goat polyclonal antibody to matrix metalloproteinase 3 (stromelysin) (MMP-3; Abcam, Cambridge, UK) diluted 1:1,000 in 5% (w/v) non-fat milk at 4°C overnight. After washing, membranes were incubated for two hours at room temperature with a secondary anti-goat antibody (1:10,000; Dako, Cambridge, UK). Membranes were washed and chemiluminescence detected using ECL+ on a Typhoon Trio+ Variable Mode Imager (both GE Healthcare, Little Chalfont, UK). Densitometric quantification of MMP-3 bands was performed using Image J software. Relative band intensity in comparison to controls was measured for samples from each animal.\n\nData were statistically analyzed using a one-way analysis of variance (ANOVA) with Tukey’s post hoc test (GraphPad InStat, version 3.05, GraphPad Software Inc., La Jolla, CA). Statistical significance was set at p<0.05. Graphs were plotted with GraphPad Prism (version 4, GraphPad Software Inc).\n\nFor chondrocyte viability quantification, results are expressed as the mean number of dead cells per field of view per treatment ± standard error of the mean (SEM). PG release percentage and PGE2 values are reported as means of combined animals ± SEM. For MMP-3 quantification analysis, relative intensity values were reported as means of 3 combined animals ± SEM.\n\n\nResults\n\nUntreated controls retained a mean cell death percentage of less than 1% at 24 hours, 48 hours and five days (Figure 1). DMSO controls and the NSAID, carprofen, did not significantly increase cell death compared to controls at all time points. SNP effectively induced cell death (p<0.001, all time points) with mean cell death of 92.95 ± 2.29% at 24 hours, 99.6 ± 0.17% at 48 hours and 100 ± 0.00% at five days. Curcumin significantly increased cell death compared to controls after 24 hours at 50μM (71.75 ± 7.25%, p<0.001) and 100μM (99.55 ± 0.12%, p<0.001). After 48 hours a significant increase in toxicity compared to controls was seen at 25μM (30.67 ± 8.94%, p<0.001), 50μM (95.9 ± 0.96%, p<0.001) and 100μM (99.59 ± 0.18%, p<0.001). After five days, curcumin (25μM) caused significant increases in cell death (95.71 ± 0.72%, p<0.001), 50μM (99.4 ± 0.39%, p<0.001) and 100μM (100 ± 0.00%, p<0.001).\n\nCurcumin significantly increases chondrocyte death after 24 hours (A) at 50μM and 100μM compared to control indicated by *** (p<0.001). After 48 hours (B) and five days (C), curcumin (25μM) significantly increases chondrocyte death compared to controls. The nitric oxide donor, sodium nitroprusside (SNP) (50mM) also significantly increases chondrocyte death at 24 hours, 48 hours, and five days (p<0.001) compared to control. DMSO at concentrations found in the 100μM curcumin treatment has no effect on chondrocyte death. Results are expressed as the mean number of dead cells per field of view per treatment ± SEM.\n\nAfter five days in culture without serum supplementation, explants retained fully viable chondrocytes as indicated by the abundant green staining and lack of red staining in the controls (Figure 2). SNP induced cell death in the explants as shown by the increased red staining and fewer, less vibrant green stained cells in comparison to the controls. Curcumin (25μM)-treated explants retained large numbers of green stained cells, showing no detriment to the viability of chondrocytes within cartilage explants after five days in culture. However, the large amount of red nuclei staining to the chondrocytes in the 100μM curcumin-treated explants indicates that curcumin was highly cytotoxic at this concentration.\n\nControl (A) consists of culture media with 2% penicillin/streptomycin. Sodium nitroprusside (SNP) (50mM) (B) is a positive control for cell death. Treatments consist of curcumin in culture media at 25μM (C) and 100μM (D). Images are representative of explants in the treatments wells for each animal. Green staining indicates live metabolizing cells and red staining highlights the nuclei of dead cells.\n\nControl explants released 13.91 ± 1.13% of the total PG content of the cartilage into the media over five days (range: 159–414μg PG/ml of culture supernatant) (Figure 3). SNP (50mM) significantly increased matrix PG release to 82.01 ± 2.43% (p<0.001) after the same period. Curcumin did not significantly alter PG release from the explants after five days at 12μM, 25μM and 100μM.\n\nControl column indicates cartilage discs incubated in the culture medium alone. Values are reported as the mean of three animals per treatment ± SEM. Significance compared to control is indicated by *** (p<0.001). Sodium nitroprusside (SNP) (50mM) significantly increases PG release compared to control (p<0.001). Curcumin does not alter PG release from the explants compared to control at concentrations of 12µM, 25µM or 50µM. PG loss is expressed as sulfated glycosaminoglycan (GAG) release.\n\nControl explants released 11.38 ± 1.26% of the total PG content of the cartilage into the media over five days (range: 86–323μg PG/ml of culture supernatant) (Figure 4A). IL-1β (10ng/ml) significantly increased PG release to 44.31 ± 3.75% of total PG content compared to control (p<0.001). The NSAID carprofen (100μg/ml) significantly reduced IL-1β-stimulated PG release to 25.96 ± 3.59% of total PG content (p<0.01). Curcumin significantly decreased IL-1β-stimulated PG release in the explants to 28.98 ± 2.45% at 3µM (p<0.05), 26.84 ± 2.49% at 6µM (p<0.01), 24.51 ± 5.42% at 12µM (p<0.01), 18.91 ± 4.36% at 25µM (p<0.001) and 16.05 ± 1.82% at 50µM (p<0.001). PGE2 release into the media of unstimulated explants was 20.35 ± 3.67pg/ml (Figure 4B). These levels were significantly increased (p<0.001) to 303.3 ± 73.38pg/ml by the addition of recombinant IL-1β (10ng/ml). The NSAID significantly attenuated this effect, reducing levels to 19.72 ± 3.74pg/ml (p<0.001). Curcumin also significantly reduced IL-1β-stimulated PGE2 release to 75.48 ± 10.68pg/ml at 3μM (p<0.001), 54.72 ± 12.41pg/ml at 6μM (p<0.001), 45.65 ± 13.31pg/ml at 12μM (p<0.001), 18.36 ± 2.38pg/ml at 25μM (p<0.001) and 26.73 ± 3.52pg/ml at 50μM (p<0.001).\n\nEffect of curcumin on PG release (A) and prostaglandin E2 (PGE2) (B) from interleukin (IL)-1β-stimulated cartilage explants (dark grey columns) (n=3). Control (light grey column) indicates cartilage discs incubated in the culture medium alone. Values are reported as the mean of three animals per treatment ± SEM. Significance compared to IL-1β is indicated by * (p<0.05), ** (p<0.01) and *** (p<0.001). IL-1β significantly increases proteoglycan (PG) (p<0.001) and PGE2 (p<0.001) release compared to control. The non-steroidal anti-inflammatory drug (NSAID) carprofen (100µg/ml) significantly reduces IL-1β-stimulated PG (p<0.01) and PGE2 (p<0.001) release. Curcumin significantly reduces IL-1β-stimulated PG release at 3µM (p<0.05), 6µM (p<0.01), 12µM (p<0.01), 25µM (p<0.001) and 50µM (p<0.001). IL-1β-stimulated PGE2 release is also significantly reduced at all curcumin concentrations (p<0.001). PG loss is expressed as sulfated glycosaminoglycan (GAG) release.\n\nControl explants released low levels of MMP-3, which were significantly increased by the addition of IL-1β (p<0.001) (Figure 5). The addition of the NSAID carprofen significantly reduced IL-1β-stimulated MMP-3 levels (p<0.01) in all animals to near that of controls. Curcumin showed a dose-dependent significant effect on reducing the IL-1β-stimulated release at 12μM (p<0.01), 25μM (p<0.01) and 50μM (p<0.001). However, the concentration at which this reduction became apparent differed between animals. For example, animal C, showed a clear reduction in MMP-3 secretion at curcumin concentrations of 12μM and above, whereas in animals A and B, the equivalent reduction was not seen until curcumin concentrations of 50μM were used. The most apparent reduction in MMP-3 secretion was always seen at 50μM with levels near to that of the controls in all animals.\n\nWestern blot images and quantifying graphs of matrix metalloproteinase (MMP)-3 in the cartilage explant supernatants from three animals (A, B, and C). The lane on the left of the images shows the molecular weights (MW) of standard markers (Invitrogen, Paisley, Scotland, UK) in kilodaltons (kDa). Graph D shows the relative intensity of bands from all three animals with labeled treatment axes. Values are reported as the mean of three animals per treatment ± SEM. Significance compared to interleukin (IL)-1β is indicated by ** (p<0.01) and *** (p<0.001). All lanes contain equal volumes of protein (50µg protein per lane). Unstimulated cartilage explants release low levels of MMP-3 as shown by the controls in lane 1. Secretion of MMP-3 from explants is significantly increased (p<0.001) by IL-1β (10ng/ml) (Lane 2). The non-steroidal anti-inflammatory drug (NSAID) carprofen (100μg/ml) (Lane 3) significantly reduces IL-1β-stimulated MMP-3 secretion (p<0.01) to near control levels. Curcumin at concentrations of 3μM (Lane 4) and 6μM (Lane 5) does not significantly reduce IL-1β-stimulated MMP-3 release from the explants. However, a significant reduction is observed at 12μM (p<0.01) (Lane 6), 25μM (p<0.01) (Lane 7) and 50μM (p<0.001) (Lane 8) from explants in a dose dependent manner. The extent of reduction differs between animals.\n\n\n\n\nDiscussion\n\nRecent research has produced conflicting results regarding the efficacy and cytotoxicity of curcumin in vitro13–15. This study attempted to address the issue of curcumin cytotoxicity and its anti-inflammatory effects in equine cartilage explants and monolayer chondrocyte cultures. The results of this investigation suggest that curcumin induces chondrocyte death at concentrations of 25μM and above in monolayer chondrocytes after 48 hours and five days.\n\nThe cytotoxic effects of curcumin (50μM) have been observed and documented in other cell types, notably tumor cell lines16. Induction of apoptosis through cytochrome c release and subsequent caspase activation is thought to be a key chemopreventive effect of curcumin in cancer studies17. In agreement with this, curcumin (50μM) has also been shown to reduce the viability of an immortalized human chondrocyte cell line after 24 hours8. The results from this study suggest that this is also the case with primary equine chondrocytes. However, a recent study on primary human chondrocytes found that curcumin (50μM) did not reduce cell viability and successfully inhibited IL-1β-induced cytotoxicity as demonstrated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay18. This could be due to species-specific differences in chondrocyte susceptibility to curcumin, or equally likely, the different sources of curcumin used. This highlights the importance of assessing cytotoxicity alongside anti-inflammatory assays, with the same formulation of curcumin in each individual study.\n\nIn contrast to the monolayer cell cultures, curcumin (25μM) did not induce cytotoxicity in chondrocytes within cartilage explants after five days, although toxicity was seen at 100μM. Within cartilage, chondrocytes are embedded in a complex extracellular matrix (ECM) across which diverse diffusion gradients and fluid flow occurs19. This could account for the differing toxicity threshold between monolayer cultures and explants. Chondrocytes are more vulnerable to cell death when they are no longer encased within a matrix, as cell adhesion and integrin attachments are important factors in promoting cell survival20. Thus, without a protective ECM, monolayer chondrocytes may be more exposed and susceptible to external agents in the culture media than those within explants, as has been shown with bupivacaine21. Therefore, cytotoxicity data from monolayer cultures may not reflect cytotoxic changes in 3-dimensional culture models and explant cultures.\n\nFor the purposes of addressing cytotoxicity in this study, a live/dead stain was selected to detect chondrocyte death as it can quickly provide qualitative and quantitative data on both monolayer and 3-dimensional chondrocyte cultures. The method does not distinguish between apoptosis and necrosis as the mode of cell death and although this information was not required for this study, it could be a useful addition to future studies. It should be noted here that cell death in monolayer cells can be underestimated, as dead cells can detach and be removed with the media. However, this risk was mitigated by centrifuging the discarded media so that the resulting pellet of detached cells could be resuspended and returned to the well before the live/dead stain reagents were added.\n\nOnce the uppermost observed safe level of curcumin was determined, the effect of curcumin on cartilage catabolism and inflammation was examined in explants. No significant effect of curcumin alone on matrix PG release from normal cartilage explants at both safe and toxic concentrations was observed. This suggests that curcumin does not alter the basal level of PG released from unstimulated cartilage explants in culture, even at cytotoxic concentrations. Interestingly, in these unstimulated explants, the two treatments that caused cell death had different effects on PG release: SNP (50mM) caused extensive PG loss into the media whereas curcumin (100μM) did not alter PG release compared to controls. PG loss does not directly lead to cell death in cartilage explants22, thus the PG loss caused by SNP is unlikely to be primarily a consequence of cell death. SNP generates nitric oxide (NO), ceramide and cyanide, which aside from causing apoptosis, reduces proteoglycan and collagen synthesis by chondrocytes and increases inflammatory mediator production and MMP activity23–25 resulting in cartilage matrix loss. Conversely, although curcumin (100μM) induces cell death, it is likely to involve a mechanism that does not induce the release of any proteases. Curcumin is known to reduce the release of inflammatory mediators, MMPs and NO6,26. We have shown in this study that cytotoxic levels of curcumin reduce IL-β-stimulated PGE2 and MMP-3 release. The reduced production of inflammatory mediators and catabolic enzymes suggests that it is unlikely to induce extensive PG loss as demonstrated in both IL-1β-treated and untreated cartilage explants in this study.\n\nCurcumin at 3μM and above significantly reduced PG loss from IL-1β-stimulated explants. This reduction in PG loss was seen at concentrations that were non-detrimental to chondrocyte viability in explants (i.e. 25μM and under) as well as cytotoxic concentrations (100μM). Previously, we observed that curcumin (100μM) significantly reduced human IL-1β-stimulated PG release from cartilage explants, but this effect was not seen at 10μM27. The differences in concentration efficacy between these two studies may be linked to individual variation or the source of cytokines used. Our previous study used human IL-1β on equine explants, whereas the current study used equine recombinant IL-1β. Despite a fairly conserved sequence homology between species, the use of species-specific IL-1β is more representative of the in vivo situation and thus may explain the difference in results seen between studies.\n\nThe involvement of pro-inflammatory cytokines and MMPs in OA is well documented28,29. MMP-3, also known as stromelysin, is produced by chondrocytes in OA cartilage tissue30. MMP-3 gene expression is up-regulated in response to IL-1β stimulation in chondrocytes31. Similarly, MMP-3 protein expression is increased by the addition of IL-1β to cartilage explants32. MMP-3 was chosen as a marker of cartilage degradation in this study, as there is convincing evidence for a role for MMP-3 in cartilage destruction in OA. MMP-3 is induced by IL-1β in joint inflammation, and has the capacity to degrade collagens, proteoglycans and activate other procollagenases33. MMP-3 and two other MMPs including MMP-1 and MMP-13 can also cleave the aggregating proteoglycan aggrecan at the interglobular domain34–36. Curcumin reduced MMP-3 secretion at concentrations as low as 12μM in some animals, and as high as 50μM in others, suggesting some animal-to-animal variability. However, when treated with 50μM curcumin, IL-1β-stimulated explants from all the animals used secreted MMP-3 levels that were lower than, or equivalent to, unstimulated control explants. This is in agreement with a previous study showing that curcumin (50μM) effectively reduced MMP-3 levels in IL-1β-stimulated-human chondrocyte lysates37. However, lower curcumin concentrations have been reported as effective in cartilage from human OA patients post mortem, where curcumin reduced MMP-3 release into the media of IL-1β-stimulated chondrocytes at 15μM and in cartilage explants at 5μM38. Many variables may account for the difference in effective curcumin concentrations, including individual variation, pre-existing joint pathology and the explant model used. It should be noted that many MMPs, including collagenases, matrilysin (MMP-7), and other stromelysins (e.g. MMP-10), are involved in osteoarthritic cartilage degradation39–41. Thus, the reduction in MMP-3 secretion would contribute to, but not totally account for, the reduction in PG release from IL-1β-stimulated cartilage explants.\n\nThe reported anti-inflammatory effects of curcumin have stimulated increasing interest in its potential for the treatment of inflammatory disorders42. Curcumin is thought to exert its anti-inflammatory effects through reducing COX-143, COX-244, and microsomal PGES expression16, thus preventing PGE2 release. This is most likely due to its inhibitory effect on the upstream NF-κB-signaling pathway, which promotes PGE2 production via upregulating the COX and PGES genes45. In this study, curcumin at concentrations of 3μM and over significantly reduced PGE2 release in response to equine IL-1β (10ng/ml). This anti-inflammatory effect is consistent with previous work in other cell culture models, such as rat peritoneal macrophages where curcumin (10µM) inhibited PGE2 release by 45%46, and in BV2 microglial cells where curcumin (10µM and 20µM) significantly reduced PGE2 release in response to lipopolysaccharide (LPS) (0.5µg/ml)47. The reduction in PGE2 levels in response to IL-1β in our study may be attributed to the inhibitory effects of curcumin on the NF-κB pathway. Curcumin (50µM) has been shown to inhibit various steps of the NF-κB pathway, such as IL-1β-dependent phosphorylation of p65; nuclear-translocation of p65; and IκBα phosphorylation in IL-1β-stimulated human chondrocytes6. Thus by inhibiting NF-κB, curcumin prevents the downstream inflammatory effects of COX-2 expression and PGE2 synthesis.\n\nAlthough curcumin was able to effectively attenuate the catabolic effects of IL-1β in this model, many other biochemical and biomechanical factors are capable of inducing cartilage inflammation and degeneration; these include compressive stress caused by overloading or traumatic injury of the joint48,49 and different cytokines such as tumor necrosis factor alpha (TNF-α) alone and in combination with oncostatin M (OSM)50. Further studies are required to determine whether curcumin can reduce inflammation and degeneration generated by combinations of different catabolic stimuli.\n\nThe anti-inflammatory and anti-catabolic effects of curcumin shown in this study support the existing evidence that curcumin may be supportive to joint health13–15. However, the bioavailability of curcumin is thought to be relatively low51 and efforts to determine whether chemically modified versions of curcumin may improve bioavailability merit further investigation13,52.\n\nIn summary, although this study found that curcumin at concentrations of 25μM and above is cytotoxic to monolayer chondrocytes after five days in culture, lower concentrations effectively antagonize PG and PGE2 release in vitro and exert a potent anti-inflammatory effect on cartilage explants treated with IL-1β. Achieving micromolar concentrations of curcumin in the synovial joint, or to the pericellular matrix of chondrocytes embedded deep within the avascular articular cartilage is highly unlikely. Controversial issues associated with the metabolism and bioavailability of curcumin highlight the need for caution when extrapolating in vitro data for translational research and clinical trials. Nevertheless, our results to date suggest that a commercially available curcumin formulation at 12μM and below, has no obvious cytotoxic effects on primary chondrocytes after five days in culture. More importantly, concentrations as low as 3μM were able to effectively reduce IL-1β-stimulated cartilage degradation and inflammatory mediator production. This study supports existing evidence to suggest that curcumin may be a suitable adjunct to conventional drugs for the treatment of inflammatory and degenerative disorders such as OA. If curcumin is to be used as an anti-inflammatory supplement, further research is required to establish its bioavailability and physiologically relevant serum and synovial concentrations in vivo in humans and animals.",
"appendix": "Author contributions\n\n\n\nAll authors have made substantial intellectual contributions to the conception and design of the study, data acquisition, analysis and interpretation. AC carried out the experimental work and contributed to data collection, interpretation and analysis. AM, DA and PH conceived the study design and supervised AC. AM was the primary supervisor. All authors contributed to data interpretation and manuscript preparation. All authors have approved the final version submitted.\n\n\nCompeting interests\n\n\n\nAM is the coordinator of the D-BOARD Consortium funded by European Commission Framework 7 program (EU FP7; HEALTH.2012.2.4.5–2, project number 305815, Novel Diagnostics and Biomarkers for Early Identification of Chronic Inflammatory Joint Diseases). DA and PA are afiiliated with the WALTHAM Centre for Pet Nutrition, which partially funded this study. No other competing interests were disclosed.\n\n\nGrant information\n\nThis work received major financial support from the Biotechnology and Biological Sciences Research Council through the Industrial CASE Studentship programme (Grant Number: BBS/S/M/2006/13141) and minor financial support from the WALTHAM Centre for Pet Nutrition. The BBSRC had no involvement in the study design, data collection, analysis and interpretation. The decision to submit the paper for publication was not influenced by the funding bodies.\n\n\nAcknowledgements\n\nWe would like to thank Leigh Sylvester from the School of Biosciences at the University of Nottingham for help with the confocal microscope studies.\n\n\nReferences\n\nMaiotti M, Monteleone G, Tarantino U, et al.: Correlation between osteoarthritic cartilage damage and levels of proteinases and proteinase inhibitors in synovial fluid from the knee joint. Arthroscopy. 2000; 16(5): 522–526. PubMed Abstract | Publisher Full Text\n\nAhmed S, Anuntiyo J, Malemud CJ, et al.: Biological basis for the use of botanicals in osteoarthritis and rheumatoid arthritis: a review. Evid Based Complement Alternat Med. 2005; 2(3): 301–308. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElmali N, Baysal O, Harma A, et al.: Effects of resveratrol in inflammatory arthritis. Inflammation. 2007; 30(1–2): 1–6. PubMed Abstract | Publisher Full Text\n\nGerhauser C, Klimo K, Heiss E, et al.: Mechanism-based in vitro screening of potential cancer chemopreventive agents. Mutat Res. 2003; 523–524: 163–172. PubMed Abstract | Publisher Full Text\n\nJobin C, Bradham CA, Russo MP, et al.: Curcumin blocks cytokine-mediated NF-kappa B activation and proinflammatory gene expression by inhibiting inhibitory factor I-kappa B kinase activity. J Immunol. 1999; 163(6): 3474–3483. PubMed Abstract\n\nShakibaei M, John T, Schulze-Tanzil G, et al.: Suppression of NF-kappaB activation by curcumin leads to inhibition of expression of cyclo-oxygenase-2 and matrix metalloproteinase-9 in human articular chondrocytes: Implications for the treatment of osteoarthritis. Biochem Pharmacol. 2007; 73(9): 1434–1445. PubMed Abstract | Publisher Full Text\n\nClutterbuck AL, Allaway D, Harris P, et al.: Toxic effects of curcumin (diferuloylmethane) on equine articular chondrocytes and synoviocytes in vitro. Eur Cell Mater. 2008; 16(Suppl. 3): 43. Reference Source\n\nToegel S, Wu SQ, Piana C, et al.: Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes. Osteoarthritis Cartilage. 2008; 16(10): 1205–1212. PubMed Abstract | Publisher Full Text\n\nFinger F, Schorle C, Zien A, et al.: Molecular phenotyping of human chondrocyte cell lines T/C-28a2, T/C-28a4, and C-28/I2. Arthritis Rheum. 2003; 48(12): 3395–3403. PubMed Abstract | Publisher Full Text\n\nDvorak LD, Cook JL, Kreeger JM, et al.: Effects of carprofen and dexamethasone on canine chondrocytes in a three-dimensional culture model of osteoarthritis. Am J Vet Res. 2002; 63(10): 1363–1369. PubMed Abstract | Publisher Full Text\n\nBlanco FJ, Ochs RL, Schwarz H, et al.: Chondrocyte apoptosis induced by nitric oxide. Am J Pathol. 1995; 146(1): 75–85. PubMed Abstract | Free Full Text\n\nFarndale RW, Sayers CA, Barrett AJ: A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res. 1982; 9(4): 247–248. PubMed Abstract | Publisher Full Text\n\nHenrotin Y, Priem F, Mobasheri A: Curcumin: a new paradigm and therapeutic opportunity for the treatment of osteoarthritis: curcumin for osteoarthritis management. Springerplus. 2013; 2(1): 56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMobasheri A, Henrotin Y, Biesalski HK, et al.: Scientific evidence and rationale for the development of curcumin and resveratrol as nutraceutricals for joint health. Int J Mol Sci. 2012; 13(4): 4202–4232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHenrotin Y, Clutterbuck AL, Allaway D, et al.: Biological actions of curcumin on articular chondrocytes. Osteoarthritis Cartilage. 2010; 18(2): 141–149. PubMed Abstract | Publisher Full Text\n\nMoon Y, Glasgow WC, Eling TE: Curcumin suppresses interleukin 1beta-mediated microsomal prostaglandin E synthase 1 by altering early growth response gene 1 and other signaling pathways. J Pharmacol Exp Ther. 2005; 315(2): 788–795. PubMed Abstract | Publisher Full Text\n\nWoo JH, Kim YH, Choi YJ, et al.: Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of cytochrome c and inhibition of Akt. Carcinogenesis. 2003; 24(7): 1199–1208. PubMed Abstract | Publisher Full Text\n\nCsaki C, Mobasheri A, Shakibaei M: Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1beta-induced NF-kappaB-mediated inflammation and apoptosis. Arthritis Res Ther. 2009; 11(6): R165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarcia AM, Frank EH, Grimshaw PE, et al.: Contributions of fluid convection and electrical migration to transport in cartilage: relevance to loading. Arch Biochem Biophys. 1996; 333(2): 317–325. PubMed Abstract | Publisher Full Text\n\nCao L, Lee V, Adams ME, et al.: beta-Integrin-collagen interaction reduces chondrocyte apoptosis. Matrix Biol. 1999; 18(4): 343–355. PubMed Abstract | Publisher Full Text\n\nChu CR, Izzo NJ, Papas NE, et al.: In vitro exposure to 0.5% bupivacaine is cytotoxic to bovine articular chondrocytes. Arthroscopy. 2006; 22(7): 693–699. PubMed Abstract | Publisher Full Text\n\nOtsuki S, Brinson DC, Creighton L, et al.: The effect of glycosaminoglycan loss on chondrocyte viability: a study on porcine cartilage explants. Arthritis Rheum. 2008; 58(4): 1076–1085. PubMed Abstract | Publisher Full Text\n\nKhatib AM, Siegfried G, Messai H, et al.: Mechanism of inhibition of endothelin-1-stimulated proteoglycan and collagen synthesis in rat articular chondrocytes. Cytokine. 2002; 17(5): 254–261. PubMed Abstract | Publisher Full Text\n\nBlanco FJ, Lotz M: IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE2. Exp Cell Res. 1995; 218(1): 319–325. PubMed Abstract | Publisher Full Text\n\nMurrell GA, Jang D, Williams RJ: Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun. 1995; 206(1): 15–21. PubMed Abstract | Publisher Full Text\n\nBrouet I, Ohshima H: Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun. 1995; 206(2): 533–540. PubMed Abstract | Publisher Full Text\n\nClutterbuck AL, Mobasheri A, Shakibaei M, et al.: Interleukin-1beta-induced extracellular matrix degradation and glycosaminoglycan release is inhibited by curcumin in an explant model of cartilage inflammation. Ann N Y Acad Sci. 2009; 1171: 428–435. PubMed Abstract | Publisher Full Text\n\nShinmei M, Masuda K, Kikuchi T, et al.: Production of cytokines by chondrocytes and its role in proteoglycan degradation. J Rheumatol Suppl. 1991; 27: 89–91. PubMed Abstract\n\nChubinskaya S, Huch K, Mikecz K, et al.: Chondrocyte matrix metalloproteinase-8: up-regulation of neutrophil collagenase by interleukin-1 beta in human cartilage from knee and ankle joints. Lab Invest. 1996; 74(1): 232–240. PubMed Abstract\n\nOkada Y, Shinmei M, Tanaka O, et al.: Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium. Lab Invest. 1992; 66(6): 680–690. PubMed Abstract\n\nTung JT, Fenton JI, Arnold C, et al.: Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes. Can J Vet Res. 2002; 66(1): 19–25. PubMed Abstract | Free Full Text\n\nJulovi SM, Yasuda T, Shimizu M, et al.: Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage. Arthritis Rheum. 2004; 50(2): 516–525. PubMed Abstract | Publisher Full Text\n\nShiomi T, Lemaitre V, D'Armiento J, et al.: Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseases. Pathol Int. 2010; 60(7): 477–496. PubMed Abstract | Publisher Full Text\n\nFosang AJ, Neame PJ, Hardingham TE, et al.: Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins. J Biol Chem. 1991; 266(24): 15579–15582. PubMed Abstract\n\nFosang AJ, Last K, Knauper V, et al.: Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain. Biochem J. 1993; 295(Pt 1): 273–276. PubMed Abstract | Free Full Text\n\nFosang AJ, Last K, Knauper V, et al.: Degradation of cartilage aggrecan by collagenase-3 (MMP-13). FEBS Lett. 1996; 380(1–2): 17–20. PubMed Abstract | Publisher Full Text\n\nSchulze-Tanzil G, Mobasheri A, Sendzik J, et al.: Effects of curcumin (diferuloylmethane) on nuclear factor kappaB signaling in interleukin-1beta-stimulated chondrocytes. Ann N Y Acad Sci. 2004; 1030: 578–586. PubMed Abstract | Publisher Full Text\n\nMathy-Hartert M, Jacquemond-Collet I, Priem F, et al.: Curcumin inhibits pro-inflammatory mediators and metalloproteinase-3 production by chondrocytes. Inflamm Res. 2009; 58(12): 899–908. PubMed Abstract | Publisher Full Text\n\nMitchell PG, Magna HA, Reeves LM, et al.: Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest. 1996; 97(3): 761–768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOhta S, Imai K, Yamashita K, et al.: Expression of matrix metalloproteinase 7 (matrilysin) in human osteoarthritic cartilage. Lab Invest. 1998; 78(1): 79–87. PubMed Abstract\n\nBarksby HE, Milner JM, Patterson AM, et al.: Matrix metalloproteinase 10 promotion of collagenolysis via procollagenase activation: implications for cartilage degradation in arthritis. Arthritis Rheum. 2006; 54(10): 3244–3253. PubMed Abstract | Publisher Full Text\n\nJagetia GC, Aggarwal BB: “Spicing up” of the immune system by curcumin. J Clin Immunol. 2007; 27(1): 19–35. PubMed Abstract | Publisher Full Text\n\nHandler N, Jaeger W, Puschacher H, et al.: Synthesis of novel curcumin analogues and their evaluation as selective cyclooxygenase-1 (COX-1) inhibitors. Chem Pharm Bull (Tokyo). 2007; 55(1): 64–71. PubMed Abstract | Publisher Full Text\n\nHong J, Bose M, Ju J, et al.: Modulation of arachidonic acid metabolism by curcumin and related beta-diketone derivatives: effects on cytosolic phospholipase A(2), cyclooxygenases and 5-lipoxygenase. Carcinogenesis. 2004; 25(9): 1671–1679. PubMed Abstract | Publisher Full Text\n\nCatley MC, Chivers JE, Cambridge LM, et al.: IL-1beta-dependent activation of NF-kappaB mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. FEBS Lett. 2003; 547(1–3): 75–79. PubMed Abstract | Publisher Full Text\n\nJoe B, Lokesh BR: Effect of curcumin and capsaicin on arachidonic acid metabolism and lysosomal enzyme secretion by rat peritoneal macrophages. Lipids. 1997; 32(11): 1173–1180. PubMed Abstract | Publisher Full Text\n\nJin CY, Lee JD, Park C, et al.: Curcumin attenuates the release of pro-inflammatory cytokines in lipopolysaccharide-stimulated BV2 microglia. Acta Pharmacol Sin. 2007; 28(10): 1645–1651. PubMed Abstract | Publisher Full Text\n\nGosset M, Berenbaum F, Levy A, et al.: Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene. Arthritis Res Ther. 2006; 8(4): R135. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatwari P, Cook MN, DiMicco MA, et al.: Proteoglycan degradation after injurious compression of bovine and human articular cartilage in vitro: interaction with exogenous cytokines. Arthritis Rheum. 2003; 48(5): 1292–1301. PubMed Abstract | Publisher Full Text\n\nHui W, Rowan AD, Richards CD, et al.: Oncostatin M in combination with tumor necrosis factor alpha induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo. Arthritis Rheum. 2003; 48(12): 3404–3418. PubMed Abstract | Publisher Full Text\n\nSharma RA, McLelland HR, Hill KA, et al.: Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer. Clin Cancer Res. 2001; 7(7): 1894–1900. PubMed Abstract\n\nBisht S, Feldmann G, Soni S, et al.: Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin”): a novel strategy for human cancer therapy. J Nanobiotechnology. 2007; 5: 3. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1053",
"date": "09 Jul 2013",
"name": "Oliver Grundmann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting article submitted by Clutterbuck et al. is a well written and designed research paper. I do have a few comments that would help to clarify the research for readers. Suggested revisions: In the methods section the authors should clearly state how many repetitions have been used for each experiment and each condition – this is not clear at times. Presumably at least 3 repetitions per assay per condition were made in order to derive the statistics but this should be specified. In figures 3 and 4A, the % of proteoglycan release is mentioned and graphed – how is this related to the total PG content if the variability appears to be rather high from the 80s to over 300 μg/mL? This needs to be clarified. In figure 5, did the authors use a reference protein to ensure that the same total protein content was used for each band? If so, this should be included as a reference in Figure 5.In the discussion section, the authors mention that curcumin in different preparations can present with varying effects. Since curcumin is a pure substance isolated from Curcuma longa, it will have the same chemical properties and therefore – given that it is properly dissolved like in this study using DMSO in non-toxic concentrations – then it should not be different from any other study. If the authors refer to a Curcuma longa extract then it would be a different issue worth discussing.Furthermore, and of great importance, the authors briefly mention the low bioavailability of curcumin in vivo in humans – how does that compare to the concentrations that were used in these experiments? What would be a good comparison blood concentration one could expect in humans after oral application of curcumin? This should be included in the discussion section to provide the reader with some perspective.",
"responses": [
{
"c_id": "521",
"date": "11 Aug 2013",
"name": "Ali Mobasheri",
"role": "Author Response",
"response": "Response to Dr. Oliver Grundmann In response to Dr. Grundmann’s comments we have added some additional points to clarify the research for the readers. In response to both reviewers we have included the number of technical and biological replicates used. At least 3 biological replicates were used in each experiment and some assays included multiple technical replicates. Regarding the proteoglycan release data, it can be argued that such results should be expressed as micrograms of glycosaminoglycan (GAG) per milligram of cartilage to account for variation in explant weight, as previously shown as either wet weight or dry weight. However, percentage GAG release has been used in previous studies and was selected as a more accurate measure. Expressing the results as a percentage means that the proportion of GAG released was quantified in relation to the total amount of GAG in each explant. Obviously, this is not the case when discussing the PGE2 results but although the data are expressed as a total value of PGE2 per ml, pilot studies showed that the explant weight variation was low, with a mean wet weight (± SEM) of 4.24 ± 0.10mg. Thus, the explants were of a similar weight and size. As highlighted in our response to Dr. Toegel, we did not include a reference protein in the western blots, as there are no reliable secreted proteins that can effectively be used as a loading control in secretome samples. Instead, we performed a protein assay to ensure that protein was equally loaded on the gels before western blotting. We agree with Dr. Grundmann’s assertion that pure curcumin isolated from Curcuma longa should have identical effects when dissolved in DMSO in cell culture studies as it should retain the same chemical properties. The curcumin used in this study is a widely used formulation from Sigma-Aldrich with a reported purity of approximately 70%. It could be argued that only pure curcumin should have been tested to ensure that the effects seen in the models were from curcumin alone. However, the curcumin used in this manuscript, has been used by several investigators on other cell types, and so there was a benefit in using it to enable comparisons with the published literature. It should be noted here that many commercially available curcumin preparations, including the preparation used in this manuscript, are often a mixture of three curcuminoids; curcumin (also known as diferuloylmethane), demethoxycurcumin and bisdemethoxycurcumin. Therefore, the term ‘curcuminoid’ may be more appropriate to describe curcumin preparations of this nature. However, for comparative purposes with published studies that use the term ‘curcumin’, and in the absence of analytical data determining the activity of various fractions of the curcuminoid constituents in the curcumin used in this study, the term curcumin was used to describe the commercially available formulation. We hope that Dr. Grundmann will be satisfied with this explanation. Finally, we wish to refer Dr. Grundmann to three recent review articles in which the corresponding author has addressed the issue of low curcumin bioavailability in vivo in human subjects (please refer to citations 13, 14 and 15 in this paper). These reviews have been cited in this manuscript. The range of concentrations used in these experiments was indeed higher than those that can be achieved in vivo. However, the pharmacokinetic profile of curcumin is characterized by its low serum concentration and limited tissue distribution. The available clinical data supports the notion that the serum concentration of curcumin peaks 1-2 hours after an oral dose in human subjects with peak serum concentrations of 0.5, 0.6 and 1.8 micromolar at doses of 4, 6 and 8 g/day. These are the most reliable serum concentrations that one might expect in humans after oral consumption of curcumin. Strategies to improve bioavailability include the use of sustained released drug delivery systems, liposome delivery systems and nanoparticles. The possibility is the intraarticular injection of biologically active doses of curcumin to metabolising chondrocytes and synoviocytes. However, there are no studies that have attempted intra-articular injection of high doses of curcumin."
}
]
},
{
"id": "1061",
"date": "15 Jul 2013",
"name": "Stefan Toegel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present report, Clutterbuck and co-workers describe the cytotoxic and anti-inflammatory effects of curcumin in equine cartilage explants and chondrocytes. Their study demonstrates that curcumin is a potent inhibitor of IL1ß-induced PGE2, MMP3 and GAG release from cartilage at non-toxic concentrations. I found this article very interesting to read as it highlights important aspects of practical relevance for research on curcumin activity in cartilage. I do have a few remarks that might help to somewhat improve the manuscript.Suggested revisions The Figure legends should include detailed information on the number of technical and biological replicates used. The Methods should clarify whether curcumin and IL1ß were added simultaneously to the cells/explants or whether curcumin was used to “pretreat” the cells/explants prior to the addition of IL1ß.In the results section the authors state: “The most apparent reduction in MMP-3 secretion was always seen at 50μM with levels near to that of the controls in all animals.” This issue is also discussed in the 'discussion section later on. However, as Figure 1 demonstrates, significant cytotoxic results are expected from applying this concentration. The results should therefore be discussed in light of this finding.A reference protein (loading control) should be included in the Western Blot images in Figure 5.Did the authors check the Gaussian distribution of the data before applying ANOVA statistics?",
"responses": [
{
"c_id": "520",
"date": "11 Aug 2013",
"name": "Ali Mobasheri",
"role": "Author Response",
"response": "Response to Dr. Stefan Toegel In response to Dr. Stefan Toegel’s review, we have included detailed information on the number of technical and biological replicates used in the revised figure legends.Fig 1 - 5 biological replicates (animals) and 2 replicates per animal Fig 2 - 3 biological replicates (animals) and 1 replicate per animal Fig 3 - 3 biological replicates (animals) as already stated in the legend and 3 replicates per animal Fig 4 - 3 biological replicates (animals) as already stated in the legend and 3 replicates per animalFig 5 - 3 biological replicates (animals) as already stated in the legend and 1 replicate per animal Curcumin was not used to “pretreat” the cells/explants prior to the addition of IL-1β. Curcumin and IL-1β were added simultaneously. The revised Materials and methods section now clarifies the fact that curcumin and IL-1β were added simultaneously to the cells and explants. This is a very good point. However, we are mainly talking about curcumin at 25uM and under being non cytotoxic, yet effective. We did not include a reference protein in the western blots, as there are no proteins that can effectively be used as reference proteins in the secretome. However, we did perform a protein assay to ensure that equal quantities of protein were loaded on the polyacrylamide gels before western blotting.No, we did not check the Gaussian distribution of the data before applying ANOVA statistics."
}
]
}
] | 1
|
https://f1000research.com/articles/2-147
|
https://f1000research.com/articles/2-166/v1
|
02 Aug 13
|
{
"type": "Review",
"title": "The effects of anaesthesia on the developing brain: a summary of the clinical evidence",
"authors": [
"Clara KY Yu",
"Vivian Man Ying Yuen",
"Gordon TC Wong",
"Michael G Irwin",
"Clara KY Yu",
"Vivian Man Ying Yuen",
"Michael G Irwin"
],
"abstract": "Introduction: There is data amassing in the literature regarding the potentially adverse effects of anaesthesia exposure on the developing human brain. The purpose of this article is to summarise current relevant data from clinical studies in this area. Methods: Articles from journals written in English were searched for using PubMed, Ovid and Medline. Keywords used included: brain (newborn, infant, child and neonate), neurodegeneration, apoptosis, toxicity, neurocognitive impairment (developmental impairment and learning disorders) and anaesthesia (intravenous, inhalational and sedation).Results: From the initial search, 23 articles were identified as potentially relevant, with publication dates spanning from 1978 to 2012. Twelve studies were deemed irrelevant to the research questions. The results of neurocognitive assessment from eight of the remaining eleven studies had showed some differences in the performances of children exposed to anaesthesia. The control population in these studies was highly variable. The age at which the subjects were exposed to anaesthesia ranged from prenatal to 4 years in the majority of studies with one including children aged up to 12 years when exposed. Discussion: Although there is clinical data suggesting a possible detrimental effect, the evidence is best considered preliminary and inconclusive at this stage. Many of the outcome measures were lacking in specificity and standardization in most cases. Parents should be counselled to not avoid necessary invasive procedures for fear of a currently ill-defined risk. However, deferral of elective procedures beyond the first few years of life should be contemplated.",
"keywords": [
"Anaesthesia",
"brain",
"neurodegeneration",
"toxicity"
],
"content": "Introduction\n\nAdvances in perioperative care and imaging have resulted in more neonates, infants and children undergoing procedures requiring anaesthesia. General anaesthesia is a incompletely understood, complex pharmacological response produced by a heterogeneous class of drugs involving mechanisms on specific neuronal networks in different regions of the central nervous system. It is well known that the use of a balanced anaesthetic technique is beneficial in decreasing the neuroendocrine and metabolic stress response to surgery and altering pain processing1–4. However, increasing data from animal studies in the last decade has shown that general anaesthesia may also trigger apoptosis in the developing brain and suggest anaesthetic interactions with neurodegenerative mechanisms, including those linked to the onset and progression of Alzheimer's disease5,6. Naturally this has raised much concern regarding the safety of general anaesthesia in infants and young children. The developing brain differs from the adult brain in several different ways, which may provide a physiological basis for any enhanced vulnerability to anaesthetics. For example, the number of neurones formed in early development is significantly greater than in adult mammals, before synapses are pruned to establish behaviourally relevant connections between neurones. Apoptosis is responsible for eliminating 50–70% of developing neurones under normal circumstances and general anaesthesia-triggered apoptosis may disrupt this normal pattern of neural pruning7. The purpose of this article is to summarise the current literature concerning the effects of anaesthesia on the developing brain and to evaluated whether the available animal data can be translated to a clinical setting. The aim is to provide up-to-date information to non-anaesthetists who may need to counsel parents in the preoperative setting.\n\n\nMethod\n\nSearch engines used included PubMed, Ovid Medline and Embase, which were accessed in March 2013. Keywords used included: brain (newborn, infant, child or neonate), neurodegeneration, apoptosis, toxicity, neurocognitive impairment (developmental impairment or learning disorders) and an(a)esthesia (intravenous, inhalational and sedation). These were used in combination such that terms related to anaesthesia and brain was used together and were paired with the remaining terms in turn. Exclusion criteria were animal studies investigating anaesthesia-induced brain structural or behavioural abnormalities and articles published in non-English language journals.\n\n\nResults\n\nA total of 23 articles were identified using the above search methods. Of these 12 were deemed irrelevant to our research question and were excluded in our discussion (Table 1). Eight of these studies focused mainly on surgical diseases, their management and neurological outcome. The study groups were sick neonates and a majority had very low birth-weights, which adds to the multiple confounding factors. These studies were not designed to investigate anaesthetic exposure and its potential neurotoxicity. The anaesthetic technique and agent used were not specified and subsequent surgery requiring anaesthesia is unknown, hence they are excluded in our review. Three of the 12 studies were performed in the third trimester of pregnancy and one during the perinatal period. Because outcome measure of behaviour alterations was performed in the first few days after birth, any positive finding may be subtle and not relevant in the long-term outcome. Hence these studies were also excluded. Therefore, only 11 relevant publications on neurodevelopmental risk and anaesthesia exposure in early childhood were identified7–16, all of which were retrospective in nature (Table 2). Owing to the nature of the study question, the vast majority of the studies were either cohort or case control studies. The respective control population in these studies was, however, highly variable. In all but three studies, the results of neurocognitive assessment had showed some abnormalities in the performance of children exposed to anaesthesia. The age at which the subjects were exposed to anaesthesia ranged from prenatal to 3 years in the majority of studies with one including children aged up to 12 years when exposed.\n\nADQC= Abbreviated Depression Questionnaire for Children Self Assessment; ASO= Arterial Switch Operation; BSID= Bayley Scales of Infant Development; CBCL= Achenbach Child Behaviour Checklist Parental Assessment; CELF-R= Clinical Evaluation of Language Fundamentals-Revised; DDST= Denver Developmental Screening Test; DHCA= Deep Hypothermic Circulatory Arrest; ELBW= Extremely Low Birth Weight (<1000g); GMDS= Griffiths Mental Development Scales; INFANIB= Infant Neurological International Battery; LF-CPB= Low Flow-Cardiopulmonary Bypass; MDI Bayley= Mental Development Index; Movement ABC= Movement Assessment Battery for Children; NDI= Neurodevelopmental Impairment; PDI= Bayley Psychomotor Developmental Index; PDMS= Peabody Developmental Motor Scales; PHBQ= Vernon Post Hospitalisation Behaviour Questionnaire; PPVT= Peabody Picture Vocabulary Test; SBIS= Stanford-Binet Intelligence Scales; TOVA= Test of Variables of Attention; TRF= Teacher’s Report Form; VMI= Developmental Test of Motor Integration; WCST= Wisconsin Card Sorting Test of problem solving; WJPB= Woodcock-Johnson Psychoeducational Battery; VLBW= Very Low Birth Weight (<1500g); WIAT= Wechsler Individual Achievement Test; WISC 3= Wechsler Intelligence Scale for Children, Third Edition; WISC-RN= Wechsler Intelligence Scale for Children; WPPS-R= Revised Wechsler Preschool and Primary Scales for Intelligence.\n\nA group at the Mayo Clinic in Rochester was responsible for four of the included studies, using a birth cohort of children born in Rochester, Minnesota, USA, between 1976 and 198213–16. In one of the investigations13, 593 children with anaesthetic exposure before the age of 4 were compared with 4764 children with no anaesthetic exposure. Children receiving two, three or more anaesthetics were respectively 1.59 or 2.6 times more likely to have subsequent learning disabilities. Using the data from the same cohort, 350 children with anaesthetic exposure were compared with 5007 children with no anaesthetic exposure before the age of 2 years16. Children who had two or more exposures were 1.95 times more likely to be diagnosed with Attention Deficit Hyperactive Disorder (ADHD) than the unexposed children. From the same cohort in a matched design study, 350 children who had anaesthetic exposure before the age of 2 for were compared to 700 unexposed matched controls on the basis of known risk factors for learning disability14. Again children who had two or more anaesthetic exposures, but not single exposure, had an increased risk of subsequent learning disability (hazard ratio 2.12). The last study from the same cohort revealed that a single perinatal exposure to general anaesthesia during delivery by Caesarean section was not associated with an increased risk of learning disability15.\n\nUsing another national registry, investigators at Odense University in Denmark identified a cohort of 2689 children born between 1986 and 1990 that had a hernia repair before one year of age12. These children were compared with a randomly selected, age matched population consisting of 14575 children. The average test scores at ninth grade and test score non-attainment rate were used as marker for learning disability. After adjusting for confounding factors, no significant differences in either parameter between the two groups were found.\n\nInvestigators at Columbia University used the New York State Medicaid registry to identify a birth cohort of children who had surgery before the age of 3 years7,9. Medicaid is a health insurance program provided by US government covering approximately 25% of all children in the USA. In the first study 383 children whose insurance codes indicated surgery for inguinal hernia before age 3 years were identified9. These children were compared to a cohort of 5050 matched controls. Insurance codes were used to identify children with behavioural or development disorders. After controlling for potential confounders, children who had hernia repair before 3 years of age were more than twice as likely as controls to be subsequently diagnosed with developmental or behavioural disorder. Using the same Medicaid registry, a birth cohort of 10450 siblings was identified by the same group of investigators7. Three hundred and four children whose insurance codes indicated surgical procedures before the age of 3 years were compared to 10146 children who had no surgery before the age of 3. Similarly insurance codes were used to identify children with behavioural or development disorders. Children who had surgical exposure before the 3 years of age were 1.6 times more likely to have a subsequent behavioural or development disorder. This same group of investigators also used the Western Australian Pregnancy Cohort of 2680 children born from 1989–1992 to identify 321 children who had surgical procedures before age of 3 years10. Learning ability was assessed by more sensitive and specific neuropsychological tests. These tests include the Symbol Digit Modality Test and Raven’s Colored Progressive Matrices for assessment of cognition, the MaCarron Assessment of Neuromuscular Development for assessment of fine and gross motor control, the Clinical Evaluation of Language Fundamentals for assessment of various aspects of language ability. The Child Behavior Checklist (CBCL) was also used for assessment of behavioural problems. Children with single or multiple anaesthesia exposure were shown to have an increased risk of language and abstract reasoning deficits, but there was no association with anaesthesia exposure and behavioural or motor problems. The adjusted risk ratio (aRR) was 1.87 [95% CI 1.2–2.93] for receptive language, 1.72 [95% CI 1.12–2.64] for expressive language, 2.11 [95% CI 1.42–3.14] for total language, and 1.69 [95% CI 1.13–2.53] for abstract reasoning, a domain of the cognitive test.\n\nInvestigators at University Medical Centre Utrecht in Netherlands identified 314 children who had urological procedures under general anaesthesia before the age of 6 years8. Neurobehavioral development was assessed using Child Behaviour Checklist (CBCL) returned by parents. This study revealed no association with behavioural disturbances and anaesthesia exposure. Another study from the Netherlands (Vrije Universiteit Amsterdam) attempted to explore causality of anaesthesia exposure and learning related outcomes by using a monozygotic concordant-discordant design11. The researchers identified 1143 monozygotic twin pairs from the Netherlands Twin Registry. Data on anaesthetic exposure and learning outcomes was based on parental reports and standardized test scores respectively. The authors revealed that children from this cohort who were exposed to anaesthesia before the age of 3 years had significantly lower educational achievement scores and more cognitive problems than the unexposed children. However the un-exposed monozygotic twins did not differ from the exposed co-twin.\n\n\nDiscussion\n\nClinical observations dating as far back as 60 years have shown an association between exposure to anaesthesia in children and central nervous system dysfunction17. Numerous animal studies on rodents and even non-human primates have since been performed to investigate why this may occur18,19. Consistently it has been shown that exposure of the developing mammalian brain to most general anaesthetic drugs causes some degree of neuronal apoptosis and neurodegeneration during critical developmental periods5. General anaesthetics are powerful modulators of neurotransmission via a variety of ligand-gated ion channels. The drugs vary in their pharmacodynamic effects and receptor interaction so, to some extent, it is difficult to generalise but they mostly potentiate the gamma-amino butyric acid (GABA) receptor complex and/or inhibit glutamatergic neurotransmission principally through blockade of the N-methyl D-aspartate (NMDA) receptor (Table 3)20. Both of these neurotransmitter systems are central in determining the excitation/inhibition activity balance underlying experience-dependent sculpting of developing neural networks during the sensitive time of the neuronal “growth spurt”21. This critical period coincides with intense synaptogenesis in most cortical regions. In humans this period of synaptogenesis occurs between the third trimester of pregnancy and first few years of postnatal life; the most marked increase in synapse number occurring between birth and six months of age22. In humans, there are significant regional differences in the timing of the neuronal growth spurt. The earliest is the primary sensorimotor cortex, which occurs around birth, subsequently the parietal and temporal region (important in language and spatial attention) around 9 months and lastly the prefrontal cortex at 2–3 years23. During normal development, neurons are produced in excess by as much as 50–70% and subsequent neuronal pruning is essential for normal brain structure and function7,24. The mechanism of anaesthesia-induced cell death is not fully understood. Hence it is uncertain whether anaesthesia-induced apoptosis occurs in cells that are not meant to die i.e. pathological apoptosis, or whether it accelerates the death of cells that are meant to die at a later time i.e. premature physiological apoptosis.\n\n“-” = no known effect; “+” = weak effect; ++ strong effect; +++ very strong effect.\n\nRecent clinical studies may lead one to think this is a significant clinical issue for children undergoing surgery but a closer look at the data however, will reveal that the evidence is far from conclusive. While eight studies revealed a positive association between anaesthetic exposure and neurodevelopmental risk, the other two studies revealed the opposite. However, as studies relevant to this question are, to date, retrospective in nature, they only allowed identification of an association without establishing causality. Although there were large number of emigrates in the Rochester cohort, it did contain more than 5000 children. Complete medical and anaesthetic records were reviewed. Data collected included the type of surgery, type of anaesthetic agents, and number of anaesthetic exposures and duration of anaesthesia. Learning disability was assessed using only educational records. Consistently these studies revealed that single anaesthetic exposure during perinatal period15 or before age of 4 years13,14,16 was not associated with increased risk of learning disabilities or ADHD behaviour. Multiple exposures however were associated with significantly increased risk for learning disabilities and ADHD disorder and this may signify a dose-response relationship with a progressively increasing risk following two or more operations. Similarly data from the Danish Cohort may indicate that a single brief anaesthetic exposure is not associated with an increased risk of learning disability. The authors using the Western Australia cohort suggested that association between anaesthesia and neurodevelopmental outcome may be confined to specific domains (language and cognition) and this investigation may help to guide future studies.\n\nIn four of the investigations where a large cohort of children was identified7,9,10,12, information on surgical exposure and behavioural and development diagnosis were based solely on the administrative data of government funded health insurance program or parent questionnaires. Medical and anaesthetic or educational records were not reviewed and therefore misclassification is possible and therefore results drawn from these data are subjected to measurement error. Moreover the children from the Medicaid registry could be children with disadvantaged background and these results may not be generalized to other population groups. Nevertheless some of the imprecision should only lead to an under-estimation of true association. In the study by Kalkman et al.8 the authors commented that this study may be underpowered to reveal any significant differences. Moreover the CBCL may be an insensitive tool to detect neurodevelopment disability. This result is consistent with the finding from investigators at Columbia University as they have shown anaesthetic exposure is not associated with behavioural or motor disabilities10.\n\nIn the study involving twins11, the authors concluded that anaesthesia exposure does not cause later learning-related disability. Only a small number of twin pairs were discordant for surgical exposure (130 pairs) and an even smaller numbers of twin pairs had an educational achievement score (110) and a cognitive problem scale (56) available25. Therefore the lack of difference in scores may be secondary to inadequate sample size. Moreover the number of anaesthetic exposures in this cohort was not stated and this could potentially affect the outcome of interest.\n\nIn summary the available data from various studies including large numbers of children points to a possible association between anaesthetic exposure in early childhood and learning disability. Moreover a dose-response effect may be present. However one must be cautious with the conclusions drawn from retrospective studies. Association between early anaesthesia exposure and subsequent learning disability does not indicate anaesthesia neurotoxicity. There are many known and unknown confounding factors. Known co-existing medical or surgical diseases and disruption to learning due to repeated hospitalization are examples of such confounding factors. It is not possible to delineate the effect of surgical exposure and hospitalization from anaesthetic exposure. Retrospective data is subject to imprecision or error. The cohorts represented in these studies were children who had anaesthesia and surgery two to three decades ago, there have been many advances in surgical approach and anaesthetic techniques, and hence results from previous treatment may not apply today. Neurocognitive outcome is difficult to study in children and because of the growing complexity in their neurocognitive development as they age. This warrants more types of psychometric tests to assess domains which were not applicable at a younger age, which is to say that more domains need to be tested. Adding to this problem is the fact that it is not known which domain is affected most by anaesthesia related neurotoxicity. Coarse scoring systems such as IQ or measures such as diagnosis of developmental delay may overlook any subtle effects confined to specific areas; however, more refined psychometric tests have an increased chance of finding at least one association purely by chance. Tests carried out at an early age will only uncover major neurological problems and psychometric tests carried out in young children are poor at predicting later outcome26,27.\n\nGiven the limitations inherent in retrospective studies, prospective randomized studies are clearly needed to clarify long-term cognitive effects of early anaesthetic exposure in humans. The main problem is one of confounding factors. The effects of anaesthesia cannot be dissociated from factors associated with anaesthesia, such as surgical trauma and pathology. Surgery is associated with other confounding factors such as humoral and inflammatory stress as well as metabolic, haemodynamic and respiratory events, which may all influence outcomes. Infants and children having surgery or diagnostic procedures are very likely to have pathology, which may influence neurobehavioural outcome. They may be septic, premature, have less parental interaction or have chromosomal abnormalities, all of which can also be associated with developmental delay and need for surgery.\n\nCurrently there are two large-scale studies underway that are trying to address the issue of anaesthetic neurotoxicity in children. One that will attempt to separate the effects of general anaesthesia from surgical procedure is the General Anaesthesia Study (GAS)28. This is a multi-centre randomised controlled trial involving 29 centres around the world. The primary objective of this study is to compare regional and general anaesthesia for effects on neurodevelopmental outcome and apnoea in infants requiring inguinal hernia repair. Six hundred infants below 60 weeks post-conception age are randomised to receive either general anaesthesia with sevoflurane or spinal anaesthesia without sedation. The follow-up period will be at 5 years, with evaluation performed at 2 years using the Bayley Scales for Infant Development-III and at 5 years using the Wechsler Preschool and Primary Scale of Intelligence-III and additional neuropsychological tests within NEPSY-II (A developmental NEuroPSYchological assessment). The expected date of completion is 2015/2016. The other one is the PANDA (Pediatric Anaesthesia and Neurodevelopmental Assessment) study, which is another multi-centre study that involves eight US sites. This study proposes using a bidirectional epidemiological approach where a historical cohort exposed to a single general anaesthesia for inguinal hernia repair American Society of anaesthesiologist (ASA) class 1 and 2 before 36 months of age is identified. The group will be followed up prospectively using neurocognitive testing between the ages of 6 and 10 years. This study is an attempt to reduce the genetic and environmental contributions to cognitive performance. The pilot study has been completed, which demonstrated feasibility of such an approach29.\n\nTherefore, it is clear from preclinical data that anaesthetic agents are associated with neurotoxicity in developing animals30,31. However interpretation of clinical studies that have been completed to date is less clear-cut. This is due to the retrospective nature of the studies, the lack of specific information in terms of age, duration, and dose of anaesthetics, precise agents used, the variable outcome endpoints used and the way these outcomes were assessed. Many of the outcome measures were lacking in specificity and standardization in most cases. Any change in anaesthetic practice should be evidence based. The Anaesthetic and Life Support Drugs Committee of the U.S. Food and Drug Administration held a meeting in March 2011 and concluded that they acknowledge the compelling animal data that anaesthesia exposure is neurotoxic to the developing brain. However, there is still not enough data, especially in humans, to draw any firm conclusions.\n\n\nConclusions\n\nIn conclusion although there are some data suggesting a possible detrimental effect of anaesthesia on the developing brain in children, the evidence is best considered preliminary and inconclusive at this stage. However what we do know is that it is unethical to subject infants and children to surgery without the benefits of anaesthesia and analgesia. Parents should be counselled to not avoid necessary invasive procedures for fear of a currently ill-defined risk.",
"appendix": "Author contributions\n\n\n\nClara KY Yu performed original literature search and draft both the manuscript and tables. Vivian Man Ying Yuen performed an independent literature search, and prepared tables. Gordon TC Wong prepared the manuscript and tables Michael G. Irwin conceived the idea and made critical revisions of the manuscript. All authors have read and are agreeable to the publication of the current article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nAnand KJ, Hickey PR: Pain and its effects in the human neonate and fetus. N Engl J Med. 1987; 317(21): 1321–9. PubMed Abstract | Publisher Full Text\n\nFitzgerald M, Millard C, McIntosh N: Cutaneous hypersensitivity following peripheral tissue damage in newborn infants and its reversal with topical anaesthesia. Pain. 1989; 39(1): 31–6. PubMed Abstract | Publisher Full Text\n\nJohnston CC, Stevens BJ: Experience in a neonatal intensive care unit affects pain response. Pediatrics. 1996; 98(5): 925–30. PubMed Abstract\n\nTaddio A, Katz J, Ilersich AL, et al.: Effect of neonatal circumcision on pain response during subsequent routine vaccination. Lancet. 1997; 349(9052): 599–603. PubMed Abstract | Publisher Full Text\n\nJevtovic-Todorovic V, Hartman RE, Izumi Y, et al.: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003; 23(3): 876–82. PubMed Abstract\n\nIkonomidou C, Bosch F, Miksa M, et al.: Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science. 1999; 283(5398): 70–4. PubMed Abstract | Publisher Full Text\n\nOppenheim RW: Cell death during development of the nervous system. Annu Rev Neurosci. 1991; 14: 453–501. PubMed Abstract | Publisher Full Text\n\nKalkman CJ, Peelen L, Moons KG, et al.: Behavior and development in children and age at the time of first anesthetic exposure. Anesthesiology. 2009; 110(4): 805–12. PubMed Abstract | Publisher Full Text\n\nDiMaggio C, Sun LS, Kakavouli A, et al.: A retrospective cohort study of the association of anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J Neurosurg Anesthesiol. 2009; 21(4): 286–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIng C, DiMaggio C, Whitehouse A, et al.: Long-term differences in language and cognitive function after childhood exposure to anesthesia. Pediatrics. 2012; 130(3): e476–85. PubMed Abstract | Publisher Full Text\n\nBartels M, Althoff RR, Boomsma DI: Anesthesia and cognitive performance in children: no evidence for a causal relationship. Twin Res Hum Genet. 2009; 12(3): 246–53. PubMed Abstract | Publisher Full Text\n\nHansen TG, Pedersen JK, Henneberg SW, et al.: Academic performance in adolescence after inguinal hernia repair in infancy: a nationwide cohort study. Anesthesiology. 2011; 114(5): 1076–85. PubMed Abstract | Publisher Full Text\n\nWilder RT, Flick RP, Sprung J, et al.: Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology. 2009; 110(4): 796–804. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFlick RP, Katusic SK, Colligan RC, et al.: Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics. 2011; 128(5): e1053–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSprung J, Flick RP, Wilder RT, et al.: Anesthesia for cesarean delivery and learning disabilities in a population-based birth cohort. Anesthesiology. 2009; 111(2): 302–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSprung J, Flick RP, Katusic SK, et al.: Attention-deficit/hyperactivity disorder after early exposure to procedures requiring general anesthesia. Mayo Clin Proc. 2012; 87(2): 120–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEckenhoff JE: Relationship of anesthesia to postoperative personality changes in children. AMA Am J Dis Child. 1953; 86(5): 587–91. PubMed Abstract\n\nBrambrink AM, Evers AS, Avidan MS, et al.: Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology. 2010; 112(4): 834–41. PubMed Abstract | Publisher Full Text\n\nSlikker W Jr, Zou X, Hotchkiss CE, et al.: Ketamine-induced neuronal cell death in the perinatal rhesus monkey. Toxicol Sci. 2007; 98(1): 145–58. PubMed Abstract | Publisher Full Text\n\nHemmings HC Jr, Akabas MH, Goldstein PA, et al.: Emerging molecular mechanisms of general anesthetic action. Trends Pharmacol Sci. 2005; 26(10): 503–10. PubMed Abstract | Publisher Full Text\n\nHensch TK: Critical period regulation. Annu Rev Neurosci. 2004; 27: 549–79. PubMed Abstract | Publisher Full Text\n\nHuttenlocher PR, Dabholkar AS: Regional differences in synaptogenesis in human cerebral cortex. J Comp Neurol. 1997; 387(2): 167–78. PubMed Abstract | Publisher Full Text\n\nSowell ER, Peterson BS, Thompson PM, et al.: Mapping cortical change across the human life span. Nat Neurosci. 2003; 6(3): 309–15. PubMed Abstract | Publisher Full Text\n\nRakic S, Zecevic N: Programmed cell death in the developing human telencephalon. Eur J Neurosci. 2000; 12(8): 2721–34. PubMed Abstract | Publisher Full Text\n\nFlick RP, Wilder RT, Sprung J, et al.: Anesthesia and cognitive performance in children: no evidence for a causal relationship. Are the conclusions justified by the data? Response to Bartels et al., 2009. Twin Res Hum Genet. 2009; 12(6): 611–2; discussion 613–4. PubMed Abstract | Publisher Full Text\n\nKorkman M, Kemp SL, Kirk U: Effects of age on neurocognitive measures of children ages 5 to 12: a cross-sectional study on 800 children from the United States. Dev Neuropsychol. 2001; 20(1): 331–54. PubMed Abstract | Publisher Full Text\n\nHack M, Taylor HG, Drotar D, et al.: Poor predictive validity of the Bayley Scales of Infant Development for cognitive function of extremely low birth weight children at school age. Pediatrics. 2005; 116(2): 333–41. PubMed Abstract | Publisher Full Text\n\nA Multi-site Randomized Controlled Trial Comparing Regional and General Anesthesia for Effects on Neurodevelopmental Outcome and Apnea in Infants (GAS). ClinicalTrials.gov Identifier: NCT00756600. Reference Source\n\nSun LS, Li G, DiMaggio CJ, et al.: Feasibility and pilot study of the Pediatric Anesthesia NeuroDevelopment Assessment (PANDA) project. J Neurosurg Anesthesiol. 2012; 24(4): 382–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKabra NS, Schmidt B, Roberts RS, et al.: Neurosensory impairment after surgical closure of patent ductus arteriosus in extremely low birth weight infants: results from the Trial of Indomethacin Prophylaxis in Preterms. J Pediatr. 2007; 150(3): 229–34, 234.e1. PubMed Abstract | Publisher Full Text\n\nTobiansky R, Lui K, Roberts S, et al.: Neurodevelopmental outcome in very low birthweight infants with necrotizing enterocolitis requiring surgery. J Paediatr Child Health. 1995; 31(3): 233–6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1334",
"date": "06 Aug 2013",
"name": "Caleb Ing",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a nice summary of the clinical studies that have been performed to evaluate the long-term effects of anesthetic exposure in young children. However, in order to improve the accuracy of the paper, there were a few corrections to point out:The Western Australian Pregnancy Cohort Study (Raine) consisted of 2868 children of which 2608 were evaluated in the cited study, with 321 who had surgical procedures before age 3 years. Table 2: In the Raine Cohort, the study group was composed of 321 children exposed to anesthesia and 2287 children who were unexposed. The number of children tested for each outcome however varied by individual outcome and also varied during the evaluation of single and multiple anesthetic exposures. While several studies only found a difference in cognitive outcomes after multiple anesthetic exposures, evaluation of the Raine cohort found differences even after a single exposure. This may be due to the use of more sensitive directly assessed neurocognitive measures in this cohort. Also as a clarification, in this study, the age of neurological assessment for all children was at age 10.",
"responses": []
},
{
"id": "1332",
"date": "07 Aug 2013",
"name": "Igor Luginbuehl",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 complex topic of significant importance and interest not only to anesthesiologists, but also to parents and, of course, the patients. A lot of research has been done in that regard, but we are still awaiting a definite answer, which will most likely take a long time.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-166
|
https://f1000research.com/articles/2-177/v1
|
20 Aug 13
|
{
"type": "Short Research Article",
"title": "Fecal transplantation does not transfer either susceptibility or resistance to food borne listeriosis in C57BL/6 and BALB/c/By mice",
"authors": [
"Tanya Myers-Morales",
"Kate M Bussell",
"Sarah EF D'Orazio",
"Tanya Myers-Morales",
"Kate M Bussell"
],
"abstract": "The composition of the intestinal microbiota has wide reaching effects on the health of an individual, including the development of protective innate immune responses. In this report, a fecal transplantation approach was used to determine whether resistance to food borne listeriosis was dependent on the murine gut microbiota. Transplantation of BALB/c/By feces did not increase the susceptibility of C57BL/6 mice to Listeria monocytogenes infection.\n\nLikewise, transplantation of C57BL/6 fecal matter did not enhance the resistance of BALB/c/By mice. Thus, intestinal microbiota composition is not a key factor that confers either susceptibility or resistance to food borne listeriosis in mice.",
"keywords": [
"Listeria monocytogenes is a Gram-positive",
"facultative intracellular bacterial species that can readily adapt to a variety of environmental stresses",
"including high salt concentration",
"low pH",
"and refrigeration1. In humans",
"ingestion of L. monocytogenes-contaminated food results in a wide spectrum of clinical outcomes",
"ranging from mild",
"self-limiting gastroenteritis to lethal meningoencephalitis2. The majority of serious infections occur in patients with some degree of immune compromise",
"but the exact host factors that determine susceptibility to these infections are not well understood."
],
"content": "Introduction\n\nListeria monocytogenes is a Gram-positive, facultative intracellular bacterial species that can readily adapt to a variety of environmental stresses, including high salt concentration, low pH, and refrigeration1. In humans, ingestion of L. monocytogenes-contaminated food results in a wide spectrum of clinical outcomes, ranging from mild, self-limiting gastroenteritis to lethal meningoencephalitis2. The majority of serious infections occur in patients with some degree of immune compromise, but the exact host factors that determine susceptibility to these infections are not well understood.\n\nL. monocytogenes infection can be established in almost any outbred or inbred strain of laboratory mice, but there are significant differences among mouse strains for both morbidity and mortality. C57BL/6 mice are one of the most resistant strains, and can readily clear L. monocytogenes infection, while BALB/c mice are highly susceptible to developing life-threatening systemic listeriosis. These differences in host susceptibility hold true whether mice are infected by the intravenous route3,4, via intragastric inoculation5, or by ingestion of contaminated food6.\n\nGenetic analysis of laboratory mouse strains has defined a number of alleles that can be either deleterious or beneficial to mice during L. monocytogenes infection7–9, however, no single trait appears to be entirely responsible for the susceptibility or resistance phenotype. Instead, the ability to clear a bacterial infection is likely to depend upon complex interactions between multiple host factors. It was also recently shown that a novel mechanism, unrelated to specific gene loci, could account for the majority of the difference in host susceptibility to infection between two mouse strains. Willing et al. demonstrated that the enhanced susceptibility of C3H/HeJ mice to oral Citrobacter infection could be completely transferred to resistant NIH Swiss mice10. To do this, they depleted the gut microbiota of the resistant NIH Swiss mice and repopulated the intestinal tracts of those animals with fecal matter harvested from susceptible C3H/HeJ mice. Their striking results implied that varying gut microbiota compositions could underlie many previously observed differences in host susceptibility to infection with a variety of orally acquired bacterial pathogens.\n\nThe mouse strains used by Willing et al. came from two different vendors, so it is not surprising that the composition of the gut microbiota in those animals differed significantly. However, the predominant organisms found in the gastrointestinal tract can differ, even for mice housed in the same facility, particularly when the mice are altered in specific components of the immune system. For example, the intestinal microbiota was reported to differ compared with parental control strains in mice expressing human alpha defensin11, or in mice lacking IL-1012, IL-2213, neutrophil elastase14, NLRP615, or TLR516. In fact, it has been shown that the presence or absence of a single type of bacteria in the gut can dramatically alter the development of innate immune responses and inflammatory disease17–19. In this report, we used the fecal transplantation approach of Willing et al. to test the hypothesis that the murine intestinal microbiota can at least partially mediate either the susceptibility of BALB/c/By mice or the resistance of C57BL/6 mice to food borne L. monocytogenes infection.\n\n\nMaterials and methods\n\nFemale C57BL/6/J (B6) and BALB/c/By/J (By) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 4 weeks of age and then housed at the University of Kentucky for 2 weeks in a specific-pathogen free facility with a 14 h light cycle (12 am–2 pm) and a 10 h dark cycle (2 pm–12 am). Groups of four mice were housed in microisolator cages (Inc., Allentown, NJ) lined with coarse grade Sani-Chip bedding (PJ Murphy Forest Products, Montville, NJ). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky.\n\nPrior to each fecal transfer treatment, fecal pellets were collected from 3–4 donor mice, pooled together and weighed, and then placed in 0.25–1.0 ml of transfer buffer sterile filtered 0.05% cysteine HCl (Calbiochem/EMD Millipore, Billerica, MA) in Dulbecco’s Phosphate Buffered Saline (PBS; Life Technologies, Grand Island, NY), as described previously10. The final volume was adjusted to give 120 mg feces per ml. The fecal pellets were mashed with sterile wooden toothpicks (Wesco Enterprises, Santa Fe Springs, CA), and then vortexed (Vortex Genie; Scientific Industries, Bohemia, NY) at maximum speed for 1 min. The fecal matter was centrifuged (Eppendorf 5417C; Hauppauge, NY) for 3 min. at 800 × g and the supernatant was used for transplantation into mice.\n\nThe endogenous gut microbiota of 6 week old mice was depleted by treating with a single dose of streptomycin (Sigma-Aldrich, St. Louis, MO). The antibiotic was suspended in sterile water at 500 mg/ml and 50 µl was placed directly in the oral cavity of each mouse. After the oral antibiotic treatment, mice were housed on raised wire flooring (# 3 mesh; Allentown) to prevent coprophagy. Fecal transplants were initiated 24 h later by placing 50 µl of the donor fecal matter directly into the oral cavity of the recipient mice (n=4 per group). Control groups of mice (n=4) were given 50 µl of transfer buffer alone. As shown in Figure 1B and Figure 2B, all mice received fecal transplants 24 and 48 hours after the streptomycin treatment. After the second fecal transplant, the wire flooring units were removed from the cages. Each group of mice (n=4) received a total of four additional treatments over the next 6–7 days (see Figure 1A and Figure 2A), prior to oral challenge with L. monocytogenes.\n\n(A) The experimental design for four groups of mice is shown. Mock-treated mice received oral treatments with buffer alone and maintained their original microbiota. Streptomycin-treated mice received a total of 6 fecal transfers over a 9 day period. On day 10, food was restricted and all mice were placed on raised wire flooring for the duration of the experiment. (B) The efficiency of streptomycin pre-treatment was evaluated by monitoring the amount of aerobic CFU shed in the feces. Arrows indicate days of either antibiotic treatment (Strep) or fecal transplants (FT). Mean values ± SEM for groups of 8 mice are shown. (C) Mean values ± SEM (n=8) for total L. monocytogenes CFU in spleen, liver, or gall bladder and cell-associated CFU in the colon were determined 5 days post infection. Asterisks indicate mean values significantly different from the mock-treated By group as determined by unpaired t-test. Pooled data from two separate experiments comprising 4 mice per group is shown.\n\n(A) The experimental design for four groups of mice is shown. Mock-treated mice received oral treatments with buffer alone and maintained their original microbiota. Streptomycin-treated mice received a total of 6 fecal transfers over an 8 day period. On day 9, food was restricted and all mice were placed on raised wire flooring for the duration of the experiment. (B) The efficiency of the streptomycin pre-treatment was evaluated by monitoring the amount of aerobic CFU shed in the feces. Mean values ± SEM for groups of 8 mice are shown. Arrows indicate days of either antibiotic treatment (Strep) or fecal transfers (FT). (C) Mean values ± SEM (n=8) for total L. monocytogenes CFU in spleen, liver, or gall bladder and the cell-associated CFU in the colon were determined 5 days post infection. Asterisks indicate mean values significantly different from the mock-treated C57BL/6 group as determined by unpaired t-test. Pooled data (n=8) from two separate experiments (n=4 per group in each) is shown.\n\nStool samples were collected both before and after streptomycin treatment. Three fecal pellets per mouse were placed in a microcentrifuge tube (VWR; Radnor, PA) containing 1 ml of sterile water. The pellets were mashed with a sterile wooden toothpick, vortexed at maximum speed for 1 min. and centrifuged at 800 × g for 3 min. The total number of aerobic colony forming units (CFU) was determined by plating serial dilutions of the supernatant on Brain Heart Infusion (BHI) agar (Difco; BD Diagnostics, Franklin Lakes, NJ) and incubating at 37°C overnight.\n\nAfter completing a total of 6 oral treatments with either donor fecal material or transfer buffer alone, all mice were denied food (but given unrestricted access to water) and placed on raised wire flooring (to prevent coprophagy) for 24 h. Aliquots of intestinally passaged L. monocytogenes EGDe InlAm (generously provided by Wolf Dieter Schubert, Braunschweig, Germany) were prepared and grown as previously described6. Bacteria were washed once with sterile PBS, suspended in 40% melted salted butter (Kroger; Cincinnati, OH) in PBS, and then 5 μl of this mixture was used to saturate a small cube of white bread (Kroger). At the onset of the dark cycle, each mouse was placed in an empty cage with one L. monocytogenes-contaminated bread piece and was observed until it picked up the bread and consumed it entirely as previously described20. After ingesting the L. moncytogenes-contaminated bread, each mouse was returned to its original cage and was given unrestricted access to mouse chow and water for the duration of the experiment (5 days).\n\nFive days post-infection, all mice were euthanized by cervical dislocation and the organs were harvested aseptically as previously described20. The total cell-associated L. monocytogenes in the colon was determined by extensively flushing the tissue with sterile PBS to remove lumenal bacteria and then homogenizing for 1 min. as previously described6,20. Spleens and livers were collected in 2 ml of sterile water and homogenized for 30 sec. using a PowerGen 1000 homogenizer (IKA Works, Wilmington, NC). Gall bladders were placed in microcentrifuge tubes containing 1 ml of sterile water, ruptured with sterile scissors (VWR, Radnor, PA), and vortexed at maximum speed for 1 min. Serial dilutions of each tissue homogenate were prepared in sterile water and then plated on BHI/L+G (BHI agar supplemented with 15 g/L LiCl (Sigma-Aldrich) and 10 g/L glycine (Calbiochem), a selective medium that inhibited the growth of intestinal microbiota6,20. Colony forming units (CFU) were observed after 48 h growth at 37°C and the number of colonies was multiplied by the dilution factor and the total volume of the sample to give the total number of CFU per tissue.\n\nUnpaired t tests were performed using Prism 5 software for Macintosh (Graph Pad). P values less than 0.05 were considered significant and are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.\n\n\nResults\n\nTo test the hypothesis that resistance to listeriosis could be mediated by gut microbiota, fecal matter was recovered from resistant C57BL/6 mice and used to repopulate the intestinal lumens of susceptible BALB/c/By mice (Figure 1A). Groups of BALB/c mice were given a single oral dose of streptomycin to eradicate the existing microbiota. One day later, the mice began a series of six oral treatments with C57BL/6 fecal matter. The efficacy of the antibiotic treatment was confirmed by plating stool samples from each of the recipient animals. As shown in Figure 1B, the number of bacteria that could be recovered during aerobic growth decreased by more than 5 logs. From 24 to 48 h post-antibiotic treatment, the number of CFU in the stool samples was below the limit of detection (10 CFU) in the majority of the animals (Figure 1B). By 72 h post-antibiotic treatment, after two fecal transplants, the number of CFU present in the stool samples had recovered to pre-treatment levels. To ensure that the transferred microbiota persisted, each animal received four additional fecal transplants prior to oral L. monocytogenes challenge (Figure 1A).\n\nGroups of BALB/c/By mice transplanted with C57BL/6 microbiota, or repopulated with BALB/c/By fecal matter as a control, were then infected with L. monocytogenes via the natural feeding route. The bacterial burdens in the colon, spleen, liver, and gall bladder were determined 5 days post-infection and compared to groups of BALB/c/By and C57BL/6 mice that were mock-treated (no fecal transplantation; see Figure 1A). As expected, C57BL/6 mice had 30 to 1000-fold fewer L. monocytogenes in each tissue examined compared with BALB/c/By mice (Figure 1C). In contrast, there was no significant difference between the groups of BALB/c/By mice that received C57BL/6 fecal transplants and the BALB/c/By mice that were mock treated. Thus, fecal transplantation did not cause BALB/c/By mice to become more resistant to oral L. monocytogenes challenge, suggesting that the gut microbiota alone does not contribute significantly to the resistance phenotype observed in C57BL/6 mice.\n\nSusceptibility and resistance to infection are separate traits that are often linked to distinct gene loci. To test the alternate hypothesis that bacteria present in the gut microbiota could confer susceptibility to listeriosis, BALB/c/By feces was transplanted into C57BL/6 mice (Figure 2A). Groups of C57BL/6 mice were pre-treated with streptomycin and then given 6 oral treatments with BALB/c/By fecal matter. Control groups received C57BL/6 feces or were mock treated (Figure 2A). Again, the streptomycin treatment efficiently cleared the vast majority of the aerobic bacteria present in the gut lumen within 24 h, and fecal transplantation restored the level of the intestinal microbiota within 3 days (Figure 2B).\n\nThe mice were infected with L. monocytogenes by ingestion of contaminated food, and the number of CFU present in the gut and in peripheral tissues was determined 5 days later. Mock-treated C57BL/6 mice had 1000-fold less L. monocytogenes than BALB/c/By mice in the colon (Figure 2C), the site within the intestines which was previously shown to allow for the greatest growth and persistence of L. monocytogenes after oral infection6. Transfer of BALB/c/By microbiota did not enhance the ability of L. monocytogenes to colonize the colon, as there was no significant difference between the B6 + By group and mock-treated C57BL/6 mice (Figure 2C).\n\nIn the food borne model of listeriosis, the spleen and liver are not colonized until 48 h post-ingestion and bacteria begin to appear in the gall bladder 3–4 days post-infection6. Thus, bacterial burdens 5 days post-infection reflect both the ability of L. monocytogenes to disseminate from the gut, and bacterial replication within these tissues. As shown in Figure 2C, resistant C57BL/6 mice had at least 1000-fold fewer L. monocytogenes in the spleen and liver and approximately 100,000-fold less bacteria in the gall bladder compared with mock-treated BALB/c/By mice. Transplantation of BALB/c/By fecal matter did not result in an increase in bacterial loads in either the liver or the gall bladder of C57BL/6 mice (B6 + By group; Figure 2C). Fecal transplants did result in a slight increase in the number of L. monocytogenes recovered from spleens of C57BL/6 mice, however, a similar increase was observed whether the mice were transplanted with BALB/c/By feces or re-populated with C57BL/6 fecal matter. Together, these results suggest that the gut microbiota of BALB/c/By mice cannot independently enhance susceptibility to food borne listeriosis.\n\n\nDiscussion\n\nDiffering levels of resistance to infection with bacterial pathogens such as L. monocytogenes are well documented in mice3–6. Until recently, it was assumed that specific DNA loci were responsible, and that different combinations of susceptibility and resistance alleles would result in varying degrees of infection in any given mouse strain21. A recent report by Willing et al. shattered this paradigm by using a fecal transplantation approach to demonstrate that the gut microbiota alone could confer resistance to Citrobacter rodentium infection10. In this study, we used a similar approach to determine whether the composition of the intestinal microbiota could alter the dynamics of L. monocytogenes infection in mice. Using a food borne model of listeriosis20, we showed that transplantation of BALB/c/By feces into C57BL/6 mice did not make the mice more susceptible to infection. Likewise, transplantation of C57BL/6 fecal matter was unable to enhance the resistance of BALB/c mice.\n\nA key factor required for C. rodentium infection appears to be the induction of a strong Th1 type inflammatory response in the intestines. Lupp et al. previously showed that the inflammation itself significantly altered the composition of the gut microbiota in a way that promoted the aerobic outgrowth of both endogenous members of the Enterobacteriaceae as well as the introduced pathogen C. rodentium12. L. monocytogenes is an invasive pathogen, and thus, may not need to overcome colonization resistance or compete with the microbiota for growth in the intestinal lumen in order to establish an infection. L. monocytogenes can cross the gut mucosa using M cells or via a “zipper” mechanism of uptake into enterocytes and goblet cells that is dependent on interactions between the bacteria surface protein InlA and E-cadherin on the host cell22,23. Both mechanisms result in rapid translocation to the lamina propria where the bacteria replicate extensively before disseminating to peripheral tissues to cause systemic listeriosis6. Thus, if even a few L. monocytogenes can translocate across the mucosal barrier, the infection will be maintained, even if there is very little growth of the bacteria in the gut lumen.\n\nThe fecal transplantation approach is a useful way to significantly alter the gut microbiota, but it does not completely eliminate the native bacterial populations present in any given mouse. A single dose of streptomycin can transiently reduce, by approximately 90%, the density of bacteria recovered using anaerobic culture conditions24, and the intestinal microbiota will return to normal levels within 3–4 days. Willing et al. used 16S RNA analysis to show that within 4–5 days of fecal transplantation, mice had an altered microbiota composed of microbial families from both the donor and recipient mouse strains, with ratios that strongly reflected the donor strain10. Given these parameters, the fecal transplantation approach is most informative if the presence of a transferred microbial species confers a particular phenotype, even when the original microbiota is not completely displaced. Thus, we cannot rule out the possibility that small numbers of native microbial species not eliminated by streptomycin treatment could confer some degree of susceptibility to BALB/c/By mice or resistance to C57BL/6 mice. However, the results shown here clearly suggest that the composition of the gut microbiota is not a major factor that governs susceptibility to food borne listeriosis, as is the case for C. rodentium infection in mice.",
"appendix": "Author contributions\n\n\n\nSEFD conceived the study. TMM and SEFD designed the experiments. TMM and KB carried out the research. SEFD prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests disclosed.\n\n\nGrant information\n\nThis work was supported by a grant (R01 AI101373) from the National Institutes of Health to SEFD.\n\n\nAcknowledgments\n\nWe would like to thank Hilary Denney and Christopher Glecos for technical assistance with this project.\n\n\nReferences\n\nSoni KA, Nannapaneni R, Tasara T: The contribution of transcriptomic and proteomic analysis in elucidating stress adaptation responses of Listeria monocytogenes. Foodborne Pathog Dis. 2011; 8(8): 843–852. PubMed Abstract | Publisher Full Text\n\nAllerberger F, Wagner M: Listeriosis: a resurgent foodborne infection. Clin Microbiol Infect. 2010; 16(1): 16–23. PubMed Abstract | Publisher Full Text\n\nCheers C, McKenzie IF, Pavlov H, et al.: Resistance and susceptibility of mice to bacterial infection: course of listeriosis in resistant or susceptible mice. Infect Immun. 1978; 19(3): 763–770. PubMed Abstract | Free Full Text\n\nD'Orazio SE, Troese MJ, Starnbach MN: Cytosolic localization of Listeria monocytogenes triggers an early IFN-gamma response by CD8+ T cells that correlates with innate resistance to infection. J Immunol. 2006; 177(10): 7146–7154. PubMed Abstract\n\nCzuprynski CJ, Faith NG, Steinberg H: A/J mice are susceptible and C57BL/6 mice are resistant to Listeria monocytogenes infection by intragastric inoculation. Infect Immun. 2003; 71(2): 682–689. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBou Ghanem EN, Jones GS, Myers-Morlaes T, et al.: InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice. PLoS Pathog. 2012; 8(11): e1003015. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoyartchuk V, Rojas M, Yan BS, et al.: The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice. J Immunol. 2004; 173(8): 5112–5120. PubMed Abstract\n\nBoyartchuk VL, Broman KW, Mosher RE, et al.: Multigenic control of Listeria monocytogenes susceptibility in mice. Nat Genet. 2001; 27(3): 259–260. PubMed Abstract | Publisher Full Text\n\nStevenson MM, Kongshavn PA, Skamene E: Genetic linkage of resistance to Listeria monocytogenes with macrophage inflammatory responses. J Immunol. 1981; 127(2): 402–407. PubMed Abstract\n\nWilling BP, Vacharaksa A, Croxen M, et al.: Altering host resistance to infections through microbial transplantation. PLoS One. 2011; 6(10): e26988. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalzman NH, Hung K, Haribhai D, et al.: Enteric defensins are essential regulators of intestinal microbial ecology. Nat Immunol. 2010; 11(1): 76–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLupp C, Robertson ML, Wickham ME, et al.: Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae. Cell Host Microbe. 2007; 2(2): 119–129. PubMed Abstract | Publisher Full Text\n\nZenewicz LA, Yin X, Wang G, et al.: IL-22 deficiency alters colonic microbiota to be transmissible and colitogenic. J Immunol. 2013; 190(10): 5306–5312. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGill N, Ferreira RB, Antunes LC, et al.: Neutrophil elastase alters the murine gut microbiota resulting in enhanced Salmonella colonization. PLoS One. 2012; 7(11): e49646. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElinav E, Strowig T, Kau AL, et al.: NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell. 2011; 145(5): 745–757. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVijay-Kumar M, Aitken JD, Carvalho FA, et al.: Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science. 2010; 328(5975): 228–231. PubMed Abstract | Publisher Full Text\n\nGarrett WS, Gallini CA, Yatsunenko T, et al.: Enterobacteriaceae act in concert with the gut microbiota to induce spontaneous and maternally transmitted colitis. Cell Host Microbe. 2010; 8(3): 292–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIvanov II, Atarashi K, Manel N, et al.: Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell. 2009; 139(3): 485–498. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRound JL, Mazmanian SK: Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci U S A. 2010; 107(27): 12204–12209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBou Ghanem EN, Myers-Morales T, Jones GS, et al.: Oral Transmission of Listeria monocytogenes in mice via ingestion of contaminated food. J Vis Exp. 2013; 75. PubMed Abstract | Publisher Full Text\n\nGruenheid S, Gros P: Forward genetic dissection of innate response to infection in inbred mouse strains: selected success stories. Clin Exp Immunol. 2010; 162(3): 393–401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChiba S, Nagai T, Hayashi T, et al.: Listerial invasion protein internalin B promotes entry into ileal Peyer's patches in vivo. Microbiol Immunol. 2011; 55(2): 123–129. PubMed Abstract | Publisher Full Text\n\nNikitas G, Deschamps C, Disson O, et al.: Transcytosis of Listeria monocytogenes across the intestinal barrier upon specific targeting of goblet cell accessible E-cadherin. J Exp Med. 2011; 208(11): 2263–2277. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStecher B, Robbiani R, Walker AW, et al.: Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota. PLoS Biol. 2007; 5(10): 2177–2189. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "1551",
"date": "27 Aug 2013",
"name": "Benjamin Willing",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 “Fecal transplantation does not transfer either susceptibility or resistance to food borne listeriosis in C57BL/6 and BALB/c/By mice” by Myers-Morales et al. demonstrates that the intestinal microbiota is not a major determinant in the differences in susceptibility between C57BL/6 and BALB/c/By mice. It is an important report of negative results.\n\nA couple of clarifications are requested/suggested.\n\n1)\n\nWhile not necessary, the characterization of the microbial community in transplanted mice to confirm effective transfer would strengthen the claim.\n2)\n\nThe report by Willing et al. demonstrated that susceptibility in C3H/HeJ mice could be partially transferred to resistant NIH Swiss mice, not completely. While higher colonization and increased pathology was shown, the transfer of mortality phenotype was not demonstrated. 3)\n\nThe presentation of data for CFU counts should be normalized to weight. (CFU/g). CFU/organ or CFU/fecal pellet would also be acceptable.\n4)\n\nIn the results section: Streptomycin depletes, but does not “eradicate” the microbiota.",
"responses": []
},
{
"id": "2212",
"date": "19 Nov 2013",
"name": "Laurel Lenz",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMyers-Morales et al. present results indicating that B6 or BALB mice depleted of gut flora using streptomycin do not show altered resistance when given a fecal transplant from the opposing strain. The studies suggest, but do not prove, that gut flora do not contribute to the differing resistance of these strains.A few concerns, if addressed, would strengthen the paper.It is assumed that B6 and BALB mouse strains have differences in their streptomycin-resistant flora. However, this was not actually established by the authors. A control group with streptomycin treatment alone might establish whether depletion (not \"eradication,\" as stated) of the flora itself affected resistance. If so, the reconstitution apparently restored the original phenotype regardless of donor. This or other more direct approaches could be used to establish whether the transfer was successful at modifying the flora in recipients.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-177
|
https://f1000research.com/articles/2-176/v1
|
16 Aug 13
|
{
"type": "Correspondence",
"title": "Rats maintain a binocular field centered on the horizon",
"authors": [
"Markus Meister",
"David Cox"
],
"abstract": "In this letter, we attempt to correct a potentially serious misperception arising from the paper “Rats maintain an overhead binocular field at the expense of constant fusion”. While the authors repeatedly emphasize that the animal’s binocular field is overhead, the authors’ own data show that the truth is quite different, even orthogonal: the binocular field is in fact centered dead-ahead in front of the animal, tapering to a sliver both above and below the animal. We predict that this paper will be widely cited for something that it does not demonstrate, a concern that is borne out by the paper’s earliest citation.",
"keywords": [
"We wish to correct a potentially serious misperception that arises from the paper \"Rats maintain an overhead binocular field at the expense of constant fusion\" by Wallace et al.",
"20131. The title",
"the abstract and the discussion all emphasize the principal claim that the rat's eye movements \"keep the visual fields of the two eyes continuously overlapping above the animal\". The final sentence reads",
"\"Instead",
"the movements keep the animal’s binocular visual field above it continuously while it is moving\". Many similar statements are found throughout the text and in the Editor's Summary. From this",
"a reader might easily conclude that a rat's binocular visual field is located overhead. However the truth is very different",
"and in fact orthogonal: the binocular field is primarily located dead ahead of the animal. As shown in Figure 5f of the same paper – where the vertical axis represents \"overhead\" in the conventional \"opposite-to-gravity\" sense of the word – the binocular field is a vertical sliver centered on the horizon",
"where it has the widest extent. It narrows towards the top and the bottom",
"and ends in a point at locations both above and below the animal."
],
"content": "Correspondence\n\nWe wish to correct a potentially serious misperception that arises from the paper \"Rats maintain an overhead binocular field at the expense of constant fusion\" by Wallace et al., 20131. The title, the abstract and the discussion all emphasize the principal claim that the rat's eye movements \"keep the visual fields of the two eyes continuously overlapping above the animal\". The final sentence reads, \"Instead, the movements keep the animal’s binocular visual field above it continuously while it is moving\". Many similar statements are found throughout the text and in the Editor's Summary. From this, a reader might easily conclude that a rat's binocular visual field is located overhead. However the truth is very different, and in fact orthogonal: the binocular field is primarily located dead ahead of the animal. As shown in Figure 5f of the same paper – where the vertical axis represents \"overhead\" in the conventional \"opposite-to-gravity\" sense of the word – the binocular field is a vertical sliver centered on the horizon, where it has the widest extent. It narrows towards the top and the bottom, and ends in a point at locations both above and below the animal.\n\nPresumably the authors wanted to say that \"the overhead direction is within the binocular field roughly 50% of the time\", which would be more accurate given the data in Figure 5f. Of course the same is true for a direction pointing almost straight down towards the ground. Meanwhile, the direction in front of the animal is effectively always in the binocular field. Why the singular focus on \"overhead\"? The authors speculate about the need for binocular vision overhead for detecting predatory birds. This is unconvincing. First, only about a quarter of the overhead visual field is binocular (Figure 5f). Given the high cost of missing a predator, it seems the rat must have monocular ways of detecting one. Second, a major benefit of binocular vision is the opportunity for depth measurement by parallax. This works only for nearby objects, rather than distant birds, and would thus apply primarily to the visual field ahead or below the animal.\n\nThe authors conducted a behavioral study to reinforce the idea that rat vision is specialized for processing overhead threats, showing that rats seek shelter under an arch-shaped platform when a drifting bar appeared overhead, but not when it appeared on the side of their enclosure (Figure 6). While we hesitate to engage in behavioral just-so stories, we do note that different sheltering strategies probably exist for different kinds of threats (e.g. land-based and aerial), and that the rat might be wise not enter into a confined space with open sides, when a threat approaches from the side. It is likewise unclear if a drifting bar is equally perceived as a threat when presented above or beside the animal.\n\nRegardless of such ethological speculations, we predict that the paper will be widely cited incorrectly for what it does not demonstrate, as a result of the misleading title and abstract. In fact, this has happened already in what is probably the article's first citation2.",
"appendix": "Author contributions\n\n\n\nMM wrote the first version of the letter and corresponded with the paper’s senior author for clarification. MM and DDC participated in the discussion and revision of the letter.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nWallace DJ, Greenberg DS, Sawinski J, et al.: Rats maintain an overhead binocular field at the expense of constant fusion. Nature. 2013; 498(7452): 65–9. PubMed Abstract | Publisher Full Text\n\nLand MF: Animal vision: rats watch the sky. Curr Biol. 2013; 23(14): R611–3. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1519",
"date": "19 Aug 2013",
"name": "Matteo Carandini",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary can perhaps be reconciled with the original paper by considering the meaning of 'overhead'. In the original paper, 'overhead' seems to mean literally 'above the head'. Because the head typically faces downward (compare Figures 5e and 5f), that actually means 'in front of the animal'. I agree with the commentary that this is possibly a poor word choice: it will likely be interpreted by most readers as 'above the animal'.",
"responses": []
},
{
"id": "1520",
"date": "22 Aug 2013",
"name": "Stephen Van Hooser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present letter refers to a recent (2013) Nature article by Wallace, Greenberg, Sawinski, and colleagues entitled 'Rats maintain an overhead binocular field at the expense of constant fusion'. This Nature article is a tour-de-force examination of eye movements in freely moving rats. The authors created instrumentation and analysis methods to both examine eye movements and rotations, and head movements and rotations, while the rats behaved freely. They found the surprising result that the eyes do not maintain constant fusion, as they do in previously examined animals such as primates. Further, from their analysis of eye movements and head movements, the authors determined the most common region of binocular overlap as the animals wandered about their environment. In head-centric coordinates, this region of binocular overlap reached its greatest horizontal width at a location superior and rostral to the nose (Figure 5e). The authors work also shows that rats tend to keep their heads pitched downward as they move about. Therefore, in body-centric coordinates (using the spine to determine rostral, caudal, superior, inferior directions), this region of binocular overlap reaches its greatest horizontal width at a location directly rostral to the body (Figure 5f).The present letter by Meister and Cox raises a legitimate and serious concern with the \"big picture\" language of the Wallace et al. paper. In the abstract, Wallace el al. report that \"the observed eye movements serve to keep the visual fields of the two eyes continuously overlapping above the animal during free movement, but not continuously aligned\". The coordinate system for the word \"above\" is not clear from context. Meister and Cox makes the important point that a substantial fraction of readers (perhaps an overwhelming majority) are likely to assume that \"above\" is in body-centric coordinates, and to make the erroneous interpretation that the binocular overlap is largest above the animal. That is, many readers will erroneously interpret this sentence to mean that the region of greatest binocular overlap is superior to the spine. This potential misinterpretation is made even more likely by the behavioral experiment reported in Wallace et al., where the authors provide visual stimulation above the spine. The letter writers also point out that there is no rigorous link between the behavioral experiment and the region of binocular overlap. While the behavioral result is interesting, it is unclear if the behavior has anything to do with binocular vision.This letter by Meister and Cox makes a very important contribution. In my opinion, the letter could be improved by making clearer references to the coordinate system being used when a direction is mentioned. For example, the letter writers state \"the binocular visual field is located dead ahead of the animal\". The writers assume that the reader will intuit that body-centric coordinates are intended. However, some readers (such as the authors of Wallace et al.) may find it more natural to assume that head-centric coordinates are intended. I understand what the authors mean by \"opposite-to-gravity\"; the authors assume the animal is on its 4 legs. Referring to coordinate systems linked to the animal would be clearer, in my opinion.",
"responses": []
},
{
"id": "1589",
"date": "28 Aug 2013",
"name": "Michael Land",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 approve of the authors' commentary. The mistake in the original paper comes from a confusion where the binocular field is relative to the head, rather than where it is relative to gravity - which is actually the relevant measurement. Cox and Meister are right to point this out. It is certainly true that the field moves as the head moves, because of the compensatory eye movements, but it remains only partially overhead, and has its maximal extent in the forward direction.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-176
|
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